Indian Journal of Experimental Biology Vol.49, December 2011, pp 958-963 Protocol for in vitro somatic embryogenesis and regeneration of rice (Oryza sativa L.) Dipti Verma 1, Rohit Joshi 2, Alok Shukla 1 & Pramod Kumar 2 * 1 Department of Plant Physiology, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar 263 145, India 2 Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi 110 012, India Received 3 January 2011; revised 9 August 2011 Development of highly efficient and reproducible plant regeneration system has tremendous potential to provide improved technology to assist in genetic transformation of indica rice cultivars for their further exploitation in selection. For the development of a highly reproducible regeneration system through somatic embryogenesis, mature embryos of highly popular rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (aromatic basmati) and Jaya (for irrigated areas) were used. Optimum callus formation (%) to MS medium supplemented with 2, 4-D was obtained at 12.0 µm in Govind, 14.0 µm in Jaya and 15.0 µm in Pusa Basmati-1. All the cultivars showed good proliferation on MS medium without hormone. In Govind, highest embryogenic response was observed in MS medium supplemented with 2, 4-D (0.4 µm) + kinetin (0.4 µm), while in Pusa Basmati-1 with 2, 4-D (0.4 µm) + kinetin (2.0 µm) and in Jaya on hormone-free MS medium. Excellent embryo regeneration in Govind was observed on MS medium supplemented with low concentrations (1.1 µm) of BAP or hormone-free MS medium, while in Pusa Basmati-1 and Jaya embryogenesis was observed on MS medium supplemented with higher concentration of BAP (2.2 µm). Similarly, maximum plantlets with proliferated roots were observed in Govind on hormonefree MS medium, while in Pusa Basmati-1 and Jaya on MS medium supplemented with high concentration of NAA (4.0 µm). Developed plantlets were further successfully acclimatized and grown under pot culture up to maturity. Further the yield potential of in vitro developed plants was accessed at par to the direct seeded one under pot culture. Present, protocol standardizes somatic embryogenesis and efficient regeneration of agronomically important, high yielding and diverse indica rice cultivars which can be utilized as an efficient tool for molecular studies and genetic transformation in future. Keywords: Indica rice, Mature embryo, Somatic embryogenesis Rice is an important cereal crop and it is the primary source of food and calories for about half of the world s population 1. India is among the largest rice growing countries, accounting for about one-third of the world acreage under the crop. During the past few decades, tissue culture techniques have been developed that could be used for the improvement of crop plants 2. Genetic transformation coupled with tissue culture technique is an alternative for improvement through non-conventional method 3. Mature embryo in rice is arguably one of the best explants for genetic transformation because of its availability and easy to handle as an explant compared to other tissues, but it has low frequency of plant regeneration from callus through somatic embryogenesis 4,5. Moreover, using mature embryos little information is available on the improvement of in vitro regeneration particularly in indica rice than *Correspondent author Telephone: +91-09868481643 E-mail: pramodk63@yahoo.com japonica type 6. In addition, a number of economically valuable indica varieties are recalcitrant to in vitro manipulation due to their poor callus production and regeneration ability 2. Callus induction and regeneration is still a challenging task in most indica rice varieties 7. Therefore, there is basic need to use mature embryos of indica rice to develop an efficient in vitro protocol for establishing a highly reproducible regeneration system for molecular transformation and breaking seed dormancy 8. Keeping the above facts in view, the present study was aimed to develop an efficient and reproducible regeneration system through somatic embryogenesis from mature embryo derived calli of highly diverse, popular and high yielding rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (a fine grain aromatic basmati for irrigated conditions) and Jaya (a bold grain rice for irrigated areas) and to identify most suitable concentration and combination of growth regulators for excellent callus induction, somatic embryogenesis and plant regeneration.
