Abstract
Three different regeneration systems, viz. regeneration through callus cultures using embryonic explant, direct regeneration using shoot bud/nodal segments as explant and regeneration through cell suspension culture using cotyledonary explant (for the induction of transgenic callus for suspension culture) were evaluated to see their effect on transfer of Cry1A(b) gene to Punica granatum L. cv. Kandhari Kabuli through Agrobacterium mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency of different explants. Out of different explants used (embryo, shoot bud, and cotyledon) for different regeneration systems cotyledonary explant showed highest putative transformation frequency (13.54%) inducing callus on selective medium for carrying out cell suspension culture to regenerate transgenic shoots. Despite of the highest transformation frequency obtained from the cotyledon explant, the plating efficiency of the transgenic cells generated through the transgenic callus (callus formed from the cotyledonary explant) during cell suspension culture was found to be very low (0.7%). Thus the plating efficiency has also played worth mentioning role in the regeneration of transformants following cell suspension culture. Among the three regeneration systems, regeneration through callus cultures using embryonic explant was found to be best for regeneration of transformants. The highest per cent regeneration of 23.33 was obtained from the putative transgenic embrogenic calli. Successful genetic transformation in the transformed plantlets was confirmed by PCR analysis. The transformation system thus developed is valuable and may be used to produce insect resistant trees.
Similar content being viewed by others
References
Agarwal S, Kanwar K. 2007. Comparison of genetic transformation in Morus alba L. via different regeneration systems. Plant Cell Rep. 26: 177–185
Agarwal S, Kanwar K, Saini NS, Jain RK. 2004. Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L. Sci. Hortic. 100: 183–191
Ainsley PJ, Collins GG, Sedgley M. 2001. Factors affecting Agrobacterium mediated gene transfer and selection of transgenic calli in paper shell almond (Prunus dulcis Mill.). J. Hortic. Sci. Biotech. 76: 522–528
Deepika R, Kanwar K. 2010. In vitro regeneration of Punica granatum L. plants from different juvenile explants. J. Fruit Ornamental Plant Res. 18: 5–22
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem. Bull. 19: 11–15
Du NX, Pijut PM. 2009. Agrobacterium mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration. Plant Cell Rep. 28: 915–923
Durzan DJ. 1990. Adult vs. Juvenile explants: directed totipoptency. In R Rodriuez, R Sancez-Tames, DJ Durzan, eds, Plant Aging, Basic and Applied Approaches, Vol. 186. Plenum Press, New York, pp 19–25
Ercisli S, Agar G, Orhan E, Yildirim A, Hizarci Y. 2007. Interspecific variability of RAPD and fatty acid composition of some pomegranate cultivars (Punica granatum L.) growing in Southern Anatolia Region in Turkey. Biochem. Sys. Ecol. 35: 764–769
Falco MC, Mendes BMJ, Tulmann NA. 1996. Cell suspension culture of sugarcane: growth, management and regeneration. Rev. Bras. Fisiol. Veg. 8: 1–6
Ghorbel R, Juarez J, Navarro L, Pena L. 1999. Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theor. Appl. Genet. 99: 350–358
Hammerschlag FA, Zimmerman RH, Yadava UL, Hunsucker S, Gercheva P. 1997. Effect of antibiotics and exposure to acidified medium on the elimination of Agrobacterium tumefaciens from apple leaf explants and on shoot regeneration. J. Am. Soc. Hortic. Sci. 122: 758–763
Ho CK, Chang SH, Tsay JY, Tsai CJ, Cheang VL, Chen ZZ. 1998. Agrobacterium tumefaciens mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. Plant Cell Rep. 17: 675–680
Hu Z, Wu YR, Li W, Gao HH. 2006. Factors affecting Agrobacterium tumefaciens mediated genetic transformation of Lycium barbarum L. In Vitro Cell Dev. Biol. Plant. 42: 461–466
Kakar KL, Dogra GS, Nath A. 1987. Incidence and control of pomegranate fruit borers, Virachola isocrates Fb. and Deudorix epijarbas Moore. Indian J. Agr. Sci. 57: 749–752
Kanwar K, Bhardwaj A, Deepika R. 2009. Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of Robinia pseudoacacia L. Plant Cell Tiss. Org. Cult. 96: 95–103
