Abstract
A highly efficient and reproducible regeneration system based on somatic embryogenesis in Gossypium hirsutum cv. Narasimha (NM), which has superior fiber qualities and is also used as a female parent in several hybrid cottons, has been developed. Embryogenic callus was obtained form both hypocotyls and cotyledonary leaves on Murashige and Skoog (MS) medium containing kinetin and 2,4-dichlorophenoxyacetic acid. Somatic embryogenesis was observed on hormone-free MS medium, but embryos did not grow well beyond globular stage. However, somatic embryos germinated well on MS medium containing B5 vitamins; addition of zeatin was found to be beneficial for their normal development. Most importantly, the media and culture conditions developed for NM were also found to be suitable for high-frequency somatic embryogenesis in Coker 310. In addition, the newly developed regeneration protocol has been successfully tested for genetic transformation through co-cultivation with Agrobacterium using embryogenic calli as explants. Molecular analysis confirmed the stable integration and expression of marker gene, green fluorescent protein (GFP). These results show that it is now possible to introduce foreign gene(s) directly into elite cultivar Narasimha with similar efficiency to in traditionally used Coker lines in a relatively short period of time. Development of efficient regeneration and transformation systems as demonstrated here should augment the introduction of new traits directly into cultivated varieties/hybrids, reducing the time required for back-crossing and the costs for seed production, besides aiding genomic research in cotton.
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Abbreviations
- NM/NA1325:
-
Narasimha
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- 2iP:
-
N6-(2-isopentenyl) adenine
- SE:
-
Somatic embryogenesis
- GFP:
-
Green fluorescent protein
- VIP:
-
Vegetative insecticidal protein
References
Cousins YL, Lyon BR, Lewellyn DJ (1991) Transformation of an Australian cultivar: prospects for cotton improvement through genetic engineering. Aus J Plant Physiol 18:481–494
Davidonis GH, Hamilton RH (1983) Plant regeneration from callus tissue of Gossypium hirsutum L. Plant Sci Lett 32:89–93
Firoozabady E, Deboer DL (1993) Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.). In Vitro Cell Dev Biol 299:166–173
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension culture of soybean root cells. Exp Cell Res 50:151–158
Kumar S, Pental D (1998) Regeneration of Indian cotton variety MCU-5 through somatic embryogenesis. Curr Sci 74:538–548
Kumar S, Sharma P, Pental D (1998) A genetic approach to in vitro regeneration of non- regenerating cotton (Gossypium hirsutum L.) cultivars. Plant Cell Rep 18:59–63
Kumria R, Sunnichan VG, Das DK, Gupta SK, Reddy VS, Bhatnagar RK, Leelavathi S (2003) High frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) though metabolic stress. Plant Cell Rep 21:635–639
Leelavathi S, Sunnichan VG, Kumria R, Vijaykanth GP, Bhatnagar RK, Reddy VS (2004) A simple and rapid Agrobacterium- mediated transformation protocol (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22:465–470
Merkle SA, Parrot WA, Flinn BS (1995) Morphogenetic aspects of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plant. Kluwer Academic, Dordrecht, pp 155–205
Mishra R, Wang HY, Yadav NR, Wilkins TA (2003) Development of highly regenerable elite Acala cotton (Gossypium hirsutum cv. Maxxa)- a step towards genotype independent regeneration. Plant Cell Tissue Organ Cult 73:21–35
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nobre J, Keith DJ, Dunwell JM (2001) Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium hirsutum L.). Plant Cell Rep 20:8–15
