6

II. Review of Literature Saffron is classified into Magnoliophyta Division, Class Liliopsida and Order

Asparagales. It is a member of the Iridaceae family and the Crocus L. genus.

Crocus consists of 9 species viz, Crocus cartwrightianus and its derivatives,

Crocus sativus, Crocus moabiticus, Crocus oreocreticus, Crocus pallasii, Crocus

thomasii, Crocus badriaticus, Crocus asumaniae and Crocus mathewii. Some

archeological and historical studies have indicated that domestication of saffron

dates back to 2,000-1,500 BC (Tammaro, 1987; Negbi, 1999). However, the sites

where the first saffron plants appeared differ according to the opinion of various

authors. Vavilov (1951) placed saffron into IV plant Centre of Origin, Middle East

(Minor Asia, Turkestan) whereas more recent reports indicate that the process of

saffron domestication has to be identified on Crete during the last Bronze Age

(Negbi, 1999). According to Negbi (1999), wild C. cartwrightianus herb was

harvested and used, then its mutant C. sativus was observed, selected and

domesticated on the Crete. C. cartwrightianus grows on a volcanic ash near

Akrotiri, Santorini and is harvested for local consumption by the villagers

(Mathew, 1999). However, C. thomasii Ten, or C. pallasii Herb, have also been

identified as possible saffron ancestors (Brighton, 1977; Chichiricco, 1989a;

Tammaro, 1990). The most recent taxonomic publications (Mathew, 1999) and

most of the former classifications (Tutin, 1980; Mathew, 1982) arranged C.sativus

and C. cartwrightianus to each other either as C.sativus to be a sub-species of C.

cartwrightianus or as a variety or a mutated derivative. Experiments of cross

pollinations turned C. thomasii out to produce some seeds with C.sativus

(Chichiricco, 1989a; Grilli Caiola, 2004). Further studies also defined C.

cartwrightianus and C. thomasii to be closest to saffron (Grilli Caiola and

Brandizzi, 1998). Mathew (1999) put forth taxonomy of C. sativus and its allies as

under:

Genus Crocus

I. Subgenus Crocus. Type species: C. sativus L.

A. Section Crocus. Type species: C. sativus L.

7

(a) Series Verni Mathew. Type species: C. sativus L.

(b) Series Scardici Mathew. Type species: C. vernus Hill.

(c) Series Versicolores Mathew. Type species: C. scardicus Kos.

(d) Series Longiflori Mathew. Type species: C. longiflorus Raf.

(e) Series Kotschyani Mathew. Type species: C. kotschyanus Koch.

(f) Series Crocus. Type species: C. sativus L.

B. Section Nudiscapus Mathew. Type species: C.reticulatus Stev. Ex Adams

(g) Series reticulati Mathew. Type species: C. reticulatus Stev. ex Adams

(h) Series Biflori Mathew. Type species: C. biflorus Mill

(i) Series Orientales Mathew. Type species: C. korolkovii Regel ex Maw

(j) Series Flavi Mathew. Type species: C. flavus Weston

(k) Series Aleppici Mathew. Type species: C. aleppicus Baker

(l) Series Carpetani Mathew. Type species: C. Carpetanus Bioss. & Reut

(m) Series Intertexti (Maw) Mathew. Type species: C. fleischeri Gay

(n) Series Speciosi Mathew. Type species: C. speciosus M. Beib.

(o) Series Laevigati Mathew. Type species: C. laevigatus Bory & Chaub

I. Subgenus Crociris (Shur) Mathew. Type species: C. banaticus Gay

Series Crocus includes Crocus sativus and its allies.

Species comprising series Crocus:

1. C. cartwrightianus

2. C. sativus

3. C. moabiticus Bornm. & Dinsm

4. C. oreocreticus B.L.Burtt

5. C. pallasii Gold

6. C. thomasii Ten

7. C. hadriaticus Herbert

8. C. asumaniae B. Mathew & T.Baytop

9. C. mathewii Kerndorff & Pasche

Cytofluemetry analysis of nuclear DNA amount and base composition of

C. sativus, C. cartwrightianus and C. thomasii compared to spring diploid

8

C.biflorun (Brandizzi and Grilli Caiola, 1998) have revealed that C. sativus is a

genomic clone of C. cartwrightianus from which it could have originated by

mutation or out-cross process. RAPD technique (Zanier, 2000) was used to

discriminate nuclear DNA compliment in autumnal flowering Crocus belonging to

C. sativus group. Because of complex genetic make up which includes triploid

nature, not much improvement has been brought about in saffron through

conventional breeding procedures.

Saffron with sub-hysteranthous behavior is a perennial herbaceous plant attaining

a height of 25-40 cm. Corm, foliar structure and floral organs constitute the main

parts of saffron plants (Mathew and Brighton, 1977; De Hertogh and Nard, 1993;

Nehvi et al., 2010a). Corms that are 3-5 cm in diameter covered by tunic consist of

nodes and are internally made up of starch-containing parenchymatous cells.

Apical, subapical and axillary buds protected by dark reddish scales are found in

internodes. As their diameter increases, they tend to group together, so that the

majority can be found in one, two or three internodes (Perez, 1997). Roots emerge

radically at the height of the third basal internodes. They are thin white in color,

numerous and variable in length. At the base of the daughter corm, a much thicker

root than the absorbent roots may arise, which is known as contractile root

(Khalesi et al., 2004). Each corm produces five to eleven green leaves or

monophylls. The leaves known as bristles are 1.5 to 2.5 mm wide per sprout and

can measure upto 50 cm (Dhar and Mir, 1997; Lucceno, 1999). The photosynthetic

activity of the leaves during the early winter and spring months contribute to the

formation of replacement corms at the base of the shoots. Corms that are covered

by tunic are dormant during summer and sprout in autumn producing 1 to 4

flowers in cataphyll with linear leaves. Cataphylls not only protect and strengthen

the stem in course of its appearance on the surface (Botella et al., 2002) but also

protect the corms once formed from degradation and possible lesions (Lopez,

1989). Propagation occurs every year via formation of daughter corms from the

parent after flower harvesting (Negbi et al., 1989; Negbi, 1990; De Hertogh and

Nard, 1993).The flower has an underground ovary, a style (5 to 9 cm long),

9

dividing at the top into three red trumpet like stigmas (2 to 3 cm long) which when

dried form the commercial spice-the saffron (Sampathu et al., 1984; Tammaro,

1990). Flowers with six stigmas have also been observed in the saffron fields.

However this kind of variation doesnot persist in the next flowering season and are

generally termed as freaks (Nehvi et al., 2004). The corm is a vegetative organ of

saffron. After flowering, the base of the stem enlarges producing a daughter corm

that propagates the plant (Tammaro, 1990; Jirage et al., 1994). The number of scar

like buds covered by scaly leaf present on the surface of mother corm vary from

2 to 20 depending on the corm size (1.0-6.0 cm).

Triploid nature of the species allows for vegetative multiplication but not for

regular sexual reproduction. This is because triploid meiosis and gamete

development are irregular, resulting in many anomalies in sporogenesis and

gametophyte development (Chichiricco, 1984). Saffron infertility is mainly related

to the male gametophyte (Chichiricco, 1989a, 1990; Grilli Caiola and Chichiricco,

1991). It does not produce viable seeds; therefore corms are indispensable for its

propagation. The karyotype of Crocus sativus L. was the subject of research by

many authors for genetic differentiation in relation to chromosome number, arm

ratio, arm size and centromeric position while the other species of Crocus sativus

aggregated in this respect remain poorly studied (Himmelbauer, 1926; Karasawa,

1933; Rzakuliyev, 1945; Brighton et al., 1973; Brighton, 1977; Chichiricco, 1984;

Dhar et al., 1988; Agayev, 2002; Agayev et al., 2010). Saffron triploidy has been

demonstrated by numerous authors (Karasawa, 1933; Brington, 1977). Studies on

micro and megasporogenesis in saffron confirmed that meiosis occurs in an

anomalous manner with irregular chromosome pairing, division and distribution in

the derived nuclei (Chichiricco and Grilli Caiola, 1984; Chichiricco, 1987, 1989b;

Grilli Caiola and Chichiricco, 1991). Due to triploidy, meiosis in C. sativus is

highly erratic, forming 8 trivalents and genetically unbalanced gametes which lead

to formation of sterile gametes, hence no seed formation. No fertilizable gametes

having 8 (n) or 16 (2n) chromosomes were found in saffron (Ghaffari, 1986) and

further self incompatibility has been observed (Chichiricco and Grilli Caiola,

10

1986, 1987). In rare case of fertilization there is some embryonic and endosperm

development but it terminates at an early stage (Chichiricco, 1987). In saffron, on

the basis of overall length and centromeric position, the somatic chromosomes are

assembled in seven triplets (3 metacentric, 2 submetacentric and 2 telocentric) one

pair (submetacentric) and one single chromosome (metacentric). Saffron from

Kashmir is identical in structure of karyotype with usual saffron (Crocus sativus

L.). However, some significant distinctions are present: 1) All three satellites at

three chromosomes of the second triploid are identical in size 2) The total length

of all chromosomes of triploid set 2n=3x=24 in Kashmir saffron is authentically

more than in usual Crocus sativus. In triplet IV, the homologues 1 and 2 have an

arm ratio equal to 1.50 whereas the homologue III have an arm ratio of 1.2. The

difference is significant being absent in usual Crocus sativus (Agayev et al.,

2010).

Saffron is valued for its color, taste and aroma. The compounds that give it these

properties are what define its quality. Saffron predominantly contains chemical

constituents such as crocin, picrocrocin and safranal responsible for its color,

flavor and aroma, respectively. Crocetin glycosyl esters are responsible for its

characteristic color and these compounds are found in extremely important

proportion in stigmas (Sampathu et al., 1984; Tarantilis et al., 1995; Dufresne et

al., 1997)-the part being the storehouse of the caretenoids which form the major

share. The caretenoids present in minor quantities include alpha and beta carotene,

lycopene and zeaxanthin as well as a conjugated xanthocarotenoid. Saffron

contains flavonoids and one of the general characteristic defining this extensive

group of compounds is bitterness. The first researchers to identify a flavonoid in

saffron extracts through mass spectrometry were Tarantilis et al., 1995. The

characteristic bitter taste of saffron has been postulated due to the presence of a

glycoside namely picrocrocin whose structure was established by Khun and

Winterstein (1934). Picrocrocin is a precursor of safranal, the major compound

responsible for saffron aroma. The study of saffron aroma began around the first

quarter of the 20th century with the isolation and identification of safranal, the

11

major aromatic compound. It is generated from crocetin esters and was obtained

for the first time in 1922 by Winterstein and Teleczky by means of alkaline or acid

hydrolysis of picrocrocin. Carotenoid degradation either by thermal treatment or

enzyme activity gives rise to small compounds that contribute to aroma and flavor.

Saffron is also a rich source of proteins, vitamins (riboflavin and thiamine),

potassium, iron, copper, zinc, sodium and manganese thus imparting antioxidant

property to it together with the status of functional food (Delgado et al., 2006).

Records on ancient cultures established in Mesopotamia, depict use of saffron

principally as a condiment in religious rites and celebrations, and also as a dye for

their clothes (Perez, 1995). Egyptians and Hebrews used it to carry out ablutions

in temples and sacred places (Capel and Girbes, 1988). Saffron, its extracts and

tinctures have been used in traditional medicine as an antispasmodic, eupeptic,

sedative, carminative, diaphoretic, expectorant, stomachic, stimulant, aphrodisiac,

emenagogue and abortive agents (Basker and Negbi, 1983; Rios et al., 1996). It

has also been used for the treatment of ocular and cutaneous conditions (Xuon et

al., 1999), lowering blood pressure (Abe, et al., 1999; Soeda et al., 2001), for

wounds, fractures and joint pain; to prevent plague and other epidemics; to cure

anaemia, migranes and insomnia, promoting and regulating menstrual periods

(Akhondzadh et al., 2004), sores and as a cardiotonic (Verma and Bordia,1998;

Schmidth et al., 2007; Bathaie and Mousavi, 2010) and treatment of respiratory

disorders (Xiag et al., 2006; Xi, 2007). It is known for its antigastric effects (Al-

Mofleh et al., 2006), antidiabetic activity (Liu et al., 2005; Shen and Quin, 2006),

anticonvulsion and antidepressant remedy (Zhang et al., 1994), anti-inflammatory

effect (Hosseinzadh and Sadeghnia, 2007), antigenotoxic effect (Prem kumar

et al., 2003), antioxidant activity (Chatterjee et al., 2005), antitumoural and

anticarcinogenic activity and its cytotoxic and antimutagenic effects have also

been reported (Abdullaev, 2002). It is also used as a tonic and promoter of

defenses in Ayurvedic medicine, for some disorders of the central nervous system

in Chinese medicine and for homeopathic preparations.

12

Saffron finds its use in the perfumery and cosmetic industry, besides, the most

important current use in food industry. This spice forms a part of some of the best

known traditional dishes. It is used to dye high textiles manufactured with silk,

cotton or wool (Takaoka et al., 1992; Tsatsaroni and Eleftheriadis, 1994;

Liakopoulou-Kyriakides et al., 1998; Tsatsaroni et al., 1998). As a dye, it is also

utilized in combination with hematoxylin, erythrosine and others to achieve

human and animal histological staining (Desmettre et al., 2001; Rostoker et al.,

2001; Alyahya et al., 2002; Edston, 2002). In Kashmir, saffron has a long history

of being used in culinary (Kashmiri cusine, wazwaan) and Kashmiri tea (Kehwa).

It is also widely used in confectionary, alcoholic and non alcoholic beverages,

colouring agent for sausages, oleomargarines, dairy products such as butter, cheese

and icecream for color and flavour improvement (Hosseini, 2000).

Taking into view the objectives of present investigation, the available literature

relevant to various aspects of study is reviewed in present chapter.

II.1 Micro-propagation

In 1838, Schwann and Scheilden put forward the so-called totipotency theory

which states that cells are autonomic and in principle are capable of regeneration

so as to give rise to a complete plant. Their theory was in fact the foundation of

plant cell and tissue culture (Pierik, 1999). Two concepts, plasticity and

totipotency, are central to understanding of plant cell culture and regeneration.

Particularly important aspects of this adaptation as far as plant tissue culture and

regeneration are concerned are the abilities to initiate cell division from almost any

tissue of the plant and to regenerate lost organs or undergo different

developmental pathways in response to particular stimuli. When plant cells and

tissues are cultured in vitro they generally exhibit a very high degree of plasticity

which allows one type of tissue or organ to be differentiated from another type. In

this way, whole plants can be subsequently regenerated. This regeneration of

whole organisms depends upon the concept that all plant cells, given the correct

stimuli, express the total genetic potential of the parent plant (Razdan, 2000).