VERMA et al.: SOMATIC EMBROYOGENESIS IN INDICA RICE 959 Materials and Methods Plant materials and growth conditions The seeds of three rice varieties Govind, Jaya and Pusa Basmati-1 were obtained from Crop Research Center, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand. Mature embryo culture was performed for the induction of somatic embryos. Mature rice seeds, were dehusked manually, washed with detergent Teepol (Reckitt Benckiser, India) for 2-5 min and thereafter, with sterile distilled water. Seeds were then surface sterilized for 10 min in ethanol (70%), followed by sodium hypochlorite (1% w/v) for 15 min and then with mercuric chloride (0.1% w/v) for 10 min and finally rinsed five times with sterile distilled water. The seeds were inoculated on MS 9 medium containing sucrose (30 g l -1 ) supplemented with different concentrations (4.5 to 20.3 µm) of 2,4-D and solidified with 8 g l -1 agar (Agar Type I, Himedia, India) to find the suitable auxin level to study the callogenic response in seed explants of rice. The cultures were incubated at 28 ± 2 C with 16 h light and 8 h dark with 60 µmol m -2 s -1 light intensity. The relative humidity within culture room was maintained at 60%. The callus induction frequency was measured as the percentage of seeds that produced a callus 5. However, for proliferation of calli, somatic embryo induction and its regeneration optimized dose of the growth regulators i.e., MS + 2, 4-D (0.4-1.0 µm) + kinetin (0.4-2.0 µm), MS + 2, 4-D (0.4-1.0 µm) + kinetin (0.4-2.0 µm), MS + BAP (1.1-2.2 µm) + kinetin (0.5-4.6 µm), MS + NAA (2.6-4.0 µm), for proliferation, somatic embryogenesis, regeneration and rooting, respectively were used for the present study, based on earlier work in rice 10-13. Proliferation of calli For callus proliferation, four week old calli were cut into small pieces and their weight was measured under aseptic condition. Thereafter, these calli were transferred onto proliferating medium containing control- (MS without hormone), (A)-{MS + 2, 4-D (0.4 µm) + kinetin (0.4 µm)} and (B)-{MS + 2, 4-D (0.4 µm) + kinetin (2.0 µm)}. After 30 days of proliferation, final weights of calli were recorded. Proliferation of the callus was measured on fresh weight (in mg) basis of proliferated calli per culture and calculated by subtracting the initial weight from the final weight of callus 14. Somatic embryo induction and regeneration For somatic embryo induction, selected calli were subcultured after 30 days in jam bottles and conical flasks containing 25 ml of control- (MS without hormone), (A)-{MS + 2, 4-D (0.4 µm) + kinetin (0.4 µm)} and (B)-{MS + 2, 4-D (0.4 µm) + kinetin (2.0 µm)} under above mentioned environmental conditions and percentage somatic embryogenesis was calculated as the percentage of calli form embryogenic callus 3. To obtain rapid regeneration, somatic embryos were transferred to the medium: control-(ms without hormone), (C)-{MS + BAP (1.1 µm)} and (D)-{MS + BAP (2.2 µm)}. The number of regenerated shoots was calculated 15 days after transferring. Two to three weeks old regenerated shoots were transferred to rooting medium containing MS without hormone, MS + NAA (2.6 µm) and MS + NAA (4.0 µm). In vitro regeneration response in different varieties was observed after one month. Thereafter, these plantlets were transferred to ½ MS liquid medium for root proliferation. Transplantation In vitro raised healthy plantlets were taken out from the culture bottles and washed