Biological Interventions for Enhancing Saffron Productivity in Kashmir

F.A. Nehvi, S.A. Wani, S.A. Dar, M.I. Makhdoomi, B.A. Allie, Z.A. Mir K. D. Research Station Sher-e-Kashmir University of Agricultural Sciences & Technology Kashmir India

Keywords: Crocus sativus, in vitro micropropogation, mutation, variability

Abstract The heterogeneity found in the natural saffron population for morphological, developmental and yield component traits are primarily due to the genetic and environmental factors. Development of high yielding genotypes using the existing gene pool of saffron applying the biological tools shows potential for improving the productivity of this crop. A survey undertaken to study the extent of variation revealed wide spectrum of variability in saffron flowers and corm samples collected from saffron growing areas of Kashmir, thus implying a great scope for saffron improvement. Flowers completely devoid of style & anthers and freaks with 4-5 stigmas where observed from the natural populations. Collection and evaluation of saffron clones revealed possibility of increasing yield per se and has lead to the identification of ten elite clones with distinct yield superiority. Due to absence of sexuality mutation breeding approach is another way of creating genetic variability in saffron. An attempt was made to create new variants for economic characters through the induction of mutation using Gamma irradiation. Existing saffron population was irradiated with gamma rays from a co60 source at 0.25, 0.50, 0.75 and 1 kr doses. Delayed sprouting was observed in higher dose (1 kr). Higher dose was also associated with decreased corm yield and dry pistil weight/plant due to strong reduction in percentage of flowering plants and number of flowers / plants. Tissue culture studies have shown possibility to regenerate saffron plantlets and in vitro corm development through somatic embryogenesis. Evaluation of in vitro corms under field conditions revealed that corm development and survival is directly proportional to initial corm weight. Survival percentage among in- vitro corms having average corm weight less than 1 g was only 26 %, whereas, it was above 88 % in above 1 g corms. Weight gain after one year was 0.25 to 3.54 g.

INTRODUCTION Saffron the legendary crop of J & K state has been under cultivation for so many centuries. In spite of extremely high demand and value in the market for its multiple uses in diverse fields as a dye, in perfumery, in food flavouring, limited attention has been paid for its genetic improvement. Meiotic abnormalities result in abnormal chromosome assortment and formation of an abnormal number of genetically unbalanced spores which vary in shape and size leading to complete sterility (Chichiricco, 1990). Therefore, utilization of heterogenity in the natural population which is due to genetic and environmental factors offers a tremendous scope for saffron improvement. Another approach for increasing variability for selection through irradiation has not received much attention. The use of mutagenesis could increase the natural variability for important characters such as high content of active principles per stigma, variability in morphological and flower component traits and precious emergence of flowers. Since the saffron reproduce only vegetative by the corms any attempt to modernize saffron cultivation will therefore require efficient mass production of pathogen free corms. Micro propagation of saffron has therefore been advocated to be the best alternative for its propagation (Georage et al., 1992; Ahuja et al., 1994). Present study was thus undertaken keeping in the view the basic need of enhancing saffron productivity in Kashmir involving biological intervention.

MATERIALS AND METHODS Present study was carried out at Saffron Research Sub-Station Dussu Konibal Pampore a constituent Research Station of Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir located at 34.1oN latitude, 74.89o E longitude with an altitude of 1650 m.a.m.s.l.

Extent of Variability and Identification of Elite Clones 438 flower samples each containing 100 randomly selected flowers were collected from the saffron growing area of Jammu & Kashmir during the flowering period (October-November). Parameters of variability were recorded for various floral and quality traits viz., fresh flower wt (g), fresh Perianth wt (g), fresh anther wt (g), fresh pistil wt (g), dry flower weight (g), dry perianth wt (g), dry anther wt (g), dry pistil weight (g), stigma length (cm), style length (cm), pistil length (cm) and crocin content (%). Material under study for identification of high yielding clones comprised of 500 corm samples of uniform corm weight (>10 g) collected from saffron growing area of Kashmir during the month of August-September. Each sample was based on 30 corms which were planted as single observational rows at spacing of 20 x 10 cm and were replicated twice in RBD design. Observations were recorded on various floral, yield and quality traits over 3 years. Standard procedures were used to compute analysis of variance and components of variance (Verma et al., 1987; Johnson et al., 1955).

