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GM Crops and Food: Biotechnology in Agriculture and the Food Chain 3:4, 289-295; October/November/December 2012; © 2012 Landes Bioscience

ReseARCh pApeR ReseARCh pApeR

*Correspondence to: Amjad Husaini; Email: amjadhusaini@yahoo.comSubmitted: 04/21/12; Revised: 06/29/12; Accepted: 07/04/12http://dx.doi.org/10.4161/gmcr.21365

Introduction

Saffron (Crocus sativus L.), a bulbous perennial plant, is a sterile geophyte with underground fleshy corms (Fig. 1a). It is an impor-tant spice crop grown in Kashmir in the Indian sub continent.1 One of the key centers of production, the plateaus of Pampore in the Indian Kashmir, has been cultivating saffron since 750 AD. Stigma are typically hand-picked from purple flowers in autumn, and so its production is typically favored in countries where labor is cheap such as Iran and Azerbaijan, but production also takes place in countries such as Greece, Spain, Argentina or the USA with global production exceeding 200 tons and newer areas being brought under its cultivation like new booming centers of China and Japan. Typically only four to five cormlets per mother corm per season are produced in saffron fields through natural breed-ing. Such low multiplication rates of cormlets compounded with problems of fungal infestation in fields restrain the availability of adequate planting material and hence drastically reduce produc-tivity of saffron.2 A corm survives for only one season, reproduc-ing via division into cormlets that eventually give rise to new

A complete protocol for the saffron cormlet production under in vitro conditions and subsequent flowering under greenhouse conditions is described. highest number of cormlets (70.0 ± 0.30) per corm slice (explant) could be regenerated on Murashige and skoog (Ms) half strength medium supplemented with thidiazuron (TDZ) (20 μM), Indole acetic acid (IAA) (10 μM), and sucrose (40 g/l). Maximum germination (90%) of these cormlets could be achieved on Ms medium containing 6-benzyl amino purine (BAp) (20 μM) and α-naphthalene acetic acid (NAA) (15 μM). In order to increase the size of the in vitro raised cormlets, these were cultured on Ms medium containing TDZ (15 μM) and IAA in the range of 1.5–30 μM. Maximum increase in cormlet size could be attained on TDZ (15 μM) + IAA (12.5 μM) + sucrose (30 g/l), and the average size of cormlets was 2.5g. In another experiment, apical vegetative buds of actively growing corms were cultured for cormlet development, and corms of size 2.5g could be developed on Ms medium with NAA (15 μM), BAp (20 μM), and sucrose (30 g/l). The in vitro developed cormlets were dried under shade at 25 ± 2°C for 7 d. These were then planted in small cups containing clay loam soil and kept in green house at 20 ± 2°C. In vitro developed cormlets with mean weight 2.5 g showed maximum flowering (25%) as well as vegetative growth (55%), while only 19% cormlets of 2.0 g flowered. To our knowledge this is the first report on successful flowering from in vitro raised cormlets under greenhouse.

In vitro cormlet production of saffron (Crocus sativus L. Kashmirianus) and their

flowering response under greenhouseJavid A. parray,1 Azra N. Kamili,1 Rehana hamid2 and Amjad M. husaini3,*

1Department of environmental science; Centre of Research for Development; University of Kashmir; srinagar, India; 2Department of Botany; Jamia hamdard; New Delhi, India; 3Mountain Research Centre for Field Crops; sher-e-Kashmir University of Agricultural sciences and Technology of Kashmir; Khudwani, Anantnag, India

Keywords: Crocus sativus, cormlet, micropropagation, flowering, greenhouse

abbreviations: BAP, 6-benzyl amino purine; CRD, completely randomized block design; IAA, Indole acetic acid; MS, Murashige and Skoog; NAA, α-naphthalene acetic acid; TDZ, thidiazuron

plants, and therefore corms are indispensable for saffron propaga-tion3 (Fig. 1B).

