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Influence of medium addition and agitation on the production of embryos in isolated microspore culture of hot pepper (Capsicum annuum L.)

고추의 소포자 배양 시 배지 첨가와 진탕이 배의 생산에 미치는 영향

  • An, Dong-Ju (Department of Life Sciences, Mokwon University) ;
  • Park, Eun-Joon (Department of Life Sciences, Mokwon University) ;
  • Kim, Moon-Za (Department of Life Sciences, Mokwon University)
  • Received : 2011.01.13
  • Accepted : 2011.02.07
  • Published : 2011.03.31

Abstract

The influences of the agitation as well as the addition of medium during culture on the production of embryos were invested in isolated microspore culture of hot pepper (Capsicum annuum L.). When the culture medium was added during initial liquid culture step of liquid-double layer culture, the embryo yield and quality greatly increased. The most effective time point for medium addition was 5 days after the culture commenced. On the other hand, the effect of medium addition at later double layer culture step in liquid-double layer culture on the embryo production was less compared to that of medium addition during the initial liquid culture step. Agitating the culture for 1 week during later double layer culture step in liquid-double layer culture effectively increased the production of normal cotyledonary embryos. In the case of liquid culture, agitating the culture for 1 week from 7 days after the culture commenced was also effective for embryo development. However, when the total agitation time was longer (2 to 3 weeks) during liquid-double layer culture or liquid culture, the embryos developed abnormally in both cases. The normal cotyledonary embryos obtained in this study successfully developed to plants when transferred to regeneration media. These regenerated plants were either diploid or haploid, and there was a difference in the number of chloroplasts between guard cells of diploid and haploid. These results can be used as an important data for developing an efficient microspore culture system with high quality embryo production in hot pepper.

본 연구에서는 고추의 나출 소포자 배양 시 배양 중 새배지의 첨가와 진탕이 배의 생산에 미치는 영향을 조사하였다. 액체-2층 배양에서 초기 액체배양 시에 새 배지를 첨가하면 배의 발생과 발달 모두 크게 증가하였다. 가장 효과적인 첨가 시기는 초기액체 배양을 시작한 5일 후 이었다. 한편 액체-2층 배양에서 후기 2층배양 시의 새 배지 첨가는 초기 액체배양 시 첨가 때에 비해 그 효과가 적었다. 액체-2층 배양에서 후기 2층배양 시의 1주간 진탕은 정상 자엽배 생산에 효과적이었다. 액체배양시에도 배양 1주 후의 1주간 진탕은 배의 발달에 효과적 이었다. 그러나 액체-2층배양 시와 액체배양 시 모두 진탕기간이 2 ~ 3 주간으로 길어질 때에는 배의 발달이 비정상적이었다. 본 실험 결과 얻어진 정상 자엽배들은 재분화 배지에 이식 시 용이하게 유식물체로 발달하였다. 재분화 식물체들 중에는 반수체와 배가반수체가 혼재하였으며, 이들 간에는 공변세포 내 엽록체 수의 차이가 뚜렷하였다. 이와 같은 결과들은 고추에서 다수의 정상자엽배를 생산할 수 있는 소포자 배양 시스템을 확립하는 데 중요한 기초자료가 될 것이다.

