In Vitro Propagation of Long Melon Var. Karnal Selection (Cucumis melo L.) from Shoot Tip

International Journal of Applied Agricultural Research ISSN 0973-2683 Volume 5 Number 1 (2010) pp. 55 62 Research India Publications http://www.ripublication.com/ijaar.htm In Vitro Propagation of Long Melon Var. Karnal Selection (Cucumis melo L.) from Shoot Tip Danswrang Goyary*, Neelam Gupta, Neeraj Khare, Sivalingam Anandhan, Meenal Rathore and Zakwan Ahmed Department of Molecular Biology and Genetic Engineering Defence Agricultural Research Laboratory (DARL) P.O. Arjunpur (Goraparaw), Haldwani, Nainital, Uttarakhand, India *Corresponding Author E-mail: dgoyary@yahoo.co.in Abstract An efficient protocol of in vitro propagation via direct plant regeneration in long melon, locally known as Kakri var. Karnal selection (Cucumis melo L.) was developed. The shoot tips from in vitro grown seedlings on water-agar medium were excised and cultured on MS medium supplemented with different concentrations and combinations of auxin and cytokinin for direct shoot regeneration. The best shoot was obtained on MS supplemented with BAP (0.1 mg l -1 ) and IBA (0.01 mg l -1 ). In this combination, the shoots are initiated in two days and the frequency of shoot regeneration was 90-95%. Length of the longest shoot was 5.1±0.56 cm after 15 days of culture. Root initiation was obtained efficient on MS media containing 0.5 mg l -1 IBA. About 85-90% of the regenerated plantlet hardened successfully in sand: soil: vermicompost (1:1:1). Keywords: Karnal selection, efficient protocol, shoot tip, in vitro propagation, long melon, Cucumis melo L., Cucurbit Abbreviations: MS, Murashige and Skoog; BAP, 6-benzylaminopurine; IBA, Indole-3-butyric acid Introduction Long melon (Cucumis melo L. var. Karnal selection), commonly known as Kakri in India, belong to the Cucurbitaceae family. The fruits are long straight, thin, up to 40-45 cm long; light green in colour and cylindrical in shape. Cucurbitaceae has a very important social and economical role in the tropical and subtropical regions. Species

56 Danswrang Goyary et al such as watermelon, long melon, cucumber and squash are widely cultivated in all over the world. Long melon is an important summer crop and a valuable alternative source of water that can be eaten as a fresh fruit. It is a good source of important minerals and vitamin C. Biotechnology offers tools that are capable of surpassing limitations found by traditional plant breeding techniques by adopting genetic engineering, molecular biology and tissue culture techniques (Souza et al., 2006) such as gene transfer technology, which allows the introduction of foreign genes into germplasm, without modifying the genetic background of elite varieties. This method has been used to develop resistance to cucurbit potyviruses in different cucurbit species (Krug et al., 2005; Fuchs et al., 2004, and Compton et al., 2004). The establishment of efficient in vitro plant regeneration from isolated organs, tissues and cells is a very essential step. The in vitro propagation technique is used to some degree in almost every major agronomic, vegetable and fibre crops and in generation of new cultivar through plant transgenic technology (Sultana and Bari Miah, 2003). Regeneration through callusing is difficult in cucurbitaceous plants. Regeneration of plants through callusing and somatic embryogenesis can cause somaclonal variation (Larkin and Scowcroft, 1981) which may eventually lead to changes in the phenotype of plants. Though callusing is attained from the cotyledonary leaves of cucurbits, but callus formation of somatic embryo is less frequent and also takes longer time period (Jelaska, 1974). So, there is a necessity for the development of regeneration protocol from explants, which are amenable for direct shoot regeneration, and it may avoid longer callusing cycle. There are several earlier reports on in vitro culture protocol of its closely related taxas, like water melon (Sultana et al., 2004), cucumber (Ahmad et al., 2005), squash (Rahman et al., 1993), teasle gourd (Nabi et al., 2002) and bitter gourd (Sultana and Bari Miah, 2003). However, there is no report on plant regeneration using shoot tip as an explant in long melon. This is a very fast technique for complete regeneration of plants, taking 25-30 days, for long melon. The objective of the present work was to implement an efficient regeneration protocol for long melon var. Karnal selection and for further studies of this crop in genetic transformation via Agrobacterium for abiotic/biotic stress tolerance. Materials and Methods Seeds were purchased from CCSHAU, HISAR, India. They were washed in running tape water for 3-4 times. It was then treated with detergent for 15 minutes and washed with double distilled water 4 times to make seed material free from detergent. Surfaced sterilization was carried out in aseptic condition by treating with HgCl 2 solution (0.1%) for 2-3 min and washing 4 times with autoclaved double distill water to remove traces of HgCl 2. Finally the seeds were surface sterilized with 70% ethanol for 2 min and washed with sterilized double distilled water for three times and blot dried. Then the seeds were placed on hormone free water-agar medium. The seeds sprouted within three days after inoculation. The germination rate was very high (80-90%). Shoot tips from six days old seedling were used as explant in this experiment. Explants were cultured on MS (Murashige and Skoog, 1962) medium supplemented with various concentration and combination of auxin and cytokinin

