Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Effects of a variety of treatments affecting Chinese cabbage protoplast culture, and plant regeneration from protoplast-derived callus

배추 원형질체 배양에 미치는 다양한 처리의 효과와 원형질체 유래 캘러스로부터 신초 재분화

  • Han, Jeung-Sul (National Horticultural Research Institute, Rural Development Administration) ;
  • Yoon, Moo-Kyeong (National Horticultural Research Institute, Rural Development Administration) ;
  • Jeong, Mi-Hye (National Institute of Agricultural Science and Technology, Rural Development Administration)
  • Published : 2008.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Here we describe a procedure for Chinese cabbage protoplast culture and effects of various treatments. Chinese cabbage protoplasts were isolated from different parts of young seedlings as using an enzyme mixture, of which yield was maximized in seven hours around after digestion. The highest rate of initial cell division followed by micro-callus formation was obtained in the medium with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, and 1.0 mg/L BA when the cotyledon-derived protoplasts were cultured. Initiation of cell division and micro-callus proliferation significantly depended upon Chinese cabbage genotype under a same culture circumstances. The micro-calli developed from cotyledon tissue of Norang-Bom cultivar successfully grew toward callus colonies on the solidified medium with 0.2 mg/L zeatin and 0.1 mM spermidine. The callus colonies generated de novo shoots at the maximum frequency of 4.3% on the medium with 5.0 mg/L BA and 1.0 mg/L NM. Our results might be helpful for further studies to enhance the regeneration efficiency in Chinese cabbage protoplast culture.

본 연구에서는 배추 원형질체 배양에 있어 다양한 처리의 효과를 구명함으로써 배추 원형질체 배양을 통한 재분화 체계를 확립하고자 하였다. 배추 원형질체 분리 수율은 효소액, 사용된 조직 및 효소액 처리 시간에 따라 달리 나타났는데, 효소액 처리 약 7시간 이후부터는 분리되는 원형질체의 수가 크게 증가하지 않았다. 따라서 효소 자체에 의한 부정적 영향을 고려하였을 때 최장 7시간 동안 효소처리를 하는 것이 적정한 것으로 판단하였다. 떡잎유래 원형질체를 2,4-D 1.0 mg/L, NAA 0.5 mg/L 및 BA 1.0 mg/L 첨가 액체배지에서 배양하였을 때 초기 세포분열과 이어지는 micro-callus 생장이 가장 양호하였다. 다양한 배추 인자형에 대한 검토 결과 본 연구의 배양조건 하에서는 초기 세포분열과micro-callus 생장이 배추 인자형에 따라 뚜렷하게 영향을 받는 것으로 나타났다. 노랑봄 품종의 떡잎에서 분리한 원형질체로부터 생장한 micro-callus 현탁액을 zeatin 0.2 mg/L외 spermidine 0.1 mM이 첨가된 반고체 MS 배지에 plating하였을 때 가장 왕성하게 callus colony가 형성 및 생장하였다. 이들 callus colony는 BA 5.0 mg 및 NAA 1.0 mg/L가 첨가된 배지에서 4.3%의 빈도로 재분화 신초가 유도되었다 얻어진 신초는 PGR 무첨가 MS배지에서 손쉽게 발근하였고 발근된 식군체는 기외에서 정상 식물체로 생장하였다. 본 연구의 결과는 배추 단독 원형질체 배양을 통한 고빈도 식물체 재분화 연구에 중요한 정보를 제공함으로써 유용하게 활용될 것으로 사료된다.

