Horticulture, Environment, and Biotechnology : HEB

Korean Society of Horticultural Science (한국원예학회)
권호 표지

Transformation of Chrysanthemum (Dendranthema grandiflorum Kitamuracv. Cheonsu) by Constitutive Expression of Rice OsMADS1 Gene

OsMADS1 유전자를 이용한 국화 '천수' 품종의 형질전환

Jeong, Jae -Hun;Chakrabarty, Debasis;Kim, Sun-Ja;Paek, Kee-Yoeup
정재훈;;김선자;백기엽

  • Published : 20020000
⟨ Previous Next ⟩

Abstract

A wide range of shoot regeneration media was tested to select most efficient regeneration medium for Agrobacterium-mediated transformation in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura cv. Cheonsu). Murasihge and Skoog’s medium containing 2 mgá$L^{-1}$ IAA and 2 mgá$L^{-1}$ BA was found to be the best for shoot bud initiation. Young leaf explants (5 × 5 mm) were infected with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector plasmid, pGA1209. This included neomycin phosphotransferase II (nptII) as a selection marker and OsMADS1 gene (cloned from rice) which is a member of AP1/AGL9 subgroup involved in early flowering with reduced apical dominance. Twenty-one and 15 putatively transformed /plants were obtained from 120 and 90 explants in two independent experiments, respectively. Putatively transformed shoots successfully rooted on rooting medium with 50 mgá$L^{-1}$ kanamycin. Integration and expression of OsMADS1 gene in 6 (3+3) transgenic plants were confirmed by PCR, PCR-southern blotting and RT-PCR analysis. However, distinct morphological change was not observed in these transgenic plants compared to control.

벼에서 유래된 OsMADS1과 선발표지유전자 nptII로 재조합된 pGA1209 binary vector를 가지는 A. tumefaciens LBA4404를 이용하여 국화 천수품종의 형질전환체를 얻었다. 5 ´ 5mm 크기로 절단된 국화 잎절편체는 증식된 Agrobacterium 균액에 침지하여 접종한 후 25oC 암조건에서 3일간 공조배양을 실시하였으며, 형질전환체의 선발을 위해 항생제 50mg·L-1 kanamycin 및 500mg·L-1 claforan이 첨가된 재분화배지(MS + 2mg·L-1 IAA + 2mg·L-1 BA)로 옮긴 후 3주마다 새로운 배지로 계대하여 신초를 유도하였다. 유도된 신초는 50mg·L-1 kanamycin 첨가된 MS 배지에서 뿌리 유도 후 토양에 이식하였다. 선발된 국화식물체들의 잎에서 DNA 및 RNA를 추출하여 nptII 및 OsMADS1 유전자를 증폭할 수 있는 primer들을 이용하여 PCR, PCR-southern blot 및 RT-PCR 분석을 수행하였으며 OsMADS1 유전자의 도입 및 발현여부를 확인하였다. 그러나 이들 형질전환체에서는 정상적인 식물체와 비교해서 형태적인 차이를 확인할 수 없었다.