Trecul (1853) pointed out that establishment of tissue culture involves the process

13

of cell dedifferentiation. Buvat (1944, 1945) devised a scheme according to which

dedifferentiation involves two steps: 1) regression to the cambial stage and 2)

return to the cytological structure of primary meristematic cells. The first stage

affects highly differentiated cells while the second is accompanied by the

organization of bud or root meristems. Regeneration of whole plants in tissue

cultures may occur via shoot or root differentiation. Alternatively, the cells may

undergo embrogenic development to give rise to bipolar embryos also called

embryoids.

Within the last few decades, an increasing number of bulbous and cormous

monocotyledons have been successfully cultured. Tissue culture technology was

greatly influenced by the demand and rapid multiplication and clonal propagation

of slow growing monocots. Schenk and Hildebrandt (1972) reported the

importance of medium composition and techniques for induction and growth of

monocotyledonous and dicotyledonous plants in cell culture. They found that a

high level of auxin-type growth regulating substance generally favoured cell

cultures of monocotyledonous plants while low levels of cytokinins were essential

for most dicotyledonous cell structures. Several economically important monocot

species constituting nutritional, medicinal or ornamental groups of plants were

used for in vitro clonal propagation (Sutter, 1986) and production of secondary

metabolites (Aslanyants et al., 1988).

In vitro asexual multiplication of plant tissues into new plants has been

successfully used to speed up the initial stages of saffron corm production

programmes by supplementing them with transplants obtained from pathogen free

micropropagated material. Approach offers the capability to produce large

quantities of propagating material in short time as well as the production of

commercially important chemical constituents like crocin, picrocrocin and safranal

(Mushtaq et al., 2014). Micropropagation protocols are presently underway as an

alternative route for propagation. Explants from corm tissues, lateral or apical

buds, leaf or nodal tissues and different floral parts have been used for in vitro

regeneration of saffron (Homes et al., 1987; Huang, 1987; Illahi et al., 1987; Isa

14

Ogasawara, 1988; Ahuja et al., 1993; Dhar and Sapru, 1993; Karamian et al.,

2004; Koraoglu et al., 2007).

Ding et al. (1979, 1981) was the first to report the successful tissue culture of

Crocus. They successfully regenerated callus and intact plantlets from corm

explants on culture media containing indole-3-acetic acid (IAA) and 2,4-D.

Similar results have also been reported by Illahi et al. (1987) using low levels of

2,4-D (0.1 mg l-1) and NAA (2 mg l-1).

Laneri and Lucretti (1983a) reported regeneration of shoots from young, dormant

and germinating corms. Several morphogenetic events occurred according to

explants and growth regulators used. The development of dormant axillary bud

were observed with incorporation of BAP in the culture medium alone or in

combination with NAA while the induction of adventitious shoots were observed

from young leaves and floral primordial with higher levels of BAP and NAA.

Young flower buds or corm tissues were also reported to induce cormels in liquid

medium with better response under dark conditions. Laneri and Lucretti (1983b)

reported callus formation on medium supplemented with 10 µM NAA with

subsequent formation of numerous tiny corm like structures originating from

compact callus on liquid medium containing 30 µM BAP and 2.5 µM NAA.

Sutter (1986) reported that axillay bud proliferation in presence of BAP can reduce

the formation of callus which may result in ploidy changes as is the case in other

bulb or corm producing species such as gladiolus. On the other hand, it is critical

to use the lowest possible concentration of 2,4-D to minimize the generation of

somaclonal variation in the cultures for multiplication purposes.

Infections caused by viruses, mycoplasma, bacteria, and fungi can be eliminated

using meristem-tip culture, which is one of the most useful applications of tissue

culture applied to many vegetatively propagated crops (Boxus and Druart, 1986;

Jones, 1986; Pierik, 1987; Slack and Tufford, 1995). It is based on the fact that the

extreme tip of the meristem (0.1-0.5 mm) is normally free from internal bacterial,

fungal and particularly viral contamination and plants regenerated from this zone

are usually pathogen-free (Collin and Edwards, 1998).

15

Sano and Himeno (1987) used excised young intact stigmas plus ovaries of Crocus

sativus L. for production of stigma like structures (SLS). MS medium

supplemented with either cytokinins or auxins or in combinations favoured SLS

production. Benzyladenine and kinetin at concentrations of 0.1, 1.0 and 5.0 mg l-1

supported growth together with in vitro biosynthesis of crocin in stigma. Auxins

had little effect. LS medium supplemented with kinetin at concentration of 1 or 5

mg l-1 and NAA or IBA at a concentration of 10 mg l-1 in combinations recorded

stigma like structures directly or indirectly through meristematic tissues.

Minicorm development in saffron was reported by Homes et al. (1987) after

culturing explants on combination of MS or B5 minerals and organic media

supplemented with 1 mg l-1 2,4-D and 0.1 mg l-1 kinetin and subsequent transfer of

callus to the same basic media containing 2 mg l-1 2,4-D. Similar reports of callus

formation on N6 medium (Chu, 1978) with 2 mg l-1 2,4-D and 0.5 mg l-1 BAP

were reported by Huang (1987). After transferring on MS medium supplemented

with 0.5 mg l-1 BAP and 0.2 mg l-1 NAA, calli differentiated into buds that

germinated with high frequency in half strength MS media with 1 mg l-1 NAA

after 8 months at 150C. Ilahi et al. (1987) cultured saffron corms on half strength

MS medium supplemented with different combinations of growth regulators viz.

auxin, cytokinins, and coconut milk. Callus was induced on medium containing

0.5 mg l-1 each of 2,4-D and BAP and 2% coconut milk. Increase in 2,4-D

enhanced callus formation but suppressed shoot-bud formation. Transfer of callus

to MS medium containing 0.5 mg l-1 NAA, 0.1 mg l-1 of either BAP or kinetin and

2% coconut milk gave rise to roots after 4 weeks of culture with subsequent

suppression of the shoot development.

Gui et al. (1988) reported corm development on MS medium supplemented with

2.5 mg l-1 BAP and 5 mg l-1 IAA using corm explants with dormant buds. Isa and

Ogasawara (1987, 1988) reported two efficient methods for regenerating shoots

from corm callus. One was based on the subculturing of calli induced by 0.5 mg l-1

2,4-D in combination with 0.3 mg l-1 zeatin at 250C in dark in liquid MS medium

16

with 1 mg l-1 2,4-D. Globular structures were formed which in presence of 1 or 3

mg l-1 BAP and 1 mg l-1 NAA differentiated into shoots after 3 months.

Fakhrai and Evans (1989) used explants of leaves, basal plates, petals, anthers and

ovaries of young growing corms of Crocus chrysanthus var E.P Bowlers to study

morphogenic potential. No major change was observed except on ovary explants

after sub-culturing of explants on MS basal medium with 20 different

combinations of either kinetin and NAA or BAP and 2,4-D in the dark. Corm

formation and shoot regeneration was obtained from the callus when the ovary

explants were cultured on media containing 50 mg l-1 and 10 mg l-1 BAP and 0.5

mg l-1 2,4-D. Increasing the level of 2,4-D markedly reduced the number of shoots

produced per explants.

Plessner et al. (1990) reported in vitro corm production in saffron using smaller

corms about 1 cm in diameter and apical buds isolated from larger corms as

explants using MS medium (Murashige and Skoog, 1962) supplemented with

sucrose (3%), nicotinic acid (5 mg l-1), pyrodoxine-HCL (1 mg l-1), thiamine-HCl

(0.5 mg l-1), myo-inositol (100 mg l-1), adenine sulphate (160 mg l-1) and casein

hydrolysate (500 mg l-1) and growth hormones viz. 1 mg l-1 2,4-D, 3-12 mg l-1

kinetin and 3 mg l-1 zeatin. Study revealed that cytokinin, particularly zeatin, and

auxin namely 2,4-D were essential for regular bud development in vitro. Study on

effects of ethylene and ethapon on organogenesis revealed that ethylene and

ethaphon pretreatments inhibited leaf development and induced corm production

and dormancy.

A study on in vitro production of stigma like structures from stigma explants of

Crocus sativus L. by Sarma et al. (1990) revealed that SLS were produced from

stigma explants of Crocus sativus L. under defined conditions. MS medium

supplemented with NAA (10 mg dm-3) + BAP (1.0 mg dm-3) induced the optimum

response. NAA was found to be an important addendum to achieve good response.

A culture temperature of 200C seemed better than 25oC.

George et al. (1992) obtained callus initiation from meristematic regions of corms

of Crocus sativus L. on Murashige and Skoog's (1962) medium (MS)

17

supplemented with 2,4-D (2 mg l-1) and kinetin (0.5 mg l-1). Somatic

embryogenesis was obtained on transfer of callus to MS medium supplemented

with indole-3-acetic acid (2 mg l-1), kinetin (2 mg l-1) and ascorbic acid (100 mg

l-1). The globular embryos on 1/2 strength MS liquid medium with 1 mg l-1

abscisic acid showed further differentiation. Adventitious shoots were obtained

from callus on MS medium with NAA (2 mg l-1) and kinetin (4 mg l-1). Plantlet

formation was obtained from globular callus cultured using filter paper support in

liquid medium containing MS basal salts with indole-3-acetic acid (2 mg l-1),

kinetin (2 mg l-1) and ascorbic acid (100 mg l-1).

Dhar and Sapru (1993) studied in vitro production of corm and shoot like

structures using 0.1% mercuric chloride for sterilization and MS medium

containing 2% sucrose supplemented with NAA (1-3 mg l-1), 2,4-D (2-5 mg l-1)

and kinetin (1-5 mg l-1). Results revealed that floral apices used as explants

responded to callus production on MS medium supplemented with 2 mg l-1 each of

2,4-D and kinetin. Kinetin (2 mg l-1) in combination with NAA (1 mg l-1) helped

in callus proliferation on MS medium.

Aguero et al. (1994) described two main phases for effective saffron

micropropagataion namely shoot multiplication and bulbification. Multiplication

phase was achieved through scission of lateral buds of adult corm on Murashige

and Skoog medium (MS) half strengthened in nitrogen supplemented with 1.5 mg

l-1 BAP and 30 g l-1 sucrose. After every 40 days the mass of newly developed

buds were divided into two explants which were transferred to the same medium.

The bulbification phase was achieved when explants from the multiplication phase

were cultured on the same medium but deprived of growth regulators and

supplemented with 60 g l-1 sucrose. Short day conditions (8h) under the

photosynthetic photon flux (PPF) of 50 μmolm-2s-1 and moderate temperature

(150C) produced big sized minicorms after 120-150 days of in vitro culture

showing the possibility to harvest 60-70 minicorms with a mean fresh weight of

0.5-1.25 g after one year of culture.

18

Ahuja et al. (1993) reported corm differentiation and development at the base of

excised shoots proliferated from callus cultures. Induction from cultured bulblets

was achieved on half-strength Murashige and Skoog (MS) basal medium

containing BAP (5×10-6 M) and NAA (5×10-6 M) + 2% activated charcoal

incubated at 15±10C. Microsurgery of the apical meristematic bud in corms prior

to culture increased the induction of cormogenic nodules. High concentrations of

BAP (2 mgcnt.dotl-1) and low levels of 2,4-D (0.1 mgcnt.dotl-1) were found to be

essential for the development and proliferation of cormogenic nodules. The

application of paclobutrazol and imazalil increased the induction rate of

adventitious shoots in the nodular cormogenic calli and the growth of microcorms.

Somatic embryogenesis was initiated in Crocus sativus L. from shoot meristem on

LS medium containing BAP (2x10-5 M) + NAA (2x10-5 M) by Ahuja et al. (1994).

Various stages of somatic embryogenesis were observed in the same medium and

the development was asynchronous. Somatic embryo development preceded

through well recognized sequence (globular to embryoids) with clearly

discremible bipolar regions. Matured embryos germinated on half strength MS

medium containing GA3 (20 mg l-1). Complete plantlets with well developed root

system and corm formation were obtained on transferring germinated embryos on

half strength MS supplemented with BAP (5x10-6 M) + 2% activated charcoal.

Root tip squashes of plantlets regenerated from somatic embryos had normal

(2n=3x=24) chromosomes number.

Igarashi et al. (1994) reported that nodular calli was induced with NAA in

combination with cytokinin, especially BAP. Appropriate concentrations for the

induction of nodular calli were 0.5-50 µM NAA and 0.5-50 µM BAP. Frequency

of callus induction was 10-15%. Poor callus induction and necrosis were observed

in 2,4-D containing medium with or without cytokinin. Vegetative shoots were

formed 20-40 days after transplantation at the regeneration frequency of 20-50%

in the wide range of NAA and BAP concentrations with maximal regeneration

frequency (50%) with 2.5 shoots per callus observed with 0.5-50 µM NAA in

combination with 0.5-5 50 µM BAP.

19

Piqueras et al. (1995) from their studies on micropropagation of saffron (Crocus

sativus L.) by microcorm regeneration reported 50% reduction in explant

contamination by using ultrasonic bath. Basal medium supplemented with 0.1 mg

l-1 2,4-D + 0.5 mg l-1 BAP developed nodular calli with some adventitious shoots

after 8 weeks which on transfer to basal medium developed into green plantlets in

6 weeks. The results are partially in agreement with Ilahi et al. (1987) and Isa and

Ogasawara (1988).

Direct adventitious shoot regeneration from ovary explants of Crocus sativus L.

was revealed by Bhagyalakshmi (1999) and emphasized the role of media

components, incubation conditions and age of the explants on regeneration

potential. Full strength MS medium supplemented with 0.54 μM NAA and 2.22

μM BA produced the best shoot response both in terms of leaf length and number.

Ovaries of different growth stages having stigmas of pale yellow, pale orange and

bright orange regenerated a maximum mean number of shoots per ovary. Further

development of ovary-derived shoots was influenced by the composition of basal

salts in the culture medium where full strength MS salts gave the best response of

those tested. Regenerated shoots produced normal photosynthetic leaves and

corms.

In vitro micropropagation studies by Koul et al. (1999) revealed possibility to

regenerate saffron plantlets and in vitro corm development through somatic

embryogenesis/organogenesis. Bipolar somatic embryos germinated on MS

medium containing GA3 which on transfer to half strength MS medium

supplemented with BA + NAA resulted into formation of complete plantlets. The

shoots/plantlets regenerated via organogenesis/somatic embryogenesis when

excised and incubated in medium containing BAP + NAA at 150C in dark

developed corms at the base.

