MOLECULAR AND RADIATION STUDIES ON IMPROVING THE

MOLECULAR AND RADIATIONSTUDIES ON IMPROVING THEAJMALICINE PRODUCTION IN

Catharanthus roseus.

ByISLAM MOHAMED SALAMA EL-SAYED

B. Sc. Agric. (Agric. Botany - Genetics), Fac. of Agric. Al-AzharUniversity 2001

M. Sc. (Agric Botany. - Genetics), Fac. of Agric. Al-Azhar University2006

THESISSubmitted in partial fulfillment of the

Requirements for the Degree

OfDOCTOR OF PHILOSOPHY

InAGRICULTURE SCIENCES

(Agric. Botany - Genetics)

Department of Agric. BotanyFaculty of Agriculture, Cairo

Al-Azhar University

1434 A. H.2013 A. D.

TITLE: MOLECULAR AND RADIATIONSTUDIES ON IMPROVING THEAJMALICINE PRODUCTION IN

Catharanthus roseus.NAME: ISLAM MOHAMED SALAMA EL-SAYED







Al-Azhar University

1434 A. H.2013 A. D.

Supervision committee:Prof. Dr. ABD EL-HADI IBRAHIM HASSN SAYED.Prof. of Genetics, Department of Agricultural Botany, Faculty ofAgriculture, Al-Azhar University.Prof. Dr. MOHAMED ALI ABD EL-RAHMAN.Prof. of Genetics, Department of Agricultural Botany, Faculty ofAgriculture, Al-Azhar University.

APPROVAL SHEET

NAME: ISLAM MOHAMED SALAMA EL-SAYEDTITLE: MOLECULAR AND RADIATION

STUDIES ON IMPROVING THEAJMALICINE PRODUCTION IN








Al-Azhar University1434 A. H.2013 A. D.

Approved by:Prof. Dr. Gomaah Ali Bahgat El – Fadly ………………...Prof. of Genetics, Faculty of Agriculture, Kafrelsheikh University.Prof. Dr. Shafik Ibrahim EL - Morsy El – Bosty ………………...Prof. of Genetics, Faculty of Agriculture, Al-Azhar University.Prof. Dr. Abd El-Hadi Ibrahim Hassn Sayed. ………………...Prof. of Genetics, Faculty of Agriculture, Al-Azhar University.Prof. Dr. Mohamed Ali Abd El-Rahman ………………...Prof. of Genetics, Faculty of Agriculture, Al-Azhar University.

Date: 23 / 1 / 2013 A.D.

i

CONTENTS page

LIST OF TABLES ii

LIST OF FIGURES iii

І. INTRODUCTION 1

ІІ. REVIEW OF LITTERATURE 7

II.1. Effect of Radiation on Indole alkalids biosynthesis. 7

II.2. Radiation doses effect. 14

II.3. Isomerism of Ajmalicine. 15

II.4. Effect of radiation on Isozymes banding patterns. 20

II.5. Effect of radiation on Proteins banding patterns. 30

II.6. Random amplified polymorphic DNA (RAPD). 31

ІІІ. MATERIALS AND METHODS 40

III.1. Materials 40

III.2. Methods 40III.2.1. Gamma radiation treatment. 41

III.2.2. Indole alkalids determination. 41

III.2.3. Isozymes banding patterns analysis. 43

III.2.4. Proteins banding patterns analysis. 48

III.2.5. Randomly amplified polymorphic DNA (RABD). 54

ІV. RESULTS AND DISCUSSION 63VI.1. Radiation treatment. 63

VI.5.Effect of radiation on Indole alkaloids biosynthesis. 64a- First variety LM 64b- Second variety CP3 72

VI.5.1. compare of Ajmalicine production in LM & CP3 variety in Catharanthus rouses. 78VI.3. Effect of radiation on Isozymes banding patterns. 85VI.3.1.Tryptophandecarpoxylase enzyme (TDC). 85VI.3.2.Strrictosidinesynthase enzyme (STR). 90VI.4.Effect of radiation on Protein banding patterns. 93VI.2. DNA finger print analysis. 98VI.2.1. Random amplified polymorphic DNA (RAPD) 98

. a- First variety LM 98VI.2.1.2. RAPD markers of the 11 radiation treatments with 5 RAPD primers. 107VI.2.1.3. Genetic similarity and cluster analysis based on RAPDs markers. 110

b- Second variety CP3 113VI.2.1.4. RAPD markers of the 16 Krad radiation treatments with 10 RAPD primers. 126VI.2.1.5. Genetic similarity and cluster analysis based on RAPDs markers. 128VI.2.2. Similarity and unsimilarity between (LM) & (CP3) Catharanthus roseus

Varieties in Genomic under radiation stress. 130V. SUMMARY 133

VI. REFERENCES 141

ARABIC SUMMARY

ii

LIST OF TABELS PageTable 1: Isomerism of Ajmalicine 17Table 2: stock solution for isozymes analysis. 43Table 3: List of primer names and their nucleotide sequences used in variety

LM (RAPD). 54Table 4: List of primer names and their nucleotide sequences used in variety

CP3 (RAPD). 55Table 5: Effect of the radiation treatments on the plants survivor. 63Table 6: Effect of Gamma radiation treatment on biosynthesis of Indole

alkaloids Catharanthus roseus variety LM concentration by (µg). 72Table 7: Effect of gamma radiation on the Ajmalicine content in C. roseus

variety CP3. 77Table 8: Molecular weight and Intensity of Tryptophan decarpoxylase (TDC)

and Strictosidine synthase (SSS) Enzymes in Catharanthus roseusvariety LM which was treated by Gamma radiation. 93

Table 9: SDS – Page protein analysis of the variety LM which is treated byGamma radiation. 98

Table 10: RAPD profiles of the Catharanthus roseas variety LM which weretreated by Gamma radiation amplified with primer OP-B01. 99

Table 11: RAPD profiles of the Catharanthus roseas variety LM which weretreated by Gamma radiation amplified with primer OP-B07 103

Table 12: RAPD profiles of the Catharanthus roseas variety LM which weretreated by Gamma radiation amplified with primer OP-B11 105

Table 13: RAPD profiles of the Catharanthus roseas variety LM which weretreated by Gamma radiation amplified with primer OP-B12. 106

Table 14: RAPD profiles of the Catharanthus roseas variety LM which weretreated by Gamma radiation amplified with primer OP-F06. 107

Table 15: RAPD markers of the 12 radiation treatment with 5 RAPD primers. 110Table 16: Similarity indices among the 12 radiation treatment Taxa Based on

RAPD-PCR using 5 primers. 112Table 17: RAPD profiles of the Catharanthus roseas variety CP3 which were

treated by Gamma radiation amplified with primer OP-B09. 114Table 18: RAPD profiles of the Catharanthus roseas variety CP3 which were

treated by Gamma radiation amplified with primer OP-C10. 115Table 19: RAPD profiles of the Catharanthus roseas variety CP3 which were



treated by Gamma radiation amplified with primer OP-G17. 118Table 22: RAPD profiles of the Catharanthus roseas variety CP3 which were

treated by Gamma radiation amplified with primer OP-L12. 119Table 23: RAPD profiles of the Catharanthus roseas variety CP3 which were




treated by Gamma radiation amplified with primer OP-Z03. 122Table 27: RAPD markers of the 16Krad with 10 RAPD primers. 127Table 28: Similarity indices among the 8 deferent time Taxa Based on RAPD-

PCR using 10 primers. 129

iii

LIST OF FIGURESFigures Page

Figure 1: The biosynthetic pathway of some phenolic compounds a small-dashed line means multi-steps reactions. 11

Figure 2: Summary of effects reported for various plant hormones and signalcompounds in Catharanthus roseus cell cultures. 12

Figure 3: Proposed model for UV-B mediated signal transduction pathwayleading to activation of the TIA pathway. 15

Figure 4: type of organic components isomerism. 16Figure 5: Construction of the binary plant expression vector pBDH5. 23Figure 6: Structure of the Str1 gene from C. roseas. 27Figure 7: Overview of transcription factors that can interact with the STR and

TDC promoters. 28Figure 8: Model for elicitor signal transduction leading to STR expression. 30Figure 9: Molecular architecture of the activation tagging vectors pVICE

n4HPT and pSKI015. 34Figure 10: Recently developed activation tagging vectors. 34Figure 11: Linkage map of Catharanthus roseas in the F2 population of the

cross ‘Pink Delhi’ × gsr8. 39Figure 12: Indole alkaloids determination by HPLC in Catharanthus roseas

variety LM which is treated by gamma radiation 67Figure 13: Effect of time development on the Ajmalicine production in C.

ruses variety CP3 at 16 Krad. 78Figure 14: HPLC analysis of time development on the Ajmalicine production

in C. ruses. under radiation does rate 16 Krad. 79Figure 15: Tryptophan decarpoxylase diagram for the Catharanthus roseus

Which are treatments by Gamma radiation. 87Figure 16: Strictosidine synthase diagram for the Catharanthus roseus Which

are treatments by Gamma radiation. 91Figure 17: Protein diagram for the Catharanthus roseus which were treated by

Gamma radiation. 96Figure 18: Figure (12): RAPD profiles of the Catharanthus roseas which are

treated by using gamma radiation amplified with 5 primers. 101Figure 19: Dendrogram for the genetic distances relationships among the12

Radiation treatments taxa based on similarity indices data ofRAPD analysis. 112

Figure 20: RAPD profiles of the 8 times treatment which are Gamma Irradiatedat 16 Krad. amplified with 10 primers. 122

Figure 21: Dendrogram for the genetic distances relationships among the8deferent time taxa based on similarity indices data of RAPDanalysis. 130

____________________________________________abstract

ABSTRACT

NAME: Islam Mohamed Salama EL-SayedTITLE: ‘‘Molecular and radiation studies on improving the

Ajmalicine production in Catharanthus roseus”

Elicitations are considered to be an important strategy

towards improve in vitro production of secondary

metabolites. In seedling cultures, biotic and abiotic elicitors

have effectively stimulated the production of plant secondary

metabolites. However, molecular basis of elicitor signaling

cascades leading to increased production of secondary

metabolites of plant cell is largely unknown. Exposure of

Catharanthus roseus cultures to low dose of Gamma

irradiation was found to increase the amount of catharanthine

and transcription of genes encoding tryptophan

decarboxylase (TDC) and strictosidine synthase (STR). In

the present study, the signaling pathway mediating Gamma

irradiation -induced catharanthine accumulation in C. roseus

seedling cultures were investigated.

Catharanthus roseus seedling cultures were exposed to

different low dose of Gamma irradiation in order to induce

alkaloid metabolism. The exposure to Gamma irradiation

elicitors resulted in the transcriptional activation of

tryptophan decarboxylase and in the accumulation of the

monoterpenoid indole alkaloids ajmalicine and catharanthine,

____________________________________________abstract

but not of vindoline. The inability of the seedling cultures to

produce vindoline was related to a lack of expression of the

tryptophan decarboxylase (TDC) and strictosidine synthase

(STR) genes.

Acknowledgement

DEEP THANKS TO ALLAH

The author wishes to express his sincere gratitude and

appreciation to Prof. Dr. ABD EL-HADI IBRAHIM

HASSN SAYED Professor of Genetics, Department of

Agricultural Botany, Faculty of Agriculture, AL-Azhar

University. For his supervision, encouragement, valuable

advises and his unlimited helps in writing thesis.

Special thanks to Prof. Dr. MOHAMED ALI ABD

EL-RAHMAN Professor of Genetics, Department of

Agricultural Botany, Faculty of Agriculture, AL-Azhar

University. For his supervision and help.

The other wishes to acknowledge all members of

Genetics, Department of Agricultural Botany, Faculty of

Agriculture, AL-Azhar University. And all members of

Department of Natural Product Research, National Center for

Radiation Research and Technology, Atomic Energy

Authority.

The author wishes to extend his deep thanks and

appreciation to Genetics, Department of Natural Product

Research, National Center for Radiation Research and

Technology, Atomic Energy Authority. for helping and

providing facilities during the experimental work.

________________________________________________Introduction

1

I- Introduction

Catharanthus roseas is a medicinal plant that produces

clinically useful drugs, such as ajmalicine and vinblastine.

Tryptophan decarboxylase (TDC) and strictosidine synthase

(STR) are two enzymes that act early in the biosynthetic

pathway leading to terpenoid indole alkaloids. Knowledge of

the regulation of these biosynthetic genes will be helpful for

metabolic engineering of terpenoid indole alkaloid

productivity. In suspension-cultured cells, the genes encoding

these enzymes are induced by fungal elicitors, such as

Pythium aphanidermatum culture filtrate or yeast extract. The

mRNA levels of TDC and STR in response to elicitor

treatment of the suspension cultured cells can be visualized

by Northern blotting using radioactively labeled cDNA

probes. This system is used in our laboratory as a bioassay to

help identify the elicitor in fractionated yeast extract. Here

we report the successful use of digoxigenin-labeled probes in

this system. Frank et al., (1996).

________________________________________________Introduction

2

Although the production of most of the current

medicines is based on chemical synthesis, more than 25% of

the current prescribed drugs contain at least one active

ingredient of plant origin. Examples of important plant-

derived pharmaceuticals include the antitumoral taxol and

vinblastine, the antimalarial drug quinine and artemisinin, the

analgesical morphine and codeine. In addition, it has been

estimated that more than 80% of the world’s population in

developing countries depends primarily on herbal medicine

for basic healthcare needs. There is also a revival of

traditional medicine in developed countries and an increase in

the use of herbal remedies. The world market of herbal

medicines’, including herbal and raw material, has been

estimated to have an annual growth rate between 5-15%.

Total global herbal drug market is estimated as US $ 62

billion and it is expected to grow to US $ 5 trillion by the

year 2050. At same time, there is a growing concern on loss

of genetic diversity since about 75% of the 50,000 different

________________________________________________Introduction

3

medicinal plant species in use are collected from the wild.

Moreover, to rely solely on wild spontaneous plants

Antonella et al., (2007).

Catharanthus roseas plant is still the only source for the

powerful antitumor drugs vinblastine and vincristine. Some

other pharmaceutical compounds from this plant, e.g.,

ajmalicine and serpentine are also of economical importance.

These two drugs are produced in small yields within the

plant, which makes them expensive to produce commercially.

Metabolic engineering has focused on increasing flux

through this pathway by various means such as elicitation,

precursor feeding, and introduction of genes encoding

specific metabolic enzymes into the plant. More than 130 C.

roseas alkaloids have been identified, they are sharing many

biosynthetic steps. The early stages of alkaloid biosynthesis

in C. roseas involve the formation of secologanin derived

from the terpenoid biosynthesis and its condensation with

tryptamine to produce the central intermediate strictosidine,

________________________________________________Introduction

4

the common precursor for the monoterpenoid indole

alkaloids. Over twenty enzymes steps are involved in the

biosynthesis of terpenoid indole alkaloids (TIAs) in C.

roseas. Whereas, reported these enzymes take place in at

least three subcellular compartments, the cytosol, the plastids,

and the vacuol. Furthermore, the full characterization of C.

roseas's alkaloid pathway is not yet achieved. A significant

amount of researchs has contributed to characterization of

several individual steps in the biosynthetic pathway of

medicinally valuable alkaloids. However, the available

knowledge of the regulation of this pathway is still sparse.

The conversion of L-tryptophan into tryptamine is catalysed

by the enzyme tryptophan decarboxylase (TDC). This

enzyme is regarded as a putative site for regulatory control of

alkaloid biosynthesis and operates at the interface between

primary and secondary metabolism. The stereospecific

condensation of tryptamine and secolaganin is catalyzes by

strictosidine synthase enzyme (STR, EC 4.3.3.2) to form the

________________________________________________Introduction

5

key indole alkaloid 3 alpha (S)-strictosidine. The STR gene

of C. roseas has been cloned and its nucleotide sequence was

furthermore, determined. Hussein et al., (2008) Reported

that cells of C. roseas (L.) Don. were genetically engineered

to over-express the enzymes strictosidine synthase and

Tryptophan decarboxylase. Cultures transgenic for STR

consistently showed ten fold higher STR. Two such lines

accumulated over 200 mg / L of the glucoalkaloid -1

strictosidine and / or strictosidine-derived terpenoid indole

alkaloids (TIAs), including ajmalicine, catharanthine,

serpentine, and tabersonine, while maintaining wild-type

levels of TDC activity.

In this study tryptophan decarboxylase and strictosidine

synthase genes will be manipulated in order to determine the

genes behavior in C. roseas which are treated by gamma

radiation. The effect of gene within the obtained, in terpenoid

Indole alkaloid production will be investigated. The selection

________________________________________________Introduction

6

of the best dose rate will be chosen depending on the

resistance of radiation treatments.

_________________________________________ Review of Literature

7

II. Review of Literature

II.1. Effect of radiation on Indole alkaloids biosynthesis

and other components:

Sharabash (1970). reported that no signification effect on

chlorophyll concentration in tissue on onion seedling occurred

when exposed to 50 or 5000 rad. of Gamma irradiation.

Sharabash et al., (1972). found that the dose of 10 Krad. of

Gamma irradiation induced a marked increase in the chlorophyll

contents in wheat seedlings.

Tikhonoy et al., (1980). found that irradiation of Datura

innoxia. seeds with Gamma irradiation at 0.5 – 1.0 Krad. reduced

N content.

Schmauder et al., (1985). Cell suspension cultures of

Cinchona succirubra were cultivated in shake cultures and for the

first time in airlift fermenters. Under both conditions L-

tryptophan exerts a stimulatorv effect on alkaloid formation. In

this context the regulatory pattern of some shikimate pathway

enzvmes was investigated in non-supplemented and Tryptophan

supplemented Cinchona cell cultures. A remarkable increase of

trvptophan decarboxylase (TDC) activity was observed in

Cinchona cells under the influence of tryptophan. Apparently, like

in some other indole alkaloid producing cell cultures, a high TDC

activity is a prerequisite for alkaloid formation. Growth pattern

and some enzyme activities of C. succirubra fermenter cultures at

controlled and non-regulated pH levels were followed. Optimum

growth and alkaloid formation were recorded under nonregulated


8

(normal) pH conditions. Abbreviations: TDC = tryptophan

decarboxylase, tyr = L-tyrosine, phe = L-phenylalanine, DAHP =

3-deoxy-D-arabino-heptulosonic acid-7-phosphate, trp = L-

tryptophan, E-4-P = erythrose-4-phosphate, PEP =

phospheenolpyruvate, NDH = malate dehydrogenase, G-6-PDH =

glucose-6-phosphate dehydrogenase, 6-PG-DH = 6-

phosphogluconate dehydrogenase, Ch-mutase = chorismate

mutase, AS-synthase = anthranilate svnthase, n.d. = not

determined

Georgiveva (1987). reported that increasing the Gamma

irradiation doses caused increases in the content of quiones in

pollen tubes of Lilum regali. and Beta vulgares.

Lucumi et al., (2001). A cell suspension culture of

Tabernaemontana divaricata, that had lost alkaloid production,

was still capable of producing a similar pattern of alkaloids as

directly after its initiation. When fed with early precursors, such

as tryptamine and loganin, 57% of the precursors were converted

into indole alkaloids such as strictosidine, vallesamine, O-

acetylvallesamine and voaphylline. Apparently most of the cell

factory has remained stable during the many years of sub

culturing. Only an early step of the biosynthesis the flux seems to

be diverted to other pathways.

Felipe et al., (2002). Catharanthus roseus cell cultures were

exposed to different conditions in order to induce alkaloid

metabolism. The exposure to jasmonate and fungal elicitors

resulted in the transcriptional activation of Tryptophan


9

decarboxylase and in the accumulation of the monoterpenoid

indole alkaloids ajmalicine and catharanthine, but not of

vindoline. The inability of the cell cultures to produce vindoline

was related to a lack of expression of the desacetoxyvindoline 4-

hydroxylase (D4h) gene. Southern blot analysis revealed that D4h

gene was not lost in the cell cultures.

Felipe and Victor (2003). The Scientific Research Center of

Yucatan (CICY, for its Spanish acronym) was founded in

November 1979 as part of an effort to decentralize scientific

activities from Mexico City. Several of the research programs

carried out at CICY makes use of plant tissue culture techniques

for their development. For this article, we have reviewed results

obtained in research projects oriented towards basic plant biology

questions, as well as towards the micropropagation of

economically important species, and the production of secondary

metabolites.

Young et al., (2003). Production of camptothecin (CPT)

from callus cultures of Camptotheca acuminata Decne was

affected by light and culture conditions. Among the culture media

tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L

2,4-D, 2 times of MS medium vitamins, 500 mg/L casein

hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated

charcoal, and 0.15% (w/v) gelite was used for callus induction.

The highest cell growth and CPT production were obtained in

dark and green light condition, respectively. Photoperiod has no

effect on cell growth and CPT production. Both cell growth and


10

CPT production were also influenced by combination ratio of red

and blue light. Cell growth and CPT production were the highest

in the ratio of red and blue light, 90:10.

Carolyn et al., (2004). The optimum growth stage for

enhancing ajmalicine production in Catharanthus roseus cultures

with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10

or 100 µM on day 6 gave a maximum ajmalicine production of

10.2 mg l−1, a 300% increase over that of non-elicited cultures.

Natali and Robert (2007). Besides alkaloids Catharanthus

roseus produces a wide spectrum of phenolic compounds, this

includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as

well as phenylpropanoids such as cinnamic acid derivatives,

flavonoids and anthocyanins. The occurrence of these compounds

in C. roseus is reviewed as well as their biosynthesis and the

regulation of the pathways. Both types of compounds compete

with the indole alkaloid biosynthesis for chorismate, an important

intermediate in plant metabolism. The biosynthesis C6C1

compounds are induced by biotic elicitors.


11

Fig. (1). The biosynthetic pathway of some phenolic compounds. Asmall-dashed line means multi-steps reactions.


12

Fig. (2). Summary of effects reported for various plant hormones andsignal compounds in Catharanthus roseus cell cultures. A continued-linemeans one-step reaction. A small-dashed line means multi-step reactions. Abigdashed line with + or – indicates activation or inhibition of gene(s)expression, enzyme activity or end product level. A big-dashed line with both+ and-means a concentration-dependent activation or inhibition. A strongactivation or-inhibition is indicated by ++ or – –

Antonio et al., (2008). For nutritional purposes, a survey of

the vitamin B6 levels from a variety of commercial presentations

of table olives was carried out, taking into account the three main

processing types (Spanish-style, directly brined and ripe olives).

The analysis was performed by HPLC, following the official


13

French method for vitamin B6 determination in foodstuffs. In-

house validation data for two commercial table olives showed that

themethod precision was good (coefficient of variation <6%) and

recovery was quantitative (104% on average). There was a wide

range of values for vitamin B6 in table olives (0–69.3 μg/100 g

edible portion). The highest mean content was found in directly

brined olives (33.9 μg/ 100 g edible portion) followed by Spanish-

style (14.4 μg/ 100 g) and ripe olives (4.3 μg/100 g). On average,

samples of the Gordal and Carrasqueña cultivars showed the

highest vitamin B6 content in the case of Spanish-style olives, but

in directly brined olives as well as in ripe olives the effect of

cultivar was not statistically significant (p<0.05).

Hussein et al., (2008). Suspension, calli and leaves of

Egyptian Catharanthus roseas (L.) were genetically engineered to

over-express the two enzymes Tryptophandecarboxylase and

Strrictosidine syntheses, which catalyze key steps in the

biosynthesis of terpenoid indole alkaloids, using Agrobacterium-

mediated transformation with the two corresponding genes. The

percentages of total alkaloids, vinblastine and vincristine were

recorded as relative to C. roseas intact plant. The highest values

of total alkaloids (14.47%), vinblastine (13.62 %) and vincristine

(11.5%) of transgenic leaf cell cultures were recorded with (CS7).

However, (C4) transformed leaf calli cultures gave 10.48, 8.3 and

6.19 (%) for total alkaloid, vinblastine and vincristine,

respectively. On other hand, the maximum values of total

alkaloids (16.47 %), vinblastine (18.09 %) and vincristine (14.16


14

%) were recorded with (L3) transgenic leaf in vitro derived

germinated seeds) as compared with other selected cell lines.

II.2. Radiation doses effect

Shilpa and Jayabaskaran (2007). Elicitations are

considered to be an important strategy towards improved in vitro

production of secondary metabolites. In cell cultures, biotic and

abiotic elicitors have effectively stimulated the production of

plant secondary metabolites. However, molecular basis of

elicitorsignaling cascades leading to increased production of

secondary metabolites of plant cell is largely unknown. Exposure

of Catharanthus roseus cell suspension culture to low dose of

UV-B irradiation was found to increase the amount of

catharanthine and transcription of genes encoding Tryptophan

decarboxylase (Tdc) and strictosidine synthase (STR). In the

present study, the signaling pathway mediating UV-B-induced

catharanthine accumulation in C. roseus suspension cultures were

investigated.