Keywords

References

  1. Broertjes, C. and A.M. Van Harten. 1978. Application of mutation breeding methods in the improvement of vegetatively propagated crops. An interpretive literature review. Elsevier Scientific Publishing Co. Amsterdam-Oxford, New York
  2. Bush, S.R., E.D. Earle, and R.W. Langhans. 1976. Plantlets from petal segments, petal epidermis and shoot tips of periclinal chimera, Chrysanthemum morifolium Indianapolis. Am. J. Bot. 63:729-737
  3. Chung, Y.Y., S.R. Kim, D. Finkel, M.F. Yanofsky, and G. An. 1994. Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene. Plant Mol. Biol. 26:657-665
  4. De Jong, J., M.M.J. Mertens, and W. Rademaker. 1994. Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA. Plant Cell Rep. 14:59-64 https://doi.org/10.1007/BF00233300
  5. Fukai, S., J. De Jong, W. Rademaker. 1995. Efficient genetic transformation of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) using stem segments. Breeding Science 45:179-184
  6. Irish, V.F. 1999. Patterning the flower. Developmental Biology 209:211-220 https://doi.org/10.1006/dbio.1999.9226
  7. Jeong, J.H., D.C. Yang, H.G. Jang, and K.Y. Paek. 2000. Transformation of Lettuce (Lactuca sativa L.) using cold regulated gene (BN 115). Kor. J. of Plant Tissue Cult. 27:7-12
  8. Jong, J.D., W. Rademaker, and M.F. Wordragen, 1993. Restoring adventitious shoot formation on chrysanthemum leaf explants following co-cultivation with A. tumefacience. Plant Cell Tiss. Org. Cult. 32:263-270 https://doi.org/10.1007/BF00042287
  9. Kim, J.Y., S.J. Park, B.Y. Um, C.H. Pak, Y.S. Chung, and J.S. Shin. 1998. Transformation of chrysanthemum by Agrobacterium tumefaciens with three different types of vectors. J. Kor. Soc. Hort. Sci. 39:360-366
  10. Kim, S., S.R. Kim, C.S. An, Y.N. Hong, and K.W. Lee. 2001. Constitutive expression of rice MADS box gene using seed explants in hot pepper. Mol. Cells 12:221-226
  11. Koornneef, M., C. Alonso-Blanco, A.J.M. Peeters, and W. Soppe. 1998. Genetic control of flowering time in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:345-370
  12. Ledger, S.E., S.C. Deroles, and N.K. Given. 1991. Regeneration and Agrobacterium-mediated transformation of chrysanthemum. 10:195-199
  13. Lee, Y.K., Y.J. Kwon, K.M. Lee, and N.I. Hyung. 1999. Plant regeneration from leaf segment cultures of chrysanthemum (Dendranthema grandiflora Tzvelev). Kor. J. Plant Tissue Cult. 26:59-63
  14. Levy, Y.Y. and C. Dean. 1998. The transition to flowering. The Plant Cell 10:1973-1989 https://doi.org/10.2307/3870778
  15. Lim, H.T., M.A. Zhao, J.L. Yu, J.Y. Lee, E.J. Park, I.J. Chun, J.W. Yu, and B.D. Kim. 2001. Establishment of genetic transformation system and introduction of MADS box gene in hot pepper. J. Plant Biotech. 3:89-94
  16. Lowe, J.M., M.R. Davey, J.B. Power, and K.S. Blundy. 1993. A study of some factors affecting Agrobacterium transformation and plant, regeneration of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.). Plant Cell Tissue and Organ Cult. 33:171-180 https://doi.org/10.1007/BF01983231
  17. Miller, H.N. 1975. Leaf, stem, crown and root galls induced in chrysanthemum by A. tumefaciens. Phytopathology 65:805-811 https://doi.org/10.1094/Phyto-65-805
  18. Mourgues, F., E. Chevreau, C. Lambert, and A.D. Bondt. 1996. Efficient Agrobacterium-mediated transformation and recovery of transgennic plants from pear (Pyrus communis L.). Plant Cell Rep. 16:245-249
  19. Murashige, T. and F.A. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  20. Pavingerova, D., J. Dostal, R. Biskova, and V. Benetka. 1994. Somatic embryogenesis and Agrobacterium mediated transformation of chrysanthemum. Plant Sci. 97:95-101 https://doi.org/10.1016/0168-9452(94)90111-2
  21. Renou, J.P., P. Brochard, and R. Jalouzot. 1993. Recovery of transgenic chrysanthemum (Dendranthema grandiflora Tzvelev) after hygromycin resistance selection. Plant Sci. 89:185-197 https://doi.org/10.1016/0168-9452(93)90127-L
  22. Robinson, K.E.P. and E. Firoozabady. 1993. Transformation of floricultural crops. Scientia Hort. 55:83-99 https://doi.org/10.1016/0304-4238(93)90026-M
  23. Sherman, J.M., J.W. Moyer, and M.E. Daub. 1998. A regeneration and Agrobacterium-mediated transformation system for genetically diverse chrysanthemum cultivars. J. Amer. Soc. Hort. Sci. 123:189-194
  24. Takatsu, Y., H. Tomotsune, M. Kasumi, and F. Sakuma. 1998. Differences in adventitious shoot regeneration capacity among Japanese chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) cultivars and the improved protocol for Agrobacterium-mediated genetic transformation. J. Japan. Soc. Hort. Sci. 67:958-964 https://doi.org/10.2503/jjshs.67.958
  25. Urban, L.A., J.M. Sherman, J.W. Moyer, and M.E. Daub. 1994. High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora). Plant Sci. 98:69-79 https://doi.org/10.1016/0168-9452(94)90149-X
  26. Wordragen, M.F., J.D. Jong, M.J. Schornagel, and H.J.M. Pons. 1992. The use of endotoxin genes from B. thuringiensis to introduce insect resistance in Chrysanthemum callus. Proce. Integration of in vitro techniques in ornamental plant breeding 145-149
  27. Yu, J.W. 2001. Molecular isolation and characterization of three MADS genes from hot pepper plants. PhD Thesis. Seoul National University