Loskutov et al. (1999) conducted studies to optimize the in vitro production of

stigma like structures (SLS) that produced important biochemical constituents

responsible for colour, taste and aroma naturally found in the stigmas of autumn

Crocus. The optimum proliferation of SLS was developed on B5 basal medium

20

containing NAA (5.4 µM), BAP (44.4 µM), MS organics, casein hydrolysate

(0.05%) and L-alanine (11.2 mM) after inoculation. Some explants formed other

structures (root, corms, petals, leaves), the growth and development of which

substantially reduced the development of SLS. Removal of brown tissues and

other tissues during subculture allowed continuous culture of half ovary explants

for 9-10 months. Activated charcoal (1%) added to B5 basal medium containing

NAA (5.4 µM), BA (44.4 µM) and sucrose (3%) was found to be helpful

addendum to prevent browning of explants and to accelerate the initiation, growth

and development of SLS.

Piqueras et al. (1999) while studying the development of cormogenic nodules and

microcorms by tissue culture reported that microsurgery of the apical meristematic

bud in corms prior to culture increased the induction of cormogenic nodules.

Positive effect of microsurgery is related to an increase in ethylene production

upon wounding caused by removal of the meristems since the application of this

plant growth regulator to isolated buds have been shown to induce the

development of both axillary and adventitious buds in several species as iris (Perl,

1985), hyacinth (Pierik and Steegmans, 1975), potatoes (Palmer and Barker, 1973)

and lilium (Van Aartrijk and Bloom-Barnhoorn, 1986). High concentration of

BAP (2 mg l-1) and low of 2,4-D (0.1 mg l-1) were found to be essential for

development and proliferation of cormogenic nodules. A high cytokinin/auxin

ratio has been considered necessary for shoot induction and development in plant

tissue culture (Skoog and Miller, 1957; Hussey, 1975). The application of

paclobutrazol and imazalil increased the induction rate of adventitious shoots in

nodular cormogenic calli and the growth of microcorms. The corms with

adventitious shoots were rooted in medium without growth regulators and were

able to generate dormant microcorms in vitro.

Sharma et al. (1999) reported three modes of regeneration system which include

direct organogenesis of shoot from corm upper segment, callus through shoot bud

organogenesis with corm middle segment and via callus through somatic

embryogenesis with bulblet explants induced respectively on B-5 and MS medium

21

supplemented with various levels of BAP and NAA. Complete plantlets with

slight base swelling were obtained on transferring germinated embryos and shoots

to 1/2 strength MS medium with high sucrose level (6% w/v) fortified with NAA.

A study on comparative effect of BAP and TDZ on multiplication of

micropropagated saffron (Crocus sativus L.) corms by Blazquez (2001) revealed

that TDZ (0.1 mg l-1) was significantly more efficient for production of

microcorms with fully developed leaf primordial than BAP (2 mg l-1).

Study on somatic embryogenesis in saffron (Crocus sativus L.), morphological

differentiation and the role of antioxidant enzymatic system by Blazquez et al.

(2004a) revealed that development of embryoids was characterized by the

emergence of shoot apical meristem and cotyledon (monopolar stage) and

subsequent differentiation of minicorm in the basal part of the embryo (dipolar

stage). The asynchronous mode of development observed in the embryogenic

callus of saffron has been previously described in different monocots during

somatic embryogenesis (Wang et al., 1999; Fereol et al., 2002). During

morphological differentiation, changes in the antioxidant enzymatic system of

somatic embryos were detected with increased ASOD and catalase activity during

the initial stages of the process which were in agreement with the sequence of

embryogenic differentiation reported in several cases of monocots (Ho and Vasil,

1983; Fransz and Schell, 1991; Samaj et al., 2003).

Blazquez et al. (2004b) from their studies on somatic embryogenesis in saffron:

optimization through temporary immersion and polyamine metabolism reported

that saffron embryogenic calli increased fresh weight four times when cultured in

temporary immersion system compared to those cultured on solid medium. 1 mg l-

1 paclobutrazol was effective in reducing hyperhydricity in the explants. The

development of somatic embryos was improved on solid medium supplemented

with 0.5 mg l-1 jasmonic acid (JA). Plant regeneration via somatic embryogenesis

was obtained after eight weeks of treatment with combination of JA and sucrose.

Jasmonates are involved and enhance the development of tubers, bulbs and corms

of several geophytes owing to their positive influence of carbohydrate

22

accumulation (Ravnikar et al., 1993; Koda, 1997; Santos and Salema, 2000).

Increased level of polyamines were found during embryo development.

Karamain (2004) from his studies on plantlet regeneration via somatic

embryogenesis in four species of Crocus viz. C. sativus, C. cancellatus, C.

michelsonii and C. caspicus reported that somatic embryogenesis was initiated

using shoot meristem culture on LS medium containing 4 mg l-1 NAA and 4 mg l-1

BAP or 1 mg l-1 2,4-D and 4 mg l-1 kinetin. Somatic embryogenesis was

asynchronous in all the four species and various stages of somatic embryo

development were observed when embryogenic calli with globular somatic

embryos were transferred to half strength MS medium containing 1 mg l-1 abscisic

acid. Complete plantlets were obtained by transferring germinated embryos to half

strength MS medium supplemented with 1 mg l-1 NAA and 1 mg l-1 BAP at 200C

under 16/8 hr (light/dark) cycle which are in agreement with the earlier reports of

this genus (Ebrahimzadeh et al., 2000). Many aspects can affect the maturation

and germination process such as temperature and light conditions, age of explants

and concentration of growth regulators (Firoozabday and DeBoer, 1993).

Zhigang et al. (2005) reported enhancement of cell growth in suspension cultures

by investigating the relationship between morphological transformation and cell

growth in callus and suspension cultures of saffron cells belonging to the cell line

C96 induced from Crocus sativus L. An unbalanced osmotic pressure between the

intra and extra cell regions induced large morphological transformation which

affected normal division of the saffron cells. An increase in osmotic pressure

caused by the addition of sucrose inhibited the vacuolation and shrinkage of

cytoplasm in the cells. As the sucrose concentration increased, the total amount of

accumulated biomass also increased. Besides sucrose concentration, increased

ionic strength and inoculation ratio also restrained to a large extent the vacuolation

and shrinkage of the cytoplasm in the suspended cells which resulted in increased

biomass.

To optimize an in vitro protocol for propagation of saffron through somatic

embryogenesis, effect of various concentrations of 2,4-D (0, 0.25, 0.5, 1, 2, 4 and

23

8 mg l-1) in combination with BAP (0, 0.25, 0.5, 1, 2, 4 and 8 mg l-1) were studied

by Saboora et al. (2006). Study revealed that 2.0 mg l-1 2,4-D + 1 mg l-1 BAP were

effective for induction of embryos. Comparative effect of different concentrations

of plant growth regulators in micropropagation of saffron by Taghizadeh et al.

(2006) revealed highest percentage of microcorm production with 4.5 mg l-1 2,4-D

in absence of kinetin. However, kinetin in lower concentrations induced callus

production.

Study on the effect of different hormone treatments on non-embryogenic and

embryogenic callus induction and time-term enzyme treatment on number and

viability of isolated protoplasts derived from embryogenic callus on saffron by

Darvishi et al. (2007) revealed that culturing of apical meristem of young corms

after sterilization on solid LS medium supplemented with 2 mg l-1 NAA and BAP

exhibited best effect on induction of non-embryogenic callus whereas 1 mg l-1 2,4-

D and BAP exhibited best effect on induction of embryogenic callus.

Jun (2007) reported that explant’s age according to style length, culture conditions

containing temperature and light and growth regulator were important factors

influencing in vitro flowering from styles of saffron. Style incised from 55-70 mm

length floral buds with light blue parienths as explants cultured on media

supplemented with 26.8 µM NAA and 31.1 µM BAP in dark at 200C were optimal

conditions for in vitro flowering.

Karaoglu et al. (2007) while studying various explants revealed that an

insignificant quantity of corms could be obtained by sterilization with 100%

commercial bleach for 20 minutes. Treatment with sulphuric acid or high

temperature treatments (47.50C) were damaging. Spores or dominant

microorganisms occurring as endogenic contaminants were resistant to

disinfectants under any condition of sterilization. These findings are partially in

line with Ozel et al. (2006) and Taylor et al. (1998). Stigma or leaf explant failed

to develop into cormlets even after a long period of culture. However, cormlet

regeneration via somatic embryogenesis was achieved from mature floral bases on

MS medium containing BAP (1 mg l-1) and NAA (1 mg l-1). After 3 weeks of

24

culture, the whole corms did not show any development. However the explants

consisting of eye buds showed development of single shoots with leggy

appearance after 6 week of culture which on subculturing on the same medium

showed regeneration of one or two cormlets from the main explants after 24 weeks

of culture.

To prevent low and heterogeneous morphogenic potential of meristems, a study on

enhanced plantlet regeneration from cultured meristems in sprouting buds of

saffron corms by Majourhat et al. (2007) revealed clear differences after culturing

of explants on different cytokinin types and concentrations (BAP, 2ip and TDZ)

for six weeks for number of new shoots per initial explants, their length and

quality. Higher multiplication rate (5.6) was observed with 5 mg l-1 BAP.

Raja et al. (2007) reported in vitro microcorm production by culturing leaf

segments of saffron on MS medium containing BAP (4.0 mg l-1) + NAA (0.50 mg

l-1) + 9% sucrose. Maximum explant survival was observed when leaf was used as

an explants source (48.90%) and incubated on MS medium supplemented with

BAP (1.0 mg l-1) and 2,4-D (1.0 mg l-1). Maximum proliferation of established

cultures (56.30) was obtained with BAP (2.0 mg l-1) + NAA (0.50 mg l-1). Plant

regeneration through somatic embryogenesis using regenerable embryogenic calli

was also obtained from leaf explants cultured on MS medium containing BAP (1.0

mg l-1) and 2,4-D (1.0 mg l-1). Somatic embryo formation was attained to the tune

of 8.66 embryos per culture by using MS medium supplemented with BAP (2.25

mg l-1 ) and 2,4-D (0.10 mg l-1). Matured embryos germinated after incubation on

MS medium containing GA3 (20.0 mg l-1) in combination with ABA (2.0 mg l-1)

after five days. An average of 10.82 shoots per explant of proliferated culture were

obtained when MS medium was supplemented with BAP (2.0 mg l-1) + NAA

(0.50 mg l-1). In vitro induction of embryogenesis in iridaceous genera was

reported earlier by Wang et al. (1990), Bach (1992), Kim and Kang (1992) and

Jehan et al. (1994).

Study on induction of somatic embryogenesis in saffron by Sheibani et al. (2007)

using different concentrations of TDZ (0, 0.1, 0.25 and 0.5 mg l-1) and 5 different

25

types of corm explants (terminal or axillary buds, upper or lower parts of the corm

tissue and terminal buds from pre-treated corms at 40C for 2 weeks) revealed that

TDZ concentrations affected the induction of somatic embryogenesis significantly

while different types of corm explants showed no significant effect on this process.

Among TDZ concentrations, 0.5 mg l-1 was the most effective treatment for

embryogenesis induction. Embryogenic calli (globular stage) proliferated well

when subcultured on MS medium supplemented with 0.25 mg l-1 TDZ before

transferring to hormone-free MS medium containing 6% sucrose for maturation

(scutellar or horn-shape stage). Matured embryos were transferred to half strength

MS medium without growth regulators which resulted in microcorm formation

from basal part after 3 months. Most of the matured embryos cultured on 1/2

strength MS medium without growth regulators produced plantlets with

microcorm and occasionally roots within 3 months whereas the ones cultured on

medium containing NAA (1 mg l-1) + BAP (1 mg l-1) returned to globular stage

and produced more globular embryos and callus.

Bhatti et al. (2009) reported successful ex vitro shoot regeneration from terminal

and lateral buds of saffron corms pulse treated with gibberelic acid (GA3), indole-

3-acetic acid (IAA) and thidiazuron (TDZ) at various concentrations and time

durations. Karamian and Ebrahimzadeh (2009) reported somatic embryogenesis

from shoot meristem culture on LS medium containing kinetin (4 mg l-1) and 2,4-

D (1 mg l-1). To initiate morphogenesis, embryogenic calli were transferred to

either half strength MS medium without growth regulators or with medium

supplemented with 1 mg l-1 abscisic acid. Sub-culturing of germinated embryos on

half strength MS media supplemented with 0.1 mg l-1 NAA and 1 mg l-1 BA lead

to plant regeneration. SLS were also obtained.

MalekZadeh et al. (2009) studied two culture media namely LS and MS in liquid

and solidified form supplemented with different concentrations of 2,4-D (2 mg l-1),

NAA (2 mg l-1) as auxins and BAP (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg l-1), kinetin

(1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg l-1) as cytokinins using apical and lateral

meristems of spring and autumn corms for regeneration ability of saffron.

26

Solidified LS medium supplemented with 4 mg l-1 BAP and 2 mg l-1 NAA showed

an adequate response for producing corm and shoots for cryopreservation of apical

and lateral corm meristems.

Mathe et al. (2009) conducted a study with an aim to investigate the tissue culture

potential of several members of Crocus genus within their natural habitat in

Carpathian Basin. Tissue culture was successfully established in case of Crocus

heuffelianus and Crocus scepusiensis. Embryogenic calli were induced through

sub-culturing of shoot tip meristems on MS medium supplemented with 2% (w/v)

sucrose, Gamborg’s vitamins, 0.8% (w/v) bacto-agar and the proper

auxin/cytokinin concentrations.

In vitro production of callus and microcorm formation in saffron (Crocus sativus

L.) was studied by Sharaf-Eldin et al. (2009). Protocol for callus induction and

microcorms production were established. Three media were compared using basal

MS medium containing 3% (w/v) sucrose supplemented with NAA (0.5 mg l-1) +

BAP (2 mg l-1); MS1, NAA (0.5 mg l-1); MS2 and BAP (3.5 mg l-1); MS3.

Various stages of somatic embryogenesis were observed in the same medium and

the development was asynchronous. Maximum explant response was observed

when sprouts or corms were used as explants and incubated on MS1 or MS2. The

highest percentage of callus induction or corm formation were obtained when

corm were used as explants and incubated on MS1 or MS2.