Stigmas of saffron flower constitute the commercial product “saffron” (Fig. 1c), which are used all over the world to treat dis-eases like respiratory infections (coughs and common colds), scarlet fever, smallpox, cancer, hypoxia, asthma, blood disorders, insom-nia, paralysis, heart diseases, flatulence, stomach upsets and disor-ders, gout, chronic uterine hemorrhage, dysmorrhea, amenorrhea, baby colic and eye disorders. Saffron is an aphrodisiac, a digestive stimulant and a tonic for dysentery and measles.4 Sterility in saffron limits the application of conventional breeding approaches for its genetic improvement while plant tissue culture offers a great poten-tial for its sustainable production. Work in the area of C. sativus tissue culture started in the early 1980s among which in vitro mass production of pathogen free cormlets offers a tremendous scope.5,6 Some recent reports on in vitro cormlet production,7-9 plantlet regeneration10 and somatic embryogenesis11 are indicative of initia-tives in this direction. However, these protocols for in vitro corm production shall be useful only when flowering could be induced from such corms. To make the technique commercially viable and

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cormlet production. The cormlets could be induced from corm slices on all treatments through the mode of non embryogenic callus. The highest number of cormlets (70 ± 0.33) were obtained on MS (1/2) medium supplemented with TDZ (20 μM) + IAA (10 μM) + sucrose (40 g/l) after 8 weeks (table 1 and Fig. 2a). TDZ was found to be effective for induction and production of cormlets.

TDZ, a substituted phenyl urea is responsible for biosynthe-sis of cytokinins and preserves endogenous hormones in plant tissues.12-14 It has been successfully used for multiplication of corms.10,11,15 Also increasing the concentration of sucrose to 4% was found effective in corm multiplication. In the recent past, Sharma et al.16 reported corm production at high concentration of sucrose (8%) and BAP (6 mg/dm3). High concentration of sucrose, a carbon source, plays a vital role for cormlet produc-tion in saffron16 because it results in increase in the osmotic pres-sure, inhibiting the vacuolation and shrinkage of cytoplasm and thereby increasing the amount of biomass accumulation.17

Germination of in vitro raised cormlets. The in vitro raised cormlets sub-cultured on MS (1/2) strength medium supple-mented with different phytohormones (BAP/NAA, 2, 4-D/Kn and TDZ/IAA) showed variable response to germination. Maximum germination of cormlets (90%) was observed on BAP (20 μM) and NAA (15 μM) (table 2 and Fig. 2B). The best medium when activated charcoal was used showed germination of 80% on MS + BAP (5 μM) + NAA (3 μM) + 0.5 mg/l acti-vated charcoal (table 2 and Fig. 2c). Some other concentra-tions and combinations that proved promising for germination of cormlets were MS + TDZ (20 μM) + IAA (10 μM) (75% germination) (table 2 and Fig. 2d) and MS + 2,4-D (20 μM) + Kn (15 μM) (70%) (table 2). NAA and BAP are a suitable com-bination for supporting organogenesis in C. sativus18 and shoot formation from corm buds.19,20 The importance of BAP on direct organogenesis of saffron has been stressed in earlier reports, too.13

productive there is an immediate need for refinement in these pro-tocols and testing the improved protocol for flower induction.

In the present study a complete protocol for cormlet produc-tion with the desirable size could be achieved, followed by induc-tion of vegetative growth and flowering under greenhouse. We believe that this will pave a way for the increasing the cormlet production as well as commercial production of crocin, safranal and picrocrocin.

Results and Discussion

Figure 1. Crocus sativus L. Kashmirianus. (A) sketch of saffron plant. (B) saffron corm with germinating buds. (C) saffron flowers with three stigmas each.