Keywords

References

  1. Custers JBM, Cordewener JHG, Fiers MA, Maassen BTH, van Lookeren Campagne MM, Liu CM (2001) Androgenesis in brassica, a model system to study the initiation of plant embryogenesis. In: Bhojwani SS, Soh WY (eds) Current trends in the embryology of angiosperms. Kluwer Academic Publishers, Netherlands, pp 451-470
  2. Dai XG, Shi XP, Fu Q, Bao MZ (2009) Improvement of isolated microspore culture of ornamental kale (Brassica oleracea var. Acephala): Effects of sucrose concentration, medium replacement, and cold pre-treatment. J Horticult Sci Biotechnol 84: 519-525 https://doi.org/10.1080/14620316.2009.11512559
  3. Dias JS, Correia MC (2002) Effect of medium renovation and incubation temperature regimes on tronchuda cabbage microspore culture embryogenesis. Sci Hortic 93:205-214 https://doi.org/10.1016/S0304-4238(01)00334-X
  4. Davies PA, Morton S (1998) A comparison of barley isolated microspore and anther culture and the influence of cell culture density. Plant Cell Reports 17:206-210 https://doi.org/10.1007/s002990050379
  5. Ferrie AMR, Epp DJ, Keller WA (1995) Evaluation of Brassica napus L. genotypes for microspore culture response and identification of a highly embryogenic line. Plant Cell Reports 14:580-584
  6. Gamborge O, Murashige T, Thorpe A, Vasil IK (1976) Plant tissue culture media. In Vitro 12:473-478 https://doi.org/10.1007/BF02796489
  7. Hansen M, Svinnset K (1993) Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media. Plant Cell Reports 12:496-500
  8. Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F (1992) Anther and microspore culture of Hordeum vulgare L. cv Igri. Plant Sci. 86:89-96 https://doi.org/10.1016/0168-9452(92)90182-L
  9. Hofer (2004) In vitro androgenesis in apple: improvement of the induction phase. Plant Cell Reports 22:365-370 https://doi.org/10.1007/s00299-003-0701-y
  10. Huang B, Brid S, Kemble R, Simmonds D, Keller W, Miki B (1990) Effects of culture density, conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas. Plant Cell Reports 8:594-597 https://doi.org/10.1007/BF00270061
  11. Indrianto A, Barinova I, Touraev A, Heberle-Bors E. (2001) Tracking individual wheat microspores in vitro : identification of embryogenic microspores and body axis formation in the embryo. Planta 212:163-174 https://doi.org/10.1007/s004250000375
  12. Jahne A, Lorz H (1995) Cereal microspore culture. Plant Sci 109:1-12 https://doi.org/10.1016/0168-9452(95)04149-O
  13. Joosen R, Cordewener J, Supena EDJ, Vorst O, Lammers M, Maliepaard C, Zeilmaker T, Miki B, America T, Custers J, Boutilier K (2007) Combined transcriptome and proteome analysis identifies pathways and robust markers associated with the establishment of Brassica napus microspore-derived embryo development. Plant Physiol 144:155-172 https://doi.org/10.1104/pp.107.098723
  14. Kasha KJ, Kao KN (1970) High frequency haploid production in barley (hordeum vulgare L.). Nature 225:874-876 https://doi.org/10.1038/225874a0
  15. Kim M, Kim J, Yoon M, Choi DI, Lee KM (2004) Origin of multicellular pollen embryos in cultured anthers of pepper (Capsicum annuum L.). Plant Cell Tiss Organ Cult 77:63-72 https://doi.org/10.1023/B:TICU.0000016506.02796.6a
  16. Kim M, Jang IC, Kim JA, Park EJ, Yoon M, Lee Y (2008) Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Reports 27:425-434 https://doi.org/10.1007/s00299-007-0442-4
  17. Kim M, Park EJ, Lee Y (2010) Increased embryo production by manipulation of pretreatment materials and media in isolated microspore culture of hot pepper (Capsicum annuum L.). In: Recent advances in plant biotechnology, (ed) Kumar A, I.K. International Publishing House Pvt. Ltd. New Delhi, India.
  18. Kott LS, Polsoni L, Ellis B, Beversdorf WD (1988) Autotoxicity in isolated microspore cultures of Brassica napus. Can J Bot-Rev Can Bot 66:1665-1670 https://doi.org/10.1139/b88-227
  19. Lantos C, Juhasz AG, Somogyi G, Otvos K, Vagi P, Mihaly R, Kristof Z, Somogyi N, Pauk J (2009) Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tiss Organ Cult 97:285-293 https://doi.org/10.1007/s11240-009-9527-9
  20. Lichter R (1989) Efficient yield of embryoids by culture of isolated microspores of different Brassicaceae species. Plant Breed 103:119-123. https://doi.org/10.1111/j.1439-0523.1989.tb00359.x