15

Proposed model for UV-B mediated signal transduction pathway leading toactivate

Fig. (3). Proposed model for UV-B mediated signal transductionpathway leading to activation of the TIA pathway.

II.3. Isomerism of Ajmalicine

Isomers are molecules that have the same molecular formula,

but have a different arrangement of the atoms in space. That

excludes any different arrangements which are simply due to the

molecule rotating as a whole, or rotating about particular bonds.

In structural isomerism, the atoms are arranged in a

completely different order. This is easier to see with specific

examples.

What follows looks at some of the ways that structural

isomers can arise. The names of the various forms of structural

isomerism probably don't matter all that much, but you must be


16

aware of the different possibilities when you come to draw

isomers. www.pubchemsubstance.com

Fig. (4) type of organic components isomerism


17

Isom

eris

m (4

)A

jmal

icin

e H

CL

Isom

eris

m (5

)19

-epi

-Ajm

alic

ine

Isom

eris

m (6

)Te

trahy

droa

lsto

nine

Pres

twic

k

3D n

ot a

vaila

ble

2D-S

tric

ture

Mo

lecu

lar

Wei

gh

t3

88

.88

78

[g

/m

ol]

Mo

lecu

lar

Form

ula

C2

1H

25C

lN2O

3

H-B

on

d D

ono

r2

H-B

on

d A

ccep

tor

5R

ota

tab

le B

on

d C

ou

nt

2

Exa

ct M

ass

38

8.1

55

37

Mo

no

Iso

top

ic M

ass

38

8.1

55

37

To

po

log

ical

Po

lar

Su

rfac

e A

rea

54

.6H

eavy

Ato

m C

ou

nt

27

Form

al C

har

ge

0C

om

ple

xity

60

6Is

oto

pe A

tom

Cou

nt

0

Def

ined

Ato

m S

tere

oC

ente

r C

ou

nt

0U

nd

efin

ed A

tom

Ste

reo

Cen

ter

Co

un

t4

Def

ined

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Un

def

ined

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Co

vale

ntl

y-B

on

ded

Un

it C

ou

nt

2

3D-S

tric

ture

2D-t

rict

ure

Mo

lecu

lar

Wei

gh

t3

52

.42

68

6 [

g/

mo

l]M

ole

cula

r Fo

rmu

laC

21H

24N

2O

3

XLo

gP

3-A

A2

.7H

-Bo

nd

Don

or

1H

-Bo

nd

Acc

epto

r5

Ro

tata

ble

Bo

nd

Co

un

t2

Exa

ct M

ass

35

2.1

78

69

3M

on

oIs

oto

pic

Mas

s3

52

.17

86

93

To

po

log

ical

Po

lar

Su

rfac

e A

rea

54

.6

Hea

vy A

tom

Co

un

t2

6Fo

rmal

Ch

arg

e0

Co

mp

lexi

ty6

06

Iso

tope

Ato

m C

oun

t0

Def

ined

Ato

m S

tere

oC

ente

r C

ou

nt

4U

nd

efin

ed A

tom

Ste

reo

Cen

ter

Co

un

t0

Def

ined

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Un

def

ined

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Co

vale

ntl

y-B

on

ded

Un

it C

ou

nt

1

3D-S

tric

ture

2D-t

rict

ure

Mol

ecul

ar W

eigh

t35

2.42

686

[g/m

ol]

Mol

ecul

ar F

orm

ula

C21

H24

N2O

3

XLo

gP3-

AA

2.7

H-B

ond

Don

or1

H-B

ond

Acce

ptor

5Rot

atab

le B

ond

Cou

nt2

Exac

t M

ass

352.

1786

93

Mon

oIso

topi

c M

ass

352.

1786

93To

polo

gica

l Pol

ar S

urfa

ce A

rea

54.6

Hea

vy A

tom

Cou

nt26

Form

al C

harg

e0

Com

plex

ity60

6Is

otop

e Ato

m C

ount

0D

efin

ed A

tom

Ster

eoCen

ter

Cou

nt3

Und

efin

ed A

tom

Ste

reoC

ente

r Cou

nt1

Def

ined

Bon

d Ste

reoC

ente

r Cou

nt0

Und

efin

ed B

ond

Ster

eoCen

ter

Cou

nt0

Cov

alen

tly-B

onde

d U

nit

Cou

nt1

Table No. (1) Isomerism of Ajmalicine


18

Isom

eris

m(4

)A

jmal

icin

e H

CL

Isom

eris

m(5

)19

-epi

-Ajm

alic

ine

Isom

eris

m(6

)T

etra

hydr

oals

toni

nePr

estw

ick

3D n

ot a

vaila

ble

2D-S

tric

ture

Mo

lecu

lar

Weig

ht

38

8.8

87

8 [

g/

mo

l]M

ole

cula

r Fo

rmu

laC

21H

25C

lN2O

3

H-B

on

d D

on

or

2

H-B

on

d A

ccep

tor

5R

ota

tab

le B

on

d C

ou

nt

2E

xact

Mass

38

8.1

55

37

Mo

no

Iso

top

ic M

ass

38

8.1

55

37

To

po

log

ical P

ola

r S

urf

ace

Are

a5

4.6

Heavy A

tom

Co

un

t2

7Fo

rmal C

harg

e0

Co

mp

lexit

y6

06

Iso

tope A

tom

Cou

nt

0D

efi

ned

Ato

m S

tere

oC

en

ter

Co

un

t0

Un

defi

ned

Ato

m S

tere

oC

en

ter

Co

un

t4

Defi

ned

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Un

defi

ned

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Co

vale

ntl

y-B

on

ded

Un

it C

ou

nt

2

3D-S

tric

ture

2D-t

rict

ure

Mo

lecu

lar

Weig

ht

35

2.4

26

86

[g

/m

ol]

Mo

lecu

lar

Fo

rmu

laC

21H

24N

2O

3

XLo

gP

3-A

A2

.7H

-Bo

nd

Don

or

1H

-Bo

nd

Acc

ep

tor

5R

ota

tab

le B

on

d C

ou

nt

2

Exact

Mass

35

2.1

78

69

3M

on

oIs

oto

pic

Mass

35

2.1

78

69

3T

op

olo

gic

al P

ola

r S

urf

ace

Are

a5

4.6

Heavy A

tom

Co

un

t2

6Fo

rmal C

harg

e0

Co

mp

lexit

y6

06

Iso

tope A

tom

Cou

nt

0

Defi

ned

Ato

m S

tere

oC

en

ter

Co

un

t4

Un

defi

ned

Ato

m S

tere

oC

en

ter

Co

un

t0

Defi

ned

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Un

defi

ned

Bo

nd

Ste

reo

Cen

ter

Co

un

t0

Co

vale

ntl

y-B

on

ded

Un

it C

ou

nt

1

3D-S

tric

ture

2D-t

rict

ure

Mol

ecula

r W

eigh

t352.4

2686 [

g/m

ol]

Mol

ecula

r Fo

rmula

C21H

24N

2O

3

XLo

gP3

-AA

2.7

H-B

ond D

onor

1H

-Bon

d A

ccep

tor

5Rot

atab

le B

ond C

ount

2Exa

ct M

ass

352.1

78693

Mon

oIso

topic

Mas

s352.1

78693

Top

olog

ical

Pol

ar S

urf

ace

Are

a54.6

Hea

vy A

tom

Cou

nt

26

Form

al C

har

ge

0Com

ple

xity

606

Isot

ope

Ato

m C

ount

0D

efin

ed A

tom

Ste

reoC

ente

r Cou

nt

3U

ndef

ined

Ato

m S

tere

oCen

ter

Cou

nt

1D

efin

ed B

ond S

tere

oCen

ter

Cou

nt

0

Undef

ined

Bon

d Ste

reoC

ente

r Cou

nt

0Cov

alen

tly-

Bon

ded

Unit C

ount

1

Table No. (1) Continue


19

Isom

eris

m(7

)Te

trahy

droa

lsto

nine

Isom

eris

m(8

)A

jmal

icin

e m

ethy

l ace

tal

Isom

eris

m(9

)A

jmal

icin

e A

lsto

nine

,3,4

,5,6

-tetra

hydr

o

3Dno

t ava

ilabl

e2D

-Str

ictu

reM

olec

ular

Wei

ght

352.

4268

6 [g

/mol

]

Mol

ecul

ar F

orm

ula

C21

H24

N2O

3

XLo

gP3-

AA

2.7

H-B

ond

Don

or1

H-B

ond

Acc

epto

r5

Rot

atab

le B

ond

Cou

nt2

Exac

t M

ass

352.

1786

93

Mon

oIso

topi

c M

ass

352.

1786

93

Topo

logi

cal P

olar

Sur

face

Are

a54

.6

Hea

vy A

tom

Cou

nt26

Form

al C

harg

e0

Com

plex

ity60

6

Isot

ope

Ato

m C

ount

0

Def

ined

Ato

mSte

reoC

ente

r Cou

nt4

Und

efin

ed A

tom

Ste

reoC

ente

r Cou

nt0

Def

ined

Bon

d Ste

reoC

ente

r Cou

nt0

Und

efin

ed B

ond

Ste

reoC

ente

r Cou

nt0

Cov

alen

tly-B

onde

d U

nit

Cou

nt1

3D n

ot a

vaila

ble

2D-S

tric

ture

Prop

ertie

s not

ava

ilabl

e3D

-Str

ictu

re2D

-tric

ture

Mol

ecul

ar W

eigh

t35

2.42

686

[g/m

ol]

Mol

ecul

ar F

orm

ula

C21

H24

N2O

3

XLo

gP3-

AA

2.7

H-B

ond

Don

or1

H-B

ond

Acc

epto

r5

Rot

atab

le B

ond

Cou

nt2

Exac

t M

ass

352.

1786

93

Mon

oIso

topi

c M

ass

352.

1786

93

Topo

logi

calP

olar

Sur

face

Are

a54

.6

Hea

vy A

tom

Cou

nt26

Form

al C

harg

e0

Com

plex

ity60

6

Isot

ope

Ato

m C

ount

0

Def

ined

Ato

m S

tere

oCen

ter

Cou

nt4

Und

efin

ed A

tom

Ste

reoC

ente

r Cou

nt0

Def

ined

Bon

d Ste

reoC

ente

r Cou

nt0

Und

efin

ed B

ond

Ste

reoC

ente

r Cou

nt0

Cov

alen

tly-B

onde

d U

nit

Cou

nt1

Table No. (1) Continue


20

II.4. Effect of radiation on Isozymes banding patterns.

Flaig and Schmid (1966). reported that differences in the

content on different compounds or enzyme activities have been

observed in irradiated seeds, seedling and plant organs. Thus the

content of sucrose and other sugars increased with increasing

doses of irradiation. Changes in the activities of different

enzymes, such as acid phosphatase, peroxidase, polyphenralase

has been observed, sometimes an increase in activity could be

observed using low doses. For example, respiration is often

increased and content of chlorophyll can be increased after

irradiation.

Toth et al., (1983). Said that there were differences in

isoperoxidase patterns within and between from seeds of Digitalis

lanata treated with Gamma rays and another untreated

plants.Georgieva (1987). reported that increasing the Gamma

irradiation doses enhanced peroxidase activity Lilium regali. and

Beta vulgaris.

Mollenschott and Berlin (1984). The purification of

Tryptophandecarboxylase from Catharanthus roseas (TDC, E.C.:

4.1.1.27), to apparent homogeneity, the enzyme represents a

soluble protein with a molecular weight of 115 000 + 3 000,

consisting of 2 identical subunits of 54 000 + 1 000. The pI was

estimated to be 5.9 and the K m for L-tryptophan was found to be

7.5 × 10 -5 M. Phenylalanine, tyrosine and DOPA were not

decarboxylated by tryptophan decarboxylase from Catharanthus

cells. Similar to the aromatic amino acid decarboxylase from hog


21

kidney the enzyme does not appear to be obligatorily dependent

on exogenously supplied pyridoxal phosphate, as it seems to

contain a certain amount of this cofactor. The average percentage

of TDC in the cells was found to be 0.002% in the growth

medium while the level increased up to 0.03% when indole

alkaloid biosynthesis was induced. The role of the protein as a

bottleneck enzyme of indole alkaloid biosynthesis is discussed.

Peter and Frank (1991) suspension-cultured cells of

Catharanthus roseus (L.) G. Don were immobilized on glass fibre

mats and cultivated in shake flasks. The highly-aggregated

immobilized cells exhibited a slower growth rate and accumulated

reduced levels of tryptamine and indole alkaloids, represented by

catharanthine and ajmalicine, in comparison to cells in

suspension. The increased total protein synthesis in immobilized

cells suggests a diversion of the primary metabolic flux toward

protein biosynthetic pathways and away from other growth

processes. In-vitro assays for the specific activity of tryptophan

decarboxylase (TDC) and Tryptophan synthase (TS) suggest that

the decreased accumulation of tryptamine in immobilized cells

was due to reduced tryptophan biosynthesis. The specific activity

of TDC was similar in immobilized and suspension-cultured cells.

However, the expression of TS activity in immobilized cells was

reduced to less than 25% of the maximum level in suspension-

cultured cells. The reduced availability of a free tryptophan pool

in immobilized cells is consistent with the reduced TS activity.

Reduced tryptamine accumulation, however, was not responsible


22

for the decreased accumulation of indole alkaloids in immobilized

cells. Indole alkaloid accumulation increased to a similar level in

immobilized and suspension- cultured cells only after the addition

of exogenous secologanin to the culture medium. The addition of

tryptophan resulted in increased accumulation of tryptamine, but

had no effect on Indole alkaloid levels. Reduced biosynthesis of

secologanin, the monoterpenoid precursor to Indole alkaloids, in

immobilized cells is suggested. Immobilization does not appear to

alter the activity of Indole alkaloid biosynthetic enzymes in our

system beyond, and including, Strrictosidinesynthase.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28)

catalyses a key step in the biosynthesis of terpenoid indole

alkaloids in C. roseus by converting tryptophan into tryptamine.

Hardly any tdc mRNA could be detected in hormone-independent

callus and cell suspension cultures transformed by the oncogenic

T-DNA of Agrobacterium tumefaciens. Oscar et al., (1995).

Supply of tryptamine may therefore represent a limiting factor in

the biosynthesis of alkaloids by such cultures. To investigate this

possibility, chimaeric gene constructs, in which a TDC cDNA is

linked in the sense or antisense orientation to the cauliflower

mosaic virus 35S promoter and terminator, were introduced in C.

roseus cells by infecting seedlings with an oncogenic A.

tumefaciens strain. In the resulting crown gall tumour calluses

harbouring the tdc sense construct, an increased TDC protein

Level, TDC activity and tryptamine content but no

significant increase in terpenoid indole alkaloid production was


23

observed compared to empty-vector-transformed tumor calluses.

In turnout calluses containing the TDC antisense construct,

decreased levels of TDC activity were measured. Factors which

might be responsible for the lack in increased terpenoid indole

alkaloid production in the tdc cDNA overexpressing crown gall

calluses are discussed.

Fig. (5). Construction of the binary plant expression vector pBDH5 andgeneration of transcriptional fusions between an artificially completed tdccDNA sequence and the CaMV 35S promoter and terminator in pBDH5.Construct pTDCs leads to overexpression of tdc mRNA, while pTDCa leadsto expression of antisense tdc RNA. Abbreviations, M.C.S., multiple cloningsite; LB, RB, left and right T-DNA border repeats; P35S and Pnos refer to theCaMV 35S and nopaline synthase promoter sequences; T35S and Trios referto the CaMV 35S and nopaline synthase terminator sequences; NPTII,neomycin phosphotransferase gene; Km, bacterial kanamycin resistanceselection marker; Cb, bacterial carbenicillin resistance selection marker.


24

Serap et al., (1998). The transgenic cell line of

Catharanthus roseus (L.) G. Don S10 was used to study the effect

of the presence of the synthetic auxins naphthalene acetic acid and

2,4-dichlorophenoxyeacetic acid in the culture medium on the

accumulation of terpenoid indole alkaloids. Line S10 carries a

recombinant, constitutively over-expressed version of the

endogenous strictosidine synthase gene. The experiments were

carried out using a two-stage culture system, consisting of a

growth phase of 7 to 10 days and a production phase of 14 or 30

days. The hormonal composition was a crucial factor when

formulating both the growth and the production media. It was

determined that the presence of naphthalene acetic acid during the

production phase led to lower levels of alkaloid accumulation.

The presence of 2,4-dichlorophenoxyacetic acid in the growth

medium reduced culture aggregation and repressed secondary

metabolism. Cultures grown in medium containing 2,4-

dichlorophenoxyacetic acid showed reduced capacity to supply

biosynthetic precursors, which resulted in low levels of

accumulation of terpenoid indole alkaloids. The expression of the

gus and strictosidine synthase transgenes, measured at the enzyme

level, was similarly high under all conditions tested.

In situ RNA hybridization and immunocytochemistry were

used to establish the cellular distribution of monoterpenoid indole

alkaloid biosynthesis in Madagascar periwinkle (Catharanthus

roseus). Tryptophan decarboxylase (TDC) and strictosidine

synthase (STR1), which are involved in the biosynthesis of the


25

central intermediate strictosidine, and desacetoxyvindoline 4-

hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase

(DAT), which are involved in the terminal steps of vindoline

biosynthesis, were localized. TDC and STR1 mRNAs were

present in the epidermis of stems, leaves, and flower buds,

whereas they appeared in most protoderm and cortical cells

around the apical meristem of root tips. Benoit et al., (1999) in

marked contrast, d4h and dat mRNAs were associated with the

laticifer and idioblast cells of leaves, stems, and flower buds.

Immunocytochemical localization for TDC, D4H, and DAT

proteins confirmed the differential localization of early and late

stages of vindoline biosynthesis. Therefore, we concluded that the

elaboration of the major leaf alkaloids involves the participation

of at least two cell types and requires the intercellular

translocation of a pathway intermediate. A basipetal gradient of

expression in maturing leaves also was shown for all four genes

by in situ RNA hybridization studies and by complementary

studies with dissected leaves, suggesting that expression of the

Vindoline pathway occurs transiently during early leaf

development. These results partially explain why attempts to

produce Vindoline by cell culture technology have failed.

Giancarlo et al., (1999). Strictosidine synthase (STR) is a

key enzyme in the biosynthesis of terpenoid indole alkaloids. This

class of secondary metabolites harbours several pharmaceutically

important compounds used, among other applications, in cancer

treatment. Terpenoid indole alkaloid biosynthesis and expression


26

of biosynthetic genes including Str1 is induced by fungal elicitors.

To identify elicitor-responsive regulatory promoter elements and

trans-acting factors, the single-copy Str1 gene was isolated from

the subtropical plant species Catharanthus roseus (Madagascar

periwinkle). Str1 upstream sequences conferred elicitor-

responsive expression to the _-glucuronidase (gusA) reporter gene

in transgenic tobacco plants. Main enhancer sequences within the

Str1 promoter region studied were shown to be located between

_339 and _145. This region and two other regions of the promoter

bound the tobacco nuclear protein factor GT-1. A G-box located

around position _105 bound nuclear and cloned G-box-binding

factors (GBFs). A mutation that knocked out GBF binding had no

measurable effect on expression, which indicates that the G-box is

not essential for the elicitor responsiveness of the Str1 promoter.

No obvious homologies with promoter elements identified in

other elicitor-responsive genes were observed, suggesting that the

Str1 gene may depend on novel regulatory mechanisms.

Fig. (6). Structure of the Str1 gene from C. roseas. a. Restriction map ofthe insert of the genomic clone pGCR18. Restriction sites are indicated forXhoI (X), BglII (B), HindIII (H), EcoRI (E) and BamHI (Ba). The outer XhoIsites are derived from the lambda vector. The region indicated with a hatchedbar was sequenced, and a detailed map is shown in b. b. Detailed map of the


27

Str1 gene and flanking sequences. The transcribed region is indicated withblack and white boxes. The black portions represent coding sequences. The50- and 30-untranslated regions as well as two introns are shown in white.The promoter region shown in Figure 2 is represented by a hatched bar.Restriction sites are indicated as in a. The XhoI site is derived from thelambda vector.

Leslie et al., (2000). Plants respond to pathogen attack by

induction of various defence responses, including the biosynthesis

of protective secondary metabolites. In Catharanthus roseus, the

elicitor-induced expression of the terpenoid indole alkaloid

biosynthetic gene Strictosidine synthase (STR) is mediated via the

plant stress hormone jasmonate. In the promoters of several

defence-related genes, cis-acting elements have been identified

that are important for transcriptional regulation upon stress

signals. Here we show that an upstream region in the STR

promoter confers responsiveness to partially purified yeast elicitor

and jasmonate. Yeast one-hybrid screening with this element as a

bait identified a MYB-like protein, which shows high homology

to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses

showed that the STR promoter fragment contained a novel

binding site for BPF-1-like proteins with higher binding affinity

than the previously described box P. CrBPF-1 mRNA

accumulated rapidly in elicitor-treated C. roseus suspension cells,

whereas no induction was observed with jasmonate. Inhibitor

studies indicated that CrBPF-1 plays a role in an elicitor-

responsive but jasmonate-independent signal transduction

pathway, acting downstream of protein phosphorylation and

calcium influx.


28

FIG. (7). Overview of transcription factors that can interact with theSTR and TDC promoters. Perception of YE leads to an increase in JA levels,which is necessary for the activation of the ORCA transcription factors.Although the cellular location of the YE receptor is unknown, it is tentativelyplaced in the plasma membrane. The ORCA transcription factors can activategene expression via interaction with the TDC promoter and the RV fragmentof the STR promoter. Although the ORCA binding site in the TDC promoterhas not been precisely mapped, it is tentatively indicated downstream of theDB fragment. In addition, YE rapidly induces the accumulation of mRNAsencoding ZCT proteins, which can repress gene expression via binding to theDB fragment of the TDC promoter and the BA and, to a lesser extent, the RVfragments of the STR promoter. Also, YE induces accumulation of mRNAencoding CrBPF1, which is putatively involved in regulation of STR viainteraction with the BA region. CrGBF transcription factors can repress STRpromoter activity via binding to the NR region.

Bea et al., (2004). In Catharanthus roseas cell suspensions,

the expression of several terpenoid indole alkaloid biosynthetic

genes, including two genes encoding strictosidinesynthase (STR)

and tryptophan decarboxylase (TDC), is coordinately induced by

fungal elicitors such as yeast extract. To identify molecular

mechanisms regulating the expression of these genes, a yeast one-

hybrid screening was performed with an elicitor-responsive part

of the TDC promoter. This screening identified three members of


29

the Cys2/His2-type (transcription factor IIIA-type) zinc finger

protein family from C. roseas, ZCT1, ZCT2, and ZCT3. These

proteins bind in a sequence-specific manner to the TDC and STR

promoters in vitro and repress the activity of these promoters in

trans-activation assays. In addition, the ZCT proteins can repress

the activating activity of APETALA2/ethylene responsefactor

domain transcription factors, the ORCAs, on the STR promoter.

The expression of the ZCT genes is rapidly induced by yeast

extract and methyljasmonate. These results suggest that the ZCT

proteins act as repressors in the regulation of elicitor-induced

secondary metabolism in C. roseas.

Elizabeta et al., (2004). Vindoline, the major alkaloid in

cultures of Catharanthus roseus shoots, reached 2 mg g−1 dry wt

after 27 d in culture. Maximal vindoline accumulation coincided

with maximum activities of deacetoxyvindoline 4-hydroxylase,

deacetylvindoline acetyl-CoA acetyl transferase and tryptophan

decarboxylase. Shoot exposure to jasmonate shortened the time

required for the maximal vindoline accumulation to 14 d.


30

Fig. (8). Model for elicitor signal transduction leading to Str expression.The model shows the position of CrBPF-1 in a JA-independent elicitor signaltransduction pathway. Protein phosphorylation and calcium influx arerequired for the activation of the ODA pathway, as well as for the induction ofCrBPF-1. The position of the TATA box and the jasmonate- and elicitor-responsive element (JERE) are indicated.

II.5. Effect of radiation on Proteins banding patterns.

El-Shebawi (1984). reported that irradiation with doses of

Gamma rays 0.05, 0.25, 0.5, 1.0 and 2.0 KGy. caused significant in

some protein bands of broad bean globulin fraction. He observed

the presence of additional bands in electrophoretic diagram of

broad bean protein.