Sharifi and Ebrahimzadeh (2009) studied globular embryo like structures and high

efficiency multiple shoot formation in saffron (Crocus sativus L.) induced by

thidiazuron. To find out suitable conditions for shoot formation from corms, the

effect of cytokinins: N6-benzyladenine and N-phenyl-1,2,3 thidiazol-5-yl urea

known as thidiazuron were compared at different concentrations. Thidiazuron at

4.54 µM induced shooting in all corm explant with an average of 39.5±5.1

(shoots/corm) whereas benzyladenine induced multiple shoot formation in only

3.6-11.4% of corm explants. To optimize further plant regeneration from the

induced adventitious shoots obtained from thidiazuron treatment, they were

transferred to MS and B5 media supplemented with different concentration and

27

combinations of NAA and BAP. Highest rate of plant regeneration was observed

on G5 media supplemented with NAA and BAP at a concentration of 2.22 µM and

2.68 µM respectively. Upon optimized hormonal condition an average of

19.55±5.75 shoots and 3.18±1.5 roots per explants were obtained.

Vatankhah et al. (2009) studied the effect of various hormonal combinations on

regeneration of shoots and roots from meristems of Crocus sativus L. The most

efficient regeneration occurred with NAA (1 mg dm-3) + thidiazuron (1 mg dm-3)

and NAA (1 mg dm-3) + kinetin (2 mg dm-3). For sprouting, regenerated shoots

were sub cultured on MS medium containing NAA (1 mg dm-3) + BAP (1mg dm-

3).

Zaffar et al. (2009a) reported that saffron calli were induced from leaf explants on

MS medium supplemented with BAP and 2,4-D at concentration of 1 mg l-1 each.

After subculturing the calli two or three times, two types of calli, a nodular one

and a friable one were induced. The nodular calli had high regeneration ability and

resulted in development of shoots after incubation on MS medium supplemented

with different concentrations of BAP and NAA. Regeneration frequency was

optimal with BAP (2.0 mg l-1) + NAA (0.50 mg l-1). Altering the concentration of

ammonical (NH4+) and nitrate (NO3

-) forms of nitrogen resulted in rooting of

proliferated cultures and both the rooting frequency and the average number of

roots per proliferated culture showed maximum values when incubated on MS

medium containing 40 mM NH4 + 20 mM NO3..

Studies on effect of media composition and growth conditions on in vitro

microcorm formation in saffron (Crocus sativus L.) by Zaffar et al. (2009b)

revealed that higher concentration of BAP and paclobutrazol in MS media resulted

in formation of bigger microcorms. Low temperature (150C) and incubation under

light appeared to be optimum for microcorm formation resulting in thickening of

the basal parts of the shoots suggesting synergistic interactions between low

temperature and light. Microcorm formation synchronized with in vivo

developmental cycle. The microcorms had a high survival rate upon transferring

28

directly into soil after preconditioning at temperature of 300C for 1 month and

storage upto planting time at 50C.

Devi et al. (2010) used Murashige & Skoog (1962) medium containing BAP and

2,4-D. Irrespective of the corm size, bud sprouting was season dependent.

Maximum bud sprouting occurred in the months from November to December and

minimum from May to August. Buds sprouted from the months of March to May

regained further growth only in the next growing phase i.e., from September to

December, indicating that the in vitro propagules followed the same natural

rhythm for bud sprouting. Young leaves from these bud sprouts were used as

explants for initiation of somatic embryos using TDZ and picloram. Lumps of

somatic embryos proliferated further to form secondary somatic embryos.

Different treatments of PGRs (ABA, GA3, BAP, 2,4-D, IAA, NAA) of varying

medium strengths (¼, ½, ¾) and sucrose concentrations (3, 6, 9, 12%) were used

for the conversion of these embryos into plantlets. Cormlets developed at the base

of these plantlets on medium supplemented with growth retardants (paclobutrazol

& CCC). These in vitro produced cormlets were transferred to green house

conditions for further growth evaluation.

Mir et al. (2010a) obtained a corm size (1.3 g) from eye bud explants cultured on

LS media supplemented with 21.6 µM NAA and 22.2 µM BAP. Microcorm

formation was influenced by external BAP concentration; also growth of corm was

improved with increased period of inoculation. Regenerated corms were kept at

5oC for 5 weeks and then transplanted to a potting mixture where the germination

percentage was very low (4%) which may be due to small corm size.

In another study by Mir et al. (2010b) for development of stigma like structures

under in vitro conditions, various explants were cultured on LS, MS, and G5

media supplemented with different combinations of phytohormones. Stigma-like

structures appeared on cultured half ovaries and the best response (60%) was

observed with half ovaries cultured on G5 media supplemented with 27 µM NAA

and 44.4 µM BAP followed by 55% on LS media supplemented with 27 µM NAA

and 44.4 µM BAP. The length of SLS ranged from 4 to 6 cm and number of SLS

29

varied from 8-12. The accumulation of pigments like safranal, crocin and

picocrocin has been confirmed by GCMS and spectrophotometrically.

Field evaluation of 8200 in vitro corms weighing <5 g and 5-7 g at Pampore,

Kashmir by Nehvi et al. (2010c) confirmed survival of above 60% among in vitro

corms weighing 5-7 g. Flowering percentage was recorded to be less than 1% in

the first year. Corm yield m-2 doubled after one year of plantation. Corm number

increased three times in 5-7 g category with an average corm number of 2.81

corms/mother corm whereas for <5 g category it was 1.5 times with 0.88

corms/mother corm.

In vitro studies on application of biotechnological tools for saffron propagation by

Parray et al., (2010) revealed maximum number of cormlets (70±3) from corm

slices cultured on half strength MS medium supplemented with thidiazuron (TDZ)

20 µM + indole-3- acetic acid (IAA) 10 µM + sucrose 40 g l-1. The prominent

increase in corm size with a weight range of 1.9-2.1 g was recorded on thidiazuron

(TDZ) 15 µM + indole-3-acetic acid (IAA) 12.5 µM + sucrose 30 g l-1 in 40% of

in vitro raised minicormlets via callus. Apical vegetative buds of actively growing

corms were cultured for cormlet development and corms of size 2.5 g were

developed on MS medium with BAP (20 µM) + NAA (15 µM) + 30 g l-1 sucrose.

50% recorded most prominent increase in corm size with a weight range of 1.5-2.5

g. Vegetative growth in in vitro corms was observed in above 90% corms

transferred in cups containing soil collected from collection sites under green

house conditions at a temperature of 200C.

Study on the effect of paclobutrazol on in vitro corm formation and enlargement in

saffron (Crocus sativus) by Zaffar et al. (2010) reported that in vitro propagation

of saffron through cormogenesis can be an efficient alternative method for large

scale propagation of pathogen free corms if a reproducible and efficient in vitro

micro-propagation protocol is available. Effects of different concentrations of

cytokinins in combination with paclobutrazol were investigated. In vitro

regenerated shoots from callus derived from corm sections were cultured on

Murashige and Skoog (MS) medium supplemented with (0.2-1.5 mg l-1) BAP,

30

paclobutrazol (2-10 mg l-1) and sucrose (3-12%). Higher concentrations of PAC (5

mg l-1) along with BAP (0.25 mg l-1) and 9% sucrose resulted in formation of

relatively larger microcorms. There was a significant interaction between

paclobutrozol and sucrose for microcorm weight.

A study on in vitro cormlet production and growth evaluation under greenhouse

conditions in saffron (Crocus sativus L.) – a commercially important crop reported

by Devi et al. (2011) described the effects of season on initial bud sprouting, direct

shoot regeneration from the base of the sprouted bud and cormlet production from

multiple shoots and provided growth evaluation of in vitro produced cormlets

under greenhouse conditions. Initial sprouting of buds from corm segments was

previously described in medium supplemented with 2,4-Dichlorophenoxyacetic

acid (2,4-D, 9.05 μM) and 6-benzylaminopurine (BAP, 26.64 μM). Maximum bud

sprouting (90%) was observed during November and December. Direct multiple

shoot primordia were initiated from the base of these sprouted buds on BAP (26.64

μM). Multiplication of shoots was achieved in BAP (26.64 μM) and α-naphthalene

acetic acid (1.0 and 5.0 μM). Growth retardants (chlorocholine chloride and

paclobutrazol) were used for cormlet production from multiple shoots and

paclobutrazol (1.7 μM) evinced maximum cormlet production (86.07%). Growth

of these in vitro produced cormlets was evaluated under greenhouse conditions and

91.66% sprouting was observed. An increase in cormlet weight (66.88%) was also

observed under in vivo conditions.

Devi et al. (2012) while studying the genome size in tissue culture raised plants of

saffron (Crocus sativus L.) revealed that genome size of tissue raised plants

remained stable. Directly regenerated shoots, somatic embryo derived plants and

mother plants i.e. plants produced under field conditions were assessed by

flowcytometery for genome size stability. Estimation of nuclear DNA content with

emphasis on nuclei preparation and testing of inhibitors for PI binding was carried

out. 3C nuclear DNA of somatic embryo derived and directly formed shoots was

9.74±0.02 and 9.73 ± 0.04 pg and 1C genome size was 4.81±0.01 x 109 bp and

31

4.80±0.01 x 109 bp, respectively. 3C nuclear DNA and IC genome size of mother

plant was 9.77±0.02 and 4.82±0.01 x 109 bp, respectively.

Study on CsSERK gene expression associated with shoot organogenesis

competence in saffron (Crocus sativus L.) by Vatankhah et al. (2012) was

conducted to study the expression of SERK gene inorganic (shooting) and non-

organic nodular calli using RT-PCR. Two hormonal treatments were used for

induction of shooting nodular calli (MS media containing NAA, TDZ and IBA,

TDZ) and two culture media namely MS and LS supplemented with 2,4-D and

kinetin for induction of non-organic nodular calli. Sequence analysis of CsSERK1

revealed high levels of similarity (85%) to AcSERK (Areca catechu SERK) gene.

Analysis of SERK expression showed that it could be detected in shooting nodular

calli before shoot development. In contrast, it was not detected in non-organic calli

thus suggesting that CsSERK expression is associated with induction of shoot

organogenesis and that it could be a potential marker for cells competent to form

shoot in saffron tissues cultured in vitro. Also SERK gene may have a broader role

in morphogenesis in cultured tissue rather than being specific to somatic

embryogenesis.

A study on influence of 2,4-D and kinetin combinations on callus induction in

saffron (Crocus sativus L.) was carried by Vahedi et al. (2012) to develop a

protocol for callus induction exploiting the influence of 2,4-D and kinetin in

different combinations. Lateral and terminal meristem of plants were collected and

inoculated on MS media supplemented with 2,4-D (1, 2 and 4 mg l-1) and kinetin

(0.5, 1, 4 and 8 mg l-1) in different combinations with 3% sucrose for callus

induction after thorough surface sterilization. The first callus was induced after 35

days of inoculation from terminal meristem explants. However, lateral meristem

was observed to be less responsive. The highest frequency of callus induction was

achieved on MS medium supplemented with 2,4-D (2 mg l-1) and kinetin (0.5 mg

l-1).

Mir et al. (2012) reported production of stigma like structures (SLS) under in vitro

conditions. Highest response was observed with half ovaries on G5 media

32

supplemented with different combinations of phytohormones. The relative

quantification through RT-PCR for expression of apocarotenoid genes like CsLYC,

CsZCD, CsBCH and CsGT-2 revealed that there was an increase in expression

from callus to SLS development. Expression pattern of CsLYC, CsZCD, CsBCH

and CsGT-2 was also studied in different flower parts and highest expression was

found in stigma followed by style and petal. Expression of the regulatory genes

responsible for biosynthesis of apocarotenoids viz. crocin, picrocrocin and safranal

was upregulated in SLS in saffron revealing that these structures are

developmentally closely related to natural stigma.

Parray et al. (2012) studied the influence of plant growth promoting rhizobacteria

(PGPR) on the size of cormlets of Crocus sativus Kashmirianus under in vitro

conditions. The study was conducted to observe the growth of bacterised tissue

cultured cormlets under in vitro conditions after 4-6 weeks of growth on MS

medium supplemented with BAP (20 µM) + NAA (15 µM). The study revealed

the effects of three plant growth promoting rhizobacteria (PGPR) i.e.

Pseudomonas sps., Bacillus subtilis and Acintobacter sps isolated from saffron

rhizosphere soil on increasing the size of cormlets under in vitro conditions. The

minicorms were subjected to three treatments; in the first treatment they were co-

cultivated with individual bacterial strains, in the second treatment co-cultivated

with combinations of three strains and in the last treatment cultured without strains

(control). The results revealed that the combination of Bacillus subtilis and

Acintobacter sps proved useful in enhancing the size of cormlets upto 8 g

compared to the control (2-4 g). Introduction of rhizobacteria to saffron cells

during in vitro micropropagation process established an early associative

interaction between the plant cells and bacteria. In the association, the

rhizobacteria provided the host plants with phytohormones and other stimulators.

To develop a protocol for in vitro microcorm formation in saffron, effects of

different concentrations of cytokinins in combination with paclobutrazol were

investigated by Zaffar et al. (2012). Embryogenic callus derived by culturing leaf

segments/sprouted apical buds/corm sections on Murashige and Skoog (MS)

33

medium supplemented with 2,4-D (1.0 mg l-1) and BAP (1 mg l -1). Shoots were

regenerated from callus by subculturing on MS medium fortified with BAP (1.0

mg l-1) and NAA (1.0 mg l-1). In vitro regenerated shoots from callus were

cultured on Murashige and Skoog (MS) medium supplemented with BAP (0.2-1.5

mg l-1), paclobutrazol (PAC) 2-10 mg 1-1 and sucrose (3-12%). Higher

concentrations of PAC (5 mg l-1) along with BAP (0.25 mg l-1) and 9% sucrose

resulted in the formation of relatively larger microcorms. There was a significant

interaction between paclobutrazol and sucrose for microcorm weight.

Ziaratnia et al. (2012) studied in vitro callus induction and production of stigma-

like structures of saffron (Crocus sativus). Callus induction was carried out by

application of different combinations of plant growth regulators to saffron explants

grown on two types of medium, MS and B5. Results showed that B5 medium

supplemented with 2,4-D (2 mg l-1) and kinetin (4 mg l-1) and MS with NAA (8

mg l-1) and BAP (1 mg l-1) exhibited better callus induction. In another

experiment, production of stigma-like structure was tested from different types of

saffron explants including stigma, style, ovary and half ovary. For that purpose,

MS medium was supplemented with several levels of auxins (2,4-D and NAA)

and cytokinins (kinetin and BAP). Results showed that all types of explants from

saffron flowers were able to produce SLS except those from flower buds. It was

also found that among different types of explants, intact ovaries were more

suitable for production of direct SLS than others while the induction of indirect

SLS was higher on styles than intact and half ovaries. The best hormonal

combination for induction of direct and indirect SLS was kinetin (2 mg l-1), NAA

(8 mg l-1) and NAA (20 mg l-1), BAP (1 mg l-1) respectively. HPLC comparison of

natural stigma and SLS showed that all the three saffron constituents are present in

SLS derived in the study but at lower levels compared to natural ones.

