Table of Content

Abbreviations.................................................................................................................................2

Introduction to Tulip......................................................................................................................4

Scientific Classification of Tulip..................................................................................................4

Horticultural Classification of Tulip............................................................................................4

Uses of Tulip...............................................................................................................................5

Why Tissue Culturing of Tulip is Important?..............................................................................5

Objective........................................................................................................................................5

Literature Review...........................................................................................................................6

Materials and Methods............................................................................................................... 16

Preparation of Stock Solution...................................................................................................16

Media Preparation...................................................................................................................16

Media Optimization................................................................................................................. 17

Procurement of Ex-plant..........................................................................................................17

Sterilization..................................................................................................................................18

Glassware.................................................................................................................................18

Culture Media.......................................................................................................................... 18

Ex-Plant.................................................................................................................................... 18

Laminar Air Flow Cabinet.........................................................................................................18

Sterilization of Hands...............................................................................................................19

Sterilization of Metallurgic Instruments...................................................................................19

Inoculation...................................................................................................................................19

Incubation Conditions..................................................................................................................20

Results and Discussion.................................................................................................................21

Appendix I....................................................................................................................................24

Bibliography.................................................................................................................................26

AbbreviationsM.S: Murashige and Skoog

mg: milligram

mg/l: milligrams per liter

µl: micro liter

NAA: Naphthalene acetic acid

BAP: Benzylaminopurine: 6-benzylaminopurine phosphate

BA: Benzyladenine: 6-benzyl adenine

IBA: Indole butaric acid

TDZ: Thidiazuron

IAA: Indole acetic acid

GA3: Gibberellicacid

2, 4 D: 2,4 Dichloroacetic acid

K: Kinetin: 6-furfural aminopurine

L: liter

M: Molar (Concentration)

mm: millimeter

w/v: percentage ―weight in volume‖ ; number of grams of constituent in 100cm3 of solution

s: second

%: percentage

psi: pounds per square inch

2

EDTA: ethylene diamine tetra-acetic acid

U.V: Ultraviolet

2, 4-D: 2, 4 dicholorophenoxy acetic acid

ELS: Embryo like structure

WAP: weeks after pollination

c.v. : Cultivar

3

Introduction to TulipTulip is a perennial ornamental flowering plant and is considered as one of the most famous

ornamental flowering plants of the world. The popularity and importance can be understood by

the fact that it is considered now as an ornamental crop because Holland, also known as tulip

Capital of the world, exports nearly 2 billion tulip bulbs every year. The tulip plant is associated

with spring and is incredibly popular during spring time. Although, Holland has become the

“tulip capital” now, but they are native of Middle East and Central Asia. They were bought to

Europe in the 16th century, where they became so popular that it became a symbol of opulence.

Scientific Classification of TulipTulip belongs to the Liliaceae family and Tulipa genus. Its common name in Pakistan is

Gul-e-Lala . الل گل If we were to give a complete classification of tulip, it would look something

like this:

Kingdom: Plantae

Phylum: Angiospermae

Class: Monocotyledonae

Order: Liliales

Family: Liliaecae

Genus: Tulipa

Species: gesneriana (1/109 species)

There are about 109 known species of Tulipa and various cultivars for each species with

gesneriana having the most cultivars.

Horticultural Classification of TulipBesides the scientific classification, the Royal General Bulbgrower’s Association has also

developed a classification, based on flower morphology and plant size, for organizing numerous

cultivars or varieties of tulips. So, in this classification tulips are divided into 15 groups.

4

One other Horticulture Classification is done on the basis of the flowering season of tulip which

is:

i. Early Flowering Tulips- Those tulips which flower in early spring.

ii. Mid-Season Flowering Tulips- It includes those divisions of tulips which blossom 2-3 weeks

after spring.

iii. Late Season Flowering Tulips- This includes those tulips which flower late during spring.

Uses of Tulip

As written above, the tulip plant can be a major export ornamental crop. So, by growing tulip,

foreign exchange is earned in countries like Holland.

Other than that, there has been evidence that during World War II, people of Holland got short

of food supplies and so ate tulip bulbs, petals and stems. One of the source describes tulip bulb

as a replacement for onion (If one is that desperate). The sepals of tulips have been reported as

edible parts.

Why Tissue Culturing of Tulip is Important?