Afify and Shousha (1988). investigated changes in the

protein patterns of five soybean cultivar, separated by sodium

dodecyle sulphate play acrylamide gel electrophoresis (SDS-

PAGE), after exposure to Gamma irradiation, and they attributed

changes in protein patterns to partial protein decemination,

scission of peptide and disulfide bands and addition to aromatic

and heterocyclic amino acid residues.

Novak et al., (1990). found that protein banding pattern of

the original Grandnain and the mutant were different. Probably


31

the most prominent differences was in the intensity quantity and

mobility of a major protein raving a molecular might of about 33

Kda. The original clone showed a densely stained band which

migrated faster (Rf = 0.44) than that of the mutant Gn = 60 Gy/A.

in addition three other bands were nat observed in the mutant, but

only in the original grand nain. Such band (i.e. proteins) were less

densely stained with an Rf value of 0.19, 0.31 and 0.64 and

molecular weight of about 94 and 26 Kda, respectively.

Cambecedes et al., (1991). found that among 20

regeneration plants from irradiation tests on Lonicera nitida

Maigrum, only one very slender mutants was characterized by the

lock of a 52 Kda. Band in the banding pattern of denaturated

soluble protein.

Kazuyuki et al., (2001). A peptidase (GICP) that cleaves the

Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a

sequence in phenylalanine oxidase, was purified from bovine

pancreas. The purified enzyme had an Mr of approximately

29,000, as determined by SDS-PAGE, and its N-terminal

sequence was identical to that of bovine pancreatic elastase II.

The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1

(Km 5 8.3 mM kcat 5 2.1 s21) and the catalytic efficiency (2.6 3

105 M21s21) was comparable to those of elastase II from porcine

pancreas and rat mesenteric arterial bed perfusate. The P1 site

specificity of GICP toward oxidized insulin A and B chains

suggested that major cleavage sites were the peptide bond at the

C-terminal side of Gln, Leu, His, and Tyr residues.


32

Rashed et al., (1997). treated high and low yielding soybean

plants with 15 Krad. of Gamma rays and analyzed then for protein

electrophoresis patterns (SDS – PAGE). The low and high

yielding treated plants were characterized by appearance and / or

the disappearance of some minor bands, which confirmed the

association between these bands and the gene expression of high

yielding trait.

II.6. Randomly amplification 0f polymorphic DNA

(RAPD)

Annemarie et al., (1993) reported that cytochrome P-450

monooxygenases are membrane-bound enzymes involved in a

wide range of biosynthetic pathways in plants. An efficient PCR

strategy for isolating cytochrome P-450 cDNA clones from plant

cDNA libraries is described. A set of degenerate primers for PCR

amplification was designed to recognize nucleotide sequences

specifying the highly conserved haembinding region of

cytochrome P-450 proteins. Using this primer set and a non-

specific primer, complementary to either the poly (A) tail of the

cDNA clones or a phage vector sequence, the others isolated 16

different cytochrome P-450 cDNA sequences from a cDNA

library of Catharanthus roseus.

Maria and Matgorzata (1999) infected Alstroemeria

seedlings with naturally infected lily 'Casablanca' with stunting

and flower bud deficiency phytoplasma resulted 3-4 weeks after

top grafting in chlorotic and/or necrotic stripes, whitening of the

leaves, shoot necrosis and die back. Flower discoloration or


33

malformation was not observed. Attempts to transmit

phytoplasma from naturally infected lily and experimentally

infected Alstroemeria to Catharanthus roseus by top grafting

resulted in stunted growth, dull yellowing and malformation of

the leaves in 4-6 weeks. Some plants were temporary entirely

vegetative and did not produce flowers. The periwinkle plants that

were bridged by Cuscuta odorata from the diseased lilies and

Alstroemerias showed similar symptoms as top-grafted ones.

With the universal primer pairs rU3/fU5 specific PCR product

with expected length -900 was amplified from samples collected

from lilies with severe symptoms and top grafted test plants. All

PCR products used for RFLP analysis after digestion with Alu I

showed the same restriction profiles. Position of three obtained

bands corresponded to the lengths of the DNA fragments of

American aster yellows (AAY) phytoplasma group.

A significant limitation of classical loss-of function screens

designed to dissect genetic pathways is that they rarely uncover

genes that function redundantly, are compensated by Helen et al.,

(2004) alternative metabolic or regulatory circuits, or which have

an additional role in early embryo or gametophyte development.

Activation T-DNA tagging is one approach that has emerged in

plants to help circumvent these potential problems. This technique

utilises a T-DNA sequence that contains four tandem copies of the

cauliflower mosaic virus (CaMV) 35S enhancer sequence. This

element enhances the expression of neighbouring genes either

side of the randomly integrated T-DNA tag, resulting in gain-of-


34

function phenotypes. Activation tagging has identified a number

of genes fundamental to plant development, metabolism and

disease resistance in Arabidopsis. This review provides selected

examples of these discoveries to highlight the utility of this

technology. The recent development of activation tagging

strategies for other model plant systems and the construction of

new more sophisticated vectors for the generation of conditional

alleles are also discussed. These recent advances have

significantly expanded the horizons for gain-of-function genetics

in plants.

Fig. (9). a, b Molecular architecture of the activation tagging vectorspVICE n4HPT and pSKI015. apVICE n4HPT contains four copies of theCaMV 35S enhancer sequence adjacent to the right T-DNA border sequence(RB), which potentially enhance expression of genes neighbouring both theleft border (LB) and RB sequences. The construct harbours an ampicillin gene(Amp) and an origin of replication (ori) which allows the isolation of flankingsequences by plasmid rescue in Escherichia coli. A hygromycin resistancegene (HPT) fused with the nopaline synthase promoter (PNOS) and the polyA sequences of gene 4 of the A. tumefaciens T-DNA (g4pA) provides aselectable marker for successful transformation. b The activation taggingvector pSKI015 (Weigel et al., 2000). derived from pVICE n4HPT. BAR:glufosinate resistance gene. The construct harbours an ampicillin gene (Amp)and an origin of replication (ori) which allows the isolation of flankingsequences by plasmid rescue in E. coli. The restriction enzyme sites indicatedcan be employed to rescue pUC19 and adjacent plant sequences fromtransformed plants. The restriction sites NotI, SpeI and BamHI can be used torescue plant sequences adjacent to the left T-DNA border. In a similar


35

fashion, KpnI, PstI, EcoRI and HindIII sites can be used to rescue plantsequences adjacent to the right T-DNA border

Fig. (10). a–c Recently developed activation tagging vectors. a ThepGA2715 vector (Jeong et al., 2002). developed for activation tagging in ricecontains four copies of the CaMV 35S enhancer (4x35S) adjacent to the leftborder (LB); the OsTubA1intron 2 carrying three putative splicing donor sites(I2); the GUSreporter gene; the NOS terminator (TNOS) sequence; theterminator of the OsTubA1 gene (TT); the hygromycin resistance gene (HPT);and the first OsTubA1 intron (I1), to increase gene expression. b The pER16vector (Zuo et al., 2002). designed for 17-β-estradiol inducible expression ofadjacent genes possesses a G10-90 promoter fused to the XVE transcriptionfactor gene and ribulose diphosphate carboxylase E9 poly A additionsequence (TE9). The G10-90promoter is a tetramer of the G-box motif; thissynthetic promoter drives constitutive non-tissue specific expression in bothdicots and monocots (Ishige et al., 1999). The chimeric XVE protein consistsof the DNA-binding domain of the bacterial repressor LEXA (X), the acidictransactivation domain of VP16 (V) and the regulatory domain of the humanestrogen receptor (Zuo et al., 2000). A second transcriptional unit iscomprised of the NOS promoter (PNOS), a kanamycin resistance gene(NPTII) and NOS terminator (TNOS). Eight copies of the LexA operatorsequence (OLexA), a binding site for the XVE transcription factor, are fusedto the minimal 46-bp CaMV 35S promoter sequence, which is adjacent to theLB. Addition of 17-β-estradiol induces XVE binding to the OLexA sites,which promotes expression of the gene(s) adjacent to the left border. c Theheat-shock tagging vector pTT101 which possesses the promoter sequence ofthe ArabidopsisHSP18.2gene (Matsuhara et al., 2000)., 93 bp from the LBsequence. This promoter contains a TATA sequence and transcriptioninitiation site and promotes the expression of neighbouring genes following 2h of heat-shock at 37°C (RBright border).


36

Ajaswrata et al., (2007) a. Plants produce secondary

metabolites in response to various external signals. Coordinated

transcriptional control of biosynthetic genes emerges as a major

mechanism dictating the accumulation of secondary metabolites

in plant cells. However, information about stress regulation of

secondary metabolites and the molecular mechanisms regulating

these specialized pathways are poorly understood. Here, we show

that terpenoid indolealkaloid (TIA) biosynthetic pathway is

differentially regulated in response to different abiotic stresses in

Catharanthus roseus a model medicinal plant producing

important anticancer and antihypertensive drugs. Semi

quantitative RT-PCR analysis of TIA and related primary

pathway genes in response to dehydration, low temperature,

salinity, UV-light and wounding revealed their negative

regulation in response to low temperature. HPLC analysis further

supports the notion that TIA biosynthetic pathway is negatively

controlled by low temperature stress. Furthermore, we report the

cloning of a C-repeat binding transcription factor from C. roseus

(CrCbf), belonging to AP2 class of transcription factor and

possessed the NLS and CBF signatures equence characteristic of

CBFs. CrCbf was found to be similar to Brassica Cbfs, whereas it

was distant to monocot Cbfs. Southern analysis of CrCbf revealed

the presence of more than one copy of CrCbf gene or other Cbf

homologues in C. roseus genome. The transcription of CrCbf was

found to be constitutive in response to low temperature but it

showed differential distribution. The need for identifying novel


37

transcription factors in understanding secondary metabolite

biosynthesis is discussed.

The understanding of the complexities and molecular events

regulating genes and the activators involved in terpenoid Indole

alkaloid (TIA) metabolism is known to a certain extent in cell

cultures of an important TIA yielding plant, Catharanthus roseas,

though it is not yet complete. Recently, the repressors of early

TIA pathway genes have also been identified. However, their

roles in the regulation of TIA pathway in C. roseas cell cultures

remains yet unknown. We have made a comparative profiling of

genes catalyzing the important steps of 2-C methyl-D-erythritol-

4-phosphate (MEP), shikimate and TIA biosynthetic pathways,

their activator and repressors using macroarray, semiquantitative

RT-PCR and northern analyses in a rotation culture system of C.

roseas comprising differentiated and proliferated cells. Our results

demonstrate that TIA biosynthetic pathway genes and their

activators show variable expression pattern, which was correlated

with the changes in the cellular conditions in these systems.

Under similar conditions, TIA pathway repressors show strong

and consistent expression. The role of repressors in the complex

regulation of the TIA pathway in C. roseas cell cultures is

discussed. The results were supported by HPLC data, which

demonstrated that the molecular program of cellular

differentiation is intimately linked with TIA pathway gene

expression and TIA production in C. roseas cell cultures.

Ajaswrata et al., (2007). b.


38

Sarika et al., (2007) an integrated genetic linkage map of

the medicinal and ornamental plant Catharanthus roseus, based

on different types of molecular and morphological markers was

constructed, using a F2 population of 144 plants. The map defines

14 linkage groups (LGs) and consists of 131 marker loci,

including 125 molecular DNA markers (76 RAPD, 3 RAPD

combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and

37 other PCR based DNA markers), selected from a total of 472

primers or primer pairs, and six morphological markers (stem

pigmentation, leaf lamina pigmentation and shape, leaf petiole

and pod size, and petal colour). The total map length is 1131.9 cM

(centiMorgans), giving an average map length and distance

between two markers equal to 80.9 cM and 8.6 cM, respectively.

The morphological markers/genes were found linked with nearest

molecular or morphological markers at distances varying from 0.7

to 11.4 cM. Linkage was observed between the morphological

markers concerned with lamina shape and petiole size of leaf on

LG1 and leaf, stem and petiole pigmentation and pod size on

LG8. This is the first genetic linkage map of C. roseas.

F


39

igure (11). Linkage map of Catharanthus roseas in the F

2 population of the cross ‘Pink Delhi’ × gsr8. Loci names andcumulative genetic distances, in cM are indicated on the right and left side ofthe vertical bars, respectively. RAPD and ISSR markers are represented as theprimer name and number is followed by band size in subscript. Other PCRbased markers (designed and designed forward/reverse + RAPD) are alsorepresented by the primer name (SGD and SSG, respectively) and numberfollowed by band size in subscript. The parental origin of each band is shownin the figure by the letter P for ‘Pink Delhi’ and letter G for gsr8 placed nextto the values of band size in brackets. The acronyms of morphologicalmarkers have been used as shown.

____________________________________ Materials and methods

٤١

III-Materials and methods

III.1-Materials:

III.1.1-plant materials:

This study was carried out by the cooperation

between genetic unit (Botany Dept., Faculty of Agriculture.,

AL-Azhar University) and (Genetic Engineering Laboratory,

Department of Natural Products Research, National Center

for Radiation Research and Technology, Atomic Energy

Authority, Nasr city, Cairo, Egypt).

Catharanthus roseus seeds of tow varieties (LM

& CP3) were obtained from Desert research center (D.R.C.),

Almatarea, Cairo, Egypt.

III.1.2- Cesium irradiation source:137Ce. was used a source of gamma rays with dose

rate 1K rad / 7.35 min. Catharanthus roseus cultivars were

exposed to gamma irradiation at National center for radiation

research and technology, Nasr city, Cairo, Egypt.

III.1.3-Media:

Water agar, free hormones were used in this work.

III.2-Methods:

Preparation seeds of tow varieties LM & CP3 of

Catharanthus roseas and culture initiation:

Seeds were washed with running after wards seeds tap

surface stabilized by soaking for 15 min in 30 % Clorox.


٤٢

Then they were thoroughly washed to assure that any

residues of Clorox had been removed. They were placed in

jars contained Water agar free hormones. The cultured jars

were incubated in growth chamber at (29.5o + 2o C) under

photoperiod of 16 h. of 1000 LUX intensity.

III.2.1-Gamma radiation treatment:

Catharanthus roseas seedling were irradiated after

four weeks from culture on the media by gamma radiation

doses 0 as a control and 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20

Krad. Cultures were incubated at the previous conditions.

Sub culturing was carried out after 4 weeks.

III.2.2. Alkaloids determination for LM & CP3

varieties:

For the determinations of terpenoid Indole alkaloids and fed

precursor(s) in the leaf, Pollets of cell were leaf ground in a

mortar with glass beads and centrifuged at 14000 g for 5 min

and 25 µl supernatant was injected directly into the HPLC

system Lucumi et al., (2001). Terpenoid Indole alkaloids,

Catharanthine, Vindoline, Vallesiachotamine(a),

Vallesiachotamine (b) Ajmalicine, Horhammericine (a),

Horhammericine (b), Vindolinine, 19-Epivindolineine,

Strrictosidinelactam, Serpentine and Vinamidine were

extracted from 100 mg freeze dried cell material with 15 ml

methanol using an Ultraturrax. After centrifugation at 3500 g


٤٣

for 30 min, the methanolic extract was evaporated under

reduced pressure, 1 ml of the Mobile phase A was added to

the dry extract and the suspension was homogenized using a

vortex mixer. After centrifugation of the alkaloid solution at

14 000 g for 5 min, 25 µl of the supernatant was injected into

the HPLC system. The identity of the analyses was

established by photodiode array detection of their UV spectra

and ESP LC-MS. ESP LC-MS was performed on a Finnegan

MAT TSQ–700 triple-stage quadropole mass spectrometer

equipped with ESI–MS electro spray ionization. A gradient

system was developed for the determination of the iridous

and the alkaloids present in the sample and in the medium.

The solvent A consisted of water/ acetonitrile / tri fluoro

acetic acid (95:5:0.01, by vol.) (pH = 3) and the solvent B of

water/acetonitrile / tri fluoro acetic acid (5:95:0.01, by vol.)

(pH = 3), linear gradient of 18.5% acetonitrile over 20 min at

1 ml min−1. The column used was a Vydac C18 4.6, 25 cm

for peptide analysis with a C-18 pre-column. 1693 Because

of lack of sufficient pure reference compounds,

quantification was based on calibration curves with alkaloids

with similar UV-spectra: Strrictosidine was quantified using

the Ajmalicine calibration curve and vallesamine, O-

acetylvallesamine, voaphylline and others above using a

tryptamine calibration curve (detection at 280 nm).


٤٤

III.2.3. Isozymes banding patterns analysis for LM

variety.

Native-polyacrylamide gel electrophoresis (Native-

PAGE) was conducted to identify isozyme variation among

studied taxa the using two isozymes systems.

Table (2) stock solution for isozymes analysis

Fresh and young leaf samples for each variety and leaf

position were used separately for isozymes extraction. The

utilized isozymes are Tryptophan decarboxylase (TDC) and

strictosidine synthase (STR) separated in 12 %

polyacrylamide gel electrophoresis according to Stegemann

et al., (1985).

III.2.3.1. Stock Solutions

a- Extraction buffer for TDC enzyme: 200 mM Tris-Cl

(pH 7.6):

Solution was prepared by dissolving 200 mMTris-Cl

pH 7.6, 10 mM ethylenediaminetetraacetic acid (EDTA), 5

mM dithiothreitol. Tris was dissolved in about 50 ml distilled

Stock Solution 8% gel

Acrlyamide (30%) 28 ml

gel buffer pH 8.6 26 ml

10 % Ammonium per sulfate 3 ml

TEMED 50.75 μl


٤٥

water and pH was adjusted to7.6 by HCl, then the volume

was completed to 100 ml with distilled water then kept at

4oC.

b- Equilibrated buffer to TDC enzyme for columns:

Solution was prepared by dissolving 50 mM Tris-Cl pH

7.5 and 28 mM ß-mercaptoethanol. About 50 ml distilled

water and pH was adjusted to7.5 by HCl, then the volume

was completed to 100 ml with distilled water then kept at

4oC.

c- Extraction buffer for STR enzyme: 200 mM Tris-Cl

(pH 7.6):

Solution was prepared by dissolving 1 m Tris-Cl pH 7.5,

4 mM dithiothreitol and 2 mM ethylenediaminetetra acetic

acid (EDTA), 5 mM dithiothreitol. Tris in about 50 ml

distilled water and pH was adjusted to7.6 by HCl, then the

volume was completed to 100 ml with distilled water then

kept at 4oC.

d- 30 % acrylamide stock:

The solution was prepared by dissolving 29.2 g

acrylamide and 0.8 g N, N, methylene bis–acrylamide in

about 70 ml distilled water, then the volume was completed

to 100 ml by distilled water. The stock solution was kept in

dark bottle at 4º C.


٤٦

e- Electrode buffer (pH 8.65):

Electrode buffer was prepared by dissolving 18.55 g

boric acid and 2.5 g sodium hydroxide in 500 ml distilled

water and mixed well with magnetic stirrer, then pH was

adjusted into 8.6 by distilled water, and then kept at 4º C.

f- Gel buffers:

Separating gel buffer (1.5 M Tris – HCl, pH 8.8):

Tris 18.15 g

H2O (distilled water) up to 100 ml

Stacking gel buffer (0.5 M Tris – HCl pH 6.8):

Tris 6.05 g


Ammonium persulfate solution (APS 10 %):

Ammonium persulfate 0.1 g


III.2.3.2. Extraction of enzymes:

a- TDC enzyme:

Enzymes were extracted from the different C. roseas taxa

by homogenizing 1g fresh leaves samples in 2.5 ml

extraction buffer using a mortar and pestle. The extract was

then transferred into clean eppendorf tubes and centrifuged at


٤٧

10000 rpm for 5 minutes. The supernatant was transferred to

new clean eppendorf tubes. Supernatant was desalted on PD-

10 columns (Pharmacia), previously equilibrated with the

equilibrated buffer. For TDC assays, the protein extract was

mixed with 50 nCi of L-[14C] H3 tryptophan (Amersham),

0.5 mM pyridoxal phosphate, and 50 mM Tris-Cl pH 7.5 in a

final volume of 120 µL. The mixtures were incubated at

37°C for 20 min and the reaction was stopped with 1.0 M

NaOH. The unreacted substrate (Rf 0.52) was separated from

the product (Rf 0.22) by thin layer chromatography (TLC)

(silica plates 0.2 mm, Merck) using 10% methanol in ethyl

acetate as solvent. The chromatograms were visualized under

UV light (366 nm) and the spots corresponding to the

product were scraped from the plates, counted and kept at –

20o C until use for the electrophoretic analysis. Felipe et al.,

(2002).

b- STR Enzyme

Enzymes were extracted from the different C. roseas taxa

by homogenizing 1g fresh leaves samples in 2.5 ml

extraction buffer using a mortar and pestle. In the presence of

polyvinylpyrrolidone (50 mg/g fresh weight). To the frozen

material one volume of extraction buffer. The material was

allowed to thaw and the extracts were clarified by


٤٨

centrifugation for 30 min at 10000 rpm. The supernatant was

desalted on Sephadex G-25 (PD-10 columns, Pharmacia,

Uppsala, Sweden) equilibrated buffer. Incubation mixture for

SSS or STR activity determination contained 25 μl of

desalted protein extract and 62.5 μl of a cocktail containing

1.6 mM tryptamine-HCL and 8.0 mM secologanin in 0.1 mM

sodium phosphate buffer pH 6.8, together with 12.5 μl of a

freshly prepared solution of 0.8 mg D(+)-gluconic acid-δ-

lactone in 0.8 mg Tris. After incubation at 30 oC for 60 min.

the reaction was stopped by adding 75 μl of 5 %

trichloroacetic acid. Blanks were made by adding

trichloroacetic acid prior to the 60-min incubation period.

After addition of 14.8 μl of internal standard (8.0 mM cold-

HCL) and centrifugation, samples were analyzed on

acrylamide gel electrophoresis.

III.2.3.3. Gel preparation:

Vertical slab gel electrophoresis apparatus was used. All

glass plates were washed with tap and distilled water, then

surface sterilized with ethanol. Spacers of 1.5 mm were used.

Separating gel was prepared by mixing the chemical

components listed in Table (2). The prepared gel solution

was poured immediately between the two glass plates and

overlaid with isopropanol and left to polymerize for at least

one hour. After polymerization, isopropanol was removed.


٤٩

Stacking gel was similarly, prepared by mixing the chemical

ingredients listed in Table (2) and then poured over the

separating gel. The comp was placed immediately. The gel

was left to polymerize. Table (2) showed the chemical

ingredients and their concentrations that used for preparing

both separating gel (resolving gel) and stacking gel.

III.2.3.4. Application of samples

A volume of 40 μl extract of each sample was mixed

with 20 μl sucrose and 10 μl bromophenol blue, then a

volume of 50 μl from this mixture was applied to each well.

III.2.3.5. Electrophoresis conditions:

The gel glasses were fixed to both lower and upper

tanks of the electrophoresis apparatus. The run (electrode)

buffer was added to both lower and upper tanks. The

apparatus was connected to the power supply. The run was

performed at 30 volt until the bromophenol blue dye has

reached the separating gel and then the voltage was increased

to 70 volt. Electrophoresis apparatus was placed inside a

refrigerator during running duration.

III.2.4. Protein banding patterns analysis for LM variety:

III.2.4.1. Stock Solutions

SDS polyacrlamid gel electrophoresis (SDS PAGE):


٥٠

Sodium dodecyl sulphate polyacrlamid gel

electrophoresis (SDS PAGE) was performed according to the

method described by Laemmli, U. K. (1970). and modified

by Studier et al., (1973).

a- The monomer solution:

29.2g Acrylamide and 0.8 g Bis-acrylamide were

dissolved in 50 ml distilled water and completed to 100 ml,

filtered and kept at 4oC in a dark bottle to be used within

three months.

b- Resolving gel buffer (1.5 m tris-base, pH 8.8):

18.2g Tris-base was dissolved in 50 ml distilled water

and completed to 100 ml. the pH was adjusted at 8.8 with

analar Hcl. The solution was filtered and kept at 4oC.

c- Stacking gel buffer (0.5 m tris- base, pH 6.8):

6 g Tris-base were dissolved in 50 ml distilled water and

completed to 100 ml. The pH was adjusted at 6.8 with anal as

Hcl. The solution was filtered kept in a dark bottle at 4oC.

d- 10 % Sodium dodecyle sulfate (SDS):

10 g SDS were dissolved in 50 ml distilled water and

completed to 100 ml. This solution should be kept at room

temperature to avoid precipitation.

e- Ammonium persulphata (10 %) initiator:

0.5 g Ammonium persulphate was dissolved in 2 ml

distilled water. This solution must be freshly prepared to


٥١

avoid disactivtion of the ammonium persulphate as a

polymerization catalyst.

f- TEMED: N,N Tetramethylenediamine:

TEMED is a strong polymerizing agent. The volume

required to carry out polymerization depends on the quality

of the used TEMED. In the present work, 50 µl and 30 μl of

TEMED were added for stacking and resolving geles,

respectively.

g- Overlay isopropyl:

50 ml Isopropyl alcohol and 5 ml distilled water.

h- Tank buffer:

6 g Tris-base, 28.8 g glycine and 20 ml of solution were

dissolved in 500 ml distilled water and completed to 2 liters.

i- Staining – Solution (1% COBB):

2g Coomassie brilliant blue (COBB, R250) were

dissolved in 200 ml distilled water, filtered and kept as a

stock solution. The staining solution was prepared as follows:

62.5 ml of stain stock, 250 ml of methyl alcohol and 50 ml of

acetic acid glacial were added, then completed to 500 ml

with distilled water.

j- Destining solution:

45 ml Methyl alcohol, 10 ml acetic acid glacial and 45

ml distilled water were added from the destining solution.