Zeybek et al. (2012) developed an efficient in vitro tissue culture system for

saffron (Crocus sativus L.) complete with roots and corms. In indirect

organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, ascorbic

acid (100 mg l-1), and the combination of 2,4-D (0.25 mg l-1) and BAP (1 mg l-1)

34

were best for callus initiation and growth while BAP (1.5 mg l-1) was excellent for

high rate of adventitious shoot formation. Indole-3-butyric acid (1 mg l-1) was

more preferable for adventitious corm and root initiation as well as growth.

Overall, 64% rooting and 33% corm production rates were achieved in indirect

organogenesis. In direct organogenesis, MS medium supplemented with 3%

sucrose, ascorbic acid (100 mg l−1) and BAP (1 mg l−1) was optimum for shoot

growth. While IBA (1 mg l−1) was best for adventitious corm formation, IBA

(2 mg l−1) promoted adventitious root initiation and growth. Overall, 36% and 57%

of explants had corms and contractile roots respectively.

Simona et al. (2013) observed that 2,4-D (1 mg l-1) and BAP (1 mg l-1)

combination was favorable for direct organogenesis and 2,4-D (0.25 mg l-1) and

BAP (1 mg l-1) combination was superior for indirect organogenesis. Cavusoglu et

al. (2013) achieved in vitro plant regeneration and daughter corm formation of

saffron (Crocus sativus L.) from corm parts consisting of meristematic region via

direct organogenesis. For direct shoot regeneration and foliation, sterile

meristematic node-containing corm explants were cultured on 1/2 strength

Murashige and Skoog (MS) Medium or MS medium supplemented with BAP.

Maximum shoot initiation to the tune of 96.7% and maximum foliation rate

(93.3%) was observed using MS medium supplemented with BAP (6 mg l-1) while

MS + BAP (1 mg l-1) gave the least result in shoot initiation (16.7%); MS + BAP

(1 mg l-1) and MS + BAP (10 mg l-1) gave the least result in foliation from the

initiated shoots in 90 days. However, daughter corm formation and rooting were

achieved on MS supplemented with IBA or IAA. They showed that MS + IAA (1

mg l-1) have the best results on daughter corm formation rate (76.7%) and daughter

corm number (1.74 corms/corm). On the other hand, rooting rate (46.7%) and root

number (1.5) were highest on MS with IBA (2 mg l-1) in 120 days.

Mir et al. (2014) studied regeneration of microcorms under in vitro conditions.

Apical bud explants were cultured on different nutrient media supplemented with

various concentrations of growth regulators. Microcorm formation was observed

on all media combinations. Maximum number (10) and weight (1.54 g) of

35

microcorms developed were observed on MS media supplemented with BAP

(2 mg l-1) + NAA (0.5 mg l-1) + paclobutrazol (1.5 mg l-1). Shoot and root

regeneration was observed in the microcorms developed under in vitro conditions.

Maximum number of shoots (11.6) and shoot length (11.4 cm) was also observed

on MS media supplemented with NAA (2.16 µM) + BAP (22.2 µM). Maximum

number of roots (11) and length of roots (11.4 cm) were obtained on G5 media

containing NAA (21.6 µM) + BAP (22.2 µM).

II.2 Mutation

Genetic variations are the basic tools to develop new cultivars with better traits like

tolerance against various environmental stresses, resistance against pests and

diseases and improved yield and quality. Thus, mutagenesis technology has been

applied to plant breeding comprehensively which allow crops to produce beneficial

varieties with good traits (Maluszynski et al., 1995; Gu et al., 2003). Although

naturally occurring mutagenesis is very simple and requires no tools to be brought

about, it occurs at very low frequency to rely alone on for accelerated plant

breeding and is in most instances very lethal to plants and thus selection is

cumbersome. Besides spontaneous mutations, it is possible to induce them

artificially using mutagens. The only option left with the interested plant breeder is

to fully utilize the “induced-mutagenesis” technique in order to feed the

burgeoning population of the world and to wage war against the alarming

environmental stresses in the current century. The usefulness of any mutagen in

plant breeding depends not only on its mutagenic effectiveness but also on its

mutagenic efficiency, efficient mutagenesis being the product of the maximum

desirable changes accompanied by the least possible undesirable changes (Tamina

and Tabash, 2010). Effectiveness and efficiency are two distinct properties of

mutagens that have been extensively discussed elsewhere (Kawai, 1969, 1975;

Shah et al., 2008; Girija and Dhanavel, 2009). Effectiveness usually means the rate

of point mutations relative to dose whereas efficiency refers to the rate of point

mutations relative to other biological effects induced by the mutagen and is

considered a measure of damage (Konzak et al., 1965). Thus, the two agents may

36

be equal in mutagenic effectiveness because at a given dose, they induce a

mutation with the same frequency. However, when they diverge in their ability to

produce undesirable changes such as sterility and lethality, they may be said to

differ in mutagenic efficiency (Tamina and Tabash, 2010). Induced mutation using

physical and chemical mutagen is one method to create genetic variation resulting

into new varieties with better characteristic.

Historically, the use of mutagenesis in breeding has involved forward genetic

screens and selection of individual mutants with improved traits and their

incorporation into breeding programmes. Over the past 70 years, more than 2500

varieties derived from mutagenesis programmes have been released, as listed in the

IAEA/FAO mutant variety database, including 534 rice lines, 205 wheat lines, and

71 maize lines. Although this approach has clearly proved very successful, there

are limitations imposed by, for example, the difficulty of identifying a small

number of individuals with novel phenotypes within a large population or by the

genetic redundancy present in many plant species as a result of gene duplication

and polyploidy, such that many mutations have no detectable effect on the plant.

Recently, reverse genetic approaches have permitted the silencing or interruption

of individual candidate genes, providing the opportunity to investigate gene

function and to relate sequence information to traits. However, these approaches

have disadvantages: methods based on post-transcriptional gene silencing such as

RNAi have variable success rates and rely on time-consuming vector construction

and plant transformation. T-DNA insertional mutagenesis is also dependent on

efficient plant transformation while insertional mutagenesis by endogenous

transposons is only available in a small number of crops, notably maize, although

there has been some success in transferring these into other species such as rice

(Kolesnik et al., 2004). In any case, these insertional methods are likely to result in

complete disruption of gene function rather than in generating allelic series of

mutants with partial loss-of-function and thus will not produce the range of

mutation strengths necessary for crop improvement. Furthermore, the insertion

sites within the genome may not be distributed randomly, increasing the number of

37

insertion lines required for full genome coverage to unrealistic levels (Zhang et al.,

2007).

Mutations may be gross, resulting in large-scale deletions of DNA or only involve

point mutations. Induced mutation is one of the most widely used techniques for

creating additional variability in flower character. Exploiting natural or induced

genetic diversity is a proven strategy for improvement of all major food crops and

the use of mutagenesis to create novel variation is particularly valuable in crops

with restricted genetic variability due to inherent problem of reproductive sterility.

It is a technique being utilized both by nature and human beings in order to

improve the qualitative and quantitative traits in plants against various biotic and

abiotic stresses Chemical or physical mutagenesis have a number of advantages

over such approaches owing to the fact that mutagens introduce random changes

throughout the genome generating a wide variety of mutations in all target genes

and a single plant can contain a large number of different mutations resulting in

manageable population sizes (Parry et al., 2009).

Mutation can be induced by irradiation with non-ionizing (e.g. UV) or ionizing

radiation (e.g. X and gamma rays, alpha and beta rays, fast and slow neutrons);

such physical mutagens often result in large scale deletion of DNA and changes in

chromosome structure. While ionizing radiation still remains the most suitable

means for inducing variability (Wit et al., 1985; Brunner, 1995; Yang, 1995;

Hayashi et al., 1996; Xu et al., 1998; Bhatia et al., 2001; Zhang et al., 2002; Irfaq

and Nawab, 2003; Chen et al., 2004; Joseph et al., 2004; Lao et al., 2004; Sangsiri

et al., 2005; Sun et al., 2005; Tah, 2006), a number of chemicals have been found

to be equally and even many times more effective and efficient mutagens (Thakur

and Sethi, 1995; Kharkwal, 1998; Solanki, 2005; Rekha and Langer, 2007; Basu et

al., 2008, Dhanavel et al., 2008; Ganapathy et al., 2008; Thilagavathi and

Mullainathan, 2009; Wani, 2009). Ionizing radiations such as γ-rays are highly

effective in inducing chromosomal aberrations (Nilan and Konzak, 1961). Gamma

rays are the most energetic form of electromagnetic radiation, possesses the energy

level from 10 keV to several hundred kilo electron volts and are considered to be

38

most penetrating in comparison to other radiation such as alpha and beta rays

(Kovacs and Keresztes, 2002). Gamma rays belong to ionizing radiation and

interact on atoms or molecules to produce free radicals in cells. These radicals can

damage or modify important components of plant cells and have been reported to

affect differentially the morphology, anatomy, biochemistry and physiology of

plants depending on the irradiation level. Chemical mutagens usually cause point

mutation but loss of a chromosome segment or deletion can also occur

(Thilagavathi and Mullainathan, 2011). Mutagens such as EMS act primarily on

base pairs of the DNA molecule and yield a higher number of gene mutations.

Because of the basic mechanistic difference between these two groups of

mutagens, chemical mutagens are generally considered to be superior to physical

mutagens for induction of mutation (Nilan and Konzak, 1961). By contrast,

chemical mutagens most often only affect single nucleotide pairs. For plants, some

of the more widely used mutagens include hydrogen fluoride (HF), sodium azide,

N-methyl-N-nitrosourea (MNU), hydroxylamine and alkylating agents like ethyl

methane sulphonate (EMS) and methyl methane sulphonate (MMS). The alkyl

group of the chemical mutagens reacts with DNA which may change the

nucleotide sequence and cause a point mutation (Broertijes and Van Harten, 1988).

The choice of chemical mutagen influences the maximum permissible mutation

rate achievable: EMS creates a larger proportion of non-sense mutations involving

the introduction of novel stop codons than a mutagen such as MNU due to the

specificity of EMS in creating mainly G–A and C–T transitions and any individual

mutations is therefore more likely to have a phenotypic effect (Parry et al., 2009).

Mutagenesis plays an important role in creating new varieties in clonally

propagated plants because mutations in such plants may easily get stabilized and

can be manipulated. Critically, mutations in important traits or genes (e.g. in

nutritional quality, resource use efficiency, architecture or phenology) can be

readily exploited by plant breeders without the legislative restrictions, licensing

costs, and societal opposition applied to GM approaches. This is despite the fact

that transcriptomic analyses have shown that large-scale plant mutagenesis may

39

induce greater changes in gene expression patterns than transgene insertion (Batista

et al., 2008).

Though induced mutations are a valuable tool in crop improvement, work related

to saffron in this aspect is limited. Hence, research work done on mutation

breeding of other vegetatively propagated crops are also reviewed here under.

Gamma rays induced mutants in Gladiolus studied by Raghava et al. (1988)

revealed that percentage of sprouting was affected significantly at 10 and 15 Krad.

LD50 was found to be between 10 and 15 Krad. Doses of 10 Krad and above

proved to be detrimental for vegetative and floral traits. Plants treated with 10

Krad did not produce flower spikes whereas the plants in 15 Krad treatment

although sprouted did not survive afterwards. When treated with 10 Krad, plant

height, leaf number and leaf-size were reduced significantly and leaves became

narrow and leathery. Flowering was delayed significantly at 5 Krad. Radiation

treatments caused decrease in spike length, number of florets per spike and floret

size. A desirable and stable mutant with shell pink floret colour was isolated from

the variety 'Wild Rose' in 1 Krad treament and it has been released as 'Shobha'.

Khan (1999) conducted studies on gamma rays induced mutations in saffron

(Crocus sativus L.) with an aim to develop mutants of saffron which can flower

regularly by irradiating the corms with 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 Krad dose of

gamma rays. Lower doses (0.5, 1.5 Krad) resulted in increased plant height with a

corresponding negative effect on plant height with higher doses (1.0, 1.5 and 2.0

Krad). Some petal mutations viz. serrated petals and 3-petal flowers were

observed.

Colchicine was applied to the emerging apical buds of saffron corms at 0.05, 0.1,

0.2 and 0.4% aqueous concentration employing cotton plugs for 48 hrs (12 hours

for four consecutive days) by Zaffar et al. (2004). Studies of C0 and C1 generation

showed that the colchiploids exhibited delayed flower and leaf emergence. The

leaves were thicker, shorter somewhat flat-ridgeless, coarser in feature and dark

green in colour with reduction in number of leaves per plant. Stomatal studies

revealed decrease in number with a concomitant increase in size of stomata in C1

40

generation. There was reduction in the number of flowers per plant, floral variants

observed included smaller sized flowers, irregularly shaped/reduced tepal number,

lobed and dentate tepals, flowers with deep pigmentation in stigmas extending to

style region and orange red pigmented anthers.

Study on induced mutagenic variability in saffron (Crocus sativus L.) by Khan

(2004) revealed delayed sprouting, slow growth in higher dose, increase and

decrease of plant height in lower and higher doses respectively is due to the

amount of auxin synthesis. Formation of serrated petal is the result of somatic gene

mutations whereas 3-petal mutant is the result of alternate inhibition of apical cell

division of petal at initial stage of flower development caused by irradiation. But

surprisingly these mutations failed to appear in VM2 generation. Results are in

confirmation with earlier reports of Gordon (1957). Stunted growth, reduction in

survival and reduced fertility was also attributed to genetic loss due to

chromosomal aberrations and gene mutations (Sparrow et al., 1961; Datta and

Gupta, 1980)

Rastegari et al. (2006) studies induced mutation in saffron (Crocus sativus L.) and

revealed that induced mutation is highly effective for enhancement of natural

genetic resources and have assisted in developing improved cultivars of many

crops. Exposure of saffron corms of different weights (6, 8, 15, 12 and 14 g) to

different doses of gamma irradiation (2.5, 5, 7.5, 10, 12 and 15 Gy) revealed

number of variations in size and colour of petals of saffron flowers. In M1V1, non-

significant differences were observed for yield. Cytogenetic studies showed

chromosomal abnormality i.e chromosome fragments, translocation and chromatid

bridges with 8-10 g of corms and 7.5-10 Gy gamma radiation. It was concluded

that the best dose of gamma radiation were 6 to 10 Gy. Higher irradiation dose (>

10 Gy) prevented corm bud sprouting.

Effects of 0~25Gy 60Co γ-rays irradiation on the development of Crocus sativus L.

corms (12~14 g weight) were investigated by Jun et al. (2007). The results showed

that irradiation at the dose of 5～10 Gy can stimulate flowering of saffron and can

improve the harvest ratio of offspring. The harvested cormlet had flowering ability

41

only after 5 and 10 Gy irradiation treatment. It was concluded that 5～10 Gy

irradiation was suitable mutation dose for corms of 12-14 g weight.