Although Tulip is easily grown plant, but the bulb of the tulip plant requires a cold storage

period during the winter in order for it to grow during spring season, a requirement which

makes it unfeasible to be grown in tropical areas. So, by propagating tulip in vitro, somaclonal

variation can be induced and plant can be given a direction in order to grow them without cold-

storage period.

Secondly, the tulip plants grow from a bulb which are exported in a large quantity which

requires large containers for transport and hence more storage space. But by making somatic

embryo culture, synthetic seeds of tulips can also be formed which can be stored in a small

space and easily transported.

Objective

5

To develop a protocol with the best media formulation for in vitro callus formation in

Tulipa gesneriana.

Literature Review

A lot of tissue culture research has been done on Tulipa and related genera of the Liliacae

Family. But some of the work is presented below.

Alderson et al., (1986) micropropagated tulip and studied bulbing of shoots in culture. The

adventitious shoots arising from the outer layer of cells of floral stem explants of tulip cultivar

Merry Widow were cultured on MS medium containing 1 mg/l NAA and BAP, resulting in

initiation of a bulb primordium at their base after incubation at 20°C for 12 to 16 weeks. It was

observed that development of this primordium was enhanced when sub-culturing was done

with media containing 1 mg/l NAA with or without 0.1 mg/l BAP. Incubation at 4°C for 12 weeks

followed by a period at 25°C on media containing 6% sucrose also enhanced bulb formation at

the base of shoots.It was also seen that bulbing of shoots was enhanced in cultures incubated

in light with a low red/far-red ratio (3.4) and after treatment with ethylene at 1 and 10 ppm

applied as a gas to the culture vessels. Shoot cultures treated at 12 weeks of age showed the

greatest response. Roots were induced on bulbs produced in vitro by manipulation of plant

growth regulators and sucrose in the culture medium.

Nischiuchi (1990) performed an experiment on organogenesis in the scale tissue culture of

tulip. It was observed that organogenetic activity in scale tissue cultures of tulip bulbs in vitro

was increased when the mother plants were treated with foliar application of growth regulators

during the growing period after flowering. It was also seen that in ABA-treated bulb scales,

adventitious shoot formation in vitro showed the best results with three cultivars, "Apeldoorn",

"Oxford's Elite", and "Oxford". Contrary to expectations there was no stimulatory effect of

adventitious shoot formation on BA-treated bulb scales. The cultivar "Red Matador" showed

acceleration of shoot formation in NAA-treated and GA-treated bulb scale cultures, but there

was no stimulation in the other three cultivars

6

Baker et al., (1990) studied and compared the precultural treatments and cultural condition on

in vitro response of tulip. Temperature and duration of bulb storage, type of explant, surface

disinfestation method, medium, and culture temperature were compared for effects on in vitro

response of explants from ‘Apeldoorn’ bulbs. It was observed that explants obtained from scale

tissue turned brown whereas those excised from the floral axis developed leaf-like structures. It

was also recorded that bulbs stored for either 56 or 63 days had superior mean visual ratings

than those bulbs which were stored for 36 days. When media compositions were compared, it

was noted that explants placed on MS medium containing NAA + BA (0.001 g/l each) had higher

mean visual rating and produced organized structures in 1 month, without callus formation. On

the other hand, the explants placed on a MS medium containing 2, 4-D and kinetin (0.001 g/l

each) produced organized structures via callus. When the leaf like structures were placed at 5°C

then 24°C, bulbs were induced. The organized structures could also be recultured to produce

increased numbers of leaf-like structures.

Custers et al., (1992) studied seedling formation and bulblet production from immature ovula

of tulip (Tulipa gesneriana L.). It was recorded that the percentage germination, on a modified

Murashige and Skoog (MS) medium, was increased by prolonging the 5°C chilling period, given

at the beginning of the culture, from 8 to 12 weeks. Culture that was kept in continuous

darkness was superior to the one placed in 16 h light in producing good quality stolon and

formation of the bulblet from the seedlings. When the stolons were prevented from entering

into the medium and growing thick and turgid by using a thin layer of medium, approximately 6

mm thick, there was an improved bulblet formation. Improvement in formation of bulblet was

also observed when the growing temperature was lowered from 24 to 12–15°C. The optimal

concentration of sucrose in the growing medium was 6% (w/v). Under the improved conditions,

up to 90% bulblet formation was obtained, with an average dry weight of approximately 50 mg.