3.2.4.2. Extraction of proteins:


٥٢

2g from leaf of the Catharanthus roseus plants ground

were to a final powder using a pestle and mortar in liquid

nitrogen. Total soluble proteins were then extracted

supernatant was taken as the total proteins extract.

a- Simple protein Determination:

Simple protein were analyzed by PAGE technology

according to Laemmi, U. K. (1970). SDS-PAGE is currently

the most commnly used electrophoretic technique in protein

analysis due to the ability of the strong anionic detergent

SDS, when used in the presence of disulfide band cleaving

reagents, to solubilize, denature and dissociate most proteins

to produce single polypeptide chains. Here, the protein

migration will be according to the molecular mass size only.

b- Preparation of protein samples:

Protein samples were prepared by mixing clear

supernatant with the sample buffer (treatment buffer) {tris-

Hcl, pH 6.8, 2% (W/V) SDS, 10% (W/V) sucrose, 0.1 %

(V/V) 2,β. Mercaptoethanol, 0.5 % (W/V) bromophenol

blue} in the ratio of 1:1 and denatured by heating in a boiling

water for 4 min, then loaded in equal amounts.

For the native gel, protein samples were prepared by

mixing clear supernatant with the treatment buffer which in

this case contains all components of denatured gel treatment

buffer except SDS.


٥٣

III.2.4.3. Gel preparation:

a- Resolving gel:

10 ml of monomer, 7.5 ml of resolving Gel buffer 0.3 ml

of 10% SDS, and 12 ml distilled water were mixed and

shaked well.300 μl of freshly prepared ammonium

persulphate (soln.5) were added and shacked well. Finally,

30μ l TEMED was added just before gel casting.

b- Stacking gel:

1.33 ml of monomer solution (soln.1), 2.5 ml of stacking

gel buffer (soln.3), 0.1 ml of 10% SDS (soln.4), 100 μl

ammonium persulphate (solen.5) and 6.1 ml distilled water

were added, respectively and shaked just before gel casting,

50 μl TEMED were added.

c- Gel casting:

Biorad multigel-long is the device which was used in

protein electrophoresis. It is a complete system with two sets

of (12.5 x 11) Cm glass plates. Straight edge while, the other

is with fixed 1.0 mm spacers. A silicon rubber seal, 1 mm

was placed around the periphery of the fixed spacers, then

the other notched glass plate was carefully placed upon it.

The two plates were together by the mean of 6 clips. Two

cobbined plates were prepared as above to carry out gel

casting.


٥٤

Resolving gel was poured in. between the two plates

leaving 2 Cm benath the plates end. Alayer of 90% isopropyl

alcohol was added over resolving gel to prevent corrugation

of the gel surface. Polymerization of the resolving gel took

from half to one hour after gel solidification, which can be

signicant by the interphase line between the alcohols arid the

solid gel, the alcohol was poured off.

Stacking gel mixture was added over the solid resolving

gel till the top of the glass plates. A comp consisted of 12

wells and 1.0 mm derision was used. The comb was placed

very gently into the liquid gel to avoid any air bubbles.

Stacking gel needed a pit longer time then resolving to be

solididfied. Once the solidification took place, the comb,

clips and the silicon rubber were removed. The two plate’s

acre installed in the electrophoresis chamber. The tank buffer

was added to immerse the wells completely. The protein

samples were pipetted in wells by automatic variable

micropipette (2-200) μ l.

III.2.4.4. Electrophoresis condition:

The run was earned out at 30 V till to loaded samples

passed the stacking gel and entered the resolving gel,

tracking dye (Bromophenol blue) reached the gel bottom.


٥٥

III.2.4.5. Staining and de staining of gels:

The gels were stained for 24 hours in the prepared

staining solution (1% COBB, R-250). To obtain a clear

background, the gels were de stained by the prepared de

staining solution.

III.2.4.6. Photo graphing and gel scaning:

Destained gels were photographed and the results were

analyzed by the GDS (Gel docurnentahon systems) – Biorad

2005.

III.2.5. Randomly amplified polymorphic DNA (RAPD).

a- First variety (LE):

In this study, RAPD was used for the

identification of markers associated with 11 radiation

treatments taxa genotypes after four weeks according to

Shilpa and Jayabaskaran (2007). Five primers random

decamers were used in this study; their names and

nucleotides sequences were presented in table (3).

Table (3): List of primer names and their nucleotide sequences used inthis study (RAPD)

NO. Name Sequence Tm1 OP-B01 5' CTGTCGTCGT 3' 322 OP-B07 5' AGGTGACCGT 3' 323 OP-B11 5' CAGCACTGCT 3` 324 OP-B12 5' CCTTGACGCA 3' 325 OP-F06 5' AGGTGCGTCC 3' 34

Tm : annealing temperature


٥٦

b- Scand variety (CP3):

In this study, RAPD was used for the

identification of markers associated with 8 times (con., 0, 2,

4, 8, 16, 48 and 186) hours which were treated by using

gamma radiation at 16 Krad taxa genotypes after four weeks

according to Ajaswrata et al., (2007). Ten random decqwers

primers were used in this study, their names and nucleotides

sequences were presented in table (4).

Table (4): List of primer names and their nucleotide sequences used inthis study (RAPD)

No. Name Sequence Tm1 OP-C09 5' CTCACCGTCC 3' 342 OP-C10 5` TGTCTGGGTG 3` 323 OP-C13 5` AAGCCTCGTC 3` 324 OP-C15 5` GACGGATCAG 3` 325 OP-G17 5' CAGCAGCAGG 3` 346 OP-L12 5` GGGCGGTACT 3` 347 OP-L13 5` ACCGCCTGCT 3` 348 OP-L16 5` AGGTTGCAGG 3` 329 OP-L20 5` TGGTGGACCA 3` 32

10 OP-Z03 5' CAGCACCCCA 3' 32Tm : annealing temperature

III.2.5.1. DNA isolation

Young and fresh leaf samples were collected separately

from C. roseas seedlings for each radiation treatments. Then

bulked DNA extraction was performed using DNeasy plant

Mini Kit (QIAGEN). Isolation protocol of DNA was as

follows:


٥٧

1- Plant tissues were ground in liquid nitrogen to a fine powder

using a mortar and pestle. Then, the powder was transferred

to an appropriately sized tube and the liquid nitrogen was

allowed to evaporate.

2- Then, 400 µl of buffer AP1 and 4 µl of RNase a stock

solution (100 mg/ml) were added to a maximum of 100 mg

of ground plant tissues and vortexed vigorously.

3- Mixture was incubated for 10 min at 65oC and mixed 2-3

times during incubation by inverting tube.

4- Then, 130 µl of buffer AP2 was added to the lysate, mixed

and incubated for 5 min on ice.

5- Lysate was applied to the QIA shredder spin column

sitting in a 2 ml collection tube and centrifuged for 2 min at

12000 rpm

6-Flow-through friction from step 5 was transferred to a new

tube without disturbing the cell-debris pellet. Typically, 450

µl of lysate was recovered.

7- Then, 0.5 volume of buffer AP3 and 1 volume of ethanol (96-

100%) were added to the cleared lysate and mixed by

pipetting.

8- Then, 650 µl of the mixture from step 7 was applied through

DNeasy Mini spin column setting in a 2 ml collection tube.


٥٨

Then, centrifuged for 1 min at 8000 rpm and flow-through

was then discarded.

9- DNeasy column was then placed in a new 2 ml collection

tube. Then, 500 µl buffer AW was added onto the DNeasy

column and centrifuged for 1 min at 8000 rpm. Flow-

through was discarded and reuse the collection tube in step

10 was reused.

10-Then, 500 µl buffer AW was added to DNeasy column and

centrifuged for 2 min at maximum speed to dry the column

membrane.

11-DNeasy column was then transferred to a 1.5 ml microfuge

tube and 100 µl of preheated (65oC) buffer AE was pipetted

directly onto the DNeasy column membrane. Then,

incubated for 5 min at room temperature and centrifuged for

1 min at 8000 rpm to elute.

12-Elution (step11) was repeated once as described. A new

microfuge can be used for first elute. Alternatively, the

microfuge tube can be reused for the second elution step to

combine the elutes.

III.2.5.2. Randomly amplified polymorphic DNA (RAPD)

procedure

PCR reactions were conducted using 15 arbitrary 10-mer

primers.


٥٩

Polymerase chain reaction (PCR) condition

Stock solutions

a- 5X Tris-borate (TBE), pH 8.0

Tris-base 5.40 g

Boric acid 2.75 g

500 mM EDTA, 8.0 0.29 g

H2O (d.w) up to 100.00 ml

b- Ethidium bromide

The stock solution was prepared by dissolving 1 g of

ethidium bromide in 100 ml distilled water and mixed well

with magnet-ic stirrer. It was transferred to a dark bottle and

stored at room temperature.

c- Sample loading dye

Na-EDTA, pH 8.0 (500 mM) 2.00 ml

Glycerol (100%) 5.00 ml

Bromophenol blue (2%) 0.75 ml

Xylene cyanole (2%) 0.75 ml

H2O (d.w.) 1.50 ml

PCR was performed in 30-µl volume tubes according

to Williams et al., (1990). that contained the following:

DNTPs (2.5 mM) 3.00 µl

MgCl2 (25 mM) 3.00 µl

Buffer (10 x) 3.00 µl

Primer (10 pmol) 2.00 µl

Taq DNA polymerse (5U/µl) 0.20 µl


٦٠

Template DNA (25 ng) 2.00 µl

H2O (d.w.) 16.80 µl

The amplification was carried out in a DNA

thermocycler (MWG-BIO TECH Primuse) Programmed as

follows:

One cycle

94oC for 5 min

45 cycles each of94 oC for 1 min

4GC + 2AT each primer as shown in tables 3 & 4 for 90 sec

72 oC for 2 min

One cycle

72 oC for 7 min, then 4 oC infinit.

III.2.5.3. Sample preparation

PCR product 15.00 µl

Loading dye 5.00 µl

III.2.5.4. Gel preparation

Agarose 1.40 g

TBE (1 x) buffer 100.00 ml

Ethidium bromide 5.00 µl

Agarose was mixed with l x TBE buffer and boiled in water

bath. Ethidium bromide was added to the melted gel after the

temperature became 5 oC.

The melted gel was poured in the tray of mini-gel

apparatus and comb was inserted immediately, then comb


٦١

was removed when the gel become hardened. The gel was

covered by the electrophoretic buffer (1 x TBE). Fifteen µl of

DNA amplified product was loaded in each well. DNA

ladder mix was used as standard DNA with size as follows:

a- For variety LM

12000, 11000, 10500, 10000, 9500, 9000, 8800, 8500,

6000, 3300, 2300, 1900, 1500, 1200, 1000, 800 600, 400,

300, 100 and 80 bp.

b- for variety CP3

10000, 9000, 8000, 7000, 6000, 5000, 4500, 4000, 2500,

1500 and 900 bp.

The run was performed for about 1 hour at 80 V in

Pharmacia submarine (20 cm X 20 cm).

III.2.5.5. Gel analysis

1-Gels were photographed and scanned with Bio-Rad video

densitometer Model 620, at a wave length of 577.

III.2.5.6. Data analysis

The similarity matrices were done using Gel works ID

advanced softwere UVP-England Program. The relationships

among rootstock genotypes as revealed by dendrograms were

done using SPSS windows (Version 10) program.

________________________________________Results and discussion

٦٣

ІV-Results and discussion

IV.1. Radiation treatments:

Table (5) presents the survivor of plants

Catharanthus roseus varieties (LM and CP3) which were

treated by using Gamma radiation doses; 0, 2, 4, 6, 8, 10, 12,

14, 16, 18, 20 and 22 Krad. The doses from 0 to 20 Krad. gave

100 % survivor, on the other hand, the survivor percentage

following dose 22 Krad. was found to be a lethal dose (20 %).

Table (5): Effect of the radiation treatments on the Catharanthus roseusplants (LM & CP3) varieties survivor.

*lethal doses

Our results are in agreement with Shilpa and

Jayabaskaran (2007). How studded UV-B-induced signal

leading to enhance production of Catharanthine in

Catharanthus roseus cell suspension cultures. Medium

alkalinization an early event occurring in elicitor-treated

No.Radiation treatments by

Krad.

No. of plants after radiationtreatments

Survivorpercentage

after 2 days after 14 days010203040506070809101112

000204060810121416182022

101010101010101010101010

101010101010101010101002

100 %100 %100 %100 %100 %100 %100 %100 %100 %100 %100 %

*020 %


٦٤

plant cell cultures, has been used as a marker of elicitor

responses in studying elicitor-binding sites in plant cells.

IV.2. Effect of radiation on Indole alkaloids biosynthesis

IV.2.1. assay in variety.

a- First variety (LM):

The TDC and STR in C. roseas variety (LM) and

over expression appeared gave a result in an increased

alkaloid accumulation but only enhanced tryptamine levels.

Therefore, in the present study we irradiated of C. roseas

variety (LM).

Total alkaloids, Catharanthine, Vindoline,

Vallesiachotamine (a), Vallesiachotamine (b) Ajmalicine,

Horhammericine (a), Horhammericine (b), Vindolinine, 19-

Epivindolineine, Strrictosidinelactam, Serpentine and

Vinamidine of were assayed and compared to their relative

percentage with those of the intact plant [Fig. (12) and Table

(6)] the highest relative percentages 6.190, 7.200, 5.940,

5.860, 7.790, 6.000, 6.672, 2.720, 7.000, 7.920, 7.230 and

6.400 μg of alkaloids respectively above , in radiation

treatments 18, 12 & 16, 14, 14, 18, 18, 18, 12, 12, 8, 12 and

16 Krad. respectively compared with other treatments.

However the Indolealkaloids of Catharanthine, Ajmalicine,

Horhammericine (a) and Horhammericine (b) were increased


٦٥

commensurate with increasing of the radiation doses up to 18

Krad the production produced of the alkaloids of Vindoline,

Vallesiachotamine (a) and Vallesiachotamine (b) [Fig. (12)

and Table (6)] were increasing up to radiation doses 12 & 16,

14 and 14 Krad respectively. On the other hands the highest

values of alkaloids, Vindolinine, 19-Epivindolineine,

Strrictosidinelactam, Serpentine and Vinamidine as a relative

percentage with those of C. roseas variety (LM) intact plant

were recorded with 12, 12, 8, 12 and 16 Krad respectively Fig.

(12).

Our results un agreed with those of Carolyn et al.,

(2004) Growth and alkaloid production with and without MJ A

typical growth and alkaloid production curve for C. roseas

suspensions with and without methyl jasmonate (MJ). In elicited

cultures, MJ was added on the inoculation day (day 0) at 100 µM;

rapid increases in Ajmalicine and serpentine occurred after 3 day,

which continued up to 7 and 11 day, respectively. Ajmalicine and

serpentine production in MJ-elicited cultures maximized at 5.4 ±

0.4 mg l−1 and 3.7 ± 0.7 mg l−1, respectively; this represents a

165% and 78% increase over the maximum Ajmalicine and

serpentine content of controls. Optimum growth stage for MJ

elicitation The optimum growth stage for inducing alkaloid

production with MJ in C. roseas suspensions was investigated by

adding MJ at 0, 10, 100, or 1000 µM on either day 0, 3, 6, 9, 12,

or 15. Resin bags containing XAD-7HP were also added to


٦٦

promote the adsorption and extra cellular recovery of

alkaloids and to potentially reduce Feedback inhibition and

product metabolism Asada and Shuler (1989) & Lee-

Parsons and Shuler (2002). Resin bags were added to

suspensions 3 d after elicitation and then exchanged every 3

d to minimize the saturation of resins. Resin was added while

metabolite production was believed to be most rapiday, i.e. 3

d after elicitation. Expression of genes involved in the C.

roseas TIA pathway reached a maximum between 4 to 24 h

after MJ elicitation Collu et al., (2001). Activity of enzymes

associated with secondary metabolism in Glycine max and

Lithospermum erythrorhizon suspensions peaked between 24

to 72 h after MJ elicitation Gundlach, et al., (1992) &

Mizukami, et al., (1993). Rapid secondary metabolite

accumulation was presumed to follow the maximum

expression of mRNA and enzymes associated with secondary

metabolism, i.e. within 72 h after elicitation. For this reason,

the experiment was designed with resin added 3 d after

elicitation when TIA accumulation was believed to be most

rapid. This variation in Indolealkaloids production in the C.

roseas cells which were treated by Gamma rays to the doses

0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 Krad. my be stable

mutation in the DNA of plant.


٦٧

No.

1

0

1

2

3

4

5

6

7

8

Strrictos

idine

lacta

m

19-Epivin

dolin

ine

Serpen

tine

Vind

oline

Vina

midine

Horham

meric

ine

Horham

meric

ine

Vind

olinin

e

Valle

siach

otam

ine

Valle

siach

otam

ine

Ajma

licine

Catharan

thine

Indole alkaloids

C0ntrol

Concentr

atin

2

0

1

2

3

4

5

6

7

8

Strrictos

idine

lactam

19-E

pivind

olinine

Serpen

tine

Vind

oline

Vina

midine

Horham

mericine

Horham

mericine

Vind

olinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicin

e

Catharanthine

Indolealkaloids

2 K rad

Concentr

ation(u

n

3

0

1

2

3

4

5

6

7

8

Strrictos

idine

lactam

19-Epiv

indolinine

Serpen

tine

Vind

oline

Vina

midine

Horham

mericine

Horham

mericine

Vind

olinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicin

e

Catharanthine

Indolealkaloids

4 K rad

Concentr

ation0n

Fig. (12) Indolealkaloids determination by HPLC in Catharanthus roseas variety (LM) which istreated by gamma radiation Concentration by (μg).


٦٨

Fig. (12) Continue4

0

1

2

3

4

5

6

7

8

Strrictosidine lactam

19-Epivin

dolinine

Serpentin

e

Vindoline

Vinamidine

Horham

mericine

Horham

mericine

Vindolinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicine

Catharanthine

Indolealkaloids6 K rad

Co

ncen

trati

on

5

0

1

2

3

4

5

6

7

8

9

Strrictos

idine

lactam

19-E

pivind

olinine

Serpen

tine

Vind

oline

Vina

midine

Horham

mericine

Horham

mericine

Vind

olinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicin

e

Catharanthine

Indolealkaloids

8 K rad

Concentr

ation

6

0

1

2

3

4

5

6

7

8


19-Epivin

dolinine

Serpentin

e

Vindoline

Vinamidine

Horham

mericine

Horham

mericine

Vindolinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicine

Catharanthine

Indolealkaloids

10 K rad

Concentr

ation


٦٩

Fig. (12) Continue

7

0

1

2

3

4

5

6

7

8

Strrictos

idine

lactam

19-E

pivind

olinine

Serpen

tine

Vind

oline

Vina

midine

Horham

mericine

Horham

mericine

Vind

olinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicin

e

Catharanthine


Co

nce

ntr

atio

n

8

0

1

2

3

4

5

6

7

8


19-E

pivin

dolinine

Serpentin

e

Vindoline

Vinamidine

Horham

mericine

Horham

mericine

Vindolinine

Vallesiacho

tamine

Vallesiacho

tamine

Ajmalicine

Catharanthine


Concentr

ation

9

0

1

2

3

4

5

6

7

8

Strrictos

idine

lactam

19-Epiv

indoli

nine

Serpen

tine

Vind

oline

Vina

midine

Horham

mericine

Horham

mericine

Vind

olinine

Valle

siach

otam

ine

Valle

siach

otam

ine

Ajmali

cine

Catha

ranthine


Co

nce

ntr

atio

n


٧٠

Fig. (12) Continue10

012

3456

789

Strrictos

idine

lacta

m

19-Epivin

dolin

ine

Serpen

tine

Vind

oline

Vina

midine

Horham

meric

ine

Horham

meric

ine

Vind

olinin

e

Valle

siach

otam

ine

Valle

siach

otam

ine

Ajma

licine

Catharan

thine


Concentr

ation

11

00.5

1

1.52

2.53

3.54

Strrictos

idine

lacta

m

19-Epivin

dolin

ine

Serpen

tine

Vind

oline

Vina

midine

Horham

meric

ine

Horham

meric

ine

Vind

olinin

e

Valle

siach

otam

ine

Valle

siach

otam

ine

Ajma

licine

Catharan

thine


Conce

ntr

atio

n


٧١

Radi

ation

trea

tmen

ts (K

rad)

Type

of I

ndol

e alk

aloid

s0

24

68

1012

1416

1820

Cath

aran

thin

e0.9

000.9

001.0

001.1

001.2

001.3

702.9

105.0

005.8

106.1

903.1

30Vi

ndol

ine

4.890

5.100

5.130

5.180

5.180

5.240

7.200

7.000

7.200

6.550

3.328

Valle

siach

otam

ine a

1.430

1.500

1.860

2.000

2.110

3.120

5.670

5.940

5.660

4.550

1.300

Valle

siach

otam

ine b

1.520

1.520

1.880

1.960

2.000

3.000

5.621

5.860

5.660

4.690

1.100

Ajm

alicin

e1.1

101.3

401.6

201.8

301.9

702.8

205.8

216.3

707.4

007.7

903.4

70Ho

rham

mer

icine

a0.1

200.1

200.9

201.3

101.5

201.9

203.9

005.1

005.7

506.0

002.8

90Ho

rham

mer

icine

b1.3

101.3

101.7

201.8

902.0

102.5

304.5

305.7

306.1

106.6

722.9

90Vi

ndol

inin

e0.2

100.2

100.4

200.3

200.4

100.9

302.7

202.4

001.4

401.0

001.0

0019

-Epi

vind

olin

eine

4.220

4.510

4.710

4.990

5.110

5.270

7.000

6.319

5.811

5.000

2.250

Strri

ctosid

inela

ctam

7.510

7.490

7.520

7.490

7.920

7.400

6.900

6.330

6.000

5.200

2.910

Serp

entin

e4.8

805.0

105.4

005.5

805.6

105.5

207.2

306.4

405.2

003.7

601.2

00Vi

nam

idin

e3.2

103.4

103.8

003.9

704.9

005.0

005.2

206.0

006.4

005.9

831.7

60

Tab

le N

o. (

6) E

ffec

t of

Gam

ma

radi

atio

n tr

eatm

ent o

n bi

osyn

thes

is o

f In

dole

alk

aloi

ds c

once

ntra

tion

by

(µg)

.


٧٢

b- Second variety CP3

Depended on the previous assay results in 4.5 and

(table 7 & fig. 13) we are design the experiment to study the

effect of the gamma radiation stress on the Ajmalicene

production on the Development of time in another C. rouses.

Sterna to understanding the gene exprassion behavior in the

cells.

Chosen the Ajmalicine especially because it is a gate

of Indole alkaloids production in a pathway system. And

chose dose rate 16 Krad. because this dose is equilibrium dose

among all Indole alkaloids components above.

In (table 7 and fig. 13) revealed that Gamma

irradiation doses were effective on reducing the content of

Ajmalicine, as compared with the control. In all cases,

raising the doses of Gamma irradiation decreased the content

of Ajmalicine production from 0 times to 48 hours but after

one week level Ajmalicine production increasing suddenly.