Induction of Genetic Variability through Mutagenesis 4 sets of saffron corms with each set containing 200 corms of uniform corm weight (>10.0 g) were irradiated with 0.25, 0.50, 0.75 and 1.0 Krad doses of Co 60 gamma rays at BARC, Srinagar. A set of 200 untreated corms was used as control. Irradiated corms were planted with plant geometry of 20 x 10 cm on raised buds. Observations were recorded on various morphological and yield related traits. Identified mutants were advanced to M2 generation for further study

In-vitro Micropropagation Hardened in vitro corms developed at Regional Research Laboratory, Jammu through somatic embryogenesis, auxillary shoot formation and direct organogenesis were evaluated under temperate conditions of Kashmir Valley for average corm weight (g), germination (%), average number of radical leaves, average weight of corms (g) after one year and gain in corm weight (g). Corms were categorized in 4 groups based on initial corm weight.

RESULTS AND DISCUSSION Extent of Variability and Identification of Elite Clones Evaluation of 438 flower samples collected from temporal sub populations of Jammu & Kashmir revealed a wide range of variability for floral, yield and quality attributes. Flowers completely devoid of style and anthers and freaks with 4 and 5 stigmas were observed from the natural population, however, such plants did not inherit the presence of supernumerary stigmas to their progenies .The presence of higher number of stigmas per flower results from physiological and/or developmental irregularities leading to 4-5 stigmas. However, detection of flowers with increased number of stigmas in natural population is a universal phenomenon (Estilai, 1978; Dhar et al., 1988). Magnitude of variability (Table 1) revealed that the mean fresh and dry weight of 100 flowers in the temporal sub-population of Jammu & Kashmir was 23.46 and 3.82 g, respectively, yielding approximately 0.759 g of laccha grade saffron (Pistil).Thus, 1Kg fresh flowers produce approximately 32 g of dried laccha saffron. Pistil weight ranged from 1.76-5.88 g on fresh weight basis revealing possibility of saffron improvement through selection. Similar results of wide range of variability in Kashmir have also been reported (Zargar, 2002). Stigma length ranged from 2.41-3.87 cm with a mean value of 2.5 cm, whereas, Crocin content (%) ranged from 9.92-14.35 percent with a mean value of 12.0 percent. Quality evaluation confirmed that saffron of Kashmir is of intrinsically high quality with respect to colouring pigment and traditional post harvest processing was responsible for poor saffron quality. The results are in general agreement with earlier reports (Nehvi et al., 2005). Evaluation of 500 saffron genotypes collected from natural population revealed wide range of variability for different traits. no. of flowers per spathe ranged from o.65-5.58 with fresh flower weight ranging from 172–355 mg. Economic product (Pistil weight) on fresh weight basis ranged from 14.37-68.42 mg, whereas, on dry weight basis it ranged from 6.00-14.60 mg. Stigma length which contributes to the yield potential varied from 1.75 cm to 3.72 cm with a corresponding variability for style length ranging from 1.70 cm – 4.25 cm. Average number of daughter corms ranged from 2.37–7.05, whereas, average weight ranged from 1.59-8.49 g. Colouring pigment (crocin content) ranged from 8.55-17.10 percent. Greater magnitude of phenotypic variance than the corresponding genotypic variance was observed which indicated greater influence of environment in the inheritance of floral, corm and quality attributes (Table 2). Heritability in the broad sense was recorded low for all traits, excepting fresh flower weight, fresh pistil weight and stigma length which revealed high broad sense heritability. Genetic gain as percent of mean was high for number of flowers/spathe, fresh flower weight, fresh pistil weight and Crocin content, whereas, it was medium for dry pistil weight, stigma length and number of daughter corms and low for rest of the traits. Components of variability indicate ample scope of saffron Improvement through selection among the temporal sub population of J&K. Grilli Caiola et al., 2001 also reported phenotypic differences in respect of flower size, tepal shape and colour intensity. Evaluation of 500 saffron genotype for yield and yield related traits over 3 years lead to the identification of 10 superior genotypes viz; SMD-3, SMD-11, SMD- 31, SMD-45, SMD-52, SMD-68, SMD-79, SMD-81, SMD-21 and SMD-224. Elite genotypes recorded saffron yield ranging from 4.0-7.6 kg/ha with a corresponding crocin content ranging from 13.89-17.10 percent. Superior performance of 3 accessions in Iran has also been reported (Parviz et al., 2004). In Kashmir elite sub- population developed from the progenies of the corms selected from extensive saffron belts have been identified and catalogued (Munshi and Zargar, 1991). With the commercial cultivation of elite genotypes the productivity level of Jammu & Kashmir is expected to enhance. Induction of Genetic Variability through Mutagenesis Mutagenesis is another approach which was applied to induce genetic enhancement for yield attributing traits. Existing saffron population was irradiated with gamma rays from a Co60 source at 0.25, 0.50, 0.75 and 1 Kr dose. 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 (Table 3). Induction of early sprouting in 0.25 Kr and 0.5 Kr and delayed sprouting in higher doses was noted. Radiation at lower doses (0.25 Kr) enhanced plant biomass, whereas, a reverse reaction was observed at higher doses. M1 mutants with 0.25 Kr radiation dose showed enhanced corm yield and saffron yield by a margin of 53.44 % and 84.28 %, respectively. Increased saffron recovery has resulted from more number of flowers/plant. Plant survival was reduced to 79 % at 1.0 Kr. 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 et al., 1983; Khan, 2004). Fifty superior mutants from M1 generation with distinct yield superiority were identified and were advances to M2 generation. In M2 generation superiority of mutants in terms of saffron recovery was maintained to the tune of 47.36 percent (Table 4). Elite mutants also revealed superiority in terms of pistil length, increased number of flowers/spathe. Delayed sprouting, slow growth in higher doses, increase/decrease plant height at lower and higher doses, respectively and induction of flowering in M1 generation at lower doses, are due to auxin synthesis, whereas, at higher doses, it is inhibited resulting into delaying, and Stunted growth. The present study is in confirmation with other reports (Gordon, 1959). Stunted growth, reduction in survival and reduced fertility is also attributed to genetic loss due to chromosomal aberrations and gene mutations (Datta and Gupta, 1980). Mutations in the biochemical pathways assist, the synthesis of flower inducing substances, leading to formation of flower.