  21. Liu W, Zheng MY, Konzak CF (2002) Improving green plant production via isolated microspore culture in bread wheat (Triticum aestivum L.). Plant Cell Reports 20:821-824. https://doi.org/10.1007/s00299-001-0408-x
  22. Maraschin SF, Gaussand G, Pulido A, Olmedilla A, Lamers GEM, Korthout H, Spaink HP, Wang M (2005) Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis. Planta 221:459-470 https://doi.org/10.1007/s00425-004-1460-x
  23. Mordhorst AP, Toonen MAJ, De Vries SC (1997) Plant embryogenesis. Crit Rev Plant Sci 16:535-576 https://doi.org/10.1080/07352689709701959
  24. Nageli M, Schmid JE, Stamp P, Buter B (1999) Improved formation of regenerable callus in isolated microspore culture of maize; impact of carbohydrates, plating density and time of transfer. Plant Cell Reports 19:177-184 https://doi.org/10.1007/s002990050730
  25. Obert B, Szabo L, Mityko J, Pre'ova A, Barnabas B (2005) Morphological events in cultures of mechanically isolated maize microspores. In Vitro Cell Dev Biol-Plant 41:775- 782 https://doi.org/10.1079/IVP2005701
  26. Park EJ, Kim JA, Kim M (2009) Influence of pretreatment medium, fresh medium addition, and culture plate size on the production of embryos in isolated microspore culture of hot pepper (Capsicum annuum L.). J Plant Biotechnol 36:184-192 https://doi.org/10.5010/JPB.2009.36.2.184
  27. Park EJ, Lee JS, An DJ, Kim M (2010) The effect of medium change after pretreating microspores, medium addition, and volume of under solid medium in double layer culture on the production of embryos in isolated microspore culture of hot pepper (Capsicum annuum L.). J Plant Biotechnol 37:494-504 https://doi.org/10.5010/JPB.2010.37.4.494
  28. Polsoni L, Kott LS, Beversdorf WD (1988) Large-scale microspore culture technique for mutation selection studies in Brassica napus. Can J Bot-Rev 66:1681-1685 https://doi.org/10.1139/b88-230
  29. Qin X, Rotino GL (1995) Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets. Plant Cell Tiss Organ Cult 41:145-149 https://doi.org/10.1007/BF00051583
  30. Raina Sk, Irfan ST (1998) High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice. Plant Cell Reports 17:957-962 https://doi.org/10.1007/s002990050517
  31. Regner F (1994) Microspore culture of Capsicum annuum. Capsicum and Eggplant Nwsl 13:72-73
  32. Regner F (1996) Anther and microspore culture in Capsicum, In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol. 3. Kluwer Academic Publ, Dordrecht the Netherlands:77-89
  33. Siebel J, Pauls KP (1989) A comparison of anther and microspore culture as a breeding tool in Brassica napus. Theor Appl Genet 78:473-47 https://doi.org/10.1007/BF00290830
  34. Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Reports 25:1-10 https://doi.org/10.1007/s00299-005-0028-y
  35. Supena EDJ, Winarto B, Riksen T, Dubas E, Lammeren A, Offringa R, Boutilier K, Custers J (2008) Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning. J Exp Bot 59:803-814 https://doi.org/10.1093/jxb/erm358
  36. Swanson EB, Coumans MP, Wu SC, Barsby TL, Beversdorf WD (1987) Efficient isolation of microspores and the production of microspore-derived embryos from Brassica napus. Plant Cell Reports 6:94-97
  37. Swanson, EB (1990) Microspore culture in Brassica : In JW Pollard and JM Walker (eds) Methods in molecular biology, Vol. 6, Plant cell and tissue culture. Humana Press New Jersey: 159-170
  38. Testillano PS, Gonzalez-Melendi P, Ahmadian P, Fadon B, Risueno MC (1995) The immunolocalization of nuclear antigens during the pollen developmental program and the induction of pollen embryogenesis. Exp Cell Res 221:41-54 https://doi.org/10.1006/excr.1995.1350
  39. Testillano P, Georgiev S, Mogensen HL, Coronado MJ, Dumas C, Risueno MC, Matthys-Rochon E (2004) Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis. Chromosoma 112:342-349 https://doi.org/10.1007/s00412-004-0279-3
  40. Touraev A, Ilham A, Vicente O, Heberle-Bors E (1996) Stressinduced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Reports 15:561-565 https://doi.org/10.1007/BF00232453
  41. Touraev A, Vicente O, Heberle-Bor E (1997) Initiation of microspore embryogenesis by stress. Trends Plant Sci 2:297-302 https://doi.org/10.1016/S1360-1385(97)89951-7
  42. Wiberg E, Råhlen L, Hellman M, Tillberg E, Glimelius K, Stymne S (1991) The microspore-derived embryo of Brassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality. Theor Appl Genet 82:515-520 https://doi.org/10.1007/BF00588608