Table 1. In vitro cormlet regeneration on Ms half strength basal medium supplemented with variable doses of TDZ, while keeping IAA (10 µM) and sucrose 40 g/l same all through the treatments

TDZ (µM) Cormlet Number (Mean ± S.D)

0 a4.0 ± 0.23

2.5 ab10.0 ± 0.1

5 b16.5 ± 0.2

7.5 be21.9 ± 0.3

10 b28.0 ± 0.3

12.5 bf33.7 ± 0.4

15 g45.7 ± 0.3

17.5 c59.5 ± 0.4

20 d70.0 ± 0.3

25 c61.0 ± 0.4

30 g50.5 ± 0.3

Data scored after 12 weeks of culture period

Values are represented as Mean ± sD (n = 10). Data was analyzed by ANOVA using sspp (17.0) by Tukey (hsD) (p < 0.005). The values having same superscript along the columns are not statistically different.

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use of sucrose at 3% for cormlets production. The highest numbers of cormlets obtained were of 1.5–2.0 g category with average per-centage response of 70% after 8 weeks of culture period. However, on control there wasn’t any increase in cormlet size.

increase in cormlet size. The in vitro raised cormlets subcultured on medium attained a maximum corm weight of 2.5 g or above on TDZ (15 μM) + IAA (12.5 μM) + sucrose 30 g/l with 60% percent-age success (table 3 and Fig. 2e). Recently, Devi et al.21 reported the

Table 2. Germination of in vitro raised cormlets on Ms medium with different growth regulator combinations/concentrations, and activated charcoal **

BAP (µM) NAA (µM) TDZ (µM) IAA (µM) 2,4- D (µM) Kn (µM) Activated Charcoal (mg/l) Percent germination*

0 0 - - - - - -

2.5 15 -

5 15 - - - - - -

7.5 15 - - - - - -

10 15 - - - - - 70a

12.5 15 - - - - - 70a

15 15 - - - - - 75b

17.5 15 - - - - - 75b

20 15 - - - - - 90c

25 15 - - - - - 65d

30 15 - - - - - 60e

1.5 1 - - - - 0.5 -

2.5 1 - - - - 0.5 -

5.0 1 - - - - 0.5 65d

1.5 2 - - - - 0.5 -

2.5 2 - - - - 0.5 -

5 2 - - - - 0.5 65d

1.5 3 - - - - 0.5 -

2.5 3 - - - - 0.5 75b

5.0 3 - - - - 0.5 80g

1.5 4 - - - - 0.5 -

2.5 4 - - - - 0.5 -

5 4 - - 0.5 -

- - 2.5 15 - - - -

- - 5 15 - - - -

- - 7.5 15 - - - -

- - 10 15 - - - -

- - 12.5 15 - - - 70a

- - 15 15 - - - 70a

- - 17.5 15 - - - 75b

- - - - 2.5 2 - -

- - - - 5 2 - -

- - - - 7.5 2 - -

- - - - 10 2 - 60e

- - - - 12.5 2 - 60e

- - - - 15 2 - 55f

- - - - 17.5 2 - 65d

- - - - 20 2 - 70a

- - - - 30 2 - 60e

*Data scored after 12 weeks of culture period. **3% sucrose was added in all treatments. Values are represented as Mean ± seM (n = 10). Data was analyzed by ANOVA using sspp (17.0) by Duncan’s multiple range test (p < 0.005). The values having same superscript along the columns are not statis-tically different.

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vegetative growth (55%) was noticed from the corms of mean weight 2.5 g while the corms of 2.0 g mean weight showed 40% vegetative growth and 19% flowering and the corms of mean weight 1.5 g exhibited only vegetative growth (25%) (table 5 and Figs. 2G–i). BAP/NAA was efficient in cormlet formation and subsequent increase in size as well as growth in green house con-dition.7 Since corms naturally grow from late autumn to early winter and that low temperature is essential for vegetative growth and flowering, the temperature of green house was maintained at about 20°C.22

In case of apical buds (obtained from actively growing field corms) cultured on medium containing BAP and NAA the max-imum increase in size of cormlets was obtained on NAA (15 μM) + BAP (20 μM) + sucrose (30 g/l) after 8 weeks of culture period (table 4 and Fig. 2F).