In Vitro Propagation of Long Melon Var. Karnal Selection 57 (Table 1) for efficient shoot regeneration. For root induction, 4-5 cm long regenerated shoots were transferred to MS medium containing various concentrations of auxins (Table 2). The ph of all media was adjusted to 5.8 ± 0.1 before autoclaving, supplemented with sucrose (3%) and solidified by agar (0.8%). The culture tubes or conical flask containing medium were then autoclaved at 121 0 C for 20 min. Following inoculation, all the cultures were maintained in tissue culture condition (16 h light / 8 h dark photoperiod, light intensity of 2000-3000 lux at temperature 25±1ºC). Data on days to shoot initiation, shoot regeneration frequency and average shoot length were recorded after 15 days of culture. Besides, data on days required for root initiation, rooting frequency after the inoculation of shoots in rooting media were also recorded. Table 1: Effect of different auxins (IBA) and cytokinins (BAP) and concentrations (0.02-1.00 mg l -1 ) on the shoot initiation, length and regeneration frequency (%) from seedlings of Cucumis melo L. (Kakri) after 15 days of culture on MS media. MS Media Conc. Shoot Mean shoot Shoot regeneration Remarks of + ( mg l - initiation length (cm) frequency (%) the shoots Hormone 1 ) (days) Plain MS - 1.1±0.35a 20-25 - IBA BAP IBA BAP IBA BAP IBA BAP 0.02 0.05 0.03 0.05 0.01 0.1 0.02 0.1 2 3.5±0.45b 61-75 * 2 3.2±0.69b 60-72 * ** 2 5.1±0.56d 90-95 4 3.2±0.55b 58-65 * IBA 0.01 - - - - BAP 0.1 5 1.5±0.25a 25-30 - *Good Shoots **Best shoots with healthy growth Different lowercase letters in a row following any two mean values of shoot length (07 replications having 30 explants for each treatment) indicate significant difference according to Student t-test (level of significance at p<0.05). [Different combination of growth hormone and supplement of single growth hormone on MS media were tried (data not shown), however, only best results found during experimentation were displayed].

58 Danswrang Goyary et al Table 2 : Effect of different auxins (IBA and IAA) and concentrations (0.01-1.00 mg l -1 ) on the root initiation, shoots with roots (mean of 07 replications having 25 explants for each treatment) and rooting frequency (%) from 15 days cultured shoots of Cucumis melo L. MS Media Conc. Root initiation No. of shoots Rooting frequency + Hormone (mg l -1 ) (Days) with roots (%) Plain MS - 12-15 12.0±0.32 48 IAA 0.2 12-14 17.0±0.25 68 IBA 0.01 IAA 0.1 12-14 17.0±0.34 68 IBA 0.03 IBA 0.15 11-13 18.0±0.46 72 IAA 0.02 IBA 0.2 11-14 19.0±0.59 76 IAA 0.03 IAA 0.5 08-09 16.0±0.18 64 IAA 0.3 11-12 16.0±0.21 64 IAA 0.1 11-14 17.0±0.36 68 IBA* 0.5 07-08 23.0±0.35 92 IBA 0.3 10-12 18.0±0.51 72 IBA 0.1 11-13 16.0±0.45 64 * Best root induction growth hormone for Cucumis melo L. Figure 1 (A-F) : In vitro regeneration of Cucumis melo L. (Kakri) plants from shoot tip explants obtained from in vitro grown seedling. Germination starts after two days of inoculation and shoot tips of six days old seedlings were taken as an explant Pre- C