Rajasekaran K (1996) Regeneration of plants from cryopreserved embryogenic cell suspension and callus culture of cotton (Gossypium hirsutum L.). Plant Cell Rep 15:859–864
Rangan TS, Rajasekaran K (1993) Regeneration of cotton plants in suspension cultures. US Patent-5244, 802
Ravindranath K (2009) Narasimha-A versatile Gossypium hirsutum cultivar and proven parent of many superior hybrid cottons in India. J Indian Soc Cotton Improve (Mumbai): 34–37
Sakhanokho HF, Zipf A, Rajasekaran K, Saba S, Sharma GC (2001) Induction of highly embryogenic calli and plant regeneration in upland (G. hirsutum L.) and Pima (G. barbadense L.) cottons. Crop Sci 41:1235–1240
Stewart JM, Hsu CL (1977) In ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.). Planta 137:113–117
Sun Y, Zhang X, Jin S, Liang S, Nie Y (2003) Somatic embryogeneis and plant regeneration in wild cotton (Gossypium Klotzschianum). Plant Cell Tiss Organ Cult 75:247–253
Sun Y, Zhang X, Huang C, Guo X, Nie Y (2006) Somatic embryogenesis and regeneration from different wild diploid cotton (Gossypium) species. Plant Cell Rep 25(4):289–296
Sunilkumar G, Rathore KS (2001) Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Mol Breed 8:37–52
Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high speed transformation of rice. Plant J 47:969–976
Trolinder NL, Goodin JR (1987) Somatic embryogenesis and plant regeneration in G. hirsutum L. Plant Cell Rep 6:231–234
Trolinder NL, Goodin JR (1988a) Somatic embryogenesis in cotton (Gossypium). I. Effects of source of explant and hormone regime. Plant Cell Tiss Organ Cult 12:31–42
Trolinder NL, Goodin JR (1988b) Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration. Plant Cell Tiss Organ Cult 12:43–53
Trolinder NL, Xhixian C (1989) Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep 8:133–136
Wang S, Just DR, Anderson PP (2008) Bt cotton and secondary pests. Int J Biotechnol 10(2–3):113–121
Wang Z, Lin H, Huang JK, Hu RF, Rozelle S, Pry C (2009) Bt cotton in China: are seconday infestations offsetting the benefits in farmer fields? Agric Sci China 8(1):83–90
Wilkins TA, Mishra R, Trolinder NL (2000) Cotton biotechnology. Crit Rev Plant Sci 15:511–550
Wilkins TA, Mishra R, Trolinder NA (2004) Agrobacterium mediated transformation of cotton. Food Agric Env 2(1):179–187
Wu JH, Zhang XL, Nie YC, Jin SX, Ling SG (2004) Factor affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of Chinese cottons (Gossypium hirsutum L.). In Vitro Cell Dev Biol Plant 40:371–375
Zhang BH (2000) Regulation of plant growth regulators on somatic embryogenesis and plant regeneration. Biochemistry 39:1567
Zhang X, Jizhong S, Jinhan L (1991) Somatic embryogenesis and plant regeneration in upland cotton. Chin J Genet 18:315–322
Zhang BH, Feng R, Li XL, Li FL (1999) Direct induction of cotton somatic embryogenesis. Chin Sci Bull 44:766–767
Zhang BH, Feng R, Liu F, Wang Q (2001) High frequency somatic embryogenesis and plant regeneration of an elite Chinese cotton variety. Bot Bull Acad Sin 42:9–16
Acknowledgments
This work was supported by funds from the Department of Biotechnology (DBT), Government of India, Indian Council of Agricultural Research under the National Agricultural Innovation Project, Component-4, Government of India and the International Centre for Genetic Engineering and Biotechnology, New Delhi, India. The pCAMBIA1390-sGFP vector from Dr. Seiichi Toki, Japan is acknowledged. T.K. is a DBT PDF fellowship recipient.
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Khan, T., Reddy, V.S. & Leelavathi, S. High-frequency regeneration via somatic embryogenesis of an elite recalcitrant cotton genotype (Gossypium hirsutum L.) and efficient Agrobacterium-mediated transformation. Plant Cell Tiss Organ Cult 101, 323–330 (2010). https://doi.org/10.1007/s11240-010-9691-y
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DOI: https://doi.org/10.1007/s11240-010-9691-y