A study on development of high yielding saffron mutants by Khan (2007) revealed

variability in respect of sprouting time, plant height, flower induction, petal shape

and number of filaments in stigma. Five fid stigmas with an average length of 2.70

cm and average dry weight of 6.950 mg were observed which may arise as a result

of gene mutations.

Nehvi et al. (2007) made an attempt to create new saffron variants for economic

characters using gamma irradiation from 60Co source at 0.25, 0.50, 0.75 and 1

Krad doses. Delayed sprouting was observed at higher dose (1 Krad). Irradiation

doses had significant effect on plant height, sprouting, survival, number of

flowering plants, number of daughter corms, corm yield, number of flowers and

pistil recovery/100 pistils. Induction of early sprouting in 0.25 Krad and 0.5 Krad

and delayed sprouting at higher doses was noted. Radiation at lower doses (0.25

Krad) enhanced plant biomass whereas a reverse reaction was observed at higher

doses. M1 mutants with 0.25 Krad radiation dose showed enhanced corm yield

and saffron yield by a margin of 53.44% and 84.28%, respectively. Increased

saffron recovery resulted from more number of flowers/plant. Plant survival was

reduced to 79% at 1.0 Krad. Radiation treatment at higher doses did not reveal any

significant effect on number of daughter corms, corm yield, number of flowers and

saffron yield. The results are in general agreement with other reports (Akhund-

Zade and Mazaferova, 1975; Laneri and Lucretti, 1983a; Khan, 2004). Superiority

of mutants in terms of saffron recovery was maintained to the tune of 47.36%.

Elite mutants also revealed superiority in terms of pistil length and increased

number of flowers/spathe. Tamina and Tabash (2010) reported higher average

effectiveness of EMS than γ-rays. Studies in wheat (Gaul and Aastveit, 1966),

Arabidopsis thaliana (Brock, 1971) and cowpea (Girija and Dhanavel, 2009) have

also shown that EMS is more effective than radiation in inducing polygenic

variability.

42

Abdullah et al. (2009) revealed that mean survival rate fell sharply from 63% at 20

Gy to 7% at 30 Gy. This decreasing trend was followed by 2% survival at 40 Gy.

Radiosensitivity test (LD50) for the Curcuma alismatifolia was approximately at

25 Gy. Gamma irradiation affected the survival rate of rhizomes, extension of days

to shoot emergence, plant height, leaves and shoot number as well as

modifications in plant morphology and flower development.

Study of possibility of mutation induction in saffron (Crocus sativus L.) was

carried out by Kamali et al. (2009). Effect of oryzalin on different saffron explants

including corm segments, callus and somatic embryos at globular stage for

mutation induction and increasing ploidy level was studied. Results showed that

different concentrations of oryzalin on corm segments prior to establishment in

culture medium resulted in decreasing the explant viability. Treatment with 12.5

µM oryzalin for 3 days caused soft and undifferentiated callus which was not able

to regenerate. By using 12.5 µM oryzalin for 1 day, deformed shoots were

obtained. Flowcytometry examination did not show any ploidy change. Treatment

with 10 µM oryzalin for 14 days on embryogenic calluses showed mixoploidy

forms. Different concentration of oryzalin on embryogenic calli at globular stage

were less effective and there were slight morphological changes in colour and

shape.

Corms at different stages of growth rate were subjected to different doses of

physical and chemical mutagens by Nehvi et al. (2010b). On the basis of

morphological and anatomical attributes, 11 variants were identified in M2

generation. Among physical mutagens, gamma radiation (0.1 and 0.2 Krad) when

given on 1st

and 15th

June induced more number of stomata ranging from 20-24 per

field. Radiation with 0.5 Krad resulted in narrow leaves exhibiting only one

stomata. Enhanced stomatal number was associated with thicker and broader

leaves. Stomata size increased with 0.2 Krad as compared to 0.1 Krad which was

comparable with control. Among chemical mutagens, similar results were

observed with application of ethyl methane sulphonate (0.1%), ethidium bromide

(0.2%) and colchicine (0.05%) when applied on 15th

September and 1st

June,

43

respectively. Treatments induced more number of stomata (>20). Colchicine

variant recorded narrow leaves (0.7 mm) with maximum leaf thickness (56.95

mm). Similar results for leaf width were also observed for ethyl methane

sulphonate (0.2%) when applied on 1st

September. Morphological variants with

increased number of stomata also recorded maximum saffron recovery on account

of increased number of flowers per corm except for colchicines that was

responsible for increased pistil length. More stomata number associated with

thicker and wider leaves evident from gamma radiation (0.2 Krad), ethidium

bromide (0.2%) and ethyl methane sulphonate (0.1%) were also associated with

increased number of heavier flowers and saffron recovery in terms of fresh pistil

weight. Study confirmed that mutagenesis made the plants photosynthetically

more efficient thus contributing to increased saffron recovery.

Tamina and Tapash (2010) reported that lower doses of mutagens were effective

and efficient in causing polygenic variability in various quantitative characters with

a negative relationship between effectiveness and mutagen dose in sesame. Similar

findings were reported by Roy Chowdhury et al. (2004) in mungbean, Dhanavel et

al. (2008) in cowpea, Ganapathy et al. (2008) in little millet and Thilagavathi and

Mullainathan (2009) in black gram.

Tiwari et al. (2010) reported reduction in survival rate, root length, number of

spike/plant, number of floret/spike, days to flower, shelf life, vase life, floret size,

number of corms/plant and weight of corms/plant with an increase in exposure of

gamma rays in gladiolus. They observed morphological abnormalities in leaf and

plants showing an increase in the number of abnormal leaves with an increase in

the gamma radiation dose.

An attempt was made by Khan et al. (2011) to create new variants for increasing

corm production per planting cycle through induction of mutations using physical

(gamma rays in Kilo-Roentgen) and chemical (ethyl methane sulphonate,

colchicine, ethidium bromide) mutagens at different growth stages of saffron using

fortnight treatments (Ist June, 15th June, 1st July, 15th July, 1st August, 15th August,

1st September, 15th September). Initially, 44 plants were selected on the basis of

44

their higher yield performances. Evaluation of elite mutant lines identified

treatment-D2T6 (15th June treatments of corms with 0.1% EMS) and D8T6 (15th

June treatment of corms with 0.05% colchicine) both producing highest number of

daughter corms (15) per mother corm followed by D2T2 (15th June treatment of

corms with 0.2 Krad) producing 12 daughter corms per mother corm as compared

to control (natural population) producing only 5 daughter corms per mother corm.

15th June was proposed as an ideal time for treatment of saffron corms in order to

induce increased number of daughter corms per mother corm. Further, 0.2 Krad

showed positive effect on number of daughter corms per mother corm.

Study on colchicine induced variability by Gowhar et al. (2012a) revealed that

corms treated with colchicines @ 1% induced morphological and anatomical

variants associated with thicker and broader leaves with maximum chlorophyll

content and stomatal size associated with reduction in stomatal number and

leaves/plant.

Chatterjee et al. (2012) reported that induced mutations often produce

abnormalities which cause morphological alterations in external form of plants

including color, shape, size etc. Study on physical (gamma rays), chemical (EMS

alone + EMS combined with gamma rays) mutagenesis to determine the effective

dose of mutagens to create plant deformities and chlorophyll (chl) mutations in

opium poppy revealed that the frequency of chl mutations were maximum for 50

Krad (0.42%) followed by 40 Krad (0.24%) in NBR1-1 while frequency was

highest for 50 Krad in combination with 0.8% EMS (0.86%) in NBR1-5. Among

the chlorophyll mutations, albino was the most frequently screened followed by

xantha type at all doses.

Variability for flower colour mutants in gladiolus induced by ethyl methane

sulphonate (EMS) for fifteen quantitative traits was studied in M1V2 generation of

gladiolus by Bhajantri and Patil (2013). A significant shift in mean values in the

positive direction was observed for all the 15 quantitative traits studied. Corm

weight and corm diameter recorded high phenotypic and genotypic coefficient of

variation at 0.50% EMS treated population in both Ethyl Cav Cole and White

45

Prosperity genotypes. The highest variability for days to spike initiation in both

the genotypes was recorded at 0.50% EMS. The moderate PCV and GCV values

for the characters like days to sprouting, days to first floret to show colour, days to

full spike emergence, spike length, rachis length, spike girth and number of florets

per spike was observed in all treated populations in both genotypes. Plant height

and spike length had moderate variability at 0.50% EMS treated populations of

White prosperity. Low PCV and GCV was recorded for characters like, leaf width,

inter floret length and floret diameter in all treated populations of Ethyl Cav Cole.

High heritability estimates along with moderate genetic advance (GA) and genetic

advance over mean (GAM) was found for most of the characters viz. number of

leaves per plant, leaf width, days to full spike emergence, days to first floret to

show colour, spike length, rachis length, florets per spike, inter floret length and

floret diameter in all treated mutant populations.

Study on induction of mutation in commercial varieties of gladiolus using physical

mutagen (60Co) by Patil (2014) revealed that percentage of sprouting and survival

was affected significantly at 1 Krad to 4 Krad. LD50 was found to be beyond 7

Krad for both sprouting and survival. 4 Krad and above proved to be detrimental

for various vegetative and floral traits. Plants treated with 6 Krad and 7 Krad did

not produce flower spikes in cv. Nova Lux and Eurovision whereas cv. American

Beauty produced few flower spikes. When corms were treated with higher doses,

plant height, leaf number and leaf size were reduced significantly and leaves

became narrow and leathery. Colour variations in florets and whole spike were

also increased with increase in dose rate along with increase or decrease in number

of floral organs. Radiation treatments at higher doses caused delay in spike

initiation with decrease in spike length, number and size of florets, vase life and

yield per plot while lower doses responded positvely. The desirable mutants with

light colours were isolated from all the three varieties with 5 Krad whereas one

mutant had bifurcated spike at 6 Krad from cv. American Beauty.

Induction of mutations in Browallia speciosa using sodium azide and

identification of genetic variation by El-Mokadem and Mostafa (2014) revealed

46

that 800 ppm sodium azide increased the number of branches and leaf, chlorophyll

content, fresh weight of vegetative growth and roots, dry weight of vegetative

growth and roots and root length in M1 and M2. All the concentration of sodium

azide produced changes in flower colour, flower shape and leaf form in both the

generations.

II.3 Components of variability

Biological characteristics of saffron makes its breeding significantly complicated.

In crop improvement, the selection of plants is made on the basis of their

phenotype and the effectiveness of selection would largely depend on the

proportion of phenotype due to the genotype which is heritable. Classification of

total variability (existing or induced) into its heritable and non-heritable

components such as phenotypic genetic advance is of paramount importance in

understanding the genetic make-up of any breeding material under improvement.

Statistically, the amount of variation is measured and expressed as variance.

Genotypic variance is a pre-requisite for an effective breeding programme.

Genetic coefficient of variation is used to measure the range of genetic variability

present in a particular character. For effective utilization of germplasm resources,

it is important to understand the amount of genetic diversity present in such

germplasm resources. Also, it is important to assess the relative magnitude of

components of variability in order to use such information together with other

selection parameters for improvement of the plant type through adoption of

effective breeding methods (Johnson et al., 1955; Hanson et al., 1956; Williams,

1964; Briggs and Knowels, 1967). It is necessary to divide the total phenotypic

variance of the entire characters into its components as these are the basis for

genetic analysis and the dimensions of these components dictate the breeding

behavior of the populations. Such selection parameters, particularly, genetic

variability helps to choose a potential genotype whereas heritability (h2) along

with genetic advance as percentage of mean (GA%) are more useful in predicting

the resultant effect from selection of best genotypes. The knowledge on the extent

of variation and diversity in the yield and quality components of germplasm

47

resources and identification of a good number of genotypes as potential donors in

yield and quality improvement programme is essential.

For most efficient mobilization of available germplasm resources, it is vital to

have better understanding of nature and magnitude of genetic variability.

Divergence studies are of paramount importance in understanding the extent of

variability and possibilities of its future utilization in subsequent crop

improvement programmes because corm multiplication in saffron does not induce

genome variation with the exception of some natural mutation that in triploid

saffron population are not easily detectable. Therefore, one of the important

considerations in the formulation of efficient selection programme is the

knowledge regarding the relative contribution of genes in the expression of a

particular trait. Genetic advance, i.e. the improvement in genotypic value of the

new population as compared to the base population depends among other things

on the magnitude of differences among genotypic values of individuals in the base

population and non-heritable agencies. Success in changing the characteristics of

population therefore depends upon the correspondence between phenotypic and

genotypic values.

Quantitative measures which provides information about choice between

genotypic variance and phenotypic variance is called heritability. The concept was

originally presented by Lush (1945) to describe the ratio between genotypic and

phenotypic variance and is now known as broad sense heritability. However, mean

genotypic value of the progeny is determined by the average effects of genes

transmitted by the parents in question. In other words, it is breeding value of the

parents which determines the genetic properties of the progeny. Hence it is the

proportion of phenotypic variance that is made up of variation attributable to the

breeding values (known as additive genetic variance) which is of considerable

practical interest to the breeder.

New variants of saffron with increased number of stigmas maintaining 2n=24 have

been reported by Estalai (1978) with a frequency 1.2 x 10-6 of a rare type flowers.

Morphological differences with flowers having higher number of stigma branches

48

and stamens have been described by Piccioli (1932) from saffron cultivation at L.

Aquali (Italy). In addition different commercial products are known suggesting the

existence of different saffron ecotypes (Tammaro, 1987) or commercial varieties

(Di Crecchio, 1960).

Munshi (1992) from his studies on saffron reported wide range of variability.

