Abstract: Multiple shoots from the immature cotyledonary nodes of two genotypes of bitter gourd were induced in MS media with GA3, IAA, IBA, NAA, KIN and BAP. The shoots continued to increase in number for 5-6 weeks. On the medium, BAP 2 mg L-1 in combination with 0.1 mg L-1 IAA + 2 mg L-1 GA3 was the most effective medium for adventitious shoot proliferation from immature cotyledonary node. BGGB1 and BGGB14 genotypes produced over 84 and 80% shoots, respectively on this medium in 6 weeks from the time at inoculation of primary explants. BGGB1 was found to be better in response than BGGB14 in most of the treatments. Rapid and reproducible transformation system for bitter gourd using Agrobacterium tumefaciens mediated gene delivery was developed. Immature cotyledonary node from green immature fruits that has been allowed to imbibe was used as explant and regeneration was achieved. Cotyledonary node was co-cultured in MS media with 2.0 mg L-1 BAP + 0.1 mg L-1 IAA + 2.0 mg L-1 GA3. Histochemical GUS assay of the explants and shoots (newly formed) revealed that they were GUS positive.
INTRODUCTION
The most potent biotechnological approach is the transfer of specifically constructed gene assemblies through various transformation techniques; this is constitutes genetic engineering. The plants obtained through genetic engineering contain a gene or genes usually from an melated organism are known as transgenic plants. Bitter gourd belongs to the Cucurbitaceae family and eaten throughout the world, specially for its medicinal properties[1]. It is used for stomach pain, diabetes, fevers, colds, headaches, menstrual disorders, hypertension and defend HIV. It also plays an important role in the agro-economy of our cmmtry and costlier compared to the other vegetable available in the season. Bitter gourd is susceptible to many diseases and insect pests that affected squash, cucumber and muslanelon. It is a host for watermelon mosaic virus and may be susceptible to other cucurbit viruses. Powdery mildew is a destructive pathogen of bitter gourd. Taking these facts rmder consideration disease resistant and pest resistant traits of bitter gourd should be developed by using transformation techniques. Therefore, the objective of the present study was to establish a protocol on Agrobacterium� mediated transformation in bitter gourd by using immature cotyledonary nodes.
MATERIALS AND METHODS
The successful transformation to plants depends on the availability of a suitable protocol which compatible with in vitro plant regeneration methods of the target plant species. Cotyledonary nodes of two genotypes of bitter gourd were transformed with Agrobacterium tumefaciens strain LBA4404 (pBxpBI121 ). Expression of neomycm phosphotransferase(npt II) encoding resistance to kanamycin was used as a selectable marker. Agrobacteriumtumefacienscultures were grown overnight in YM8 medium at 28°C on a shaker at 200 rmp and then diluted in liquid MS medium to varying concentration. The explants were then incubated in the bacterial suspension for 30 min and placed on the co-cultivation medium which consisted of MS medium supplemented with sucrose 3% (w/v), agar 0.6% (w/v), GA3, (2.0 mg L-1), BAP or KIN (0.5-3.0 mg L-1) and IAA or IBA or NAA (0.1 mg L-1). The duration of co-cultivation varied from 2-5 days. After the co-cultivation time, the inoculated explants were rinsed in MS liquid medium containing cefatoxin 100 mg L-1, blotted dry with sterile filter paper and cultured on selective medium which consisted of 100 mg L-1 cefatoxin and 0.05 mg mL-1 kanamycin per selection and incubated at 26�1 °C with a 16 h photoperiod. After 7-8 weeks, the explants showed regeneration. The green shoots were cultured in rooting medium containing MS with IAA or IBA or NAA (0.2, 0.4, 0.6 and 0.8 mg L-1). The expression of -glucuronidase gene in the transformed plants were analysed. The plant was dipped into histochemical reagent, X-gluc and incubated for 24-48 hat 37°C. After X-gluc treatment, plant was transformed to 70% ethanol for de-green. Following de-green, plant was observed rmder stereo microscope which was confirmed the transformation of GUS gene.