Kanwar K, Joseph J, Deepika R. 2010. Comparison of in vitro regeneration pathways in Punica granatum L. Plant Cell Tiss. Org. Cult. 100: 199–207
Kanwar K, Kaushal B, Abrol S, Deepika R. 2008. Plant regeneration in Robinia pseudoacacia from cell suspension cultures. Biol. Plant. 52: 187–190
Kavitah G, Taghipour F, Huyop F. 2010. Investigations of factors in Agrobacterium mediated gene transfer in Citrullus lanatus cv. Round Dragon. J. Biol. Sci. 10: 209–216
Khawale RN, Singh SK, Garg G, Baranwal VK, Ajirlo A. 2006. Agrobacterium mediated genetic transformation of Nagpur mandarin (Citrus reticulate Blanco). Curr. Sci. 91: 1700–1705
Kumar J. 2005. Evaluation of pomegranate cultivars in mid hill zone of Himachal Pradesh. Haryana J. Hortic. Sci. 34: 1–2
Lam SL. 1977. Regeneration of plantlets from single cells in potatoes. Am. Potato J. 54: 575–580
Mir MM, Sofi AA, Singh DB, Khan FU. 2007. Evaluation of pomegranate cultivars under temperate conditions of Kashmir Valley. Indian J. Hortic. 64: 150–154
Moriguchi T, Omura T, Matsuta N, Kozaki I. 1987. In vitro adventitious shoot formation from anthers of pomegranate. Hortic. Sci. 22: 947–948
Mullins MG, Tang FCA, Facciotti D. 1990. Agrobacterium mediated genetic transformation of grapevines: transgenic plants of Vitis rupestris Scheele and buds of Vitis vinifera L. Bio-Technology 8: 1041–1045
Naik SK, Chand PK. 2003. Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.). J. Plant Physiol. 160: 423–430
Naik SK, Chand PK. 2011. Tissue culture-mediated biotechnological intervention in pomegranate: a review. Plant Cell Rep. 30: 707–721
Naik SK, Pattnaik S, Chand PK. 1999. In vitro propagation of pomegranate (Punica granatum L. cv. Ganesh) through axillary shoot proliferation from nodal segments of mature tree. Sci. Hortic. 79: 175–183
Narayanan ES. 1954. The anar butterfly Virachola Isocrates. Indian Fmg. 4: 8–9
Omura M, Matsuta T, Moriguchi T, Kozaki I. 1987. Adventitious shoot and plantlet formation from cultured pomegranate leaf explants. HortScience 22: 133–134
Omura M, Matsuta N, Moriguchi T, Kozaki I. 1990. Suspension culture and plantlet regeneration in dwarf pomegranate (Punica granatum L. var. nana Pers.). Bull. Fruit Tree Res. Stn. 17: 19–33
Pena L, Perez RM, Cervera M, Jaurez JA and Navarro L. 2004. Early events in Agrobacterium mediated genetic transformation of citrus explants. Ann. Bot. 94: 67–74
Prasad R, Mali PC. 2000. Changes in physio chemical characteristics of pomegranate squash during storage. Indian J. Hortic. 57: 18–20
Salgado JM, Ferreira TRB, Biazatto FD, Dias CTS. 2012. Increased Antioxidant Content in Juice Enriched with Dried Extract of Pomegranate (Punica granatum) Peel. Plant Foods Hum. Nutr. 67: 39–43
Saxena A, Vikram NK. 2004. Role of selected Indian plants in management of type 2 diabetes: a review. J. Altern. Complement Med. 10: 369–378
Scorza R, Levy L, Damsteegt V, Yepes LM, Cordts JM, Hadidi A, Slightum J, Gonsalves D. 1995. Transformation of plum with papaya ringspot virus coat protein gene and reaction of transgenic plant to plum pox virus. J. Am. Soc. Hortic. Sci. 120: 943–952
Sharon M, Sinha S. 2000. Plant regeneration from cotyledonary node of Punica granatum L. Indian J. Plant Physi. 5: 344–348
Srikandharajah S, Goodwin P. 1998. Conditioning promotes regeneration and transformation leaf explants. Plant Cell Tiss. Org. Cult. 53: 1–11
Terakami S, Matsuta N, Yamamoto T, Sugaya S, Gemma H, Soejima J. 2007. Agrobacterium mediated transformation of the dwarf pomegranate (Punica granatum L. var. nana). Plant Cell Rep. 26: 1243–1251
Touchet B, Duval Y, Pannetier C. 1991. Plant regeneration from embryogenic suspension cultures of oil palm (Elaeis guineensis Jacq.). Plant Cell Rep.10: 529–532
Vidal N, Mallon R, Valladares S, Meijomin AM, Vieitez AM. 2010. Regeneration of transgenic plants by Agrobacterium-mediated transformation of somatic embryos of juvenile and mature Quercus robur. Plant Cell Rep. 29: 1411–1422
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Verma, V., Kanwar, K., Tufchi, M. et al. Agrobacterium-mediated Cry1A(b) gene transfer in Punica granatum L. cv. Kandhari Kabuli using different In Vitro regeneration pathways. J. Crop Sci. Biotechnol. 17, 1–10 (2014). https://doi.org/10.1007/s12892-013-0033-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12892-013-0033-6