In Vitro Micropropagation Since the saffron reproduce only vegetatively by the corms nay attempt to modernize saffron cultivation will therefore require efficient mass production of pathogen free corms. For mass propagation of corms tissue culture is the only effective alternative (George et al., 1992). Tissue cultured studies have shown possibility to regenerate saffron plantlets and in vitro corm development through Somatic embryogenesis, auxiliary short formation and direct organogenesis. Development of tissue cultured cormels at RRL, Jammu was asynchronous and corms of different size and shape were produced. 201 corms produced through Somatic embryogenesis averagely weighed from 0.04-2.73 g. For field evaluation of tissue- cultured corms, 4 sub-groups were made based on the average weight of Corms. Majority of the in-vitro corms (165) weighed less than 1g, whereas, 22 corms weighed from 1-1.5g. An average weight of 1.5-2.0 g was exhibited by 9 corms and only 5 corms showed an average corm weight above 2 g. The hardened tissue cultured cormels were planted for field evaluation. Field evaluation studies of in vitro corms revealed importance of corm weight. Survival percentage among the corms having average corm weight less than 1g was only 26 %, whereas, it was 83.66 % among the corms having average corm weight above 1 g. Maximum number of radicle leaves was observed among the corms having average corm weight above 2.0 g. Corms having average corm weight between 1.01 to 1.96 g behaved equally with regard to average number of radicle leaves. However, corms having average corm weight 1.58 to 1.96 were superior in length of radicle leaves. Corm development of tissue cultured corms was directly proportional to initial corm weight. Average corm weight after one year of plantation ranged from 0.80 (Group 4) to 5.97 g (Group 5) showing a weight gain ranging from 0.25 g (Group 3) to 3.54 g (Group 5). For corms weighing 1.58 to 1.96 g the average corm weight on uprooting was 3.22, however, with further decline in initial weight the corm development was slow (Table 5). The protocol needs to be revised in view of corm size so as to make it commercially viable and acceptable.

ACKNOWLEDGEMENTS The authors acknowledge the financial support and guidance provided by the Indian council of Agricultural research, National Horticulture Board (Government of India) and SKUAST-K.