The occur of the biosynthesis of the indole alkaloids,

and Ajmalicine specially was reduced in C. roseus seedling

as reported previously seedling cultures of C. roseus

typically exhibit a growth-dissociated accumulation of

secondary products. As shown in (Fig. 13 and table 7),

Ajmalicine levels began to decrease directly after irradiated

on the other hand increase during the deceleration of linear


٧٣

growth and reached plateau levels early in the stationary

phase. The accumulation kinetics of the Ajmalicine-derived

alkaloids succeeded the increase in into cellular, Ajmalicine

concentration (table 7 & Fig. 13 and 14). In contrast, product

levels failed to increase significantly in seedling during the

exponential growth period. For example, the maximum level

of Ajmalicine in cells was approximately 2.1801 µg,

compared with a maximum concentration of greater than

2.8991 µg after 168 hour. Alkaloids were not detected in the

culture medium of either system indicating that product

release was not responsible for low intracellular

concentrations. The accumulation kinetics suggest that

alkaloid biosynthesis is reduced in cells, but not completely

inhibited, since the intracellular product levels, expressed as l

µg / g (fresh weight), remained relatively constant during the

growth cycle. These results are consistent with those reported

for the effects of other non-alginate immobilization methods

on Indole alkaloid accumulation in C. roseus cells. The

decreased biosynthesis of Ajmalicine was initially suggested

as responsible for the reduced accumulation of indole

alkaloids in cells of C. roseus. In order to test this hypothesis,

invitro assays were performed to determine the specific

activity of enzymes involved in Ajmalicine biosynthesis in

cells. Preliminary evidence from these experiments suggested


٧٤

that the biosynthesis of total extractable proteins was

enhanced in immobilized cells relative to cells in suspension.

Since this phenomenon was in contrast to the otherwise

reduced primary metabolism of immobilized cells,

manifested as a decreased growth rate, formal experiments

were performed to determine the total extractable protein

level in both systems. The kinetics of total extractable

Ajmalicine biosynthesis established that irradiated cells were

synthesizing more extractable Ajmalicine than control cells.

Ajmalicine levels per gram (fresh weight) of cells increased

rapidly in both systems reaching peak values within 7 days of

inoculation into plant. The synthesis of total proteins

decreased by day 7 as seedling entered the linear growth

phase. In combination with a rapidly increasing age.

Irradiated seedling may stimulate increased Ajmalicine

biosynthesis, at least in some species. Maximum specific

activity of TDC was similar in seedling cells Fig. (15) and

(table 8). The specific activity of TDC in seedling cells was

greatest on dose rate 16 Krad and declined continuously to

undetectable levels by dose rate 20 Krad The increased

accumulation of Ajmalicine beginning after day 7 at dose

rate 16 Krad (Fig. 13 & 14) is consistent with high TDC

activity during this period. Although the time-course of the

specific activity for TDC during the growth of seedling in 4.6


٧٥

was altered relative to that of seedling in 4.5 maximum

activity levels were similar in both systems. However,

increased accumulation of Ajmalicine was not observed in

seedling in other times suggesting that the limited availability

of an Ajmalicine precursor was responsible for the reduced

Ajmalicine pool. TDC has been characterized and purified to

homogeneity from cell seedling of C. roseus. The

relationship between the expression of enzyme activity under

certain conditions and the accumulation of Ajmalicine

implicated TDC expression as the controlling factor for

alkaloid production in C. roseus seedling.

Workers in several laboratories have observed the

presence in a number of tissues of enzyme systems which are

active in the synthesis of Indole alkaloids. The result

agreement to the result in the assay in (LM). Our result

agreement with Shilpa et al., (2007) who irradiated C.

roseas by UV and studied enzyme (TDC) and (STR) Since

the UV-B-induced early cellular responses, medium

alkalinization and ROS production were inhibited by

suramin, we investigated whether. The effect of UV-B

irradiation on expression of TIA biosynthetic genes, Tdc and

Str, and Indole alkaloids production has been reported

previously in C. roseus leaves. The transcription factor GT-1

binds to the promoter region of Tdc in vitro. The functional


٧٦

importance of GT-1 in the induction of Tdc expression by

UV light has been demonstrated by point mutations in the

GT-1 binding site. However, the molecular basis of UV-B

signaling cascades leading to the induction of expression of

Tdc and Str genes and the production of TIAs is largely

unknown. It has been observed that the polypeptide wound

signal, systemin- specific cell surface receptors initiate a

signal transduction cascade upon UV-B irradiation in L.

peruvianum cell suspension cultures. In the present study, the

signaling pathways mediating UV-B-induced catharanthine

accumulation in C. roseus suspension cultures were

investigated. UV-B induced alkalinization of the culture

medium, generation of hydrogen peroxide, activation of

CDPK and MBPK as well as accumulation of catharanthine

and stimulation of transcription of Tdc and Str genes were

studied. Inhibitors of binding of ligand-cell surface receptors,

protein kinases and phosphatases, calcium fluxes and H2O2

were used to dissect the UV-B signaling cascade.

The oxidative burst, a rapid consumption of oxygen

and production of reactive oxygen species (ROS) such as

H2O2, is a typical early event in plant defense responses with

5 min of UV-B irradiation of C. roseus cells H2O2 production

increased six-fold compared to control cells. We next

examined effects of suramin, an inhibitor of G-protein


٧٧

inhibitor, N-acetyl cysteine, a putative ROS scavanger,

verapamil, a calcium channel blocker and staurosporine, a

serine-threonine kinase inhibitor, SB 203580, a P38 MAPK

inhibitor, PD 98059, an ERKK inhibitor and SB 600125 JNK

inhibitor. The UV-B induced H2O2 production was

suppressed by all the inhibitors except the MAPK cascade

inhibitors. This indicated that upon receiving the UV-B

signal by a putative receptor in C. roseus cells, calcium

influx and activation of serine/threoine kinases are required

to induce H2O2 production. However, activation of the

MAPK cascade occurs downstream of H2O2 production.

Table No. (7) Effect of gamma radiation on the Ajmalicine content in C. roseus.

Parameters Time Area Height Con. µg/g

tissue

Standard

Control

000 time

002 hour

004 hour

008 hour

016 hour

048 hour

168 hour

14.42

14.39

14.22

14.22

14.00

14.42

14.21

14.32

14.40

0864

1410

0727

0926

0701

0682

0600

0612

1993

3.8961

1.9974

1.4011

1.5388

1.0001

1.0116

0.7101

0.7147

4.9691

2.0000

2.1801

2.0001

1.8777

1.4062

1.2001

1.0006

0.9963

2.8991


٧٨

Fig. 13 effect of time development on the Ajmalicine prodction in C. ruses. under

radiation doas 16 Krad.

VI.2.2. compare of Ajmalicine production in LM &

CP3 variety in Catharanthus rouses.

Data obtained from HPLC experiment in LM & CP3

variety are similarity at dose 16 Krad., in LM variety we are

see concentration of Ajmalicine 7.4 µg after four weeks

compare the control 1.11 µg (table 6). on the other hand seen

concentration of Ajmalicine in CP3 variety which are treated

by Gamma radiation at 16 Krad after 168 hours (one week)

were 2.899 µg compare standard 2 µg table (7) so we can

see compatibly in gene expiration in Ajmalicine production

in both variety LM & CP3.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 8 16 48 168

Time / hoars

Conc

en./u

g


٧٩

Fig. 14 HPLC analysis of time development on the Ajmalicine production in

C. ruses. under radiation dose 16 Krad.

control 0 hour

2 hour 4 hour

8 hour 16 hour

48 hour 168 hour


٨٠

This system provided alternating conditions of cellular

growth and proliferation and thus allowed to study the

transcriptional changes of TIA pathway genes and regulators

simultaneously under changing culture conditions. The

results were compared with TIA production data by HPLC.

This study demonstrates the complexities of molecular

regulation of TIA metabolism in C. roseus cell culture and

indicates the probable bottleneck for scale-up production of

TIAs in this system.

Understanding the Terpenoid indole alkaloids (TIAs), an

important group of secondary metabolites are produced by C.

roseus (L.) G. Don (Madagascar periwinkle), a member of

the apocynaceae family. Some of these alkaloids have high

therapeutic values such as, the antihypertensive alkaloids,

ajmalicine, serpentine and anticancer alkaloids, vincristine,

vinblastine. The monoterpenoid moiety of TIAs originates

from the 2-C methyl erythritol phosphate (MEP) pathway

whereas the indole skeleton of TIAs is derived from the

shikimate pathway. The biosynthesis of TIAs in C. roseus is

reported to be under strict regulation. Their metabolism has

been found to be restricted to certain tissues and it is

modulated by developmental and environmental

mechanisms. Despite significant efforts, TIA biosynthesis in

plant cell cultures remains poorly characterized. Regulation


٨١

of two important TIA biosynthetic pathway genes, namely,

strictosidine synthase Str and tryptophan decarboxylase Tdc

has been studied in great detail, which revealed thereof

coordinate regulation and elicitor-mediated induction in cell

cultures. The regulatory mechanisms controlling many of the

remaining steps of the TIA pathway are yet unknown.

Mustafa and Verpoorte (2007).

the understanding of which may reveal the complex

molecular program that governs TIA metabolism in cell

culture. Functional analysis of Str and Tdc gene promoters

led to the identification of cisregulatory sequences involved

in elicitor and Me JAinduced responses. Octadecanoid-

derivative responsive Catharanthus AP2-domain (ORCA3)

transcription factor was found to be jasmonateresponsive

transcriptional regulator of C. roseus primary and secondary

metabolism. Furthermore, G-box binding factors (GBF1,

GBF2 and GBF3) and zinc finger proteins (ZBF1, ZBF2 and

ZBF3) were shown to act as repressors in the regulation of

elicitor-induced TIA metabolism in C. roseus. Two

downstream vindoline pathway genes, desacetoxyvindoline

4-hydroxylase (D4h) and deacetoxyvindoline 4-O-

acetyltransferase (Dat) are known to be transcriptionally

blocked in C. roseus in vitro grown cultures. Many aspects of

such key regulatory events have been recently explored, but


٨٢

understanding of the regulation of complete TIA pathway in

cell cultures is still lacking for which it has not yet become a

viable alternative for improved production of TIAs. The

differential expression of genes in different state of cultures

is expected to be different and will also be associated with

different levels of secondary metabolites. In order to

demonstrate the status of similar changes in expression of

TIA pathway genes and regulators in C. roseus cell culture.

The expressions of two feeder primary (MEP,

shikimate) and committed TIA metabolite pathway genes

were analyzed in C. roseus rotation culture system along

with TIA pathway regulators using a Gamma radation stress.

The database search for C. roseus genome revealed six genes

encoding selected enzymes for each of the primary (MEP and

shikimate) and TIA pathways along with eight TIA pathway

regulator genes. A single amplified product obtained for each

target gene was radiated and identities of putative RAPD-

PCR which showed 95–99% homology with the available

sequences in database. The genes were studding and analysis

with CP3 variety RAPD-PCR (Table 23). Consequently, 495

fragments were spotted in agarose gel to facilitate the

expression analysis and their expressions were monitored in

the tissues originating from cultures. Each membrane was

hybridized simultaneously under radiation stress sample.


٨٣

There were significant changes in the expression levels of

primary as well as TIA pathway genes and the regulators in

the samples originating. TIA pathway repressor genes were,

however, relatively highly expressed in the same tissues.

This system revealed a high degree of reproducibility.

Gene, activator and repressor transcripts of TIA and

related primary pathways in seedling culture system We

known the transcript profiles of three TIA pathway

repressors each of G-box binding factor type, namely, Gbf1,

Gbf2 and Gbf3, and zinc finger protein type, namely, Zct1,

Zct2 and Zct3, one master regulator, Orca3, one MYB

transcription factor, CrBpf1 and TIA and related primary

pathway genes in cells initially. The appropriate number of

cycles for PCR amplification of each target gene was

determined so that it can be quantified and that the

amplification lies in the exponential range before reaching

the plateau. PCR amplification of 30 cycles was found to be

optimum for all the target genes studied and their

amplification was found to be in the exponential range. The

mRNA levels of primary pathway genes, TDC and STR were

higher in cells-induced by Gamma radiation in cultured

seedling, compared to that of the control as below. The

transcript levels of the latter two genes, TDC and STR

remained undetected in seedling cultured. The mRNA levels


٨٤

of early TIA pathway genes, Tdc and Str, showed changes

with changing conditions of irradiated and thus were higher

in 16 Krad but low in other doses.

Ajmalicine level showed low or undetected level of

expression under similar conditions. The levels of

expressions of late TIA pathway transcripts, TDC and STR,

Data changed from high to low with changing culture

irradiated. Some of these show restoration of expression in

seedling cultures. Similar study was carried out using UV

irradiated cell cultures, which revealed similar pattern of

expression for specific TIA as well as primary pathway genes

except G10h, which was faintly detected in this system. One

gene from each pathway, namely G10h (MEP pathway), Tdc

(shikimate pathway), Str (early TIA pathway) and Dat (late

TIA pathway) were used as probes for northern analysis,

which revealed biphasic nature of expression of Tdc and Str

transcripts in cell cultures noted by a reduction in the level of

expression in equilibrium doses but again the level of

expression was restored in high doses.

The transcript level of TDC must be confirmed to be

low in seedling cultured without any treatments and Data

transcript remained undetected by HPLC analysis. There was

significant induction of the repressor Ajmalicene in seedling

cultured. Some of these transcripts, Gbf1, Gbf3, Zct1 were


٨٥

upregulated in seedling cultured. Similarly, the TDC levels,

which play a role in elicitor responsive signal transduction

pathway of TIA biosynthesis, were high in 16 Krad. dose.

Under similar conditions, the STR, the master regulator for

TIA pathway biosynthesis, were relatively high. However, in

seedling cultures, the expression of this STR was reverse in

10 Krad. The steady state TDC levels were confirmed by gel

electrophoresis analysis in seedling cultures, but STR

remained undetected because this enzyme is very low in

molecular Weight.

IV.3. Effect of radiation on Isozymes banding patterns.

VI.3.1. Tryptophandecarpoxylase enzyme (TDC):

The results of electrophoretic patterns of Tryptophan

decarpoxylase isozyme extracted from the seedling variety

(LM) bush of the different doses 0, 2, 4, 6, 8, 10, 12, 14, 16,

18 and 20 Krad in the radiation treatments taxa, Catharanthus

roseas are shown in Figure (15) and are diagrammatically

illustrated in Figure (15) and table (8).The Figures showed a

maximum number of eleven bands.

In taxa with (bands 0 to 18) showed that one bands, the

20 exhibited bands (4) with lower intensity than the treated

samples, the radiation effects on gene expression. Band 1

presents and very faint in the control untreated but was


٨٦

contradictory in the intensity and molecular Weght in the

other doses treatments. Therefore, irradiation modified gene

expression in taxa.

In 20 Krad (lanes 1 to 4) showed that, the enzyme

degradation while complete in control and all doses, so that

should be considered as a conferring modification of gene

expression by irradiation.

As kown Irradiation induces the expression of the

Kinase proteins genes via posttranslational modification

which further interacts with the Tdc and Str promoter

enhancing the gene expression, many protein kinases are

known to respond to both biotic and abiotic stresses. Two

kinases, MAPKs and CDPKs, have been implicated to play

pivotal roles in response to diverse stimuli. Previous studies

have demonstrated that C. roseas variety (LM) cells also

respond to UV-B irradiation by expressing biosynthetic genes

and production of TIAs. To establish a functional link

between these processes, we first examined the possible

activation of MAPK and CDPK in cells irradiated with UV-

B. MBP is known to be a conventional MAPK substrate and

MAPK homologs also have MBP kinase activity. To

determine if a MAPK is associated with the UV-B signaling

the activation of MBP kinase was investigate Shilpa et al.,

(2007).


٨٧

The obtained results are in agreement with Shilpa et

al., (2007). Who analyzed a C. roseas which are treated by

UV throw isoenzyme (TDC) The enzyme was estimated to be

approximately 49 kDa. The 49-kDa MBPK activity increased

by UV-B irradiation in cells compared with that of the un-

irradiated control.

Figure (15): Tryptophan decarpoxylase histogram for the Catharanthusroseus variety (LM) which is treatments by Gamma radiation.

Tryptophan decarboxylase (TDC) M. W. K. Da.Bands

0 1 2 3 4 5 6 7 8 9 10 11 12

M. W

. K. Da.

100

102

104

106

108

110

112

114

116

118

Tryptophan decarboxylase (TDC) IntenesityBands

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nesi

ty

0

5

10

15

20

25


٨٨

The maximum MBPK activity was observed at 10 min

after UV-B treatment. In all the in vitro experiments carried

out with MBP as substrate, the phosphorylation peaked at 10

min; these results were consistently obtained when the

experiments were repeated with different batches of cells.

Therefore, in all further experiments the MBPK activity was

assayed at 10 min after irradiation. To further characterize

the MBPK activity induced by UVB, immunoprecipitation

and in-gel kinase assays were used. The protein extracts were

incubated with anti-phosphotyrosine monoclonal antibody

and immunoprecipitated with protein A-agarose. The

immunoprecipitated proteins were separated on a SDS-

polyacrylamide gel containing MBP as a substrate and

MBPK activity was assayed in the gel in the presence of 32P-

ATP. As shown in Figure 3c, a 49 kDa protein kinase was

again detected in the immunoprecipitated from UV-B-

irradiated cells. Co-incubation with phosphotyrosine

prevented immunoprecipitation of the 49 kDa protein kinase

with antiphosphotyrosine antibody, but co-incubation with

phosphothreonine did not. These results indicate that only

phosphotyrosine and not phosphothreonine could act as a

competitor during immunoprecipitation, showing that MBP

phosphorylating kinase was specifically phosphorylated on a

tyrosine residue. Till date MAPK are the only known plant


٨٩

kinases to be phosphorylated on tyrosine residues. Calcium

dependent protein kinases (CDPKs) belong to the unique

family of calcium-regulated kinases and histone IIIS was one

of the best exogenous substrates for assaying CDPKs to

characterize the kinase (s) induced by UV-B, the activities

were assayed using histone IIIS as a substrate in protein

extracts from cells irradiated with UV-B, as well as the

controls. The protein extracts from 5-min UV-B irradiated

cells, assayed in the presence of calcium using histone IIIS as

substrate showed that, the kinase activity increased

significantly peaking at 4 min after UV-B irradiation and

remained high even at 20 min after UV-B irradiation. The

protein extracts from 5-min UV-B irradiated cells assayed by

in- gel kinase assay in the absence and presence of calcium

using histone IIIS as substrate demonstrated that the

phosphorylation of histone IIIS was calcium dependent in

both UV-B irradiated and un-irradiated cells. CDPK

activities were identified at two positions with an apparent

molecular weight of 55 kDa and 40 kDa. One of the CDPK

activated had an apparent molecular weight of 40 kDa and

was constitutive, as it was observed to phosphorylate histone

IIIS to a similar extent in both un-irradiated and irradiated

cells whereas the 55 kDa kinase activity showed UV-B

dependence and peaked at 4 min. Therefore, the


٩٠

phosphorylation of histone IIIS observed in vitro experiments

was both due to the activities of the 55 and assayed by in-gel

kinase assay containing histone IIIS as substrate. Figure 4c

shows that the 55 and 40 kDa kinases identified by in-gel

kinase assay were both phosphorylated on serine residues and

that the activity of 40 kDa kinase was constitutive in our cell

cultures. In all the in vitro experiments carried out with

histone IIIS as substrate, the phosphorylation peaked at 4

min. These results were consistently obtained when the

experiments were repeated with different batches of cells.

Therefore, in all further experiments the CDPK activity was

assayed at min after irradiation.

IV.3.2. Strrictosidinesynthase enzyme (STR).

Zymograms of Strrictosidinesynthase enzyme

(STR) for the Catharanthus roseas variety (LM) taxa are

shown in Figure (16) and Table (8) and are

histogrammatically illustrated in Figure (16).

In taxa with (lanes 0 to 18) showed that one bands, the

20 exhibited no bands with lower intensity than the treated

samples, the radiation effects on gene expression. Bands

from control to 10 Krad. presents and very dark but was

contradictory in the intensity and molecular Weght in the


expression in taxa. On the other hand radiation treatments 12,


٩١

14, 16, and 18 Krad. were feint or very feint and contradictory

in the molecular Weght so that should be considered as a

conferring modification of gene expression by irradiation.

Our results agreed with those of Shilpa et al., (2007)

who irradiated C. roseas by UV and studied enzyme (TDC)

and (STR) since the UV-B-induced early cellular responses

viz., medium alkalinization and ROS production were

inhibited by suramin, we investigated whether

Figure (16): Strictosidine synthase histogram for the Catharanthusroseus variety (LM) which is treatments by Gamma radiation.

Strictosidine synthase(SSS)Bands

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nesi

ty

0

5

10

15

20

25

Strictosidine Synthase(SSS) M. W. K. Da.

Bands

0 1 2 3 4 5 6 7 8 9 10 11 12

M. W

. K. D

a.

0

10

20

30

40


٩٢

suramin could inhibit the UV-B induced other cellular

responses related to synthesis of TIAs. When the cells were

pretreated for 10 min with 0.1 and 1 mM suramin

concentrations and subsequently irradiated with UV-B for 5

min, the UV-Binduced MBPK and CDPK activities,

accumulation of Tdc and STR transcripts and Catharanthine

was strongly inhibited. However, the UV-B-induced MBPK

activity could not be completely inhibited by suramin. To

rule out the possibility that the inhibitory effects of suramin

on responses triggered by UV-B are not due to the unspecific

binding to cell surface components, we used heparin a

structurally similar molecule viz., heparin that possesses

sulfonic acid groups similar to that of suramin for inhibition

of UV-B responses.


٩٣

Table (8) M. W. and Intensity of Tryptophan decarpoxylase (TDC) andStrictosidine synthase (STR) Enzymes in Catharanthus roseus variety

(LM) which is treatment by Gamma radiation.

IV.4. Effect of radiation on Protein banding patterns.

The results of electrophoretic patterns of protein

extracted from the seedling of Catharanthus roseus variety

(LM) bush of the different doses of γ-rays control, 2, 4, 6, 8,

10, 12, 14, 16, 18 and 20 Krad in the taxa, the obtained of

results show in table (9) and figure (16) and are

histogrammatically illustrated in Figure (16).The Figures

showed a maximum number of twelve bands.

In taxa with lanes 1 showed that two bands, the control

exhibited band 1 with lower intensity than the treated

samples, the radiation effects on gene expression. Bands 1

and 2 presents in the control and sample 2 Krad treated but

Symbol

No.

Radiation

treatment

TDC STR

M. W. KDa Intensity M. W. KDa Intensity

1 0 101 5 31 6

2 2 113 8 32 8

3 4 105 9 37 9

4 6 115 10 37 10

5 8 109 9 36 10

6 10 110 9 38 10

7 12 112 8 30 7

8 14 112 7 29 6

9 16 116 8 28 2

10 18 107 3 34 1

11 20 113 2 00 0


٩٤

was absent in the other doses treatment. Therefore,

irradiation modified gene expression in these taxa.

In the radiation treatments 2 Krad lanes 2 showed that,

band 3, 4, 5 and 6 was absent in control while present in 6

and 8 Krad doses, so that should be considered as a conferring

modification of gene expression by irradiation.

The pattern of 4 Krad lanes 3 exhibited five bands,

band 7 to 12 were present. Indicating the effect of 6 Krad

irradiation treatments on modification of gene expression in

C. roseas variety (LM) Bands 3 and 5 were presents only in

the 6 Krad treatment but were absent in control and 4, 12 and

14 Krad treatments. Therefore, irradiation treatments effected

on modification of gene expression in C. roseas variety (LE).

treated.

The protein Zymograms of lanes 5 to 11 showed nine

bands, which are absent in the some treatments and were

present in the others irradiation treatments affected on gene

expression in C. roseas variety (LM).

Our results agreed with those of Kazuyuki et al.,

(2001) in the preliminary experiments, we found that a

TPCK-treated bovine trypsin (Sigma, type XIII) was

contaminated with GICP. Therefore, we tried to purify GICP

from 116 mg trypsin. Using a combination of several


٩٥

chromatographic procedures, we obtained 0.09 mg enzyme

(the specific activity of 12 n molls/min/mg proteins). SDS-

PAGE pattern of the enzyme showed a major band with a Mr

of approximately 29,000. The N-terminal sequence of major

band was homologous to that of chymotrypsinogen, starting

from Ile-16 Meloun et al., (1966). Therefore, we tested

whether chymotrypsin isozymes, a-, d-, and p-chymotrypsins

have the activity to cleave T-1 in the presence of TPCK.

However, none of these chymotrypsins had such activity.

These results suggest that the amount of GICP is very small

in the commercial preparations of trypsin. Thus, we decided

to purify GICP from bovine pancreas.

Seedling to affect on both Tdc and STR genes, where

STR enzyme wills condensate tryptamine and secologanin to

produce Strrictosidine. The obtained results showed that the

following experiments of C. roseas were used as seedling

bush for extra Tryptophan decarpoxylase and

Strrictosidinesynthase genes


٩٦

Figure (16): Protein histogram for the Catharanthus roseus variety(LM) which are treated by Gamma radiation

M. W. of protein bandsBands

0 1 2 3 4 5 6 7 8 9 10 11 12

M. W

. K. D

a.