Growth of cormlets under green house conditions. The cormlets produced under in vitro were transplanted into small pots containing the clay loomy soil collected from traditional site of saffron cultivation (Pampore) and watered ocassionally to avoid possibility of water stress. The flowering (25%) and

Figure 2. In vitro cormlet development and inhouse flowering of Crocus sativus L. Kashmirianus. (A) Multiple corms on Ms (1/2)- TDZ (20 μM) + IAA (15 μM) + 4% sucrose. (B) Germination of cormlets on Ms+ BAp (20 μM) + NAA (15 μM). (C) Germination of cormlets on Ms + BAp (5 μM) + NAA (3 μM) + 0.5 mg/l activated charcoal. (D) Germination of cormlets on Ms +TDZ (20 μM) + IAA (10 μM). (e) Increase in size on Ms + TDZ (15 μM) + NAA (12.5μM +3% sucrose. (F) Increase in size on Ms + BAp (20 μM) + NAA (15 μM +3% sucrose. (G) Dried cormlets. (h) Flowering under Greenhouse conditions. (I) Vegetative growth under greenhouse conditions.

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and washed five times with sterilized double distilled water. All the chemicals, unless otherwise specified, were obtained from Hi-Media Mumbai Pvt. Ltd.

culture media. MS basal medium23 supplemented with dif-ferent concentrations of sucrose (Hi-media), Difcobacto agar (Qualigens, India) and different concentrations of plant growth regulators (PGRs; Hi-media) were prepared. The pH was adjusted to 5.6–5.8 with 1 N HCl or 1 N NaOH and finally dispensed into 100 ml Erlenmeyer flasks (borosilicate glass) plugged with non-absorbent cotton. The media were sterilized in an autoclave for 15 min at 121°C and cultures were incu-bated at 25 ± 2°C under 16 h photoperiod, illuminated with cool white fluorescent tubes, at irradiance of 100 μmol m-2s-

1. The experiments were performed in completely randomized block design (CRD), repeated three times; each treatment had 10 replicates.

There experimental sets were designed as below.(1) The sterilized corms were cut with a sharp scalpel into

slices and these were cultured on MS (1/2) medium with differ-ent concentrations of auxins and cytokinins. The different treat-ments used for cormlet induction/ production using corm slices as explants were designated as T0 = MS basal medium (control); T1 = TDZ (2.5 μM) + IAA (10 μM); T2 = TDZ (5 μM) +

The main achievement of the present study is successful development of a complete protocol for in vitro cormlet develop-ment of saffron and their successful flowering under green house (Figs. 2i and 3).

Material and Methods

plant material. Corms of Crocus sativus L. Kashmirianus were collected from Pampore area of Kashmir, J&K, India and were thoroughly washed with detergent Extran (0.5%) and Tween-20 (surfactant) with tap water followed by rinsing with double distilled water. Subsequently these were surface disinfected with 70% ethanol for 1 min followed by 0.5% HgCl2 (w/v) for 6 min

Table 3. Growth of in vitro cormlets of Crocus sativus L. Kashmirianus on Ms medium supplemented with TDZ (15 µM) and IAA (0–30 µM)*

MS+TDZ (15µM)+IAA (_ µM)Min. cormlets

weight (g)Percent cormlets

above the min. weight

Control (Basal medium) No response No response

1.5 1.5 50a

2.5 1.5 50a

5.0 1.5 55b

7.5 1.5 70d

10.0 2.5 60c

12.5 2.0 55b

17.5 2.0 60c

20.0 1.5 30e

25.0 1.5 30e

30.0 1.5 30e

Data scored after 12 weeks of culture period *plus sucrose (3%). Values are represented as Mean ± seM (n = 10). Data was analyzed by ANOVA using sspp (17.0) by Duncan’s multiple range test (p < 0.005). The values having same superscript along the columns are not statistically different.