Information on coefficients of variation, heritability and genetic advance was

derived from data on 10 yield components in 11 diverse saffron genotypes (mostly

from Jammu and Kashmir with some exotic varieties) grown during kharif 1986-

89. Significant genotypic differences were observed for all characters except for

days to 100% flowering. The coefficients of variation was highest for number of

daughter corms/mother corm and number of flowers per spathe. High values of

heritability and genetic advance were observed for number of daughter

corms/mother corm and yield/plant.

Lattoo (1997) reported appreciable differences in the coefficient of variation for all

floral characters at different locations. Observations on floral characters (fresh

flower weight and size, stigma length, stigma fresh weight, dry weight and saffron

percentage) were recorded from 200 flowers collected from 4 locations in Kashmir

Valley (Chrar-Sharief, Sanatnagar, Pampore and Malabagh) during October-

November 1988. Due to clonal propagation in saffron, the population is expected

to be genetically similar. However, the variability observed appeared to be due to

varied microclimates at different sites.

Survey conducted in Pampore kerawa by Gohil (1999) revealed some interesting

and promising variants. The promising variants were recommended to be

multiplied through clonal selection. Latto and Dhar (1999) studied temporal

saffron populations of Kashmir for six floral characters of saffron viz. number of

flowers per spathe, fresh flower weight, flower size (perianth area), stigma length,

fresh stigma weight and dry stigma weight. Appreciable differences in coefficient

of variation for all the characters were observed. Maximum coefficient of variation

was recorded for flowers per spathe (59.15) whereas minimum coefficient of

variations (0.42) was recorded for stigma length. For fresh flower weight, flower

49

size, fresh stigma weight and dry stigma weight, coefficient of variation ranged

from 12.16-127.46, 13.32-31.22, 17.36-51.03 and 23.27-36.17 at four different

locations respectively. Studies on status of saffron cultivation in Kashmir and

strategies for improving productivity through crop improvement by Zargar (1999)

revealed that genetic variability existing in the natural population of saffron in

Kashmir has been an outcome of spontaneous mutations that have occurred over

centuries and need to be collected, evaluated and propagated.

In order to verify the possible phenotypic and genotypic variation in saffron,

corms from different countries (Italy, Israel, Spain and Holland) have been

examined (Grilli Caiola et al., 2001). Analysis of phenotypic variation revealed

differences in flower size, tepal shape and colour intensity with lobbed tepal in

plant from Israel and more intense colour of tepal in plant from Sardinia (Italy).

Cytofluorimetric analysis on nuclear DNA was carried to detect genome size and

base pair composition and results revealed no differences in DNA content and

composition in saffron corms from different countries (Brandizzi and Grilli Caiola,

1998).

Zargar (2001) from the studies on genetic variation in saffron and importance of

seed corm reported that significant variability exhibited for floral traits in eleven

populations of saffron (including 3 exotic genotypes from Spain, Iran and

Netherlands). Co-heritability estimates indicated that perianth size, number of

flowers per perianth and saffron recovery per flower had high co-heritability and

would help as a selection index for increasing the saffron yield. Mean fresh and

dry weight of 100 flowers in the temporal sub-population of district Budgam and

Srinagar (new plantation) was 29.03 and 4.39 g, respectively, yielding

approximate 8.01 dg of lacha grade saffron. The range and phenotypic coefficient

of variation was high. The analysis of sub-population of main saffron growing area

(district Pulwama) revealed that the fresh and dry weight of 100 flowers was only

23.28 and 3.29 g, respectively, which represented about 19.0 and 9.0 percent

decrease, respectively as compared to mean of the sub-populations of district

Budgam. The recovery of lacha grade saffron was 7.34 dg/100 flowers, which was

50

about 8.36% less as compared to the mean recovery from sub-populations of

district Budgam. The possible reason was attributed to the fact that the crop fields

in district Pulwama were exhausted as compared to new plantations in district

Budgam. The magnitude of phenotypic variability and its coefficients of

variability were comparable. On 100 g flower basis (sub-population of district

Pulwama), the mongra and lacha grade saffron recovery was 18.04 and 25.26 dg,

respectively. Thus 1dg fresh flower produced approximately 18 and 25 g of dried

mongra and lacha saffron grade, respectively.

Nehvi (2003) and Nehvi et al. (2004 and 2006) reported wide spectrum of

variability for floral and corm attributes in temporal subpopulation of Kashmir and

the results implied a great scope for saffron improvement. Perianth size, number of

flowers per spathe and saffron recovery per flower were identified as selection

index criteria for increasing saffron yield. Temporal subpopulation of district

Pulwama exhibited highest range of variability. Flowers completely devoid of

style and anthers, freaks with 4 and 5 stigmas were observed from natural

population. The presence of more number of stigmas per flower (4-6) were due to

physiological and or developmental irregularities. However, the detection of

flowers with increased number of stigmas in natural population is a universal

phenomenon. Magnitude of variability revealed that the mean fresh and dry weight

of 100 flowers in temporal sub-population of Jammu and Kashmir was 23.46 and

3.82 g, respectively, yielding 0.75 g of lacha grade saffron. Stigma length ranged

from 2.41-3.87 cm with a mean value of 2.5 cm. Number of flowers per spathe

ranged from 0.65-5.58 with fresh flower weight ranging from 172-355 mg.

Economic product on fresh basis ranged from 14.37-68.42 mg whereas on dry

weight basis, it ranged from 6.0-14.60 mg. Greater magnitude of phenotypic

variance than corresponding genotypic variance was observed with low values of

broad sense heritability for all the traits except for fresh flower weight, fresh

perianth weight and stigma length. Genetic gain as percent of mean was high for

number of flowers per spathe, fresh flower weight, fresh pistil weight and crocin

content, whereas, it was medium for dry pistil weight, stigma length and number

51

of daughter corms and low for other studied traits.

Makhdoomi (2006) studied components of phenotypic variability after taking

genotype x environment interaction into account and indicated that a wide range

of variability existed for number of flowers per corm (0.21-5.59), fresh flower

weight per corm (154.7-529.5 mg), fresh perianth weight per corm (100.00-468.3

mg), fresh pistil weight per corm (11.63-40.35 mg), fresh stigma weight per corm

(5.9-33.98 mg), fresh style weight per corm (1.85-7.80 mg), fresh stamen weight

per corm (11.28-47.9 mg), stigma length (1.7-3.8 cm), style length (1.23-1.3 cm),

pistil length (3.48-7.68 cm), dry flower weight per corm (33.88-78.25 mg), dry

perianth weight per corm (33.58-52.00 mg), dry pistil weight per corm (4.15-15.30

mg), dry stigma weight per corm (2.5-13.5 mg), dry stamen weight per corm (4.73-

16.1 mg), number of daughter cormels per mothercorm (7.5-50.2), average weight

of daughter cormels per mother corm (18.8- 41.4 mg), leaf length (255.5-1487.3

cm), number of radical leaves per corm (2.38-10.81) and dry leaf weight per corm

(1.55-12.54 mg). Similar results of wide range of phenotypic variability were also

reported in gladiolus by Lal et al. (1985) and Khanna and Arora (1986). High

value of heritability accompanied with high genotypic and phenotypic variation

were reported for weight of cormels per corm, total number of florets per spike,

spike length and spike weight, whereas, plant height, number of leaves and days to

flower exhibited heritability with low genetic advance. High heritability and high

expected genetic gain was observed for number of cormels. Similar results in

gladiolus for number of flowers, spike length, spike weight, weight of daughter

corms and number of daughter corms were reported by Misra and Saini (1988),

Soorianathasundaram and Nambisan (1991), Mahanta and Paswan (1993),

Ashwath and Parthasarthy (1994), Prasad et al. (1994), Sarangi et al. (1994) and

Sheikh et al. (1995) whereas Gowda (1989) and Anuradha and Gowda (1994)

reported low heritability with low value of genetic gain for plant height, days to

flowering and spike length. Sharief-ud-Din et al. (2000) reported medium value

for corm weight and corm size.

52

Studies on productivity, growth and qualitative attributes of 10 Iranian saffron

accessions under climatic conditions of Charra Mahal Bakhtiari, Central Iran by

Parviz et al. (2004) revealed superior performance of 3 accessions including

Shahr-Kord, Birjand and Ghaen with productivity level of 3.26, 2.67 and 2.66/ha

respectively in terms of dry stigma yield. Data collected for different traits in the

first year, in general and for stigma yield in particular were highly variable.

Treatment effects were statistically significant for stigma yield, total dry matter,

corm number and weight. For most of traits, majority of variation due to the

treatment effect arose from the differences of the Birj and Ghaen and Shahr-Kord

with the rest of accessions.

Agayev (2006) reported that metaphase chromosomes of saffron can be arranged

according to their size and morphological features in 8 triplets ranging in size from

11.58 ± 0.13µm (triplet 1) to 4.57 ± 0.13µm (triplet 8). Triplet 1 and 2 consist of

subacrocentric, 3, 4 and 8 metacentric and 6 and 7 submetacentric chromosomes.

Three chromosomes in the specified triplet, as a rule are similar although in some

triplets one of them is slightly distinguishable from the other two. Triplet 5 shows

an extreme difference so that it always contain 2 kinds of chromosomes:

chromosome 5(1) and chromosome 5(2,3). Chromosome 5(1) is metacentric

(r=1.49) and 6.04±0.13µm in length but chromosome 5(2,3) are subacrocentric

(r=3.49) and noticeably smaller (5.41±0.09µm). Application of C-banding

technique revealed heterochromatin segments: the sharpest, sharp and weak. New

specific and sharpest heterochromatin segment using acto-iron-hematoxylin stain

was revealed on satellite chromosomes (triplet 2) on the proximal part of the long

arm. It was assumed that the species C. sativus is obviously a clone of one triploid

plant originated spontaneously in nature through crossing between 2 closely

related species with participation of n and 2n gametes.

Genetic variability studies by Verma et al. (2006a) revealed that mean squares

were highly significant for all the characters viz. flower weight, fresh pistil weight,

dry flower weight, dry pistil weight, parienth length, parienth breadth, stigma

length, style length and total length of stigma and style indicating enough

53

variability for all the traits. Fresh flower weight ranged from 0.60-0.85 g and total

length of stigma and style varied from 39.09 to 97.10 mm which showed a good

range of variation between the genotypes. Style length exhibited highest values of

genotypic coefficient of variation (18.56) and phenotypic coefficient of variation

(24.77). Stigma length and total length of style + stigma showed highest estimate

of broad sense heritability. High estimate of heritability (broad sense), genotypic

coefficient of variation and genetic advance was observed for total length of style

+ stigma which showed possibility of improvement through selection.

A study on exploration of Kashmir (Budgam & Srinagar) valley for the selection

of superior genotypes of saffron by Verma et al. (2006b) revealed a high degree of

variability in the plant age (1 to 30 years), number of cormels/mother corm (1.22-

11.8), average weight of cormels (1.65-10.16 g), average length of cormels (1.40-

1.92 cm), corm girth (1.27-2.66 cm) and corm size (1.426-5.107 cm). Floral

characters of 141 samples also revealed marked variability in fresh and dry weight

of flower, fresh and dry weight of pistil, parienth length and breadth and length of

stigma and style.

Agayev et al. (2007) showed that within each weight group of corms, number of

flowers were differing in large extent in different clusters (holes). Roughly

35-50% of clusters had no flowers at all. 20-25% had one flower, 13-15% had two,

5-10% had three, 2-7% had four, 1-2% had five flowers, etc. Unique clusters had

8-10 and even up to 12-13 flowers. Results revealed heterogeneity of experimental

populations.

Comparison of saffron clones, each grown from one corm of the same weight,

resulted in the identification of ‘‘superior’’ clones in terms of exceptionally large

numbers of flowers and large (≥ 10g) corms (Agayev et al., 2009). Based on the

number of flowers and number of large corms-the two most economically

important attributes of saffron, the clones were classified as extraordinary,

superior, ordinary, inferior and declining clones. The first two classifications of

clones, which had the highest numbers of flowers and largest corms have been

chosen for use in a saffron breeding program aimed at developing new high

54

yielding cultivars of saffron. Those clones have been found to be suitable for

facilitating the mechanization of saffron agriculture in terms of the lifting, sorting,

corm plantation, weeding and flower harvesting. Clone 27F-4 had the highest

number of flowers (27) with an initial corm weight of 8 g. It was characterized by

the highest parameters of corm set: total weight of corms increased upto 304.6 g;

‘‘Big corm index’’ was 92.3%, number of large corms was very high (16); average

weight of one big corm was desirable (17.6 g); ‘‘Flower creating index’’ was

satisfactory (11.3 g) and ‘‘Multiplication index’’ was high (38.19). On the whole,

the clone unified high positive qualities.

A preliminary characterization of saffron germplasm from the CROCUS BANK

collection was done by De-Los-Mozos-Pascual et al. (2009) for phenology (date

of sprouting, flowering, duration of flowering), floral morphology (tepals length

and width and length of stamen filament) and saffron production (number of

flowering corms, number of flowers/corm, saffron spice weight/flower). Stamen

filament length was the only measured parameter identical in all the accessions.

Concerning other parameters, there were significant differences with proportion of

the total variance due to differences among accessions ranging from 53% for the

duration of the flowering period to 24% for the number of flowers per corm. For

some characters like precosity, a part of the observed variation was due to small

differences in the initial weight of the mother corms. The variation in other

characters like dry saffron weight per flower or the duration of the flowering

period were independent of this factor and was of interest for selection and

breeding.

Study on genetic difference among wild Greek Crocus taxa and cultivated forms

(Crocus sativus L.) currently being maintained in ex situ conservation at the

Balkan Botanic Garden of Kroussia (Greece) by Tsoktouridis et al. (2009)

reported significant variation for the wild accessions, identifying several

transitions, transversions and indels whereas no nucleotide difference was

observed for 10 accessions of C. sativus L.

55

Khan et al. (2010) reported that utilization of heterogenity in the natural

population which is due to genetic and environmental factors offers a tremendous

scope for saffron improvement. Germplasm conservation and evaluation is

underway for identification of elite clones with distinct yield superiority.

Evaluation of 24 genotypes with distinct superiority over natural population for

yield stability recorded superiority of SMD-87, SMD-61, SMD-9, SMD-102,

SMD-98 and SMD-170 with average saffron yield ranging from10.41 to 12.00

kg/ha.