0

10

20

30

40

50

60

70

80

Intenesity of protein bandsBands

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nesi

ty

0

1

2

3

4

5

6

7


٩٧

Table (9): SDS – Page protein analysis of the Catharanthus roseasvariety (LM) which is treated by Gamma radiation.

M: molecular Weght by K da. I: intensity

M. W. of bands Total number of

bands70 65 44 40 37 36 35 27 25 20 18 15 10 7 5

Rad

iatio

n tr

eatm

ents

Kra

d.

00 M + + - - - - - - + + + + - - + 7

I V.f d - - - - - - v.f v.f v.f v.f - - d

02 M + + + + + + - - + + + + + - + 12

I d f d f v.f v.f - - d d f d d - vd

04 M - - - - - - - - + + + + - - + 5

I - - - - - - - - f f f f - - Vd

06 M - - - + - + - - + + + + + - + 8

I - - - d - d - - vd d d d d - Vd

08 M - - + - + - + - + - + + + + + 9

I - - f - f - f - vd - f f f f Vd

10 M - - - + - + - - + - + + + - + 7

I - - - vf - vf - - vd - f f f - Vd

12 M - - - - - - - - + - + + + - + 5

I - - - - - - - - f - f f f - Vd

14 M - - - - - - - - + - + + - - + 4

I - - - - - - - - f - f f - - Vd

16 M - - - - - + + + + - + + - - - 6

I - - - - - d d d d - vf vf - - -

18 M - - - - + - + - + - + - - + + 6

I - - - - d - d - vd - d - - d vd

20 M - - - - + - + - + - + - + - + 6

I - - - - f - f - vd - f - f - vd


٩٨

IV.5. DNA finger print analysis

Random amplified polymorphic DNA (RAPD).

a- First variety (LM):

Result obtained from RAPD PCR analysis with 5

primers were used for the identification of markers associated

with 11 radiation treatments taxa genotypes after four weeks

presented as follows;

1- Primer OP-B01

(Table 10) shows the effect of gamma radiation

doses on Catharanthus roseas genome throw PCR

techniques, Primer OP-B01 gave 2 monomorphic fragments

with molecular sizes ranging from 80 to 12000 bp. (Figure

17). with 17 polymorphic fragments (80.9 %) with numbers

4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20 and 21

with corresponding molecular sizes of 11000, 10500, 10000,

9500, 9000, 8800, 8500, 6000, 3300, 2300, 1900, 1200,

1000, 800, 600, 400, 300, 100 and 80 bp. were observed. On

the other hand unique bands were 11000 bp. whereas,

monomorphic were at 1 and 13 with corresponding 12000

and 4500 bp. radiation treatment 16 and 18 Krad. exhibited the

maximum number 12 of fragments, while the lowest number

6 appeared in radiation treatment 0 and 2 Krad.

This primer showed that a fragments with 11000

and 10500 bp. appeared exclusively in radiation treatment20


٩٩

Krad., the fragments with 9500 bp. appeared exclusively in

radiation treatment 4 and 6 Krad. but was absent in all other

taxa. So it could be used as molecular marker for both above

tretments. the fragments with 300 bp. appeared exclusively

in radiation treatment16 and 18 Krad., The fragments with 80

bp. appeared exclusively in radiation tretments 12 or 14 Krad.

respectively, so the fragments could be used as molecular

markers for both above tretments.

Table (10): RAPD profiles of the Catharanthus roseas which were treated by Gammaradiation amplified with primer OP-B01.

BandNo.

size(bp.) M.

Radiation treatments Krad

0 2 4 6 8 10 12 14 16 18 201 12000 - - - - - - - - - - -2 11000 +3 10500 +4 10000 + + + + + +5 9500 + +6 9000 + + + + + +7 8800 + + + + + +8 8500 + + + + + + + +9 6000 + + + + + + +

10 3300 + + + + + + + +11 2300 + + + + + +12 1900 + + + + + + + + +13 1500 - - - - - - - - - - -14 1200 + + + + + + + + + +15 1000 + + + + + + +16 800 + + + + + + + + +17 600 + + + + +18 400 + + + +19 300 + +20 100 + + +21 80 + +Unique bands: 2 (9.5) % Polymorphic: 17 (80.9) % Monomorphic: 3 (14.3) %

Total bands / column 5 6 7 8 11 11 12 9 12 12 9Total bands: 102


١٠٠

2- Primer OP-B07

Table (11) Figure (17) were presented the results

obtained by using primer OP-B07 gave 4 monomorphic

fragments with molecular sizes ranging from 80 to 12000 bp.

with 16 polymorphic fragments (76.2 %) with numbers 1, 2,

3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 18 and 21 with

corresponding molecular sizes of 12000, 11000, 10500,

10000, 9500, 9000, 8800, 8500, 6000, 3300, 2300, 1900,

1500, 1200, 1000, 800, 600, 400, 300, 100 and 80 bp. were

observed on the other hand unique band with numbers 20

corresponding 2000 bp. (4.8 %).

Specific markers were 6 polymorphic fragments (8

%) and unique band fragments on the other hand unspecific

markers were 10 polymorphic fragments (13 %). Radiation

treatment 16 and 18 Krad exhibited the maximum number 11

of fragment; while the lowest number 4 appeared in radiation

treatment 10 and 20 Krad., So it could be used as molecular

markers for both above tretments. The fragment with 300 bp.

appeared in radaition treatment4 Krad. only but was absent in

all other taxa. So we could consider this primer as molecular

marker for radaition tretments 4, 10, 16, 18 and 20 Krad.


١٠١

Fig. (17) Continue

PO-B01

PO-B07

PO-B11


١٠٢

PO-B12

PO-F06

Figure (17): RAPD profiles of the Catharanthus roseas which are treatment by

Gamma radiation amplified with 5 primers (PO-B01, PO-B07, PO-B11, PO-B12, PO-

F06)


١٠٣


BandNo.

size(bp.)M.


0 2 4 6 8 10 12 14 16 18 20

1 12000 + +2 11000 + +3 10500 + + +4 10000 + + + +5 9500 + + + +6 9000 + + + +7 8800 + + + + + +8 8500 + + + + + + +9 6000 + + +10 3300 + + + + + + +11 2300 + + +12 1900 + + + + + + + +13 1500 + + + + + + + + + +14 1200 + + + +15 1000 - - - - - - - - - - -16 800 - - - - - - - - - - -17 600 - - - - - - - - - - -18 400 + + + + +19 300 +20 100 - - - - - - - - - - -21 80 + +

Unique bands: 1 (4.8) % Polymorphic: 16 (76.2) % Monomorphic: 4 (19) %

Total bands / columns 5 5 7 5 5 4 9 9 11 11 4Total bands: 75

3- Primer OP-B11

Result obtained from RAPD PCR analysis was

presented in Table (12) Primer OP-B11 gave 9 (42.9)

monomorphic fragments with molecular sizes ranging from

100 to 12000 bp. (9 %) figure (17). With 11 polymorphic

fragments (52.4 %) with numbers 5, 6, 7, 8, 10, 11, 12, 13,

18, 19 and 20 with corresponding molecular sizes of 9500,

9000, 8800, 8500, 3300, 2300, 1900, 1500, 400, 300 and 100

bp. were observed. Unique band was number 21 at 80 bp. on

the other hand specific markers didn’t observed, unspecific


١٠٤

markers were 54 polymorphic fragments (98.2 %). Whereas,

the numbers 1, 2, 3, 4, 14, 15, 16 and 17 were monomorphic.

Radiation treatment 20 Krad. exhibited the maximum number

9 of fragments, while the lowest number 2 appeared in

radiation treatments 0 and 2 Krad.

This primer showed that a fragment with 80 bp.

appeared exclusively in radiation treatment18 Krad., only but

was absent in all other taxa so the fragment could be used as

molecular markers for this doas.


BandNo.

size(bp.)M.

Radiation treatments Krad.

0 2 4 6 8 10 12 14 16 18 20

1 12000 - - - - - - - - - - -2 11000 - - - - - - - - - - -3 10500 - - - - - - - - - - -4 10000 - - - - - - - - - - -5 9500 + + +6 9000 + +7 8800 + + + +8 8500 + + + + + +9 6000 - - - - - - - - - - -

10 3300 + + +11 2300 + + +12 1900 + +13 1500 + + + + + +14 1200 - - - - - - - - - - -15 1000 - - - - - - - - - - -16 800 - - - - - - - - - - -17 600 - - - - - - - - - - -18 400 + + + + + + + + +19 300 + + + + + + + + +20 100 + + + + + + +21 80 +Unique bands: 1 (4.8) % Polymorphic: 11 (52.4) % Monomorphic: 9 (42.9) %



١٠٥

4- Primer OP-B12

As shown in Table (13) and fig. (17) Primer OP-

B12 gave 9 monomorphic fragments with molecular sizes

ranging from 80 to 12000 bp. with 11 polymorphic fragments

(52.4 %) with numbers 8, 9, 10, 11, 12, 13, 14, 15, 18, 19 and

20 with corresponding molecular sizes of 8500, 6000, 3300,

2300, 1900,1500,1200, 1000, 400, 300 and 100 bp. were

observed. Unique bands was 80 bp. at 20 Krad. On the other

hand specific markers were one polymorphic fragments (9.1

%) and unspecific markers were 10 polymorphic fragments

(99.9 %). Radiation treatment 20 Krad exhibited the

maximum number 12 of fragments, while the lowest number

1 appeared in radiation treatments 2 Krad while radiation

treatments 0 Krad didn’t give any fingerprint.


appeared in 2 and exclusively in radiation treatment20Krad.,

so the fragment could be used as molecular markers for thes

tretment.

5- Primer OP-F06

The effect of different doses on the Genomic DNA

was cleared in Primer OP-F06, it was gave 7 monomorphic

fragments with molecular sizes ranging from 80 to 12000 bp.

(7 %) (figure 17) and (table 14). with 11 polymorphic

fragments (52.4 %) with numbers 6, 8, 11, 12, 13, 14, 15, 16,


١٠٦


Band No.size(bp.)M.


0 2 4 6 8 10 12 14 16 18 20

1 12000 - - - - - - - - - - -2 11000 - - - - - - - - - - -

3 10500 - - - - - - - - - - -4 10000 - - - - - - - - - - -

5 9500 - - - - - - - - - - -6 9000 - - - - - - - - - - -7 8800 - - - - - - - - - - -8 8500 + + +

9 6000 + +10 3300 + + + +11 2300 + + + + + + +12 1900 + + + + + + +13 1500 + + + + + + + + +14 1200 + + + + + + + + +15 1000 + + + + + + + + +16 800 - - - - - - - - - - -17 600 - - - - - - - - - - -18 400 + + + + + + + + +19 300 + + + + + + + + +20 100 + + + + +21 80 +Unique bands: 1 (4.8) % Polymorphic: 11 (52.4) % Monomorphic: 9 (42.6) %


17, 18, 19, 20 and 21 with corresponding molecular

sizes of 9000, 8500, 2300, 1900, 1500, 1200, 1000, 800, 600

and 400 bp. unique band was 3 fragments at 2, 3 and 9

corresponding 11000, 10500 and 6000 bp. (14.3 %) were

observed. On the other hand specific markers were 1

polymorphic fragments (9.1 %) and unspecific markers were

10 polymorphic fragments (99.1 %). radiation treatments 18

Krad. exhibited the maximum number 11 of fragments, while

the lowest number 3 appeared in radiation treatments 0 Krad.


١٠٧


appeared exclusively in radiation treatment10 and 14 Krad.,

So the fragment could be used as molecular marker for both

above tretments.

Table (15): RAPD profiles of the Catharanthus roseas which were treated by Gammaradiation amplified with primer OP-F06.

BandNo.

size (bp.)M.


0 2 4 6 8 10 12 14 16 18 20

1 12000 - - - - - - - - - - -2 11000 +

3 10500 +4 10000 - - - - - - - - - - -

5 9500 - - - - - - - - - - -6 9000 + + +7 8800 - - - - - - - - - - -8 8500 + + +

9 6000 +10 3300 - - - - - - - - - - -11 2300 + + + + + + + + + + +12 1900 + + +13 1500 + + + + + + + + +14 1200 + + + +15 1000 + + + + + +16 800 + + + + + +17 600 + + + + + + +18 400 + + + + + +19 300 + + + + + + + + + + +20 100 + + + + +21 80 + +Unique bands: 3 (14.3) % Polymorphic: 11 (52.4) % Monomorphic: 7 (33.3) %


VI.5.1.1. RAPD markers of the 11 radiation treatments

with 5 RAPD primers:

Data of the amplified fragments using the

aforementioned five 10-mer arbitrary primers OPB-01, OP-

B07, OP-B11, OP-B12, and OP-F06 for the 11 radiation


١٠٨

treatments controlled with 0 Krad; 2, 4, 6, 8, 10, 12, 14, 16, 18

and 20 Krad indicated successful amplification of PCR

products. Polymorphism levels differed from one primer to

the other .The main results was as following table (16).

Data in Table (16) showed that the five primers

showed polymorphic differences among the radiation

treatments, while some primers exhibited high polymorphism

such as OPB-01 (80.9 %) and OP-B07 (76.2 %) on the other

hand, some primers exhibited medium levels of

polymorphism such as OP-B07, OP-F06 and OP-B01 (52.4

%).

The Randomly Amplified Polymorphic DNA (RAPD)

technique is suitable for developing the molecular markers

because of its technical simplicity and the modest cost of

generating a large number of markers. Furthermore, the

technique dose not require knowledge of the genome and

RAPD marker have been adopted as a convenient means of

tagging genes of interest in Catharanthus roseas Quarta et

al., (2001).

PCR-based multi-locus DNA fingerprints represent

one of the most informative and cost-effective measures of

genetic diversity and are useful population-level biomarkers

of toxicological and other anthropogenic impacts. However,

Concerns about reproducibility of DNA fingerprints have


١٠٩

limited their wider use in environmental biology. We

assessed polymorphism and reproducibility of common

fingerprinting techniques, RAPD (randomly amplified

polymorphic DNA).

The results that by excluding bands that comprised

less than 1% of total intensity, and by excluding the largest

and smallest 10% of the bands, we could achieve nearly

100% reproducibility of RAPD fingerprints. Similar

application of band exclusion criteria to RAPD fingerprints

did not significantly enhance their reproducibility, and at

least 15% of RAPD bands were not fully repeat table, heir

table, or transmit table. Our results are in agreement with

Sarika et al., (2007) whom analyzed a C. roseas by

isoenzyme and RAPD markers in order to assess their genetic

relationships. They stated that RAPD technique, through

discriminating among all the species and distinguishing

among 144 F2 plants of C. roseas, developed by crossing the

accession ‘Pink Delhi’ (pink colored flower petals and stem,

dark green leaf lamina bearing elliptic apex and borne on

small petiole, long pods, tall habit, less salt and drought

sensitivity, and high in alkaloid yield).


١١٠

VI.5.1.2 Genetic similarity and cluster analysis

based on RAPDs markers

The RAPD data were used to estimate the genetic

similarity among 11 radiation treatments of seedling taxa by

Table (16): RAPD markers of the 11- radiation treatment with 5 RAPD primersRadiation

treatments Krad.

Primers Totalamplifiedfragments

OPB-01

OP-B07 OPB-11 OP-B12 OP-F06

0 Uniq.* 0 0 0 0 0 0Poly. ** 5 5 2 0 3 15Mono*** - - - + - 0

2 Uniq.* 0 0 0 1 0 1Poly. ** 6 5 2 0 4 17Mono*** 0 0 0 0 0 0

4 Uniq.* - 0 0 0 0 0Poly. ** 7 7 8 7 5 34Mono*** 0 0 0 0 0 0

6 Uniq.* 0 0 0 0 0 0Poly. ** 8 5 8 7 4 32Mono*** 0 0 0 0 0 0

8 Uniq.* 0 0 0 0 0 0Poly. ** 11 5 7 7 5 35Mono*** 0 0 0 0 0 0

10 Uniq.* 0 0 0 0 0 0Poly. ** 11 4 7 7 9 38Mono*** 0 0 0 0 0 0

12 Uniq.* 0 0 0 0 0 0Poly. ** 11 9 2 7 9 38Mono*** 0 0 0 0 0 0

14 Uniq.* 0 0 0 0 0 0Poly. ** 9 9 2 7 9 36Mono*** 0 0 0 0 0 0

16 Uniq.* 0 0 0 0 0 0Poly. ** 12 11 7 10 10 50Mono*** 0 0 0 0 0 0

18 Uniq.* 0 0 0 0 0 0Poly. ** 12 11 6 10 11 50Mono*** 0 0 0 0 0 0

20 Uniq.* 0 0 0 0 0 0Poly. ** 9 4 9 12 10 44Mono*** 0 0 0 0 0 0

*Unique bands: 1 **Polymorphic: 389 ***Monomorphic: 0Total bands: 390


١١١

using Biorad softwere computer analysis was shown in (table

17). The highest similarity index recorded was 0.998, which

was observed between the two taxa control and radiation

treatment 20 Krad while the lowest similarity index recorded

was 0.91, which was observed between radiation treatments

2 and 6 Krad

A dendrogram for the genetic relationships among

the control and 10 radiation treatments was carried out as in

Fig. (18). The 11 taxa were separated into two clusters;

cluster one included 0, 20, 12, 16, 18, 8, 2 and 4 Krad., while

the cluster two included 10, 14 and 6 Krad.

Within the cluster one, three sub clusters were found;

the first one contained 2 Krad. and 4 Krad., the second sub

clusters was divided into sub-sub-sub clusters the first

contain 0 and 20 Krad. The while the third sub clusters contain

12, 16 and 18 Krad. (in the first division), and 8 Krad. (in the

second division). The second cluster divided into two sub

clusters the first one contained 6 and 14 Krad. , while the

second sub cluster contained 10 Krad. only.


١١٢

Figiure No. (18 )

Table (17): Similarity indices among the 11 radiation treatment TaxaBased on RAPD-PCR using 5 primers

Radiationtreatment

Krad0 2 4 6 8 10 12 14 16 18 20

0

2 143

4 500 286

6 636 091 318

8 286 500 571 182

10 737 105 368 864 211

12 875 125 438 727 250 842

14 667 095 333 955 190 905 762

16 824 118 412 773 235 895 441 810

18 929 154 538 591 308 684 813 619 765

20 998 143 500 636 286 737 875 667 824 929


١١٣

b- Second variety (CP3):Depended on the above results, chosen the dose 16

Krad. because this dose appears of change on the Indole

alkaloids production, the result obtained from RAPD PCR

analysis with 10 primers were used for the identification of

markers associated with 8 time hours (con, 0, 2, 4, 8, 16, 48

and 168) hours which were radiated at 16 Krad. taxa

genotypes after four weeks presented as follows:

1- Primer OP-C09

Figure (19) and Table (18) showed that primer OP-C09

gave 11 polymorphic fragments with molecular sizes ranging

from 10000 to 900 bp. (100%) with numbers 1, 2, 3, 4, 5, 6,

7, 8, 9, 10 and 11 with corresponding 10000, 9000, 8000,

7000, 6000, 5000, 4500, 4000, 2500, 1500 and 900 bp. were

observed. Specific markers were 3 polymorphic fragments

(27%) and unspecific markers were 8 polymorphic fragments

(73%) this primers showed that a fragment with 10000, 9000

and 8000 bp. appeared exclusively after 168 hours on the

other hand no monomorphism. So we can concider these

fragments as molecular marker for radiation treatment 16

Krad. after 186 hours.


١١٤

2- Primer OP-C10

Primer OP-C10 exhibited eleven DNA fragments

ranging in molecular sizes from 10000 to 900 bp. (Figure 16

and Table 19). Five polymorphic fragments (45%) with

numbers 7, 8, 9, 10 and 11 with corresponding molecular

sizes of 4500, 4000, 2500, 1500 and 900 bp., respectively,

were observed. Whereas, the remaining bands were

monomorphic (55%).

Table (18): RAPD profiles of the 8 time treatment which are irradiated at 16 Krad.

with primer OP-C09.

Band No. M.W (bp)Time by hours

Con. 0 2 4 8 16 48 168

1 10000 0 0 0 0 0 0 0 12 9000 0 0 0 0 0 0 0 13 8000 1 1 1 1 1 1 1 04 7000 1 1 0 1 0 0 0 15 6000 0 1 0 0 0 0 0 06 5000 0 1 0 1 0 0 0 17 4500 0 1 0 1 0 0 0 08 4000 0 1 0 1 0 0 0 09 2500 1 1 0 1 1 1 1 010 1500 1 1 0 1 0 1 0 111 900 0 1 0 1 0 1 1 1

Total bands 4 9 1 8 2 4 3 61= present 0= absent


with primer OP-C10.


١١٥

3-

Primer

OP-C13

Pri

mer OP-

C13

exhibite

d 11

DNA fragments ranging in molecular sizes 10000 to 900 bp.

(Figure 19 and Table 19). Sex fragments were polymorphic

with number 1, 2, 3, 4, 6 and 9 with corresponding molecular

sizes of 1000, 9000, 8000, 7000, 5000 and 2500 bp. (82%)

meanwhile bands number (5, 7, 8, 10 and 11) were

monomorphic(18%). This primer showed that a fragment

with 9000, 5000 bp. after 168 hours and 2500 bp. after 8

hours appeared exclusively so we can concider these

fragments as molecular marker for radiation treatment 16

Krad. after 8 and 186 hours.


Con. 0 2 4 8 16 48 168

1 10000 0 0 0 0 0 0 0 02 9000 0 0 0 0 0 0 0 03 8000 0 0 0 0 0 0 0 04 7000 0 0 0 0 0 0 0 05 6000 0 0 0 0 0 0 0 06 5000 0 0 0 0 0 0 0 07 4500 1 1 0 1 0 1 0 18 4000 1 1 0 1 0 1 1 19 2500 1 1 1 1 1 1 1 1

10 1500 1 1 0 1 0 1 0 111 900 1 1 0 0 0 1 0 1



with primer OP-C13.

Band No. M.W (bp) Time by hours


١١٦

4-Primer OP-C15

Pr

imer

OP-

C15

resulted in 11 DNA fragments with molecular sizes from

10000 to 900 bp. (Figure 19 and Table 20). Four

polymorphic fragments (36%) with numbers 1, 2, 3 and 4

with corresponding molecular sizes of 10000, 9000, 8000

and 7000 bp. were observed, while the other bands were

monomorphic. This primer showed that a fragment with 7000

bp. appear in 4 time hour. but was absent in all other taxa. So

the fragment could be used as molecular marker for 4 time

hour.


with primer OP-C15.


Con. 0 2 4 8 16 48 168

Con. 0 2 4 8 16 48 168

1 10000 0 0 0 0 1 0 0 02 9000 1 1 1 1 1 1 1 03 8000 0 0 0 0 1 1 1 14 7000 0 0 0 0 1 1 1 15 6000 1 1 1 1 1 1 1 16 5000 0 0 0 0 0 0 0 17 4500 0 0 0 0 0 0 0 08 4000 0 0 0 0 0 0 0 09 2500 1 1 1 1 0 1 1 1

10 1500 0 0 0 0 0 0 0 011 900 0 0 0 0 0 0 0 0



١١٧

1 10000 1 1 1 1 0 0 0 02 9000 1 1 1 1 0 0 0 03 8000 1 1 1 1 0 0 0 04 7000 0 0 0 1 0 0 0 05 6000 1 1 1 1 1 1 1 16 5000 0 0 0 0 0 0 0 07 4500 0 0 0 0 0 0 0 08 4000 1 1 1 1 1 1 1 19 2500 0 0 0 0 0 0 0 0

10 1500 0 0 0 0 0 0 0 011 900 0 0 0 0 0 0 0 0


5- Primer OP-G17Primer OP-G17 gave 4 monomorphic fragments with

molecular sizes 7000, 6000, 5000 and 4000 bp. (36%) and 7

polymorphism fragments (64%) (Figure 19 and Table 21).

The fragment with 4500 and 900 bp. appeareds exclusively

in 4 and 0 time hours fluctuated but was absent in other taxa.

So it could be used as molecular marker for 4 and 0 time

hours.


with primer OP-G17.


