Transformation of Gourd through Leaf Explant Regeneration

잎 절편의 재분화에 의한 참박 형질전환

  • Published : 2006.10.01

Abstract

In order to develop a disease-resistant root stock for the growth of watermelon, an efficient regeneration system of the gourd(Lagenaria leucantha Duch.) inbred line GO701-2 via organogenesis was established in this experiment. Using proximal parts of cotyledon explant excised from germinated seedling in vitro, maximum adventitious shoot formation (39%) was achieved on MS medium where cytokinin (BA) and auxin (IAA) were added at a concentration of 3mg/L and 0.1mg/L, respectively. Roots of the elongated shoots were successfully formed on MS medium without adding any plant growth regulators. The cucumber CsGolS1 gene known as a resistance gene against biotic and abiotic stresses, was constructed into the binary vector pBI121 under the control of CaMV 35S promoter. When the gene was introduced into the genome of gourd by Agrobacterium-mediated transformation, putative transgenic plants were obtained with the transformation efficiency of approximately 20 percent.

수박의 대목으로 사용되고 있는 참박의 재분화조건을 확립함으로서 병저항성 형질전환 식물체를 얻어 보고자 본 실험을 실시하였다. 참박의 자엽을 잘라 재분화에 미치는 식물생장조절물질의 효과를 조사하였고 오이 galactinol synthase (CsGolS1) 유전자를 참박에 형질전환하였던 결과는 다음과 같다. 참박 신초의 재분화는 7일째 된 유묘의 자엽부위 중 전반부위에서 기장 높은 효율을 나타내었고, cytokinin으로 BA나 zeatin을 단용처리하는 것보다 옥신으로 IAA를 혼용처리하는 것이 신초 분화에 더 효과적이었다. 복합스트레스 내성 유전자인 오이 CsGolS1유전자를 pBI121 binary vector에 재조합하여 아그로박테리움을 이용, 참박에 형질전환하였던 결과, 형질전환체는 kanamycin이 첨가된 배지에서 생장하였고 PCR 및 Southern blot 분석에 의해 유식물체의 20% 정도가 형질전환체임을 확인할 수 있었다.

Keywords

References

  1. Blackman, S.A., R.L. Obendorf and A.C. Leopold. 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100: 225-230 https://doi.org/10.1104/pp.100.1.225
  2. Brenac, P., M. Horbowicz, S.M. Downer, A.M. Dickerman, M.E. Smith and R.L. Obendorf. 1997. Raffinose accumulation related to desiccation tolerance during maize (Zea mays L.) seed development and maturation. J. Plant Physiol. 150: 481-488 https://doi.org/10.1016/S0176-1617(97)80102-2
  3. Compton, M.E. 1999. Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon. Plant Cell Tissue Organ Cult. 58: 185-188 https://doi.org/10.1023/A:1006364013126
  4. Curuk, S., G. Ananthakrishnan, S. Singer, X. Xia, C. Elman, D. Nestel, S. Cetiner and V. Gaba. 2003. Regeneration in vitro from the hypocotyls of Cucumis species produces almost exclusively diploid shoots, and does not require light. HortSci. 38: 105-109
  5. Dirks, R. and M. van Buggenum. 1989. in vitro plant regeneration fromleaf and cotyledon explants of Cucumis melo L. Plant Cell Rep. 7: 626-627 https://doi.org/10.1007/BF00272045
  6. Dong, J.Z and S.R. Jia. 1991. High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad.) Plant Cell Rep. 9: 559-562
  7. Ezura, H., H. Amagai, K. Yoshioka and K. Oosawa. 1992. Highly frequent appearance of tetraploidy in regenerated plants, a universal phenomenon, in tissue culture of melon (Cucumis melo L.). Plant Sci. 85: 209-213 https://doi.org/10.1016/0168-9452(92)90117-5
  8. Gambley, R.L. and W.A. Dodd. 1990. An in vitro technique for the production de novo of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.). Plant Cell Tissue Organ Cult. 20: 177-183
  9. Gonsalves, C., B. Xue, M. Yepes, M. Fuchs, K. Ling and S. Namba. 1994. Transferring cucumber mosaic virus-white leaf strain coat protein gene intoCucumis melo L. and evaluating transgenic plants for protection against infection. J. Am. Soc. Hort. Sci. 119: 345-355
  10. Kim, M.S., J.S. Chung, Y.J. Im, S.M. Cho, H. Hwangbo, Y.C. Kim, C.M. Ryu, K.Y. Yang, K.Y. Kim, G.C. Chung, M.Y. Eun and B.H. Cho. 2006. Galactinol as a novel priming component on plant innate immunity elicited by Pseudomonas chlororaphis O6 . Plant Cell 18: in press
  11. Kim, S.G., J.R, Chang, H.C. Cha and K.W. Lee. 1988. Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.). Plant Cell Tissue Organ Cult. 12: 67-74 https://doi.org/10.1007/BF00043109
  12. Lee, Y.K., W.I. Chung and H. Ezura. 2003. Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.). Plant Sci. 164: 413-418 https://doi.org/10.1016/S0168-9452(02)00429-6
  13. Mohiuddin, A.K.M., M.K.U. Chowdhury, C. Abdullah-Zaliha and S. Napis. 1997. Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tissue Organ Cult. 51: 75-78 https://doi.org/10.1023/A:1005814514409
  14. 박경석 남상현 김충회. 1994. 수박대목용 참박에 발생한 Monosporascus cannonballus에 의한 검은점뿌리썩음병(黑点根腐病). 한국식물병리학회지. 10(3): 175-180
  15. 박진우, 천정욱, 최홍수. 2000. 종자전염바이러스발생생태 및 방제에 관한 연구.수원농업기술연구소.작물바이러스관리기술연구보고서
  16. Sambrook, J. and D.W. Russell. 2000. Molecular cloning: A laboratory manual Ed 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  17. Srivastava, D.R., V.M. Andrianov and E.S. Piruzian. 1989. Tissue culture and plant regeneration (Citrullus vulgaris Schrad, cv, Melitopolski). Plant Cell Rep. 8: 300-302
  18. Taji, T., C. Ohsumi, S. Inchi, M. Seki, M. Kasuga, M. Kobayashi, K. Yamaguchi-Shinozaki and K. Shinozaki. 2002. Important roles of drought-and cold inducible genes for galactinol synthase in stress tolerance inArabidopsis thaliana. Plant J. 29: 417-426 https://doi.org/10.1046/j.0960-7412.2001.01227.x
  19. Tabei, Y., S. Kitade, Y. Nishizawa, N. Kikuchi, T. Kayano, T. Hibi and K. Akutsu. 1998. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea), Plant Cell Rep. 17: 159-164 https://doi.org/10.1007/s002990050371