Table 4. effect of Ms medium with NAA (15 µM) and BAp (5–30 µM) on growth of active vegetative buds of Crocus sativus L. Kashmirianus

MS + NAA(15µM) +BAP(- µM) Edible sugar (g/l) Sucrose (g/l) Minimum cormlet weight (g) Percent cormlets above the minimum weight

Control (Ms medium) - 30 - -

5 - 30 1.5 a 80

10 - 30 1.5 a70

15 - 30 2.0 b60

20 - 30 2.5 b50

25 - 30 2.0 c45

30 - 30 1.5 b50

5 30 - - -

10 30 - - -

15 30 - 1.5 c45

20 30 - 1.5 c40

25 30 - - -

30 30 - - -

Data scored after 12 weeks of culture period;

Values are represented as Mean ± seM (n = 10). Data was analyzed by ANOVA using sspp (17.0) by Duncan’s multiple range test (p < 0.005). The values having same superscript along the columns are not statically different.

Table 5. Growth evaluation of cormlets of Crocus sativus L. Kashmirianus in greenhouse after 6 weeks

Minimum cormlet size (g)Percent

GerminationPercent Flowering

2.5 55 a 25 a

2.0 40 b 19 b

1.5 25 c 0

Values are represented as Mean ± seM (n = 10). Data was analyzed by ANOVA using sspp (17.0) by Duncan’s multiple range test (p < 0.005). The values having same superscript along the columns are not statistically different.

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(3) The in vitro raised cormlets were categorized into three groups, with a minimum weight of 2.5 g, 2.0 g and 1.5 g. These cormlets were taken out from culture vials and after shade drying (7 d) in a well ventilated room with room temperature (25°C), were transplanted into small pots containing clay loomy soil. Misting of the cormlets was done as per the need until the ger-mination of cormlets. These pots were kept under green house conditions for germination and flowering at a temperature 20 ± 3°C, humidity 60–70% and monitored regularly upto 6 weeks.

statistical analysis. Results were reported as means ± SD. Significant differences for multiple comparisons were deter-mined by one-way analysis of variance (ANOVA) followed by Duncan’s/Tukey’s test with (p ≤ 0.005) by SPSS statistical pack-age (ver.17.0).

Conclusion

Saffron being an important crop of commercial significance is highly priced due to its limited production. Micropropagation of saffron holds promise only when we are able to induce produc-tion of flowers on a commercial scale. The present study describes a complete protocol for cormlet production with desirable size,

Figure 3. Flowchart for complete in vitro cormlet development and flowering under greenhouse.

IAA (10 μM); T3 = TDZ (7.5 μM) + IAA (10 μM); T4 = TDZ (10 μM) + IAA (10 μM); T5 = TDZ (12.5 μM) + IAA (10 μM); T6 = TDZ (15 μM) + IAA (10 μM); T7 = TDZ (17.5 μM) + IAA (10 μM); T8 = TDZ (20 μM) + IAA (10 μM); T9 = TDZ (25 μM) + IAA (10 μM); T10 = TDZ (30 μM) + IAA (10 μM). In all above treatments, the concentration of IAA (10 μM) was kept unaltered.

(2) The in vitro regenerated cormlets were sub-cultured on MS half strength basal medium supplemented with different combinations of auxins and cytokinins (BAP/NAA, 2,4-D/Kn and TDZ/IAA) and percentage germination was recorded. Besides activated charcoal too was added in some.

The in vitro regenerated cormlets were also sub-cultured on MS medium augmented with TDZ (2.5–30 μM), IAA (10 μM) and 4% sucrose and were evaluated for increment in cormlet size. Besides, the apical vegetative buds from active corms (obtained from field) were also cultured on MS medium supplemented with varying concentration of BAP (5–30 μM) and different carbon sources (sucrose and edible sugar), in order to note the most effective combination for production of larger sized cormlets. The concentration of auxin (NAA 15 μM) was kept same in all these treatments.

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followed by vegetative growth and flowering under greenhouse. This will pave a way for increasing the cormlet producton as well as commercial production of saffron stigma and its bioactive components viz. crocin, safranal and picrocrocin.

disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed

acknowledgments

The present piece of research work has been performed under centrally funded project for which the authors are highly thank-ful to Ministry of Science and Technology, Department of Biotechnology, New Delhi, India for providing funds.

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