gently with sterile water to remove the adhering medium completely. Thereafter, they were transferred to disposable plastic glasses containing autoclaved soil mixture and vermiculite (3:1 v/v) and irrigated with 50 ml of Hoagland solution, supplemented with 100 mg l -1 bavistin (BVN) to prevent fungal contamination. The plantlets were maintained in a greenhouse (Vista Biosciences, India) at temperature 28 ±2 C for 5-7 days. Complete plantlets were transferred to pots filled with 1:1 v/v mixture of soil and organic manure and were successfully established in field conditions. Plant height, total biomass, seed yield and its attributes were recorded per plant after harvesting. The experiments were set-up in a completely randomized block design and each treatment was replicated five times, and the average values were estimated. Results Amongst the different rice cultivars i.e., Gaurav, Jaya and Pusa Basmati-1, the significant genotypic differences in callus induction from the cultured mature embryo on MS medium supplemented with 2, 4-D were observed after 8-10 days (Fig. 1a). Optimum callus induction response in terms of fresh weight to 2, 4-D concentrations varied from one cultivar to another cultivar (Fig. 2). Govind showed optimum response in MS medium at 2, 4-D (14.0 µm), Jaya at 2, 4-D (12.0 µm) whereas, Pusa Basmati-1 at
960 INDIAN J EXP BIOL, DECEMBER 2011 2, 4-D (16.0 µm) concentration. Healthy four to five week old calli were transferred onto callus proliferating medium containing MS basal medium (hormone-free), MS + 2, 4-D (0.4 µm) + kinetin (0.4 µm) and MS + 2, 4-D (0.4 µm) + kinetin (2.0 µm) Fig. 1 Primary callus induction, somatic embryogenesis and plant regeneration from mature embryos of rice. (a) Proliferation of calli in MS basal, MS + 2,4-D (0.4 µm) + kinetin (0.4 µm) and MS + 2,4-D (0.4 µm) + kinetin (2.0 µm). (b) Somatic embryo development in MS basal, MS + 2,4-D (0.4 µm) + kinetin (0.4 µm) and MS + 2,4-D (0.4 µm) + kinetin (2.0 µm). (c-d) Regeneration of shoots in MS basal, MS + BAP (1.1 µm) and MS + BAP (2.2 µm). (e) Root induction of shoots in MS basal, MS + NAA (2.6 µm) and MS + NAA (4.0 µm), (f) Normal fertile plantlet regenerated from mature embryo growing in greenhouse, (g) Hardening of regenerated plantlets in pot culture. Fig. 2 Influence of different levels of 2, 4-D on callus induction from mature embryos of rice. (Table 1). Significant genotypic differences were observed in callus proliferation among different media combinations. All the cultivars showed good proliferation on hormone free MS medium as compared to MS medium supplemented with 2,4-D + kinetin. Highest proliferation was observed in cultivar Jaya in MS basal medium followed by Govind. Well proliferated one month old calli were further transferred for somatic embryogenesis in the MS without hormone, MS + 2, 4-D (0.4 µm) + kinetin (0.4 µm) and MS + 2, 4-D (0.4 µm) + kinetin (2.0 µm) (Fig. 1b). After 15 days the calli were subcultured in the same medium and per cent somatic embryogenesis in different rice cultivars was estimated (Table 1). Significant genotypic differences were observed in frequency of somatic embryogenesis in different media combinations. Govind showed the maximum frequency of somatic embryogenesis in MS medium supplemented with 2, 4-D (0.4µM) + kinetin (0.4µM) and in Pusa Basmati-1 with 2, 4-D (0.4µM) + kinetin (2.0µM) while in Jaya MS basal medium without growth hormone. Significant genotypic differences in number of shoots