It was noted that transfer to soil became possible only if bulblets were used, but these

7

frequently developed abnormal or scierotic. The percentage of second-year bulblet production

after transplantation was 12–30%, depending on the genotype used.

Hulsher et al., (1992) devised a technique for micropropagating tulip shoots and bulbs. For

generating shoots, stem explants and axillary buds were cultured on media with a-

naphthaleneacetic acid (NAA), N6-[2-isopentenyl] adenine (2iP) and 6-benzylaminopurine (BAP)

or Zeatine. Shoots with a meristem were selected. Propagation of these shoots was possible by

cutting them longitudinally. The micropropagated single shoots formed bulbs after a cold

treatment on a basal medium with 70 g/l sucrose. These propagated bulbs were planted in soil.

They emerged for 30–100% depending on the weight of the bulbs. After the growing season

new bulbs were harvested from almost every sprouted.

Wilmink et al., (1995) formed adventitious shoots on explants taken from young floral stems of

tulip (Tulipa gesneriana L.). Direct regeneration of shoots was recorded without an

intermediate callus phase. It was observed that more shoots developed on explants taken from

bulbs which were dry-stored than those bulbs stored on ice. At the basis of the shoots a

meristem was formed that developed into a new bulb.8 cultivars were compared for their

ability to form the bulb meristem, and was highest in Lucky Strike and Monte Carlo.

Adventitious shoot formation was initiated in the first two subepidermal cell layers and a

number of cells of the original explant contributed to the shoot formation. An optimal selection

system was established in order to avoid the formation of chimeric transformed shoots in

future transformation. For selection purposes, it was seen that the aminoglycoside antibiotics

were not very effective in inhibiting shoot formation as tulip tissue showed a high tolerance for

kanamycin. On the other hand, G418 and hygromycin induced severe necrosis of the explants at

low concentrations. In contrast, the herbicides phosphinothricin and glyphosate were very

effective and offer good perspectives to be used for selection of transformed shoots.

Hulscher and Krijgsheld (1995) proposed and compared two methods of micropropagation of

tulip. In the first method, adventitious shoot was regenerated from stem slices and in the

second method; shoot was regenerated from axillary buds of the mother bulb. It was observed

that regeneration from stem slices was very variable. Although many shoot-like structures were

8

formed, only a small percentage of these shoot-like structures proved to be real shoots with

meristems that could be used for further propagation. Comparatively, it was seen that shoot

development was better in the shoots regenerated from axillary buds than the regeneration of

shoots on stem slices. Furthermore, the rate of propagation of shoots from axillary buds proved

to be higher than the rates of propagation of shoots from stem slices. These two factors make

the axillary bud system more useful than stem slices to start micropropagation of tulip.

Gude and Dijkema (1996) proposed a method for somatic embryogenesis is tulip which would

be a rapid in vitro propagation alternative to the micropropagation through adventitious shoot

formation in stem explant which has a low propagation factor, is laborious and expensive. The

long term aim of the study was to develop a propagation method, consisting of a cell

suspension phase, and a regeneration phase, during which the cell clusters regenerate through

somatic embryogenesis, a system which would enable the rapid production of large numbers of

bulblets at low costs. For the suspension culture, friable callus was obtained from bulb scale

and stem explants cultured on a nutrient medium containing the auxins 2, 4-D or Picloram at a

concentration range from 0.5–50 μM. Undifferentiated and ‘meristematic’ type of callus on

bulb scale explants was induced. The meristematic type seemed the most suitable to start a

liquid culture. It was seen in liquid medium that the meristematic nodules divided into smaller

units, which could be released from the explant by shaking. The stem explants, cultured on

medium with 5 or 50 μM 2, 4-D or Picloram resulted in formation of somatic embryos. It was

also concluded from the results that embryo-tissue from the somatic embryos can be cultured

for the production of embryogenic callus.

Tribulato et al., (1997) described a protocol for somatic embryogenesis and plant regeneration

in Lilium longiflorm. For this purpose, friable callus was obtained from styles and flower

pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige

and Skoog (MS) media containing either 2 µM dicamba or 2 µM picloram. Friable callus was used

to establish cell suspension cultures by suspending the callus of L. longiflorum Snow Queen in

liquid medium containing 2 µM dicamba. Through a purification process, a fine fast-growing cell

suspension was obtained. This suspension was composed of a homogenous population of small

9

dense cells, which tended to organize into embryo like structures (ELS). In the liquid culture

containing the auxin dicamba, the ELS underwent continuous callus formation. When it was

transferred to solidified hormone-free MS medium, the ELS germinated, forming complete

plantlets. When histological analysis was done, the results showed that in the ELS both shoot

and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact

somatic embryos.