In Vitro Propagation of Long Melon Var. Karnal Selection 59 cultured for two days on MS medium containing BAP (0.1 mg l -1 ) and IBA (0.01 mg l -1 ) Regeneration and elongation of shoot tips in the precultured media Root formation on MS medium containing IBA (0.5 mg l -1 ) E-F. Hardening of well-rooted plantlets in sand: soil: vermicompost (1:1:1) and fruits bearing plants in glasshouse facility Results and Discussion Efficient and reproducible regeneration protocol development from cells or tissues of plant is a pre-requisite for the successful application of genetic manipulation techniques for the improvement of important crop plants. There is no universally applicable method of culture, regeneration and transformation systems for all species. Tissues from different genotypes will differ in their response to culture. In the present investigation, we have made an attempt to standardize a reproducible protocol for in vitro regeneration of long melon var. Karnal Selection (Cucumis melo L). Choice of explant plays an important role that makes an absolute difference between success and failure in inducing in vitro regeneration of any crop. Seedling age and the type of explant used were reported to be an important factor for shoot regeneration (Choi et al., 1994; Compton, 2000; Ananthakrishnan et al., 2003). It has also been reported that the composition of induction media is an important factor for adventitious bud development; the presence of cytokinin is critical for shoot induction and for differentiation from cotyledonary explants of watermelon (Dong and Jia, 1991). Our experiment indicated that more than 6 days old explants decrease the regenerative capacity. Therefore, we have chosen shoot tip of 6 days old seedlings germinated under sterile condition (Fig. 1A) as an explant, which is a meristamatic tissue with high regenerative capacity. The shoot tips are able to regenerate a whole plant within 25-30 days without any phenotypic variation, which makes it a very efficient protocol for in vitro propagation for development of true to type and may be used in genetic transformation experiments. Different concentrations and combinations of auxin (IAA and IBA) and cytokinin (BAP) were used on MS medium for obtaining the best shooting from the shoot tip explant obtained from in vitro grown Cucumis melo L. Among the various growth regulator supplements used, the best response for shoot regeneration was observed on MS medium fortified with BAP (0.1 mg l -1 ) and IBA (0.01 mg l -1 ) where the frequency of shoot regeneration was 90-95% and the length of the longest shoot was observed 5.1±0.56 cm after 15 days culture (Table 1 and Fig. 1C). This finding coincides with the previous report that BAP is highly effective for cucurbit organogenesis inducing multiple adventitious shoot bud differentiation (Srivastava et al., 1989; Compton and Gray, 1993). It is also reported that the lower growth regulator concentration (cytokinin + coconut water) favoured morphogenesis and resulting in a higher number of explants with shoots and plantlets avoiding somaclonal variation in watermelon (Krug et al., 2005). 2-isopentenyladenine (2iP) acts synergistically with BAP improving organogenesis rates in watermelon cv. sugar baby (Chaturvedi and Bhatnagar, 2001). In cucurbits, regeneration is reported through somatic embryos derived from cotyledons and hypocotyls but organogenesis through callusing cycle is difficult in cucurbits (Larkin and Scowcroft, 1981). Besides,

60 Danswrang Goyary et al regeneration through callusing cycle takes longer time compared to the shoot development from the shoot tips because of well-developed merismatic tissue. Different types of auxins at different concentrations were used on MS medium to regenerate root induction. IBA is a widely used plant growth regulator for root induction in some cucurbits (Sarowar et al., 2003; Thomas and Sreejesh, 2004), while NAA is also used (Kathiravan et al., 2006). In our experimentation, among different concentration of auxins, IBA was found to give comparatively better response than IAA and IAA+ IBA in combination for inducing roots. The best rooting was obtained using MS medium supplemented with 0.5 mg l -1 IBA (Table 2 and fig. 1D). The differences in rooting response may be a result of genotype or cultural conditions (Pal et al., 2007). The root was initiated within 7-8 days and the rooting percentage was observed to be 90-95%. Although rooting was observed in different combinations of auxin and at varying concentrations, the percentage of root formation was lower and roots were also malformed. The plantlets with well-developed roots were successfully hardened in sand: soil: vermicompost (1:1:1) (Fig.1E-F) and the percentage of survivability was 85-90%. Acknowledgment Authors thank DRDO, Ministry of defence, Govt. of India for the financial support for the project work (RD-P1-2004/DARL-65). References [1] Ahmad, N., and Anis, M., 2005, In Vitro Mass Propagation of Cucumis Sativus L. from Nodal Segments, Turk. J. Bot., 29, 237-240. [2] Ananthakrishnan, G., Xia, X., Elman, C., Singer, S., Paris, H.S., Galon, A., and Gaba, V., 2003, Shoot Production in Squash (Cucurbita pepo) By in Vitro Organogenesis, Plant Cell Rep., 21: 739-746. [3] Compton, M.E., Gray, D.J., and Gaba, V.P., 2004, Use of Tissue Culture and Biotechnology for the Genetic Improvement of Watermelon, Plant Cell Tiss. and Org. Cult., 77: 231-243. [4] Compton, M.E., and Gray, D.J., 1993, Shoot Organogenesis and Plant Regeneration from Cotyledons of Diploid, Triploid, and Tetraploid Watermelon, J. Am. Soc. Hort. Sci., 118: 151-157. [5] Compton, M.E., 2000, Interactions between Explant Size and Cultivar Affects Shoot Organogenic Competence of Watermelon Cotyledons, Hort. Sci., 35: 749-750. [6] Choi, P.S., Soh, W.Y., Kim,Y.S., Yoo,O.J., and Liu, J.R., 1994, Genetic Transformation and Plant Regeneration of Watermelon using Agrobacterium Tumefaciens, Plant Cell Rep., 13: 344-348. [7] Chaturvedi, R., and Bhatnagar, S.P., 2001, High-Frequency Shoot Regeneration from Cotyledon Explants of Watermelon CV. Sugar Baby, In Vitro Cel. and Dev. Biol. Plant, 37: 255-258.