Con. 0 2 4 8 16 48 168

1 10000 0 0 0 1 0 0 0 0


١١٨

2 9000 0 0 1 1 0 1 0 03 8000 1 1 1 1 0 1 0 04 7000 1 1 1 1 1 1 1 15 6000 1 1 1 1 1 1 1 16 5000 1 1 1 1 1 1 1 17 4500 1 1 1 0 1 1 1 18 4000 1 1 1 1 1 1 1 19 2500 0 1 1 1 0 1 0 1

10 1500 1 0 1 0 1 0 0 111 900 0 1 0 0 0 0 0 0


6- Primer OP-L12Primer OP-L12 resulted polymorph in 6 DNA fragments

with molecular sizes 1000, 9000, 8000, 7000, 6000 and 900

bp. (Figure 19 and Table 22) (55%). Three polymorphic

fragments (Number 3, 4 and 5) with corresponding molecular

size of 8000, 7000 and 6000 bp were observed exclusively in

control, while the other bands were monomorphic So, it

could be used as molecular marker for control.

7- Primer OP- L13Primer OP-L13 gave 7 monomorphic fragments with

molecular sizes 10000, 9000, 7000, 5000, 4500, 4000 and

2500 bp. and 4 polymorphism with molecular size 8000,

6000, 1500 and 900 bp. (Figure 19 and Table 22).


with primer OP-L12.


Con. 0 2 4 8 16 48 168

1 10000 0 1 1 1 1 0 1 1


١١٩

2 9000 0 1 1 1 1 0 1 13 8000 0 1 1 1 1 1 1 14 7000 0 1 1 1 1 1 1 15 6000 0 1 1 1 1 1 1 16 5000 1 1 1 1 1 1 1 17 4500 1 1 1 1 1 1 1 18 4000 1 1 1 1 1 1 1 19 2500 1 1 1 1 1 1 1 1

10 1500 1 1 1 1 1 1 1 111 900 1 0 1 0 0 0 0 0


The fragments 8000 and 6000 bp. were observed exclusively

in control and 48 time hours fluctuated, but were absent in

other taxa. So it could be used as molecular marker for

control and 48 time hours.


with primer OP-L13.


Con. 0 2 4 8 16 48 168

1 10000 0 0 0 0 0 0 0 02 9000 0 0 0 0 0 0 0 03 8000 0 1 1 1 1 1 1 14 7000 1 1 1 1 1 1 1 15 6000 0 0 0 0 0 0 1 06 5000 1 1 1 1 1 1 1 17 4500 0 0 0 0 0 0 0 08 4000 1 1 1 1 1 1 1 19 2500 1 1 1 1 1 1 1 1

10 1500 0 1 0 0 0 1 0 011 900 1 0 0 0 1 1 0 0


8- Primer OP- L16


١٢٠

Primer OP-L16 showed 2 monomorphic fragments

with molecular sizes 10000 and 4000 bp. with 9

polymorphism corresponding molecular size of 9000, 8000,

7000, 6000, 5000, 4500, 2500, 1500 and 900 (Figure 19 and

Table 24).The fragments 7000, 6000, 5000, 4500 and 900bp.

were observed in 0 time hour, on the other hands the 1500

bp. were observed exclusively in control but this fragments

were absent in other taxa. So it could be used as molecular

marker for control and 0 time hours.

9- Primer OP-L20

Primer OP-L20 resulted in 6 monomorphic DNA

fragments with molecular sizes from 6000 to 1500 bp.

(Figure 19 and Table 25). On the other hands their fife

polymorphic fragments (45%) with numbers1, 2, 3, 4 and 11


with primer OP-L16.


Con. 0 2 4 8 16 48 168

1 10000 0 0 0 0 0 0 0 02 9000 1 0 1 1 1 0 1 13 8000 1 0 1 1 1 0 1 14 7000 1 0 1 1 1 1 1 15 6000 1 0 1 1 1 1 1 16 5000 1 0 1 1 1 1 1 17 4500 1 0 1 1 1 1 1 18 4000 1 1 1 1 1 1 1 19 2500 1 1 1 1 0 1 1 1

10 1500 0 1 1 1 0 1 0 011 900 0 1 0 0 0 0 0 0



١٢١

with corresponding molecular size of 10000, 9000, 8000,

7000 and 900 bp. were observed, while the band of 7000 was

appeared exclusively in 48 time hours but this fragments was

absent in other taxa. So it could be used as molecular marker

for 48 time hours.


with primer OP-L20.


Con. 0 2 4 8 16 48 168

1 10000 1 1 1 1 1 0 0 12 9000 1 1 1 1 1 0 0 13 8000 1 1 1 1 1 0 0 14 7000 1 1 1 1 1 1 0 15 6000 1 1 1 1 1 1 1 16 5000 1 1 1 1 1 1 1 17 4500 1 1 1 1 1 1 1 18 4000 1 1 1 1 1 1 1 19 2500 1 1 1 1 1 1 1 1

10 1500 1 1 1 1 1 1 1 111 900 1 0 0 0 0 1 1 0


10- Primer OP-Z03

The results of Primer OP-Z03 gave 11 DNA fragments

ranging in molecular sizes from 10000 to 900 bp. (Figure 19

and Table 26). All fragment were polymorphic (100 %) were

observed. Whereas, on the other hands was no monomorphic

(0 %). This primer showed that the fragment with 8000, 7000

and 4500 bp. appeared in 16, 8 and control time hours

fluctuated exclusively only, but it were absent in all other

taxa. So it could be used as molecular marker for 16, 8 and

control time hours.


١٢٢


with primer OP-Z03.


Con. 0 2 4 8 16 48 168

1 10000 1 1 1 1 0 0 1 12 9000 1 1 1 1 0 0 1 13 8000 1 1 1 1 1 0 1 14 7000 1 1 1 1 0 1 1 15 6000 0 1 1 1 1 1 1 16 5000 0 1 1 1 1 1 0 17 4500 0 1 1 1 1 1 1 18 4000 0 1 1 1 1 1 0 09 2500 1 1 0 1 0 1 0 0

10 1500 1 1 0 1 0 1 0 011 900 0 1 0 1 0 1 0 0


OP-C09

Figure (19): RAPD profiles of the 8 times treatment which are Gamma Irradiated at 16 Krad.

amplified with primers (OP-C09, OP-C10, OP-C13, OP-C15, OP-G17, OP-L12, OP-L13,OP-L16, OP-L20 and OP-Z03).

Figure 19 continue,


١٢٣

OP-C10

OP-C13

OP-C15

Figure 19 continue,


١٢٤

OP-G17

OP-L12

OP-L13

Figure 19 continue,


١٢٥

OP-L16

OP-L20

OP-Z03


١٢٦

VI.5.2.1. RAPD markers of the 16 Krad radiation

treatments with 10 RAPD primers

Data of the amplified fragments using the

aforementioned five 10-mer arbitrary primers OP-C09, OP-

C10, OP-C13, OP-C15, OP-G17, OP-L12, OP-L13, OP-L16,

OP-L20 and OP-Z03 for 8 times (con., 0, 2, 4, 8, 16,

48 and 168) which are treated by using gamma radiation on

the dose 16 Krad. controlled with con. Which is none treated

by gamma ray; 0, 2, 4, 6, 8, 10, 12, 14, 16, 48 and 168 hours

indicated successful amplification of PCR products.

Polymorphism levels differed from one primer to the other

.The main results was as following table (27).

Data in Table (27) showed that the tine primers

showed polymorphic differences among the development

time by hours, while some primers exhibited high

polymorphism such as OP-C09 (73 %), OP-C13 (82 %), OP-

L13 (82%), OP-L16 (82%) and OP-Z03 (100 %) on the other

hand, some primers exhibited medium levels of

polymorphism such as OP-C10 (45 %), OP-C15 (63 %), OP-

G17 (64%), OP-L12 (55) and OP-L20 (45%).


١٢٧

Our result agreement with Ajaswrata et al., (2007)

who’s studying the terpenoid indole alkaloid pathway

genes and regulators reveals strong expression of

repressors in Catharanthus roseus cell cultures. Since

TIA biosynthetic pathway is reported to be stress induced in

C. roseus, we wanted to know the response of different TIA

and related pathway genes to different a biotic stressors. The

expression profiles of twelve genes that encode enzymes for

Table (27): Cultivars-specific RAPD markers of the 16 Krad with 10

RAPD primers

Time by hours Primers Total amplified

fragments

OP-

C09

OP-

C10

OP-

C13

OP-

C15

OP-

G17

OP-

L12

OP-

L13

OP-

L16

OP-

L20

OP-

Z03

Con. AF 4 5 3 5 7 6 5 8 11 6 60

SM - - - - 1 - - - - - 1

0 AF 9 5 3 5 8 10 6 4 10 11 71

SM - - - - - - - 1 - - 1

2 AF 1 1 3 5 9 11 5 9 10 8 59

SM - - - - - - - - - - -

4 AF 8 4 3 6 8 10 5 9 10 11 64

SM - - - 1 - - - - - - 1

8 AF 2 1 5 2 6 10 6 7 10 4 53

SM - - - - - - - - - - -

16 AF 4 5 5 2 8 8 7 7 8 8 62

SM - - - - - - - - - - -

48 AF 3 2 5 2 5 10 6 8 7 6 54

SM - - - - - - 1 - - - 1

168 AF 6 5 5 2 7 10 5 8 10 7 65

SM 2 - 1 - - - - - - - 3

AF: amplified fragmentsSM: specific markers


١٢٨

TIA and related primary (MEP and Shikimate) pathways

were monitored in leaf tissue separately exposed to 4_C,

dehydration, 200 mM NaCl, UV-light and wounding

conditions by semi quantitative RT-PCR analysis and

compared with the untreated control after 6 h treatment.

VI.5.2.2. Genetic similarity and cluster analysis

based on RAPDs markers

The RAPD data were used to estimate the genetic

similarity among 8 time hours which are treated by using

Gamma radiation bush taxa by using Concort softwere

computer analysis was shown in table (28). The highest

similarity index recorded was 0.906, which was observed

between the two taxa 8 hour and 48 hour while the lowest

similarity index recorded was 0.341, which was observed

between 0 hour and 8 hour.

A dendrogram for the genetic relationships among

the 8 different times was carried out as in Fig. (20). The 8

taxa were separated into two clusters; cluster one included 2,

4, 8, 16 and 168 hours while the cluster two included con., 0

and 48 hours.

Within the cluster one, four sub clusters were found;

the first one contained 2 and 8 hours, the second sub clusters


١٢٩

8 and 168 hours, the three sub clusters was 186 and 16 hours

the four sub clusters was 16 and 4 hours.

The second cluster divided into two sub clusters the

first one contained 4 and 48 hours, while the second sub

cluster contained 0 and control.

Table (28): Similarity indices among the 8 different time Taxa Based on RAPD-PCR using 10 primers

Time byhours Con. 0 2 4 8 16 48 168Con.

0 3972 729 4614 642 802 7428 652 342 838 507

16 586 517 738 580 74348 474 341 812 582 906 781168 679 595 719 721 821 818 819

Figure (20): Dendrogram for the genetic distances relationships among the8

different time taxa based on similarity indices data of RAPD analysis.


١٣٠

VI.6. Similarity and unsimilarity between (LM) & (CP3)

Catharanthus roseus Varieties in Genomic under

radiation stress.

The results obtained from RAPD-DNA in two C. roseus

variety (LM) & (CP3) appeared complementary in both it

especially at treatment 16 Krad we are shown in variety (LM)

primers OP-B11 & OP-B12 appeared low specific markers

one fragment only on the other hand variety (CP3) which are

treated by using gamma radiation at 16 Krad after 168 hours

given the same result low specific markers two and one

fragments in primers OP-C09 and OP-C13 respectively.

But all the remaining primers don’t give any specific

fragments in both two variety LM (at 16 Krad) & CP3 (at 168

hours).

There is suggested between both genomes LM & CP3

varieties after radiation treatment especially at 16 Krad. and

complimentary in gene expression to indole alkaloids

production, this are appeared clearly Suffix in HPLC results.

This results agreement with Sarika et al., (2007) that

analyzed a C. roseas by isoenzyme and RAPD markers in

order to assess their genetic relationships. The molecular

marker mapped in this study was of the RAPD types. The

fingerprinting RAPD markers are known to be useful in

increasing the map density in both eu chromatic and hetero


١٣١

chromatic regions. Indeed, 86 (68.8%) of the 125 DNA

markers placed on the C. roseus map are of RAPD kinds. The

sequence specific and EST-SSR markers are known to cover

the euchromatic regions of genome/map. A total of 12 such

markers were placed on the map. Thirty markers have their

origin in RAPD combinations. The combinations apparently

gave markers by promoting new amplifications in regions not

covered by a single RAPD.

RAPD markers are also expected to have covered the

chromatic regions of C. roseus map. This study showed that

combining of RAPD with anther kinds of primers can be a

reliable means to generate new markers.


١٣٢

______________________________________________Summary

١٣٣

Summary:

This work is a cooperation between Agriculture

Botany Department; Faculty of Agriculture, Al-Azhar

University, Nasr city, Cairo, Egypt; and Natural products

department; National center for radiation research and

technology, Atomic Energy Authority, Nasr city, Cairo,

Egypt. During the period (2007 – 2012), to study the gamma

irradiated effects on the Indole alkaloids production, protein

marker, TDC & STR enzymes, and DNA content in


The present work was aimed also to obtain the

maximum values of Indole alkaloids content using ionizing

radiation in tow variety (LM & CP3). Quantity & quality of

Indole alkaloids, protein, enzymes TDC & STR and DNA

determinations during expression of Indole alkaloids which

helped to achievement of these objectives.

I- Indole alkaloids determination by HPLC:

a- variety (LM):

Gamma rays effects on Catharanthus roseus (L.) were

genetically engineered to over express the two enzymes

Tryptophandecarboxylase and Strrictosidinesynthase, which

catalyze key steps in the biosynthesis of terpenoid Indole

alkaloids, using gamma radiation effect with the two

______________________________________________Summary

١٣٤

corresponding genes. The percentages of total alkaloids,

Catharanthine, Vindoline, Vallesiachotamine a (a),

Vallesiachotamine b (b) Ajmalicine, Horhammericine (a),


Strrictosidinelactam, Serpentine and Vinamidine were

recorded as relative to C. roseus intact plant. The highest

values of alkaloids were 6.190, 7.200, 5.940, 5.860, 7.790,

6.000, 6.672, 2.720, 7.000, 7.920, 7.230 and 6.400 μg of

alkaloids respectively above , in radiation treatments 18, 12

& 16, 14, 14, 18, 18, 18, 12, 12, 8, 12 and 16 K rad

respectively compared with other treatments.

b- Variety (CP3):

Gamma irradiation at 16 Krad was effective on

reducing the content of Ajmalicine, as compared with the

control. In all cases, raising the doses of Gamma irradiation

decreased the content of Ajmalicine production from 0 times

to 48 hour this content were 2.1801, 2.0001, 1.8777, 1.4062,

1.2001, 1.0006 and 0.9963 µg / g leaf tissue fluctuated. But

after one week 168 hours, Ajmalicine level production

increasing suddenly 208991 µg / g leaf tissue. ; this results

corresponds rationalize the cell surface receptor(s), Ca2+

influx, medium alkalinization, CDPK, H2O2 and MAPK

play significant roles in Gamma ray signaling leading to

stimulation of Tdc and Str genes and the accumulation of

______________________________________________Summary

١٣٥

catharanthine in C. roseus seedling cultures. Based on these

findings, a model for signal transduction cascade has been

proposed.

II- Enzyme analysis verity (LM)

a- TDC enzyme:

The radiation with Gamma rays effects on TDC. In

taxa with (lanes 0 to 18) showed that one bands, the 20

exhibited bands (4) with lower intensity than the treated

samples, the radiation effects on gene expression. Band 1

presents and very faint in the control untreated but was

contradictory in the intensity and molecular Wight in the


expression in taxa.

In 20 K rad (lanes 1 to 4) showed that, the enzyme de

dig ration while complete in control and all doses, so that


expression by irradiation. That we could be considered as a

positive molecular marker associated with irradiation

treatments.

b- STR or SSS enzyme:

STR or SSS, enzyme activity. In taxa with (lanes 0 to

18) showed that one bands, the 20 exhibited no bands with

lower intensity than the treated samples, the radiation effects

______________________________________________Summary

١٣٦

on gene expression. Bands from control to 10 Krad presents

and very dark but was contradictory in the intensity and

molecular Wight in the other doses treatments. Therefore,

irradiation modified gene expression in taxa. On the other

hand radiation treatments 12, 14, 16, and 18 Krad were feint

or very feint and contradictory in the molecular Wight so that


expression by irradiation. That we could be considered as a

positive molecular marker associated with irradiation

treatments.

III- SDS- protein Page electrophoresis in variety

(LM):

The effect of radiation treatments on the protein

banding were 2 Krad. lanes 2 showed that, band 3, 4, 5 and 6

was absent in control while present in 6 and 8 K rad. doses,

so that should be considered as a conferring modification of

gene expression by irradiation. The pattern of 4 Krad. lanes 3

exhibited five bands, band 7 to 12 were present. Indicating

the effect of 6 K rad. irradiation treatments on modification

of gene expression in C. roseas Bands 3 and 5 were presents

only in the 6 K rad treatment but were absent in control and

4, 12 and 14 Krad. treatments. Therefore, irradiation

treatments effected on modification of gene expression in C.

roseas treated.

______________________________________________Summary

١٣٧

IV-RAPD-PCR analysis showed that:

a- first verity (LM):

All the 5 primers successfully exhibited DNA

fragments with 11 radiation treatments taxa genotypes after

four weeks presented as follows; a total number of 72

fragments were visualized across the taxa. Different levels of

polymorphism were observed from one primer to the other.

Some primers exhibited high levels of polymorphism as:

OB-P01 primer showed that, fragment with 5500 bp

could be used as molecular marker for 6 Krad. Fragment with

4500 bp could be used as molecular marker for 8 K rad.

Fragment with 400 bp. could be used as molecular marker for

14 Krad.


could be used as molecular marker for 12 Krad. Fragment

with 250 bp. could be used as molecular marker for 20 Krad.


could be used as molecular marker for16 Krad. Fragment with

5000 bp. could be used as molecular marker for 2 Krad.


10 Krad. Fragment with 300 bp. could be used as molecular

marker for 4 Krad. Fragment with 100 bp. could be used as

molecular marker for 18 Krad.

______________________________________________Summary

١٣٨

OB-P12 primer showed that, fragment with 8500 bp.


with 4600 bp. could be used as molecular marker for 2 K rad.


12 Krad.

OP- F06 primer showed that, fragment with 11000 bp.


with 10000 bp. could be used as molecular marker for 18

Krad.

b- Second verity (CP3):

the result obtained from RAPD PCR analysis with

10 primers were used for the identification of markers

associated with 8 time hours deferens (con, 0, 2, 4, 8, 16, 48

and 168) hours which are radiated at 16 Krad taxa genotypes

after time above presented as follows; a total number of 495

fragments were visualized across the taxa. while some

primers exhibited high polymorphism such as OP-C09 (73

%), OP-C13 (82 %), OP-L13 (82%), OP-L16 (82%) and OP-

Z03 (100 %) on the other hand, some primers exhibited

medium levels of polymorphism such as OP-C10 (45 %),

OP-C15 (63 %), OP-G17 (64%), OP-L12 (55%) and OP-L20

(45%).

Primer OP-C09 this primer showed that a fragment

with 10000, 9000 and 8000 bp. appeared exclusively after

______________________________________________Summary

١٣٩

168 hours So we can concider these fragments as molecular

marker for radiation treatment 16 K rad. after 186 hours.

Primer OP-C13 This primer showed that a fragment with

9000, 5000 bp. after 168 hours and 2500 bp. after 8 hours

appeared exclusively so we can concider these fragments as

molecular marker for radiation treatment 16Krad. After 8 and 186

hours.

Primer OP-C15 This primer showed that a fragment with

7000 bp. appear in 4 time hour. but was absent in all other taxa.

So the fragment could be used as molecular marker for 4 time

hour.

Primer OP-G17 The fragment with 4500 and 900 bp

appeareds exclusively in 4 and 0 time hours flectuated but was

absent in other taxa. So it could be used as molecular marker for

4 and 0 time hours.

Primer OP-L12 The fragments 8000 and 6000 bp. were

observed exclusively in control and 48 time hours flectuated, but

were absent in other taxa. So it could be used as molecular

marker for control and 48 time hours.

Primer OP- L16 this primer showed that a fragment with

900 bp was observed in 0 time hour So it could be used as

molecular marker for control and 0 time hours.

On the other hands Primers OP-C10, OP- L13, OP-L20 and

OP-Z03 didn’t gave any molecular markers with samples above.

_____________________________________________ Reference

141

Reference

Afify, M. R. and Shousha, M. (1988). Effect of low dose

irradiation on soybean protein salubilit, typsin

inhibitor activity and protein patterns separated by

polyacrylamide gel electrophoresis, J. Agric. Food

Chem., 36: 810-813.

Ajaswrata, D.; Digvijay, S.; Sushil, K. and Jayanti, S.

(2007) a. Transcript profiling of terpenoid indole

alkaloid pathway genes and regulators reveals

strong expression of repressors in Catharanthus

roseus cell cultures. Plant Cell Rep. 26:907–915

Ajaswrata, D.; Jayanti, S. and Renu, D. (2007) b.

Downregulation of terpenoid indole alkaloid

biosynthetic pathway by low temperature and

cloning of a AP2 type C-repeat binding factor

(CBF) from Catharanthus roseus (L). G. Don.

Plant Cell Rep. 26:1869–1878.

Annemarie, H. M.; Erik, S.; Robert, V.; and J. Harry, C.

H. (1993) Isolation of cytochrome P-450 cDNA

clones from the higher plant Catharanthus roseus

by a PCR strategy. Plant Molecular Biology 22:

379-383.

Antonella, L.; Stefania, G.; Luigi M. and Teodoro, C.

(2007): Molecular tailoring and boosting of

_____________________________________________ Reference

142

bioactive secondary metabolites in medicinal plants

P. Ranalli (ed.), Improvement of Crop Plants for

Industrial End Uses, 471–507.

Antonio, L. and Alfredo, M. (2008): Survey of Vitamin B6

Content in Commercial Presentations of Table

Olives Plant. Foods Hum Nutr. 63:87–91.

Asada, M. and Shuler, M. (1989): Stimulation of

ajmalicine production and excretion from

Catharanthus roseus: effects of adsorption in situ,

elicitors, and alginate immobilization. Appl.

Microbiol. Biotechnol. 30: 475–481.

Bea, P.; Frederique, A. O. H.; Virginia, S. M.;

Guillaume, C.; Cocky, J. F.; Antony, C.;

Martial, P.; Bertvan, D.; Jan, W.; Kijne, L. F.

and Johan, M. (2004): Zinc Finger Proteins Act as

Transcriptional Repressors of Alkaloid

Biosynthesis Genes in Catharanthus roseus The

journal of biologecal chemistry vol. 279, No. 51,

17, pp. 52940–52948.

Benoit, S.; Felipe, A. V. and Vincenzo, D. (1999)

Multicellular Compartmentation of Catharanthus

roseus Alkaloid Biosynthesis Predicts Intercellular

Translocation of a Pathway Intermediate. The

Plant Cell, Vol. 11, 887–900.

_____________________________________________ Reference

143

Cambecedes, J.; Duron, M., Decourtye, L. and jalouzot,

R. (1992). Methodology of vitro Gamma

irradiation from lonicera species, mutant

description and biochemical characterization. Acts

Hort, 320, 119-126.

Carolyn, W.T. L.; Seda, E. and Jennifer, T. (2004):

Enhancement of ajmalicine production in

Catharanthus roseus cell cultures with methyl

jasmonate is dependent on timing and dosage of

elicitation. Biotechnology Letters 0: 1595–1599.

Collu, G.; Unver, N.; Peltenburg, A.; Heijden, R.;

Verpoorte, R. and Memelink, J. (2001): Geraniol

10-hydroxylase, a cytochrome P450 enzyme

involved in terpenoid indole alkaloid biosynthesis.

FEBS Lett. 508: 215–220.

Elizabeta, H. D.; Freddy, C. and Felipe, V. (2004).

Vindoline synthesis in vitro shoot cultures of

Catharanthus roseus. Biotechnology Letters 26:

671–674.

El-Shebawi, K. (1984). Biochemical changes in protein of

some food stuffs by ionizing radiation. M. Sc.

Thesis, faculty of Agriculture, Cairo University.

Felipe, V.; Vincenzo, D.; Mildred, C.; Adriana, C. and

Maria, M. (2002): Vindoline Biosynthesis Is

_____________________________________________ Reference

144

Transcription ally blocked in Catharanthus roseus

Cell Suspension Cultures Molecular biotechnology

Volume 22.

Felipe, V. and Victor, M. L. (2003): in vitro plant cell

culture as the basis for the development of a

research institute Mexico: Centro de Investigacio N

Cientifica de Yucata N. In Vitro Cell. Dev. Biol.

Plant 39:250–258.