Famelaer et al., (1997) studied how cold treatment of seeds, obtained from crosses between

cultivars of T. gesneriana L., affects the developmental stage of embryos, which in turn

influences the frequency of callus induction and the development of different callus types. For

the research, cold-treated, mature embryos and basal segments of in vitro-derived bulblets,

were used as explants for the initiation of regenerative callus on medium with 2,4-

dichlorophenoxyacetic acid. The bulblets were initiated on flower-stalk segments from cold-

stored bulbs of T. gesneriana ‘Christmas Marvel.’ When the regenerative callus was analyzed

histologically, the regeneration of bulb-like structures was recorded.

Suh (1997) analyzed and studied the stem elongation and flowering response of Tulipa cultivars

as influenced by cold treatment duration, plant growth regulator application, and light quality

during tulip forcing. It was observed that the flowering percentage of ‘Apeldoorn’ and ‘Golden

Apeldoorn’ was more than 90%, regardless of low temperature and GA3 treatments. It was also

seen that flowering in both cultivars was accelerated by gibberellins (GA3 and GA4+7) or

Promalin injected into bulb-scale tissue and the flower bud after bulbs had been stored at 5°C

for 6–12 weeks. The results also showed that the total stem length was shorter after injection

of bulbs and flower buds with GA than in control plants. When compared with ‘Apeldoorn’, it

was seen that the flower stem length was slightly reduced when GA3, GA4+7 or Promalin were

injected into bulb-scale tissue. When light quality was tested, it was found that blue and far-red

light, as well as darkness increased the length of the 1st and 2nd internode in ‘Apeldoorn’ tulips

when compared with control plants or other light treatments. Growth of the last internode was

stimulated by darkness and by red and blue light treatments.

10

van Rossum et al., (1998) investigated the effect of different O2 levels on regeneration of

adventitious organs in various systems, viz., shoot regeneration from tulip bulb scale and stalk

explants, bulblet regeneration from lily scale explants and root regeneration from apple stem

slices. The explants were exposed to 2% O2 (low O2), 100 % O2 (high O2) or ambient air (20 %

O2). It was noted that the culture under continuous high O2 conditions had a moderate adverse

effect in all systems. It was also observed that the continuous culture at a low level of O2 had

strong inhibitory effects in tulip and apple, but was promotive in lily. The results also showed

that when low O2 was applied to tulip stalks only concurrent with the wound reaction, it had no

or even an inhibitory effect. When High O2 was applied directly to apple stem slices after

cutting, there was a slight inhibitory effect on formation of adventitious roots. Once the

regenerated organs were formed, low O2 levels inhibited growth (in lily and tulip), and high O2

levels were stimulating in tulip and inhibitory in lily on organ growth. The results showed that

the alledgedly toxic effect of O2 does not exist or is very small.

Van Creij et al., (2000) studied the effect of several media components on the germination

percentage of ovules in intraspecific T. gesneriana L. crosses. Two embryo rescue techniques

were used; one was viz. ovary-slice culture followed by ovule culture and the other was direct

ovule culture. By the addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at

5 weeks after pollination (WAP), showed significant improvement in the germination

percentage as compared to 5% sucrose. The germination percentage did not differ between

both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with

ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained

with media containing the full or half of the concentrations of micronutrients and

macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule

culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between

ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or

0.1 mg/l) had no effect on the germination percentage. The use of liquid-shaken culture

resulted in germination percentages which were similar to those on agar-solidified media.

Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-

slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose.

11

The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice

culture, whereas the total concentration of carbohydrates did not decrease remarkably in

media for ovule culture.

Bach and Ptak (2002) found a new method to micropropagating tulip from ovary tissue. The

object of this study was the possibility of improving the multiplication rate of micropropagated

tulips (Tulipa gesneriana L.). Explants of ovary were isolated from chilled (12 weeks) and

unchilled bulbs of ‘Apeldoorn’ and ‘Red Matador’ cultivars and were exposed to various

concentrations of Picloram and BAP. Plants were generated through somatic embryogenesis via

an intermediary callus phase. Callus was observed to be formed on all medium types but the

embryogenic callus was formed only on the media containing a higher concentration of

Picloram than BAP. Embryos were induced in high frequency in explants isolated from chilled

bulbs. Efficiency of embryogenesis depended on the genotype. ‘Apeldoorn’ cv. produced more

somatic embryos then the ‘Red Matador’ cv. (16.7 and 9.7 embryos per one explant,

respectively). Cytological analysis did not reveal any changes in chromosome number of

regenerated explants.