Literature Cited Akhund-Zade. and Muzaferova, R.S. 1975. Studies of the effectiveness of gamma irradiation of the saffron. Radiobiologiya. 15(2):319-322. Ahuja, A., Koul, S., Ram G. and Kaul, B.L. 1994. Somatic embryogenesis and regeneration of plantlets in saffron. Ind.J.Exptl. Biol. 32:135-140. Chichirricco,G.1990. Sterlity and improvement of saffron Crocus:In Tammaro and Marra (eds): Lo Zafferano: Proceeding of the International Conference in saffron (Crocus sativa L.) L” Aquila (Italy) 27-29 October 1989. Universita Degli Studi L’ Aquila e Academia Italiana della Cuema, L’Aquila. Datta, S.K. and Gupta, M.M.1980. Effect of gamma irradiation on rooted cuttings of small flowered chrysanthemum, New Bot. VII:7 3-85. Dhar, A.H., Sapru, R. and Rebha. K.1988. Studies on saffron in Kashmir. Variation in natural population on cytological behaviour. Crop Improv. 15(1): 48-52. Estilai, A. 1978. Variability in saffron (Crocus sativus L.). Experientia 34:725-727. Gordon, S. A.1957. The effect of ionizing radiation on plants: Biochemical and physiological aspects, Quart. Rev. Biol. 32:3-14. George, P.S., Visavanath, S., Ravishankar, G.A. and Venkataraman L.V. 1992. Tissue culture of saffron: Somatic embryogenesis and shoot regeneration. Food Biotech. 6:217-223. Grilli Caiola, M., Di Somma, D. and Lauretti, P.2001. Comparative study on pollen and pistil of Crocus sativus L. (Iridaceae) and its allies species. Ann. Bot. (Roma) 1:93-103. Khan, I.A. 2004. Induced mutagenic variability in saffron (Crocus sativus L.) Acta Hort. 650:281-283. Johnson, H.W., Robinson, H.F. and Constock, R.E. 1995. Estimates of genetic and environmental variability in soyabean. Agron. J. 47:314-318. Laneri, U. and Lucretti, S. 1983. Propagazione in vitro di Crocus sativus L. rapporto dei resultati ottenuti nel 1982. ENEA. Rome, RT/FARE-SIN (83). Munshi, A.M. and Zargar, G.H. 1991. Variation in the natural population of saffron (Crocus sativus L.) crop in Kashmir and performance of some selected sub- populations. Phytobreedon. 71 (1 & 2):62-67 Nehvi F.A., Agarwal, S.G., Mir, M.A., Dar, S.A., Mir, Z.A. and Nusrat Nabi.2005.Quality drying of Saffron SKUAST J Res. 7(2):343-346 Parviz, E., Yadollahi. A. and Maibodi, M. 2004. Productivity, growth and quality attributes of 10 Iranian saffron accessions under climatic conditions of Chaha- Mahal Bakhtiari central Iran. Acta Hort. 650:183-88. Verma, M.M., Gill, K.S. and Vivk, D.S. 1987. Genotype-Environment interaction, its measurement and significance in plant Breeding. Communication Centre Punjab Agricultural University Ludhiana. Zargar, G.H. 2002. Genetic variation in saffron and importance of quality seed corms. National Seminar Cum Workshop Srinagar. August, 2002. Table 1. Nature and magnitude of phenotypic variability for flower and its economic components in the temporal sub-population of saffron crop in J&K. Characters Mean Components of variability Range CV % Fresh flower wt (g) 23.46 ± 3.31 15.86-36.69 14.14