Flaig, W and Schmid, G. (1966). Comparison of the effect

of chemical compounds and low doses of radiation

on plant metabolism. International Atomic Energy,

Vienna, 64:26.

Frank, L. H. M.; JAN, W. K.; and Johan, M. (1996):

Digging for Gene Expression Levels in

Catharanthus roseus Non radioactive Detection of

Plant mRNA Levels Biochemical. No. 2.

Georgieva, I. (1987). Cytochemical investigation of pallem

and pollen tubes after Gamma irradiation. II. Effect

of the irradiation on quinine formation.

Phytomorphology, 37: 203, 159-163.

Giancarlo, P.; Alexandra, S.W. E.; Pieter, B. F. O.;

Frank, L.H. M. and Johan, M. (1999): The

promoter of the strictosidine synthase gene from

periwinkle confers elicitor-inducible expression in

_____________________________________________ Reference

145

transgenic tobacco and binds nuclear factors GT-1

and GBF. Plant Molecular Biology 39: 1299–1310.

Gundlach, H.; Muller, M. J.; Kutchan, T. M. and Zenk,

M. H. (1992): Jasmonic acid is a signal transducer

in elicitor-induced plant cell cultures. Proc. Natl.

Acad. Sci. USA 89: 2389–2393.

Helen, T.; Xinwei C.; Pedro, N.; John, J. G.; Marjorie,

Santa, M. A. C.; Eleanor, G.; Paul, R. J. B. and

Gary, J. L. (2004) Activation tagging in plants: a

tool for gene discovery Funct Integr Genomics 4:

258–266.

Hussein, S. T.; Salah, E. M. A.; Ola, I.M. El-H.; Nahla, S.

A.; Naglaa, M. N.; Mohamed, K. E. and

Medhat, M. S. E. (2008): In vitro studies on

Egyptian Catharanthus roseus (L.) G. Don: III.

Effects of Extra Tryptophan Decarboxylase and

Strictosidine Synthase Genes Copies in Indole

Alkaloid Production. Research Journal of Cell and

Molecular Biology, 2 (2): 18-23.

Ishige, F.; Takaichi, M.; Foster, R.; Chua, N. H. and

Oeda, K. (1999). A G-box motif

(GCCACGTGCC) tetramer confers high-level

constitutiveexpression in dicot and monocot plants.

Plant J 18:443 – 448.

_____________________________________________ Reference

146

Jeong, D. H.; An, S.; Kang, H. G.; Moon, S.; Han, J. J.;

Park, S.; Lee, H. S.; An, K. and An, G. (2002). T-

DNA insertion mutagenesis for activation tagging

in rice. Plant Physiol 130:1636–1644.

Kazuyuki, A.; Yasuko, B. and Haruo, S. (2001): Bovine

Pancreatic Elastase II Cleaves Gln-Ile BondJournal

of Protein Chemistry, Vol. 20, No. 7

Laemmli, U. K. (1970). Cleavage of structural protein

during assembly of head bacteriophage T4. Nature,

227: 680-686

Lee, C. W. T. and Shuler, M. L. (2002): The effect of

ajmalicine spiking and resin addition timing on the

production of indole alkaloids from Catharanthus

roseus cell cultures. Biotechnol. Bioeng. 79: 408–

415.

Leslie, V.; Hui Z.; Frank, L.H. M,; Magdalena, D. and

Johan, M. (2000): A Catharanthus roseus BPF-1

homologue interacts with an elicitor-responsive

region of the secondary metabolite biosynthetic

gene Str and is induced by elicitor via a JA-

independent signal transduction Pathway. Plant

Molecular Biology 44: 675–685, 2000.

_____________________________________________ Reference

147

Lucumi, E.; Luczkiewicz M.; Vera A.; Hallard D.;

Heijden R. and Verpoorte R. (2001): Alkaloid

formation in cell suspension cultures of

Tabernaemontana divaricata after feeding of

tryptamine and loganin Biotechnology Letters 23:

1691–1696.

Maria, K. and Matgorzata, K. (1999): Graft and dodder

transmission of phytoplasma affecting lily to

experimental hosts. physiologiae plantarum Vol.

21. No. 1. 21-26.

Matsuhara, S.; Jingu, F.; Takahashi, T. and Komeda, Y.

(2000). Heat-shock tagging: a simple method for

expression and isolation of plant genome DNA

flanked by T-DNA insertions. Plant J 22:79 – 86.

Meloun, B.; Kluh, I.; Kostka, V.; Moravek, L.; Prusik,

Z.; Vanecek, J.; Keil, B.; and Sorm, F. (1966):

Biochim. Biophys. Acta 130, 543–546.

Mizukami, H.; Tabira, Y. and Ellis, B. E. (1993): Methyl

jasmonate-induced rosmarinic acid biosynthesis in

Lithospermum erythrorhizon cell suspension

cultures. Plant Cell Rep. 12: 706–709.

Mollenschott, W. N. C. and Berlin J. (1984): Tryptophan

decarboxylase from Catharanthus roseus cell

suspension cultures: purification, molecular and

_____________________________________________ Reference

148

kinetic data of the homogenous protein. Plant

Molecular Biology 3:281-288.

Mustafa R. N. and Verpoorte R. (2007): Phenolic

compounds in Catharanthus roseus Natali Rianika

Mustafa Robert VerpoortePhytochem Rev (2007)

6:243–258.

Novake, F. J.; Afza, R., Duren, M. V. and Omar, M. S.

(1990). Mutation induction by Gamma rays in vitro

cultured shoot tips of banana and plantain (Musa

Cvs). Tropical Agriculture, 67: 1-21.

OSCAR, J. M. G.; ED, J. M. P.; PETER, V.; ROB, A. S.;

ROB, V. and J. HARRY, C. H. (1995)

Overexpression of a tryptophan decarboxylase

cDNA in Catharanthus roseus crown gall calluses

results in increased tryptamine levels but not in

increased terpenoid indole alkaloid production

Transgenic Research 4,315-323

Peter, J. F. and Frank, D. (1991) Secondary metabolite

biosynthesis in cultured cells of Catharanthus

roseus (L.) G. Don immobilized by adhesion to

glass fibres Appl Microbiol Biotechnol 35:382-

392.

Saghai, M. A.; Soliman, K. M.; Jorgensen, R. A. and

Allard, R.W. (1984): Ribosomal DNA spacer-

_____________________________________________ Reference

149

length polymorphism in barley: Mendelian

inheritance, chromosomal location, and

populationdynamics. Proc. Natl. Acad. Sci. USA

81, 8014–8018.

Sarika, G.; Sashi, P.; Suchi, S.; Subhas, C. N.; Manoj, P.

and Sushil, K. (2007) Construction of genetic

linkage map of the medicinal and ornamental plant

Catharanthus roseus. Journal of Genetics, Vol. 86,

No. 3.

Schmauder H. P.; GrOger D.; Koblitz H.; and Koblitz D.

(1985): Shikimate pathway activity in shake and

fermenter cultures of Cinchona succirubraPlant

Cell Reports 4: 233- 236.

Serap, W.; Robert, V. and Camilo, C. (1998) Influence of

auxins on alkaloid accumulation by a transgenic

cell line of Catharanthus roseus. Plant Cell, Tissue

and Organ Culture 53: 135–141.

Shilpa, R. and Jayabaskaran, C. (2007): UV-B-induced

signaling events leading to enhanced-production of

catharanthine in Catharanthus roseus cell

suspension cultures. BMC Plant Biology 2007,

7:61

Stegmann, H.; Affify A. M. R. and Hussein K. R. F.

(1985). Cultivar identification of dates (Phoenix

_____________________________________________ Reference

150

dectylifera) by protein patterns. 2nd international

symposium of Biochemical Approaches to

identification of taxa. Braunschweig, West

Germany, 44pp.

Studier, R.; Rajkumar, W.; Dicosmo, Q. and Misawa, S.

(1973). Photoautotrophic cell suspension culture of

periwinkle (Catharanthus roseus) transition from

heterophic to photoautotrophic growth. Plant cell

Rep. 3: 195-198.

Toth, E.; Ghiorghita, G. and Gille, E. (1983). Some

biochemical peculiarities in plants of Digitalis

lanata Ehrh. Modified after successive treatments

with Gamma rays and ehylmethanosulfonate.

review roumaine de Biologie Vegetale, 28: 1, 41-

47.

Weigel, D., Ahn, J. H., Blazquez, M. A., Borevitz, J. O.,Christensen, S. K., Fankhauser, C., Ferrandiz,C., Kardailsky, I., Malancharuvil, E. J., Neff, M.M., Nguyen, J. T., Sato, S., Wang, Z. Y., Xia, Y.,Dixon, R. A., Harrison, M. J. Lamb, C. J.,Yanofsky, M. F. and Chory, J. (2000) Activationtagging in Arabidopsis. Plant Physiol 122:1003–1013.

Williams, J. G. K.; Kublik, A. R.; Livak, K. J., Rafalsky,

J. A. and Tingey, S. V. (1990). DNA

_____________________________________________ Reference

151

polymorphisms amplified by arbitrary primers are

useful as genetic markers. Nucl. Acid.Res.(18):6531-

6535.

www.pubchemsubstance.com

Young, G. M. H. K.; Jae, K. Y.; Young, G. C. and

Myung, S. C. (2003): Light-susceptibility of

Camptothecin Production from In Vitro Cultures of

Camptotheca acuminata Decne Biotechnology and

Bioprocess Engineering 8: 32-36.

Zuo, J; Niu, Q. W.; Frugis, G. and Chua, N. H. (2002).

The WUSCHEL gene promotes vegetative-to-

embryonic transition in Arabidopsis.Plant J 30:349

– 359.

Zuo, Z.; Niu, Q. W. and Chua, N. H. (2000). An estrogen

receptor-based transactivator XVE mediates highly

inducible gene expression in transgenic plants.

Plant J 24:265 – 273.

الملخص العربي_____________________________________________

1

الملخص العربى

–كلیة الزراعة –قسم النبات الزراعيبالتعاون بینأجریت ھذه الدراسة

بمدینة نصر ومعمل بحوث الھندسة الوراثیة بقسم بحوث –الازھرجامعة

بھیئة الطاقة -المنتجات الطبیعیة بالمركز القومي لبحوث وتكنولوجیا الاشعاع

دراسة لوذلك٢٠١٢-٢٠٠٧ن خلال الفترة مالذریة بمدینة نصر بالقاھرة

تأثیر الاشعاع الجامي على أنتاجیة قلویدات الاندول و حزم البروتین و أنزیمات

وكذلك المحتوى من ) التربتوفان دي كاربوكسیلیز و مخلق الاستركتوسیدین(

.المادة الوراثیة في نبات الونكا

ت الاندول والدراسة الحالیة تھدف الي الحصول علي قیم عالیة من قلویدا

وتم أجراء تحالیل كمیة في صنفین من نبات الونكابأستخدام الاشعاع المؤین

التربتوفان دي كاربوكسیلیز و (البروتین وأنزیمي ووكیفیة لقلویدات الاندول

وكذلك المادة الوراثیة وذلك أثناء التعبییر الجیني لانتاج ) مخلق الاستركتوسیدین

.الھدف من الدراسةقلویدات الاندول وذلك لتحقیق

I -تحلیل قلویدات الاندول باستخدام جھازHPLC:

: (LM)الصنف -ا

أظھرت المعاملات الاشعاعیة علي نبات الونكا تغیرات وراثیة وبالتالي

التربتوفان دي كاربوكسیلیز تغیرات علي التعبییر الفائق لانزیمیین ھما

ستركتوسیدین والمعروف اختصارا مخلق الاو (TDC)والمعروف أختصارا

(SSS or STR) وھما الانزیمان الاساسیا ن اللاذمان لحفز خطوات عملیة

التخلیق الحیوي لقلویدات الاندول و بأستعمال الاشعاع الجامي كان ھناك تأثیر

.علي تداخل فعل كلا الجینین المسؤلین عن انتاج كلا من الانزیمین السابقتین

:التي قدرت ھيوكانت القلویدات

الملخص العربي_____________________________________________

2

Catharanthine, Vindoline, Vallesiachotamine a (a),

Vallesiachotamine b (b) Ajmalicine, Horhammericine (a),


Strrictosidinelactam, Serpentine and Vinamidine

: عالیة ھي علي التوالي ونسبة ھذة القلویدات التي سجلت قیم

و2.720و6.672و6.000و7.790و5.860و5.940و7.200و6.190

وذلك .جرام نسیج ورقة/ میكرو جرام 6.400و7.230و7.920و٧٫٠٠٠

:للجرعات الإشعاعیة

18, 12 & 16, 14, 14, 18, 18, 18, 12, 12, 8, 12, 16 K rad

.وذلك مقارنة بباقي الجرعات الإشعاعیة

:(CP3)الصنف -ب

ملحوظا في اتأثیرKrad 16بجرعةأظھر تأثیر الإشعاع الجامي

فقد لوحظ أن المحتوى من قلوید الاجمالایسن وذلك مقارنة بالكنترول

فيساعة ٤٨المحتوى من القلویدات بدایة من عقب التشعیع مباشرة وحتى

فكانت القیمتدھور في إنتاجیة الاجمالایسین

1.0006و1.2001و1.4062و1.8777و2.0001و2.1801

ساعة أو ١٩٦ولكن بعد مرور .جرام نسیج ورقة/ میكرو جرام 0.9963

میكرو 2,08991لیصبح أسبوع فأن مستوى إنتاج الاجمالایسین قد أرتفع فجأة

)S(تفسیر بأن المستقبلھذه النتائج تتماشى مع ال، . جرام نسیج ورقة/ جرام

على سطح الخلیة و وتدفق أیونات الكالسیوم وقلویة البیئة وفوق أكسید

الكینیز تلعب دور ملموس مع الإشعاع الجامي في تالھیدروجین وبروتینیا

تحفیز كلا من جیني إنزیمي نازع الكربوكسیل ومخلق الاستركتوسیدین كما

الملخص العربي_____________________________________________

3

نسین في بادرات نبات الونكا و اعتمادا یساعد على تراكم وتجمیع مادة الكزارا

. نقل الإشارة بانتظام ھو النموذج المثاليلعلى الدراسات یعتبر مود ی

II- تحلیل الأنزیمات للصنف(LM):

: نازع مجموعة الكربوكسیل من التربتوفانإنزیم-ا

أظھر تحلیل نشاط انزیم التربتوفان دي كاربوكسیلیز والمعروف

٠وذلك بعد المعاملة بالاشعاع الجامي ان المعاملات من (TDC)أختصارا

حزم٤كانت بھا ٢٠احتوت علي بند واحدة بینما المعاملة الاشعاعیة ١٨الي

ذات كثافة خفیفة مقارنة مع باقي المعاملات وكان ھناك تأثیر للا شعاع علي

ولكن یلو راد ك٠التعبییر الجیني متمثلا بوجود بند واحدة خفیفة في المعاملة

كانت علي النقییض من ذلك في الكثافة والوذن الجزیئي في باقي المعاملات

الجیني نتیجة المعاملة بالاشعاع التعبیرفي تحویرولذلك یمكن ملاحظة وجود

كیلو راد اظھرت ان الانزیم ٢٠في الجرعة الاشعاعاعیة حزموالاربع . الجامي

لجرعات الاشعاعیة ولذلك من الممكن مقارنة مع الكونترول وباقي امتحلل

.بالإشعاعمعلمات موجبة للمعاملة حزماعتبار ھذة ال

:إنزیم مخلق الاستركتوسیدین-ب

أظھر تحلیل نشاط انزیم مخلق الاستركتوسیدین والمعروف اختصارا

(SSS or STR)كیلو ) ١٨(وحتي ) ٠(من ان مختلف الجرعات الاشعاعیة

تعطي اى لاكیلو راد ) ٢٠(دة اما الجرعة الاشعاعیة حزمة واحاعطت راد

. مع انخفاض ملحوظ في الكثافة مقارنة بباقي المعاملاتحزم

كیلو راد وحتي ) ٠(من حزماظھر تأثیر الاشعاع على التعبییر الجیني ان ال

كیلو راد كانت موجودة وذو كثافة كبیرة مقارنة مع الكثافة والاوزان ) ١٠(

ومن . الجینيالتعبیریر في الجرعات الاخري ولذلك حدث تحوالجزیئیة في

كیلو راد خفیفة او ١٨، ١٦، ١٤، ١٢جھة اخري كانت المعاملات الاشعاعیة

الملخص العربي_____________________________________________

4

ات المدروسة ولذلك المعطیحزممتناقضا مع الاوزان الجزیئیة لنفس الخفیفة جدا

اع الجامي یر في التعبیر الجیني بواسطة المعاملة بالاشعدلت علي ان ھناك تحو

ھناك تلاذم واضح بین المعلامات الجزیئیة الموجبة ولذلك نستطیع القول بأن

.الإشعاعیةوالمعاملات

III -الصنف تكنیك الالكتروفوریسیس في متحلیل الحزم البروتینیة باستخدا

(LM) .

ظاھرا في علي الحزم البروتینیةالمعاملات الاشعاعیةتأثیركان

K 2الجرعة الاشعاعیة rad. 3فظھرت حزم, 4, 5, بینما كانت ھذة الحزم 6

K 0البروتینیة غائبة فى المعاملة rad. بینما ھذة الحزم كانت موجودة في

,6المعاملات الاشعاعیة 8 K rad. ولذلك من الممكن استعمال تحلیل الحزم

ي التعبیر البروتینیة بأستخدام تكنیك الالكتروفوریسیز للاستدلال علي التغییر ف

K 4في الجرعة الاشعاعیة اما الجینیي rad. الي ٥ھي من حزمظھرت خمس

K 6واستحدثت المعاملة الاشعاعیة ٧ rad. تغییر في التعبییر الجیني في نبات

١٤و ١٢و ٤و٠بینما كانت غائبة في المعاملات ٥و ٣حزمالونكا فظھرت ال

املات الشعاعیة أحدثت تغییر في التعبییر كیلو راد ولذلك یمكن القول بأن المع

.الجیني في نبات الونكا

VΙ- أظھر تحلیل تفاعل البلمرة المتسلسل باستخدام البادئات العشوائیة

: آلاتي

):LM(الصنف الأول -ا

RAPDتقنیة الـ باستخدامأظھر التحلیل – PCR من ٥وعدد

، وقد اختلف مستوى التباین من حزمة ٧٢البادئات العشوائیة تم الحصول على

من التباین وكان ذلك عاليحیث أظھرت بعض البادئات مستوى . لاخر بادئ

: كما یلي

الملخص العربي_____________________________________________

5

جزیئيوتعتبر كشاف .pb 5500حزمةOP-BO1البادئأظھر

جزیئيتعتبر كشاف .pb 4500وأظھر حزمة Krad 6.الإشعاعیةللجرعة

تعتبر .pb 400جزیئيزمة بوزن وكذلك أظھر حKrad 8.الإشعاعیةللجرعة

.Krad 14الإشعاعیةكشاف جزیئي للجرعة

تعتبر كشاف والتي.pb 10000حزمة OP-B07البادئأوضح

من الممكن .pb 250واظھر حزمة Krad 12الإشعاعیةللجرعة جزیئي

.Krad 20الإشعاعیةاعتبارھا كشاف جزیئي للجرعة

والتى تعتبر كشاف جزیئى .pb 8500حزمةOP-B11البادئ أوضح

تعتبر كشاف جزیئي .pb 5000واظھر حزمة.Krad 16للجرعة الاشعاعیة

من الممكن اعتبارھا كشاف جزیئي .pb 500واظھر حزمة.Krad 2للجرعة

والتي من الممكن .pb 300وظھرت حزمة .Krad 10للجرعة الاشعاعیة

.pb 100وظھرت حزمة.Krad 4ة اعبارھا كشاف جزیئي للجرعة الاشعاعی

. .Krad 18من الممكن اعتبارھا كشاف جزیئي للجرعة

والتى تعتبر كشاف .pb 8500حزمة OP-B12البادئ أظھر

وتعتبر كشاف .pb 4600واظھر حزمة . Krad 16جزیئي للجرعة الاشعاعیة

كشاف یمكن اعتبارھا .pb 400كما ظھرت حزمة Krad 2جزیئي للجرعة

.Krad 12جزیئي للجرعة الاشعاعیة

وتعتبر كشاف جزیئى .pb 11000حزمة OP-F06البادئ بین

والتى تعتبر .pb 10000ظھر حزمة أكذلك Krad 20للجرعة الاشعاعیة

.Krad 18كشاف جزیئى للجرعة الاشعاعیة

: )CP3(الصنف الثاني -ب

RAPDأظھرت النتائج المتحصل علیھا من تحلیل – PCR١٠عدد ل

أوقات مختلفة ٨للتعریف كمعلم وذلك لثماني عینات تمثل من البادئات العشوائیة

الملخص العربي_____________________________________________

6

ساعة والتي عوملت بالجرعة ) ١٦٨و ٤٨و ١٦و ٨و ٤و ٢و ٠كنترول و (

كیلو راد وكان المظھر العام للحزم على الجل بعد الأوقات ١٦الإشعاعیة

:السابق ذكرھا ظھرت كما یلي

وكان ، حزمة منظورة ٤٩٥ن المجموع الكلي للحزم المتحصل علیھا كا

%) ٧٣(OP-C09ھناك بعض البادئات التي أعطت تباین عالي مثل

و %) ٨٢(OP-L13و %) ٨٢(OP-C13و

OP-Z3)ومن جھة أخرى نجد أن نعض البادئات أعطت مستوى %) ١٠٠

و %) ٦٣(OP-C15و %) ٤٥(OP-C10:باین مثلمتوسط من الت

OP-G17)٦٤ (% وOP-L12)٥٥ (% وOP-L20)٤٥.(%

و ٩٠٠٠و ١٠٠٠٠وزنھا الجزیئي حزمOP-C09أظھر البادئ

١٦٨كیلو راد بعد ١٦زوج نیوكلیوتیدي فقط مع المعاملة الإشعاعیة ٨٠٠٠

.ھساعة ولذلك یمكن اعتبارھا معلمات جزیئی

زوج ٥٠٠٠و ٩٠٠٠وزنھا الجزیئي حزمOP-C13أظھر البادئ

وكذلك أظھر١٦٨كیلو راد بعد ١٦نیوكلیوتیدي فقط مع المعاملة الإشعاعیة

ساعات ولذلك ٨زوج نیوكلیوتیدي في العینة بعد ٢٥٠٠بوزن حزمة واحدة

.ا الوقتیمكن اعتبار ھذا البادئ معلم جزیئي مع المعاملة الإشعاعیة بعد ھذ

زوج نیوكلیوتیدي ٧٠٠٠بوزن حزمة واحدة OP-C15أظھر البادئ

.ساعات ولذلك یمكن اعتبار ھذه الحزمة معلم جزیئي٤مع العینة المأخوذة بعد

زوج ٩٠٠و ٤٥٠٠وزنھا الجزیئي حزمأعطىOP-G17البادئ

م في ساعة وقد اختفت ھذه الحز٤نیوكلیوتیدي مع المعاملات الوقتیة صفر و

.باقي المعاملات ولذلك تعتبر معلمات جزیئیھ لھذه المعاملات

الملخص العربي_____________________________________________

7

٦٠٠٠و ٨٠٠٠حزم وزنھا الجزیئي أعطىOP-L12البادئ

ساعة بالترتیب ولذلك یمكن ٤٨زوج نیوكلیوتیدي حصریا في الكنترول و

.معلمات جزیئیھ خاصة بھذه المعاملاتحزماعتبار ھذه ال

زوج ٩٠٠مة بوزن جزیئي حزأعطى OP-L16البادئ

ولذلك تعتبر ھذه ) عقب التشعیع مباشرة(نیوكلیوتیدي في المعاملة صفر ساعة

.الحزمة معلم جزیئي لھذه المعاملة

,OP-C10ومن جھة أخرى البادئات OP- L13, OP-L20 and

OP-Z03لم تعطي أي معلمات جزیئیھ ممیزة مع المعاملات الإشعاعیة.

جامعة) - () (٢٠٠١

جامعة ) -(ة ي) (٢٠٠٦

) ()-(

–

هـ١٤٣٤٢٠١٣

عية

جامعة) - () بكال(٢٠٠١

جامعة ) ل-() (٢٠٠٦

) ()-(

–

هـ١٤٣٤٢٠١٣

:. / ...................................

.جامعة -ب. / ....................................

-.

جامعة) - () (٢٠٠١

جامعة ) -() (٢٠٠٦

) ()-(

جامعة –

هـ١٤٣٤٢٠١٣

:./..............................

-../.............................

-.. /..............................

-../...............................

-.

:٢٠١٣/ ٢٣/١

Documents

MOLECULAR AND RADIATION STUDIES ON IMPROVING THE