Podwyszynsky and Sochacki (2003) conducted a research and described the effects of TDZ and

Paclobutrazol on the primary regeneration on tulip flower stalk explants of six cultivars and

subsequent shoot multiplication. The explants used were flower stalk slices which were excised

from cooled and subsequently forced bulbs. The explants were incubated for two months in

darkness on medium containing NAA and cytokinins, 2iP and BAP, as control, or TDZ (0.5-4

mg/l) and paclobutrazol (0.05-0.4 mg/l). After two months, the regenerating explants were

subcultured on a medium with TDZ and NAA which were applied at low concentrations. The

results showed different regeneration capabilities which depended on cultivar and growth

regulators. It was seen that the percentage of explants forming leaf-like structures ranged, on

the control medium, from 80% in 'Blue Parrot' and 'Prominence' to below 30% in 'Apeldoorn'

and 'Mirjoran'. It was also noted that TDZ applied at optimum for each cultivar concentration

greatly increased the regeneration potential up to 70-100%. When Paclobutrazol was added to

the TDZ-containing medium, the explant responded significantly resulting in a high number of

12

leaf-like structures formed per explant. The structures developed gradually into characteristic

forms: the growing up cotyledonary leaf, the probable root primordium formed at its base, the

growing downwards stolon and the shoot meristem developed finely on its tip. It was suggested

by the study that such primary regeneration may have a nature of somatic embryogenesis.

After that, the adventitious shoots developed and formed clusters, which were divided into 2-3

smaller ones every two months. It was recorded that the growth regulators, used at initial stage

markedly influenced subsequent shoot multiplication. As a result, the most intensive shoot

formation was noted with TDZ at concentrations of 0.5-2 mg/l and paclobutrazol of 0.05-0.1

mg/l.

Ghaffoor et al., (2004) conducted a study on in vitro response of tulips to various growth

regulators. Different explant sources of three cultivars (Beauty of Apeldoorn, Page Polka and

Toronto) of tulip were used for study for their responses to different growth regulators on MS

medium. It was noted that there was maximum shoot formation in culture media with NAA (2

mg/l) and BAP (1mg/l). It was also seen that bulblets formation was better on the medium that

contained 2 mg L-1 NAA combined with 0.5 mg L-1 BAP.

Minas (2007) proposed a method for in vitro Micropropagation of Akama tulip via adventitious

organogenesis from bulb slices. For sterilization, five bulbs of Akama tulip (Tulipa cipria

akamantis) were sliced and soaked in 20% commercial bleach solution with a few drops of

Tween-20 for 10 min and were then washed 3 times in distil sterile water. The explants

obtained from bulb scale slices were placed on MS medium resulting in production of micro-

embryonic shoots. The growth medium was composed of MS basic salts and vitamins,

supplemented with 3% sucrose, 4.5 mg/l BAP, 0.009 mg/l IBA, and 55.7 mg/l ascorbic acid and

solidified with 2.5 g L-1 phytagel. Cultures were incubated in a growth room for two weeks in

the dark and constant temperature of 15±2°C after which they were transferred to a growth

room with 16 hours light of 600 lux provided by white and red light fluorescent lambs (50-50%)

and 8 hours dark at constant temperature of 15±2°C. Proliferation was on average 4-fold per 2

weeks and rooted in vitro, in the same medium, micro-plants were produced.

13

Ptak and Bach (2007) conducted a study for developing a protocol for in vitro somatic

embryogenesis in tulip flower stem culture. Somatic embryogenesis was initiated on flower

stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs

had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured

with exogenous auxins 2, 4-dichlorophenoxyacetic acid (2, 4-D), 4-amino-3,5,6-trichloropicolinic

acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine

(BA) and zeatin (ZEA) at 0.5–50 μM. It was observed that increasing the concentrations of auxin

caused an intensive enlargement of the explant parenchyma, which changed into homogenous

colorless callus and the vein bundles on the same media developed into yellowish, nodular

callus. The induction of nodular embryogenic callus was more efficient with Picloram than 2,4-

D, whereas the latter stimulated formation of colorless callus. It was also noted that callus

formation was best in the explant taken from the base of the lower part of the flower stem

isolated from bulbs chilled for 12 weeks. The results showed that the highest number of

somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Adventitious

roots were developed in the presence of 2, 4-D. Globular embryos developed into torpedo

stage embryos under the influence of BA (5 μM) and NAA (0.5 μM).