Fresh Perianth wt (g) 16.73 ± 2.15 11.03-28.22 12.89

Fresh Anther wt (g) 3.21 ± 0.62 1.43-4.90 19.31

Fresh Pistil w t(g) 3.52 ± 0.47 1.76-5.88 13.42

Dry flower wt (g) 3.82 ± 0.71 1.41-6.41 18.78

Dry Perianth wt (g) 2.37 ± 0.34 1.41-3.58 14.66

Dry Anther wt (g) 0.692 ± 0.11 0.230-1.61 16.51

Dry Pistil wt (g) 0.759 ± 0.13 0.39-1.59 18.32

Stigma length (cm) 2.5 ± 0.46 2.41-3.87 18.49

Style length (cm) 2.0 ± 0.56 1.50-3.10 28.20

Pistil length (cm) 4.5 ± 0.69 3.90-6.70 15.44

Crocin % 12.01 ± 1.25 9.92-14.35 10.44 Table 2. Components of variability for different floral, morphological, corm and quality attributes in saffron genotypes Components No. of Fresh Fresh Dry Stigm Style No. of Avg. Crocin flower flower pistil Pistil a lengt daughter corm (%) / wt(mg) wt wt length h corms wt.(g) spathe (mg) (mg) (cm) (cm) 1 2 3 4 5 6 7 8 9 10 Range 0.65 – 172 - 14.37- 6.0 1.75- 1.70- 2.37- 159- 8.55- 5.58 355 68.42 -14.60 3.72 4.25 7.05 8.49 17.10 62 g 0.218 1924.2 16.60 0.85 0.053 0.05 0.66 0.57 4.64 7 62 p 0.937 2984.0 20.7 2.56 0.085 0.55 2.85 6.9 6.52 1 GCV 32.24 17.02 19.91 14.9 10.35 7.14 19.65 12.66 17.47 PCV 67.18 21.2 22.24 25.9 13.22 16.0 38.71 44.06 20.70 2 h2 0.23 0.65 0.80 0.33 0.61 0.20 0.23 0.08 0.712 G.A 0.47 72.56 7.52 1.09 0.37 0.30 0.81 0.45 3.75 G. gain (% 32.21 28.16 36.78 17.6 16.70 6.55 18.48 7.46 30.4 of mean)

Table 3. Effect of radiation doses on saffron development in M1 generation Characters Control 0.25 krd 0.5 krd 0.75 krd 1.0 krd Plant height(cm) 21.00 + 1.30 43.00 + 3.10 39.0 + 2.8 19.5 + 1.9 22.00 + 1.9 Days to sprouting 50+1.90 45 + 1.80 46 + 2.00 60 + 1.400 61 + 2.00 Survival Plants 97.00 99.00 98.00 86.5 79.00 (%) Flowering plants 98.0 100.00 89.00 81.00 72.00 (%) No. of daughter 1.9 + 0.6 256 + 1.50 2.42 + 2.0 2.16 + 2.9 1.98 + 1.5 corms/plant Corm yield/plant 5.80 + 1.4 8.96 + 1.80 7.05 + 1.9 6.84 + 1.9 5.65 + 1.4 (g) No. of 1.46 + 0.30 1.87 + 0.20 1.55 + 0.25 1.54 + 0.30 1.44 + 0.15 flowers/plant Fresh pistil wt 4.200 + 1.500 5.790 + 1.30 4.800 + 0.790 4.750 + 4.180 + 0.900 (100 pistils) g 1.00 Dry pistil 0.700 + 0.200 1.290 + 0.320 1.00 + 0.100 1.00 + 0.100 0.680 + 0.250 (wt/100 pistils) g Table 4. Performance of mutants in M2 generation Population Plant Days to No. of Fresh Dry Stigma Style Pistil height sprout flowering pistil wt. pistil length length length (cm) plants (100 wt. (cm) (cm) (cm) (%) pistils) (100 (g) pistil) (g) Natural 35.0 45.00 0.97 3.900 0.950 2.950 3.220 6.170 population Mutant 49.0 47.00 2.15 8.670 1.400 3.110 13.520 6.630 population

Table 5. Performance of hardened in vitro corms under field conditions of Kashmir No. of Total Initial Range Germination Avg. Avg. After 1 year corms corm avg. (%) No. of Length of Avg. Grain wt. (g) corm radicle radical Wt. of in wt. (g) leaves leaves corms weight (cm) (g) (g) 5 12.13 2.43 2.13-2.73 60.0 5.0 22.17 5.97 3.54 9 16.45 1.83 1.58-1.96 100.0 3.78 25.83 3.22 1.45 22 26.12 1.19 1.47-1.61 91.0 3.40 23.78 2.19 0.25 165 67.16 0.41 0.04-0.88 26.0 2.23 18.13 0.80 0.34 201 121.86 1.46 0.04-273 69.25 3.60 22.47 3.04 1.39