were observed 15 days after transferring in different media combinations i.e., MS medium (hormone free), MS + BAP (1.1 µm) and MS + BAP (2.2 µm) (Fig. 1c-d). In Govind, maximum number of shoots per embryogenic calli was observed on MS + BAP (1.1 µm) or MS basal medium, while in Pusa Basmati-1 and Jaya maximum number of shoots per embryogenic calli on MS + BAP (2.2 µm) (Table 1). On transferring the elongated shoots to rooting media i.e., MS medium (hormone free), MS + NAA (2.6 µm) and MS + NAA (4.0 µm) (Fig. 1e) maximum plantlets with proliferated roots were observed in Govind on hormone free MS medium, while in Pusa Basmati-1 and Jaya on MS + NAA (4.0 µm) (Fig. 3). The regenerated plants over 90 per cent were successfully established in the pot culture conditions. Further, the fully developed in vitro regenerated plants after transferring in soil, showed healthy growth and normal seed yield after harvesting (Table 2; Fig. 1f-g). Discussion In the present study in vitro, significant genotypic response to 2, 4-D was observed in callus induction. For primary callus induction and embryogenic callus
VERMA et al.: SOMATIC EMBROYOGENESIS IN INDICA RICE 961 Table 1 Influence of MS medium supplemented with different concentrations of growth hormones on callus proliferation, % frequency of somatic embryogenesis and number of shoots developed per embryogenic calli. [Values are mean ± SEM of five replicates] Treatment Fresh weight (mg) of proliferated callus in rice cultivars Govind Jaya Pusa Basmati-1 Mean Control 67.67±1.06 75.17±1.80 62.80±2.42 64.58 A 55.78±2.10 61.86±1.44 56.68±1.53 62.07 B 57.02±1.34 52.59±2.15 51.81±2.13 53.81 Mean 60.16 63.22 57.10 S.Em.± 1.98 CD at 5% Treatment (T) 2.71 Cultivar (C) 3.15 T X C 5.87 Frequency of somatic embryogenesis (%) Control 51.18±2.32 65.99±2.16 54.76±2.65 57.31 A 46.83±1.55 59.84±1.26 67.77±2.33 47.78 B 69.03±3.42 46.70±2.91 49.80±2.80 55.18 Mean 55.68 57.51 57.44 S.Em.± 1.48 CD at 5% Treatment (T) 3.62 Cultivar (C) 4.18 T X C 7.25 Number of shoots per embryogenic calli Control 9.27±0.35 7.47±0.24 6.53±0.18 7.76 C 9.63±0.88 4.03±0.45 7.37±0.15 7.01 D 6.73±0.35 9.30±0.36 10.10±0.38 8.71 Mean 8.54 6.93 8.00 S.Em.± 0.38 CD at 5% Treatment (T) 0.55 Cultivar (C) 0.64 T X C 1.11 Treatment: (A)- 2, 4-D (0.4µM) + Kinetin (0.4µM); (B)- 2,4-D (0.4µM) + Kinetin (2.0µM); (C)- BAP 1.1 µm; (D)- BAP 2.2 µm; (Control)-Phytohormone free formation from immature or mature embryo, 2, 4-D is considered critical at concentrations between 10-5 to 10-7 M in cereals 6. Moreover, differences in 2, 4-D concentration required for callus proliferation may be due to the genetic variation in endogenous levels of phytohormones which influence the callus proliferation 15. However, at higher concentration of 2, 4-D all the cultivars showed similar tendency of decrease in callus induction. In our study, all the cultivars showed good proliferation on hormone free MS medium as compared to MS medium supplemented with 2,4-D + kinetin. Jaya showed highest proliferation in MS basal medium followed by Govind. The present results were similar to the earlier reports 12. In the present study, Govind and Pusa Basmati-1 had maximum frequency of somatic embryogenesis in MS medium supplemented with 2, 4-D and kinetin, while Jaya showed good embryogenic response in hormone free MS medium. The hormone, 2, 4-D, Fig. 3 Number of shoots having roots in different rice cultivars on MS media supplemented with different concentrations of NAA.