Xin-Ying et al., (2007) performed an experiment in look for a method of micropropagation and

also for establishing a base for research of molecular level. The effect of six factors on tissue

culture of bulb scales of four tulip cultivars was studied. The results showed that bulbs sterilized

with 0.1%HgCl2 for ten minutes and 5% NaClO for twenty minutes proved to be best for

sterilization. Induction of callus of cv.'Parade' was observed to be best in MS + NAA 1.0 mg/l

+BA 1.0mg/l. The rate of callus induction was up to 100%. The best culture media for shoot

regeneration of cv.'Parade' and cv.'Merry Widow' were MS+NAA 2.0mg/l + BA 0.5mg/l and

MS+NAA 0.5mg/l + BA 1.0mg/l respectively. The overall rate of shoot regeneration was 15.4%.

It was also observed that the middle-layer scales of bulb were easier to induce callus, with the

rate of 8.0% and inner-layer scales of bulb proved better for direct shoot induction, with the

rate of 26.7%. Adding 50-200mg/l ascorbic acid in culture medium proved advantageous

regeneration of shoot but was disadvantageous in callus induction. The rate of shoot

14

regeneration was up to 12.5% and 10.3% when bulb scales were incubated in darkness for five

days and fifteen days after inoculating respectively.

Podwyszynsky and Sochacki (2010) formulated a new tulip micropropagation method based on

the cyclic shoot multiplication in presence of the Thidiazuron (TDZ), which enables the

production of virus-free stock plants, speeds up breeding, and provides new genotypes for the

market. In their novel protocol, cyclic shoot multiplication can be performed for 2-3 years by

using TDZ instead of other cytokinins, as 6-benzylaminopurine (BAP) and N6-(-

isopentyl)adenine (2iP). This protocol makes it possible to produce 500-2,000 microbulbs from

one healthy plant. Six main stages of tulip micropropagation were studied. Stage 1 is the

selection of true-to-type and virus-free plants, confirmed by ELISA. Initially, explants were

obtained from fragments of flower stems isolated from bulbs. Shoot multiplication was based

on the regeneration of adventitious shoots, which were sub-cultured after every 8 weeks. In the

third stage, the specially prepared shoots were induced by low temperature treatment to at

20˚C on a sucrose-rich medium to form bulbs. After that, the bulbs were dried for 6 weeks and

rooted in vivo. It was concluded that to reduce the risk of mutations, the number of

multiplication subcultures should be limited to 5-10 cycles. Also, virus indexing should be

repeated 3-4 times, at the initial stage and also during shoot multiplication.

15

Materials and Methods

Preparation of Stock SolutionThe Medium used for this project was Murashige and Skoog (MS) media, for which the

following stock solutions were prepared.

1. Macronutrients (20X)

Macronutrients were prepared at 20X i.e. at 20times the strength of the normal solution,

according to the composition stated in Appendix 1A. The stock solution was poured in

brown bottle for storage and later use.

2. Micronutrients (100X)

The stock solution for Micronutrients was prepared at 100X i.e. at 100 times the strength of

the normal solution, prepared according to the composition stated in Appendix 1B. After

preparation, it poured in brown bottle for storage and later use.

3. Iron EDTA (200X)

Stock solution of Iron EDTA was prepared at 200X, according to the composition stated in

Appendix 1C, and was stored in brown bottle for later use.

4. Vitamins (200X)

Stock Solution for Vitamins was also prepared at 200X, according to the composition stated

in Appendix 1D. The stock solution was poured in a brown bottle for storage and later use.

16

Media PreparationFor preparing 250ml MS media, following procedure was implied.

1. First I took a 250ml clean glass beaker and rinsed it with distilled water.

2. Then added about 80-100ml water in it.

3. After that, I added 12.5ml Macronutrients, 2.5ml Micronutrients, 1.25ml Iron EDTA and

0.25ml Vitamins, each with a separate clean pipette.