RESULTS AND DISCUSSION
The physiological state of explants appears to be the key point for the success of regenerating plants. In this study, regeneration system of bitter gourd has been established from immature cotyledonary node, the main advantage of immature cotyledonary node explants being their availability through the whole year. The cotyledonary nodes were cultured on MS media with BAP, KIN, GA,, IAA, IBA and NAA for induction of multiple shoots. The shoots continued to increase in number for 5-6 weeks. MS medium supplemented with 2.0mgL-1 BAP, 0.1 mg L-1 IAA with2.0mg L-1 GA3 was the most effective medium for shoot proliferation of bitter gourd. BGGBl and BGGB14 genotypes of bitter gourd produced over 84 and 80% shoots, respectively on the medium six weeks from the time of inoculation of primary explants and was followed by 80 and 78% in 2.0 mg L-1 BAP + 0.1 mg L-1 IBA + 2.0 mg L-1 GA3, respectively (Table 1).
| Table 1: | Effects of different concentrations and combinations of growth regulators on multiple shoot regeneration from immature cotyledonary node |
To have a continuous supply of shoots sub-culturing on fresh medium was reported at 2 weeks intervals. However, the addition of low concentration of auxin (IBA and IAA) to BAP supplemented media enhanced shoot regeneration efficiency and IAA was formd superior to IBA. BGGB1 was formd to be better in response than BGGB14 for most of the treatments. The average total number of shoots formed from one explant was about 12.7�0.38andl0.7�0.27 inBGGBl andBGGB14, respectively (Table 1).Similar results were observed by Ahad et al. [2] in watermelon, in their experiment they observed that the MS medium withBA (0.5-1 mg L-1), 0.1 mg L-1 IAA was suitable for adventitious shoot regeneration and with addition of 100 mg L-1 CH improved shoot regeneration efficiency. Islam et al.[3]reported that maximum regeneration occmed on 1.0 mg L-1 BA and 0.1 mg L-1 IAA in Aegle mormelons Corr. Sarker et al.[4]reported that the MS medium supplied with 0.5 mg L-1 BAP, 0.5 mg L -1 KIN and 0.1 mg L-1 GA, and 5.5 mg L-1 tyrosine was the best combination for multiple shoot regeneration from cotyledonary nodes in lentil. The immature cotyledonary nodes are also good source for initiating shoot formation as has been shown by Arnin andAkhtar[5], Maity et al.[6] and Banerjee et al.[7].
Aseptically cotyledonary node explants of bitter gourd were transformed withAgrobacterium tumefaciens strain LBA4404 (pBI121). Expression of neomycin phosphotransferase (npt-II) encoding resistance to kanamycin was used as a selectable marker. The best growth of cotyledonary node was observed in MS with 2.0mgL-1 BAP+O.I mg L-1 IAA + 2.0mg L-1 GA,.
| Table 2: | GUS assay and regeneration ability of transformed cotyledonary |
The optimal procedure involved exposing the explants to a bacterial suspension (1.47 OD) followed by 3 days cocultivation period (Table 2).
After co-cultivation, infected explants were transferred to the selection media for 5-6 weeks after infection and some explants were subjected to histochemical assay to verify the transient expression of GUS. The grovvth of the newly appeared cultures were slow at first but become vigorous within 3-4 weeks. At that time, in some culture media high bacterial growth was detected which was prevented by frequent sub-culturing (3 days) of the explants on the selected media. Most of the explants for cotyledonary node produced a few shoots on selected media (kanamycin containing) were GUS positive.
The expressiOn of -glucuronidase gene m transformed explants and regenerated shoots were analyzed as described by Jefferson et al.[8] Histochemical GUS assay of the explants and shoots (newly formed) revealed that they were GUS positive. This study also provides a basis for further research to generate transgenic bitter gourd.
Agrobacterium based vectors has been successfully employed to transfer gene into a number of dicotyledonous plants and a few grain and legurnes[9,10] Similarly reported by Nehra et al.[11]l in strawberry, Sarker and Islam[12] in peanut, Southgate et al.[13] in maize, Ahmed et al.[14] in tobacco, Lolananhakim[15] in potato, Peng et al.[16] in rice, Cardi[17] in Solanum commersonii DW1, Frankiin et al.[18] in greenbean and Noorain et al.[19]m nee. But Geetha et al.[20]failed to successfully transfer of Agrobocterium strain LBA4404 (pB x pBI121) in blackgram.