962 INDIAN J EXP BIOL, DECEMBER 2011 Table 2 Comparative account of different growth and yield parameters of control and in vitro developed somaclones Cultivars Parameters Govind Jaya Pusa Basmati-1 CD at 5 % DS TC DS TC DS TC Plant height (cm) 91.33 88.23 79.34 82.00 94.55 91.33 6.52 Tiller number/plant 8.12 8.53 11.33 11.67 9.33 9.07 2.98 Panicle number/plant 6.73 6.55 8.30 8.50 7.36 7.12 1.58 Shoot weight (g) 50.84 53.26 68.47 71.25 64.00 62.67 5.98 Length of panicle (cm) 21.58 21.33 22.92 22.17 25.67 23.25 0.18 Weight of panicle 3.97 3.10 5.12 4.75 4.17 4.30 0.064 Number of grain/panicle 94.89 94.53 209.33 173.67 123.33 122.50 5.99 1000-grain weight 25.39 25.86 38.33 33.00 29.63 26.24 2.43 Grain straw ratio 0.39 0.37 0.61 0.57 0.45 0.42 0.064 Grain yield/plant 6.91 6.85 12.80 12.35 9.26 8.17 1.59 (DS= Direct seeded plants, TC= Tissue culture developed somaclones) has been reported to promote somatic embryogenesis and plays important role in dedifferentiation and cell division in rice 3,16 and also influence endogenous IAA in explants tissues 15. These genotypic variations are probably due to differential expression depending upon the spatial and temporal distribution, their physiological and developmental stages 17. Somatic embryogenesis and organogenesis both are triggered by auxins and cytokinins 6,18,19. In the present investigation, kinetin was found to be effective for induction of somatic embryos. Role of cytokinins on somatic embryogenesis has been explained by enhanced cell division of pre-embryogenically determined cells 20. In addition to this, kinetin enhances callus proliferation and regeneration by influencing mitosis, cytokinesis, total protein synthesis, lignin biosynthesis, vascular differentiation and differentiation of mature chloroplasts from protoplastids 21. Marked genotypic variations in number of shoots on MS medium supplemented with different concentrations of BAP and proliferated roots on MS medium supplemented with varying levels of NAA are in agreement with earlier reports 2,22,23. Stimulatory effect of BAP in combination with NAA has previously been reported to facilitate regeneration in indica rice callus cultures 22,23. Similarly, in indica rice cultivars HKR-46 and HKR-126 have shown maximum plantlet regeneration in MS + kinetin (2.0 mg l -1 ) and NAA (0.5 mg l -1 ) 24. This may be because cytokinin causes development of single pole and formation of meristematic dome from which the shoot primordium arises. High auxin concentration leads to development of root primordium leading to root development 25. However, one of our cultivar Govind showed decrease in root development at high auxin concentration. This, in turn, suggested that genotypes also possessed variability in their capability of regeneration as reported earlier 7. An important aspect of the present protocol was that all the plantlets regenerated from mature embryo derived calli developed good root system. Moreover, after transferring to pots, in vitro regenerated plants of all cultivars survived and showed seed yield parameters at par to the direct seeded one in pot culture which in turn indicated good adaptability of somaclones in ambient environmental conditions. The present work reported a successful indica rice high frequency regeneration protocol from mature embryo through somatic embryogenesis in three diverse and highly productive cultivars Govind, Jaya and Pusa Basmati-1, belong to Indian subcontinent, which showed yield potential of in vitro developed plants at par to that of direct seeded plants in ambient conditions. The present protocol would be useful for genetic transformation in elite genotypes of rice using any selected gene transfer method in future. References 1 Vega R, Vásquez N, Espinoza A M, Gatica A M & Valdez- Melara M, Histology of somatic embryogenesis in rice (Oryza sativa cv. 5272), Int J Trop Biol, 57 (2009) 141. 2 Ramesh M, Murugiah V & Gupta A K, Efficient in vitro plant regeneration via leaf base segments of indica rice (Oryza sativa L.), Indian J Exp Biol, 47 (2009) 68. 3 Panjaitan S B, Abdullah S N A, Aziz M A, Meon S & Omar O, Somatic embryogenesis from scutellar embryo of Oryza sativa L. var. MR219, Pertanika J Trop Agric Sci, 32 (2009) 185. 4 Chen J Y, Yue R Q, Xu H X & Chen X J, Study on plant regeneration of wheat mature embryos under endosperm supported culture, Agric Sci China, 5 (2006) 572.