Study on genetic variability in saffron (Crocus sativus L.) was undertaken by

Makhdoomi et al. (2010) to generate information on the nature and magnitude of

components of phenotypic variability including heritability, genetic gain, nature of

interrelationship among components of economic worth, contribution of different

morphological and yield component traits in 240 saffron populations collected

from natural saffron growing areas of Kashmir. Significant variations among

populations were observed for all the traits indicating presence of high level of

variability. G x E interaction was also observed to be significant for all the traits

indicating differential behaviour of populations over years. Wide range of

variability was observed for all the traits implying considerable scope for saffron

improvement through clonal selection. Range of variability was observed to be

high in second year estimates and phenotypic variances were observed to be

higher than the corresponding estimates of genotypic variances. Estimates of

phenotypic and genotypic coefficients of variation demonstrated similar trend. For

most of the traits, genotypic coefficient of variation ranged from 8.76 to 32.24,

whereas, style length, pistil length, dry flower weight and dry perianth weight

recorded medium to high values of genotypic coefficient of variation (GCV).

Heritability in broad sense was observed to be high for all traits in both the years.

However, on the basis of pooled analysis over years, it was medium for all the

traits except fresh flower weight, fresh perianth weight, fresh pistil weight, fresh

stigma weight, stigma length and average weight of daughter corms revealing a

strong influence of environment on the performance of populations. Expected

56

genetic gain (% of mean) ranged from 5.15-40.68 and was observed to be high for

all the traits except plant height, pistil length, dry perianth weight and style length.

Study on stability analysis in saffron (Crocus sativus L.) by Nehvi et al. (2010d)

revealed that significant G x E interaction was observed for all the traits except

days to 50% sprouting and 50% flowering thereby revealing that genotypes

perform differently for traits under study at different locations. Highly significant

mean squares for environments except for flowering traits indicated that

environment selected were random and were different in agro climatic conditions.

Genotypes observed average stability for all the characters except for number of

radical leaves and corm yield per plot.

Agayev et al. (2012) gave new specific scientific concepts of clonal selection of

saffron (Crocus sativus L.) viz. initial corm weight, corm set formulae, big corm

index, ultra-extraordinary clone, extraordinary clone, superior clone, selective

(elite) clone, low grade clones, ordinary clone, inferior clone, declining clone,

flower creating index and multiplication index.

The study of clonal selection of saffron was undertaken with an aim of searching,

identification and separation of genetically superior clones of saffron from existing

population with higher number of flowers and higher quantity of large sized corms

(Gowhar et al., 2012b). Samples of saffron population from most ancient

cultivating regions of Kashmir were collected from 500 sites for the study with >

2000 corms comprising the sampled population from each region. The corms were

divided into two weight groups viz. < 8 gm and > 8 gm and then planted into pits

separately with pit to pit distance of 50 cm. Superior clones in terms of production

of large number of daughter corms both in terms of weight and number associated

with maximum number of radical leaves with increased plant height were

identified.

Nehvi et al. (2012) carried out genetic resource management to explore and utilize

available genetic variability present in natural sub-populations of Jammu and

Kashmir. About 2500 saffron accessions collected from saffron areas of Jammu

and Kashmir during 2002-2011 were subjected to variability and divergence

57

studies at genetic and molecular level. Selected lines from natural sub-population

exhibited a wide range of variability for economic, morphological and corm

attributing traits indicating considerable scope for saffron improvement through

clonal selection.

Morphological characterization of saffron clones by Sheikh et al. (2012) revealed

significant genotypic differences for number of flowers per corm, fresh pistil

weight per corm, pistil length, stigma length, number of daughter corms, average

weight of daughter corms, plant height, number of radical leaves per plant,

stomatal size, stomatal frequency and chlorophyll content. Components of

variability indicated a wide range of variability for the traits under study.

Estimates of phenotypic variance were higher than corresponding genotypic

variance thereby indicating influence of environment in the expressions of these

traits. High values of heritability were recorded for all the traits. Estimates of GCV

were higher in magnitude though similar in direction for all the traits.

II.4 Genetic divergence

Geographic diversity among parents as an index of genetic diversity has been

acclaimed and disclaimed in numerous published reports. Murty and Arunachalam

(1965) hypothesized the Mahalanobis (1928) generalized distance a measure of

metric distance between population centroids that can be a very useful multivariate

statistical tool for effective discrimination among parents on the basis of genetic

diversity. Precise information about genetic divergence is critical for productive

breeding programme as genetically diverse parents are known to produce high

heterotic effects consequently increasing yield in desirable segregants. High

yielding parents with greater genetic diversity are required to develop productive

hybrids. For identifying genetically diverse parents for hybridization, multivariate

analysis (Mahalanobis D2 statistics, 1936) has been used in almost all crop species.

D2 statistics gives a result based on the magnitude of divergence dependent on the

sample size. Genetic diversity in biological populations has been found to occur

due to several causes. Human selection has led to quite a big array of varieties

grown for the same end product and thus effected their diversity, whereas, stress

58

conditions, natural selection and genetic drift maintained divergence (Ram and

Panwar, 1970; Das and Borthakur, 1973). Studies in a number of crop species with

different breeding systems by means of D2 statistics suggested that genetic

diversity need not be directly related to geographical diversity (Murty and

Arunachalam, 1965, 1966). Experimental evidences in Drosophila (Brunica, 1954;

Wallse, 1955) have demonstrated that crosses of strains of diverse origin exhibited

greater heterotic response than crosses of strains of the same origin.

Nature and magnitude of genetic divergence was assessed in different genotypes

and species of gladiolus using D2 statistics by several workers (Avishai and

Zohary 1980; De and Misra 1993; Arya et al., 1999; Desh and Misra 1999;

Nimbalkar et al., 2002). These workers have grouped the genotypes into different

clusters. D2 statistics revealed that clustering behavior and mean performance of

genotypes of individual clusters were not consistent over the environments

because of large genotype x environment interactions. Number and weight of

corms and cormels plant-1, number of florets plant-1, and plant height contributed

considerably to divergence.

Brandizzi and Grilli Caiola (1998) while studying DNA content of saffron corm

received from Italy, Israel, Spain and Holland reported no difference in DNA

content and composition of saffron corms. Zanier (2000) while analyzing nuclear

DNA by RAPD technique revealed that saffron corms from different cultivation

areas did not identify genomic redundant differences. There were no differences in

DNA present in 10 corms from saffron in L-Aquila. The RAPD data were in

accordance with the DNA content and base pair composition among different

Crocus sativus cultivars as revealed by the previous cytofluorimetric studies.

Pardo et al. (2004) investigated the distinctive variability of Crocus sativus from

several geographical areas (Italy, Israel, Greece and Spain) using 38 random

amplified polymorphic DNA (RAPD) markers. Low percentage of primers were

suitable for differentiating four saffron samples from different origin. However

other studies with triploid species with apomictic reproduction offered a large

genetic variation in the genetic structure of populations.

59

Grilli Caiola et al. (2004) carried out Random Amplified Polymorphic DNA

(RAPD) analysis on DNAs from 5 Crocus sativus (saffron) accessions cultivated

in different countries (Italy, Israel, Spain and Holland) and on 6 closely related

Crocus species (C. asumaniae, C. cartwrightianus, C. hadriaticus, C.

oreocreticus, C. pallasii and C. thomasii) to determine whether cultivated saffron

has maintained a constant genomic organization and to clarify its relationship with

possible ancestor species. For the 15 primers, which produced positive results, the

DNA of saffron corms from different accessions presented the same amplification

pattern in accordance with the similar DNA content and base composition pointed

out in previous studies. The amplification of the DNA of the 7 Crocus species

with 21 primers provided 217 repeatable and interpretable fragments which were

scored for presence/absence of band and employed for cluster analysis. The results

indicated that C. sativus is very closely related to C. cartwrightianus and is similar

to C. thomasii. This result concurring with part of the previous evidence rules out

the hypothesis of close relationships between C. sativus and C. pallasii. Genetic

relationships among populations of Crocus hyemalis Boiss. and Blanche collected

from different regions of Jordan were studied using Random Amplified

Polymorphic DNA analysis. C. hyemalis was compared to the cultivated species

C. sativus and C. vernus. RAPD and cluster analysis indicated high degree of inter

and intra-population variation within the wild C. hyemalis population. High

genetic association was found among some wild populations originating from the

same collection sites.

DNA molecular comparison using RAPD markers on six species (C. pallasii

subsp. Hausknechtii, C. cancellatus, C. speciosus, C. caspius, C. michelsonii, C.

sativus) by Tarazi and Rashid Mohassel (2006) revealed that the origin of

cultivated saffron in Iran is Western Iran possibly Zagros area.

Taghizadeh (2006) conducted a study on molecular genetic assessments in Crocus

sativus L. and reported that DNA polymorphism based AFLP method confirmed

close relationship between species of Crocus sativus L. from several geographical

areas using random amplified polymorphic DNA (RAPD) markers.

60

Soheilivand et al. (2007) while studying diversity in flowering rate of two saffron

(Crocus sativus) populations of Iran revealed that in cluster analysis diagram,

populations planted with different weight of corms, based on 5 traits related to

flowering rate, constituted 3 groups at a distance of 10 units. Gonabad population

with 3, 4, 5, 6 and 7 g corm weight were placed in group I. The result showed that

cluster analysis could distinguish plants with lower maternal corm weight of one

specific population from the other specific population. The results also showed

that the maternal corms with 3 to 7 g had non-significant effect on flowering rate.

Gonabad populations with 8 and 9 g corm weight and Ghaen population with 3, 4,

5 and 6 g corm weight were placed in group II thereby showing different

flowering rates between corm derived from Gonabad and Ghaen. Two populations

of 10 g weight of Gonabad and 7 g weight of Ghaen were placed in cluster III. The

results showed that cluster analysis could completely distinguish the corms of

different weight. It also depicted the difference in the number of flowers produced

by heavy and light corms and emphasized the fact that the heavier maternal corms

compared to the lighter corms produce more flowers. The results were in

consistent with results of other investigators (Pandey et al., 1979; De-mastro et al.,

1993; Omidbeigi et al., 2003).

Studies in relation to molecular variability in saffron by Imran et al. (2007)

revealed considerable amount of genetic diversity among tested genotypes.

Similarity index based on Jaccards coefficient ranged from 0.375 to 0.834 with

maximum similarity coefficient (0.834) observed between the genotype SMD-45

and SMD-79. The dendrogram based on molecular data divided the tested

genotypes in two clusters. However, genotypes viz. SMD-3, SMD-45, SMD-79

and SMD-68 formed cluster I and genotypes viz. SMD-11, SMD-52, SMD-81,

SMD-211, SMD-224 and control formed cluster II at similarity coefficient of 44%

which showed a high level of genetic diversity between two clusters containing

different genotypes. A different level of variability was observed among different

genotypes within each cluster.

61

Fluch et al. (2009) reported that out of the 6803 available saffron ESTs, 200

sequences proved to contain SSR region. Out of these, for 38 sequences, primers

could be developed and tested on 29 different saffron accessions. In addition to the

nuclear SSRs, 8cp regions were investigated. None of the region revealed any

genetic difference among the investigated probes. Sequencing of 5 genes was

conducted in order to identify single base pair mutations in the respective genomic

areas.

Henna et al. (2009) studied genetic divergence in saffron under temperate

conditions of Kashmir. Cluster analysis revealed sixteen clusters in pooled

analysis, three in year 1 and two in year 2. Highest intercluster distance was

observed between cluster XV and cluster XVI followed by cluster V and XVI

respectively. The characters viz. fresh stamen weight, plant height, fresh flower

weight, pistil length and fresh pistil weight contributed maximum towards

divergence.

Genetic divergence in saffron studied by Maqhdoomi et al. (2009) grouped all the

genotypes in eleven clusters in pooled analysis with majority of genotypes (229) in

cluster I. All other clusters were monogenotypic except cluster II. Percent

contribution of different floral, morphological, corm and quality attributes towards

divergence in saffron germplasm lines revealed strong influence of fresh pistil

weight, stigma length, and crocin content. Hence, these characters qualify to be a

selection criteria for identification of divergent lines.

Nemati et al. (2009) studied genetic diversity of 51 accessions of Iranian saffron.

56 novel polymorphic microsatellite loci were isolated and characterized.

Preliminary analyses revealed that the number of polymorphic alleles ranged from

3 to 6 and heterozygosity was determined from 0.17 to 0.39. Genetic variation was

observed among studied accessions and the isolated microsatellites markers can

provide an efficient tool for diversity assessment in saffron.

Fougat et al. (2012) carried investigation to assess genetic diversity among

commercially available brands of saffron through DNA based SSR markers. Study

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points to conclusion that SSR markers could be successfully utilized to assess

genetic diversity and to check clonal fidelity of various saffron cultivars.

Study of Gaikwad et al. (2012) on molecular diversity analysis in saffron (Crocus

sativus) using AFLP markers demonstrates the efficacy of AFLP markers in

identification of polymorphism. Genetic variability among 13 different clonal

accessions of saffron collected from different areas of Jammu and Kashmir was

determined by employing nine AFLP primer pairs. A total of 154 amplicons were

generated, 98.7% of which were polymorphic. No two accessions analysed in the

study were found to be identical using AFLP markers. The average heterozygosity

revealed was 0.33% and values of polymorphism information content (PIC) varied

from 0.09 to 0.43.

Izadpanah et al. (2012) studied variations in saffron (Crocus sativus L.) accessions

and wild species by RAPD analysis. Five accessions of cultivated saffron from

five areas in Khorasan and Esfahan, namely, Gon-Abad, Ferdows, Ghaen,

Estahbanat and Golpaygan were used. Out of nine species of saffron naturally

growing in Iran, two wild species (C. caspius & C. speciosus) from the north of

Iran (Gilan province) were selected for study. RAPD markers were used to classify

these species and to find the relationship between them. In the results of the study,

cluster analysis showed 2 main groups. Also, maximum similarity value was seen

between wild (C. caspius & C. speciosus) genotypes (0.82) and minimum was

between Estahbanat, Ferdows accessions and wild (C. speciosus) genotype (0.33).

Abedi et al. (2012) conducted a study to assess and classify 65 different saffron

accessions for morphological attributes at reproductive and vegetative stages. The

accessions were collected from various climatic areas of Khorasan, Razavi and

South Khorasan provinces. UPGMA cluster analysis using morphological traits at

vegetative stage grouped the accessions into four distinct groups. The third and

fourth clusters contained the accessions with high mean for leaf number and leaf

length. Grouping the accessions using morphological traits at reproductive stages

produced different classification.

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Laura et al. 2013 demonstrated that saffron corms of different origins grown in the

same experimental field produced daughter corms with different dimensions and

produce stigma samples with different pigment profiles. Furthermore, daughter

corm dimensions and pigment profile even more so could be related to the origin

of the sample and therefore pigments can be used as chemotaxonomic markers.