4. After that, I added 7.5g sugar (sucrose) in it and mix the sugar on the magnetic stirrer.

5. When sugar was completely mixed, PGRs (which and how much stated in the “Media

Composition” section) were added to the solution.

6. The solution was given a slight stir and poured in a 250ml measuring cylinder after which

distilled water was poured in measuring cylinder to make it 250ml.

7. The liquid media was then transferred to a 1 liter glass beaker (to preventthe media from

boiling and overflowing) in which 2.5g (1% w/v) was added.

8. After that, the beaker was kept in microwave oven for 4-5 minutes to boil the mixture in

order to dissolve agar.

9. Lastly, after the dissolution of agar, beaker was taken out of the microwave oven and

culture media was poured into the culture tubes, which were then sealed with

polypropylene sheets using rubber band.

Media OptimizationThe growth media was optimized with different PGRs and increasing and decreasing the

nutrients in the culture media. The following media formulations were made for this project.

1. MS + 10mg/l 2,4-D

2. MS + 0.5uM 2,4-D + 5uM BAP

3. MS + 1mg/l NAA + 1mg/l BAP

4. MS + 1mg/l 2,4-D + 1mg/l Kin

5. MS + 8mg/l BAP

6. MS + 2mg/l Kin + 4mg/l IBA + 60g/l sugar + 0.5ml Vitamins

17

Procurement of Ex-plantEx-plants used for the purpose of this project were tulip bulbs, leaves and tulip shoot. The ex-

plants were provided to us by our professor Dr. Kanwar Shoaib, which were stored for later use

according to their condition stated as follows:

i. Leaves and stem were submerged in plenty of water in beaker to prevent desiccation and

kept in a refrigerator at 4-5 C,

ii. Bulbs were also placed in the refrigerator at 4-5 C without removing their outer, brown

covering.

Sterilization

GlasswareGlassware which includes beakers, pipettes, petri-plates, glass bottles, etc. was sterilized by implying the

dry heat method. All the glassware was washed, dried and finally wrapped in brown paper. Then the

glassware was kept in an oven at 160 oC for 2 hours.

Culture MediaAfter preparation and pouring of growth culture media in the culture tube, it was sterilized by placing

the culture tubes in an autoclave at 121 oC at 15psi for 15mins. This makes the culture media absolute

germ free, even the microspores and extremophiles do not survive at this temperature and pressure.

Ex-PlantThe procedure for sterilization of ex-plant is written below.

First, I added about 2-3 spoons of liquid detergent and 1-2 drops of Tween-20 in a beaker containing

150-200 ml water. Afterwards, I put my ex-plant (leaf or bulb) in this mixture and scrubbed it gently with

the tips of the finger. In case of stem, the stem was scrubbed quite rigorously with finger tips. After

washing for about 2 minutes, I drained the water and then washed the beaker and the ex-plant, 3-4

times with distilled water very thoroughly to remove the detergents. After washing the ex-plant, I

sterilized the ex-plant in laminar flow cabinet by putting it in sterilized petri-plates containing Mercuric

Chloride, HgCl2 (0.1%-1%). The ex-plants were dipped in mercuric chloride for only 30secs to 1minutes

18

maximum. Then the ex-plant was washed 3-4 times with Autoclave Water in the laminar flow cabinet.

After that, the ex-plant was suspended in autoclave water in petri-plate till inoculation.

Laminar Air Flow CabinetPrior to sterilizing the ex-plant and inoculation, the laminar air flow cabinet was sterilized. For that, the

base and the sidewalls of the air flow cabinet (Fig. 1) were first thoroughly swapped with cotton dipped

in ethanol. Then UV lamp was switched ON for 30-40 minutes.

Fig. 1: Laminar Air Flow Cabinet

Sterilization of HandsBefore performing inoculation, hands and forearms were first washed with soap and then sprayed with

ethanol to disinfect them of microorganisms.

Sterilization of Metallurgic InstrumentsThe metal instruments like forceps and scalpels were kept in a beaker containing alcohol and were flame

sterilized before using.

Inoculation

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The ex-plant was inoculated under germ-free, aseptic environment (created by following the above

stated sterilization protocol). Before inoculation, the UV light was turned off (UV radiations are

carcinogenic) and the fluorescent light and air flow was switched ON. The ultra-filtered, pressurized air

flows towards the person using airflow cabinet, thus preventing micro-organisms from entering.