Keywords

References

  1. Bannerot H, Boulidard L, Cauderon Y, Tempe J (1974) Transfer of cytoplasmic male sterility from Raphanus sativus to Brassica oleracea. In: Proc EUCARPIA Meeting Cruciferae. Dundee. pp 52-54
  2. Bengochea T, Dodds JH (1986) Plant protoplasts: A biotechnological tool for plant improvement. Chapman and Hall. New York
  3. Chi GL, Pua EC, Goh CJ (1991) Role of ethylene on de novo shoot regeneration from cotyledonary explants of Brassica campestris ssp. Pekinensis (lour) Olsson in vitro. Physiol Plantarum 96: 178-183 https://doi.org/10.1104/pp.96.1.178
  4. Curuk S, Ananthakrishnan G, Singer S, Xia X, Elamn C, Nestel D, Cetiner S, Gaba V (2003) Regeneration in vitro from the hypocotyls of Cucumis species produces almost exclusively diploid shoots, and does not require light. HortScience 38: 105-109
  5. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirement of suspension cultures of soybean root cells. Exp Cell Res 50: 151-158 https://doi.org/10.1016/0014-4827(68)90403-5
  6. Han EJ, Kim HI (1990) Root & shoot regeneration from the protoplast of Brassica campestris ssp. pekinensis. Korean J Plant Tiss Cult 17: 285-289
  7. Heyn FW (1978) Cytoplasmic genetic male sterility in Brassica napus. EUCARPIA Cruciferae News 1. 3: 34-35
  8. Huxter TJ, Thorpe TA, Reid DM (1981) Shooting initiation in light- and dark-grown tobacco callus: the role of ethylene. Physiol Plantarum 53: 319-326 https://doi.org/10.1111/j.1399-3054.1981.tb04507.x
  9. In DS, Song MJ, Jang IC, Min BW, Nahm SH, Shin JS, Lee SW, Harn CH (2008) Regeneration of symmetric protoplast fusion between cabbage (Brassica oleracea L.) and radish (Raphanus sativus L.). J Plant Biotechnol 35: 121-126 https://doi.org/10.5010/JPB.2008.35.2.121
  10. Jain RK, Chowdhury JB, Sharma DR, Friedt W (1988) Genotypic and media effects on plant regeneration from cotyledon explant cultures of some Brassica species. Plant Cell Tiss Org Cult 14: 197-206 https://doi.org/10.1007/BF00043410
  11. Jee SO, Chung JD (1989a) Protoplast culture of flower cabbage (Brassica oleracea L. var. acephala DC. forma tricolor Hort.), I. Factors affecting cell division in protoplast culture. J Kor Soc Hort Sci 30: 96-107
  12. Jee SO, Chung JD (1989b) Protoplast culture of flower cabbage (Brassica oleracea L. var. acephala DC. forma tricolor Hort.), II. Effect of plant growth regulators on plant regeneration. Korean J Plant Tiss Cult 16: 15-24 https://doi.org/10.1007/BF00044068
  13. Jee SO, Chung JD (1989c) Protoplast culture of flowering cabbage (Brassica oleracea L. var. acephala DC. forma tricolor Hort.), III. Plant regeneration from fused protoplasts. Korean J Plant Tiss Cult 16: 79-92
  14. Jourdan PS, Earle ED, Mutschler MA (1989) Synthesis of male-sterile, triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male-sterile B. oleracea and atrazine-resistant B. campestris. Theor Appl Genet 78: 445-455
  15. Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126: 105-110 https://doi.org/10.1007/BF00380613
  16. Kirti PB, Banga SS, Prakash S, Chopra VL (1995) Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion. Theor Appl Genet 91: 517-521
  17. Kumar PP, Reid DM, Thorpe TA (1987) The role of ethylene and carbon dioxide in differentiation of shoot buds in excised cotyledons of Pinus radiate in vitro. Physiol Plantarum 69: 244-252 https://doi.org/10.1111/j.1399-3054.1987.tb04282.x
  18. Leshem B, Ronen R, Soudry E, Lurie S, Gepstein S (1995) Cytokinin and white light coact to enhance polypeptide metabolism and shoot regeneration in cultured melon cotyledons. J Plant Physiol 145: 291-295 https://doi.org/10.1016/S0176-1617(11)81892-4
  19. Lian YJ, Lim HT (2001) Production and characterization of somatic hybrids between Brassica campestris L. ssp pekinensis and Brassica oleracea L. var capitata. J Plant Biotechnol 3: 33-38
  20. Menczel L, Wolfe K (1984) High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH. Plant Cell Rep 3: 196-198 https://doi.org/10.1007/BF00270199
  21. Murashige T, Skoog FA (1962) Revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plantarum 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  22. Murata M, Orton TJ (1987) Callus initiation and regeneration capacities in Brassica species. Plant Cell Tiss Org Cult 11: 111-123 https://doi.org/10.1007/BF00041844
  23. Narasimhulu SB, Chopra VL (1988) Species specific shoot regeneration response of cotyledonary explants of Brassicas. Plant Cell Rep 7: 104-106 https://doi.org/10.1007/BF00270115
  24. Nitch JP, Nitch C (1969) Haploid plants from pollen grains. Science 163: 85-87 https://doi.org/10.1126/science.163.3862.85
  25. Ogura H (1968) Studies on the new male sterility in Japanese radish with special reference to utilization of this sterility toward the practical raising of hybrid seeds. Mem Fac Agric. Kagoshima Univ. 6: 39-78
  26. Pelletier G, Primard C, Vedel F, Chetrit P, Remy R, Rousselle, Renard M (1983) Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion. Mol Gen Genet 191: 244-250 https://doi.org/10.1007/BF00334821
  27. Pierik RLM (1987) In vitro culture of higher plants. Martinus Nijhoff Pubishers, Dordrecht, The Netherlands
  28. Pua EC (1993) Cellular and molecular aspects of ethylene on plant morphogenesis of recalcitrant Brassica species in vitro. Bot Bull Acad Sin 34: 191-209
  29. Ren JP, Dickson MH, Earle ED (2000) Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea. Theor Appl Genet 100: 810-819 https://doi.org/10.1007/s001220051356
  30. Saha S, Mori H, Hattori K (2007) Synergistic effect of kinetin and benzyl adenine plays a vital role in high frequency regeneration from cotyledon explants of bottle gourd (Lagenaria siceraria) in relation to ethylene production. Breeding Sci 57: 197-202 https://doi.org/10.1270/jsbbs.57.197
  31. Sakai T, Imamura J (1990) Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion. Theor Appl Genet 80: 421-427
  32. Songstad DD, Duncan DR, Widholm JM (1988) Effect of 1-aminocyclopropane-1 carboxylic acid, silver nitrate and non-bornadiene on plant regeneration from maize callus cultures. Plant Cell Rep 7: 262-265 https://doi.org/10.1007/BF00272538
  33. Williams PH, Heyn FW (1981) The origins and development of cytoplasmic male sterile Chinese cabbage. In: Talekar NS, Griggs TD (eds), Chinese Cabbage, Asian Vegetable Research and Development Center, Tainan, pp 293-300

Cited by

  1. High frequency somatic embryogenesis through leaf explant-derived callus culture in Muscari armeniacum cv. 'Early Giant' vol.39, pp.1, 2012, https://doi.org/10.5010/JPB.2012.39.1.069