VERMA et al.: SOMATIC EMBROYOGENESIS IN INDICA RICE 963 5 Lee K W, Choi G J, Kim K Y, Ji H C, Park H S, Yoon S H & Lee S H, High frequency plant regeneration from mature seed derived callus of Italian ryegrass (Lolium multiflorum) cultivars, African J Biotech, 8 (2009) 6828. 6 Jia X X, Zhang J W, Wang H N & Kong W P, Efficient maize (Zea mays L.) regeneration derived from mature embryos in vitro, Maydica, 53 (2008) 239. 7 Kumria R, Waie B, Pujni D & Rajam M V, Biotechnology of rice: Present limitations and future prospects, Plant Cell Biotech Mol Biol, 1 (2000) 1. 8 Hoque M N, Rahman L & Hassan L, Effect of culture media on seed dormancy and callus induction ability of some wild and cultivated rice genotypes, Biotechnology, 6 (2007) 61. 9 Murashige T & Skoog F, A revised medium for rapid growth bioassays with tobacco tissue culture, Physiol Plant, 15 (1962) 473. 10 Joshi R, Physiological and molecular evaluation of field grown rice varieties and in vitro developed rice somaclones for aerobic situations. Thesis submitted to G.B. Pant University of Agriculture and Technology, Pantnagar, India. (2007). 11 Verma D, In vitro development of drought tolerant lines in rice (Oryza sativa L.). Thesis submitted to G.B. Pant University of Agriculture and Technology, Pantnagar, India. (2009). 12 Kaur D, Shukla A & Pant R C, In vitro development of low phosphorus tolerant lines in rice (Oryza sativa L.), J Plant Biol, 36 (2009) 65. 13 Joshi R, Shukla A & Sairam R K, In vitro screening of rice genotypes for drought tolerance using polyethylene glycol. Acta Physiol Plant, (2011) DOI 10.1007/s11738-011-0760-6. 14 Agarwal R S, Formulas of arithmetics, (S.Chand Publication, New Delhi) 2003, 6. 15 Deo P C, Harding R M, Taylor M, Tyagi A P & Becker D K, Somatic embryogenesis, organogenesis and plant regeneration in taro (Colocasia esculenta var. esculenta), Plant Cell Tissue Organ Cult, 99 (2009) 61. 16 Meneses A, Flores D, Muñoz M, Arrieta G & Espinoza A M, Effect of 2,4-D, hydric stress and light on indica rice (Oryza sativa) somatic embryogenesis, Rev Biol Trop, 53 (2005) 361. 17 Indra A P & Krishnaveni S, Effect of hormones, explants and genotypes in in vitro culturing of Sorghum, J Biochem Tech, 1 (2009) 96. 18 Liu M, Yang J, Lu S, Guo Z, Lin X & Wu H, Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.), In vitro Cell Dev Biol-Plant, 44 (2008) 100. 19 Chaudhury A & Qu R, Somatic embryogenesis and plant regeneration of turftype bermudagrass, effect of 6-benzyladenine in callus induction medium, Plant Cell Tissue Organ Cult, 60 (2000) 113. 20 Kintzios S, Sereti E, Bluchos P, Drossopoulos J B, Kitsaki C K & Tsakalidis L A, Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Cucurbita pepo L.) and melon (Cucurbita melo L.), Plant Cell Rep, 21 (2002) 1. 21 Wan Y, Sorensen E L & Liang G H, The effects of kinetin on callus characters in alfalfa (Medicago sativa L.), Euphytica, 39 (1988) 249. 22 Mandal A B, Maiti A & Biswas A, Somatic embryogenesis in root derived callus of Indica rice, Plant Tissue Cult, 13 (2003) 125. 23 Karthikeyan A, Pandian S T K & Ramesh M, High frequency plant regeneration from embryogenic callus of a popular indica rice (Oryza sativa L.), Physiol Mol Biol Plant, 15 (2009) 371. 24 Saharan V, Yadav R C, Yadav N R & Chapagain B P, High frequency plant regeneration from desiccated calli of indica rice (Oryza sativa L.), African J Biotech, 3 (2004) 256. 25 Khan T, Singh A K & Pant R C, Regeneration via somatic embryogenesis and organogenesis in different cultivars of cotton (Gossypium spp.), In Vitro Cell Dev Biol-Plant, 42 (2006) 498.