Sneeze guard was also worn in order to prevent contamination of ex-plant and culture medium by either

talking or sneezing. Even in the presence of sneeze guard during the inoculation procedure, talking with

any group member was avoided or kept to minimum

After that, the scalpel dipped in alcohol was given a slight jerk to remove alcohol and was kept on flame

to sterilize it. Then the petri-plate containing ex-plant was opened and ≈1 cm pieces of ex-plant were

made using the sterilized scalpel. After cutting ex-plant, the polypropylene sheet sealing the culture

tubes was removed and the neck of the tube was kept on flame. Then using forceps (also flame

sterilized), the ex-plant was placed on the culture media in the culture tube i.e. the ex-plant was

inoculated. As soon as the inoculation was done, the culture tube was sealed using the same

polypropylene sheet which was taken off before.

Incubation ConditionsAfter performing inoculation, the culture tubes were kept in a culture room (Fig. 2). The conditions in

the culture room were:

i. Temperature: 26 ± 2˚C

ii. Light Intensity: 4000 lux. Light provided was cool fluorescent white light.

iii. Dark condition was provided by putting the culture tubes in a box.

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Fig. 2 Culture Room showing racks with culture tubes, fluorescent light and boxes for providing dark treatment

Results and DiscussionThe main observation regarding the tulip is that it is very recalcitrant towards callogenesis and

embryogenesis. During the 2-3 months of working, only 2 test tubes showed some callogenesis and one

tube showed caulogenesis.

The MS + 10mg/l media, inoculated with leaf ex-plant were kept in dark and light. The leaf ex-plants in

all the MS + 10mg/l tubes kept in light showed complete dedifferentiation in 2 weeks and became clear

white (Fig. 3), so much so that they were hard to distinguish from the white color of the media. One of

the leaf explant showed some swelling but it was not very noticeable.

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Fig. 3: Dedifferentiated leaf ex-plant which has become white

On the other hand, the ex-plant placed in dark was de-differentiating but very slowly. During

the course of two months of incubating in dark, the color of the leaf changed only slightly

turning pale green.

The ex-plants cultured on the MS + 0.5uM 2, 4-D + 5uM BAP media, showed some hyaline

colored callus as shown in Fig. 4. Stem was also regenerated on the same bulb explant (Fig. 5)

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Fig. 4: Ex plant showing hyaline callus formation

Fig. 5: Microscopic image showing caulogenesis in Tulip Bulb

As there was not very evident callogenesis and regeneration in the above two media, and no

activity in any of the other media, it was thought that the PGR’s in the media might be diluted

23

thus showing slow or no activity. So the concentration of the PGR was increased 2-3 times the

normal concentration. As a result, the ex-plant showed callogenesis in about 2-3 weeks (Fig. 6).

Fig. 6: Bulb Explant showing callus formation in two weeks

The rest of the 4 media showed no visible signs of activity. The culture tubes containing the

stem ex-plant was contaminated with microorganisms that resulted in breakage of the solid

media in the tubes. Some of the bulb explants turned brown after a period of 2-3 weeks after

inoculation and incubation.

The results stated above can bring us to a conclusion that for regeneration and callogenesis of

tulip, an increase in the concentration of the PGR results in rapid activity in the explants of

“recalcitrant” tulip.

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Appendix IMurashige and Skoog (1962) Medium Preparation

Stock MS Media(mg/L)

A Macronutrients 20X(mg/L) (50cm3 SS gives)

N

H4N

33,000 1,650

K

N

39,800 1,900

CaCl2.2H2O 8,800 440

MgSO4.7H2O 7,400 370

K

H2P

3,400 170

B Micronutrients 100X(mg/L) (10cm3 SS gives)

MnSO4.4H2O 2,230 2.3

ZnSO4.4H2O 860 8.6

H

3B

620 6.2

K

I

83 0.83

Na2MoO2.2H2O 25 0.25

CuSO4.5H2O 2.5 0.025

CoCl2.6H2O 2.5 0.025

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C Iron EDTA 200X(mg/100cm3) (5cm3 SS gives)

N

a2ED

672 37.6

FeSO4.7H2O 556 27.6

D Vitamins 200X (5 cm3 SS gives)

Glycine 20 0.2

Nicotinic Acid 5 0.05

Pyridoxine HCl 5 0.05

Thymine HCl 1 0.01

Sugar(Sucrose) = 30g/L

Agar = 10g/L

PH should be 5.8+ 0.1

26

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