In Vitro Propagation of Long Melon Var. Karnal Selection 61 [8] Dong, J.Z., and Jia, S.R., 1991, High Efficiency Plant Regeneration from Cotyledons of Watermelon (Citrullus vulgaris Schrad.), Plant Cell Rep., 9: 559-562. [9] Fuchs, M., Chirco, E.M., McFerson, J.R., and Gonsalves, D., 2004, Comparative Fitness of a Wild Squash Species and Three Generations of Hybrids between Wild Virus-Resistant Transgenic Squash, Environ. Biosafety Res., 3: 17-28. [10] Jelaska, S., 1974, Embryogenesis and organogenesis in pumpkin explants, Physiol. Plant, 31: 257-261. [11] Kathiravan, K., Vengedesan, G., Singer, S., Steinitz, B., Paris, H., and Gaba, V., 2006, Adventitious Regeneration in Vitro Occurs Across a Wide Spectrum of Squash (Cucurbita pepo) Genotypes, Plant Cell Tiss. Org. Cult., 85: 285-295. [12] Krug, M.G.Z., Stipp, L.C.L., Rodriguez A.P.M., and Mendes, B.M.J., 2005, In Vitro Organogenesis in Watermelon Cotyledons, Pesq. agropec. Bres., 40(9): 861-865. [13] Larkin, P.J. and Scowcroft, W.R., 1981, Somaclonal Variation - a Novel Source of Variability from Cell Cultures, Theor. Appl. Genet., 60: 197-214. [14] Murashige, T., and Skoog, F., 1962, A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures, Physiol. Plant, 15: 473-497. [15] Nabi, S.A., Rashid, M.M., Al-Amin, M., and Rasul, M.G., 2002, Organogenesis in Teasle Gourd (Momordica dioica Roxb.), Plant Tiss. Cult., 12: 173-180. [16] Pal, S.P., Alam, I., Anisuzzaman, M., Sarker, K.K., Sharmin, S.A., Alam, M.F., 2007, Indirect Organogenesis in Summer Squash ( Cucurbita pepo L.), Turk. J. Agric., 31: 63-70. [17] Rahman, S.M., Hossain, M., Islam, R., and Joarder, O.I., 1993, Plant Regeneration from Internode Segments of Cucurbita Maxima Duch. Cucurbita Moschata Duch, Curr. Sci., 65: 562-564. [18] Sarowar, S., Oh, H.Y., Hyung, N.I., Min, B.W., Harn, C.H., Yang, S.K., Ok, S.H., and Shin, J.S., 2003, In Vitro Micropropagation of a Cucurbita Interspecific Hybrid Cultivar a Root Stock Plant, Plant Cell Tiss. Org. Cult., 75: 179-182. [19] Souza, F.V.D., Garcia-Sogo, B., Silva, A., San-Juán, A.P., and Moreno, V., 2006, Morphogenetic Response of Cotyledon and Leaf Explants of Melon (Cucumis melo L.) CV. Amarillo Oro, Braz. arch. biol. technol., 49(1): 21-27. [20] Sultana, R.S., and Bari Miah, M.A., 2003, In Vitro Propagation of Karalla (Momordica Charantea Linn.) from Nodal Segment and Shoot Tip, Biol. Sci., 3(12): 1134-1139. [21] Sultana, R.S., Bari, M.A., Rahman, M.H., Rahman, M.M., Siddique, N.A., Khatun, N., 2004, In Vitro Rapid Regeneration of Plantlets from Leaf Explant of Watermelon (Citrullus lanatus Thumb.), Biotechnol., 3(2): 131-135. [22] Srivastava, D.R., Andrianov, V.M. and Piruzian, E.S., 1989, Tissue Culture and Plant Regeneration of Watermelon (Citrullus vulgaris schrad. CV. Melitopolski), Plant Cell Rep., 8: 300-302.

62 Danswrang Goyary et al [23] Thomas, T.D. and Sreejesh, K.R., 2004, Callus Induction and Plant Regeneration from Cotyledonary Explants of Ash Gourd ( Benincasa Hispida L.), Sci. Hortic., 100: 359-367.