Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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In vitro micropropagation of M.26 (Malus pumila Mill) apple rootstock and assessment of the genetic diversity of proliferated plantlets using simple sequence repeat markers

사과 대목 M.26 (Malus pumila Mill)의 기내 대량번식 및 simple sequence repeat 마커를 이용한 증식된 식물체의 유전적 다양성 평가

  • Cho, Kang Hee (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Han, Bong Hee (Foundation of Agri. Tech. Commercialization and Transfer) ;
  • Han, Jeom Hwa (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Park, Seo Jun (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Kim, Se Hee (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Lee, Han Chan (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Kim, Mi Young (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Kim, Myung-Su (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration)
  • 조강희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 한봉희 (농촌진흥청 농업기술실용화재단) ;
  • 한점화 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 박서준 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 김세희 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 이한찬 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 김미영 (농촌진흥청 국립원예특작과학원 과수과) ;
  • 김명수 (농촌진흥청 국립원예특작과학원 과수과)
  • Received : 2018.10.24
  • Accepted : 2018.11.20
  • Published : 2018.12.31
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Abstract

The objective of this study was to determine the most effective medium condition of shoot proliferation and root formation for the efficient in vitro micropropagation of M.26 (Malus pumila Mill). Simple sequence repeat (SSR) markers were used to analyze the genetic diversity of micro-propagated and greenhouse grown M.26. Shoot proliferation was carried out in MS (Murashige and Skoog) containing benzyladenin (BA, $0.5{\sim}5.0mg{\cdot}L^{-1}$) and thidiazuron (TDZ, $0.01{\sim}0.1mg{\cdot}L^{-1}$). The highest number of shoots (10.67 shoots per explant) was induced by adding BA at a concentration $1.0mg{\cdot}L^{-1}$. TDZ treatments caused higher hyperhydricity rate in cultured explants than in BA treatments. There was no significant effect of both BA and auxin on shoot proliferation, and the optimum proliferation medium for M.26 was MS medium containing $1.0mg{\cdot}L^{-1}$ BA. To find a suitable medium composition for shoot rooting, we tested different concentrations indole-3-butyric acid (IBA) and ${\alpha}$-naphthaleneacetic acid ($0.5{\sim}5.0mg{\cdot}L^{-1}$), MS medium (1/4-1), sucrose ($0{\sim}30g{\cdot}L^{-1}$). The shoots showed good rooting on half-strength MS medium containing $1.0mg{\cdot}L^{-1}$ IBA and $15-20g{\cdot}L^{-1}$ sucrose. The rooting rate (100%), number of roots (10.45 ~ 13.60 roots per explant), root length (7.41 ~ 8.33 cm), and shoot length (4.93 ~ 5.38 cm) were good on this medium. Fifteen SSR primers were detected in a total of 30 alleles in 20 micro-propagated plantlets, all SSR profiles from micro-propagated plantlets were monomorphic and similar to greenhouse grown control plantlet M.26 plant. The results indicated that M.26 micro-propagated plantlets were genetically stable.

본 연구는 사과 대목 M.26 (Malus pumila Mill))의 효과적인 기내 대량번식하기 위해 신초 증식과 뿌리 형성에 적합한 배지조건을 확립하고, simple sequence repeat (SSR) 마커를 이용하여 증식된 소식물체의 유전적 다양성을 분석하고자 수행하였다. MS (Murashige and Skoog) 기본배지에 benzyladenin (BA, $0.5{\sim}5.0mg{\cdot}L^{-1}$)와 thidiazuron (TDZ, $0.01{\sim}0.1mg{\cdot}L^{-1}$)을 첨가하여 신초를 배양한 결과, $1.0mg{\cdot}L^{-1}$ BA 처리에서 절편체 당신초 수가 10.67개로 가장 많았으며 과수화 발생률은 BA 처리구보다 TDZ 처리구에서 높았다. M.26 신초 증식에 BA와 auxin과의 혼용처리 효과는 없었고, $1.0mg{\cdot}L^{-1}$ BA가 첨가된 MS 기본배지가 적합하였다. 신초 발근에 적합한 배지를 구명하고자 auxin인 indole-3-butyric acid (IBA)와 ${\alpha}$-naphthaleneacetic acid의 농도($0.5{\sim}5.0mg{\cdot}L^{-1}$), MS 배지의 무기염류(1/4 ~ 1배) 및 sucrose 농도($0{\sim}30g{\cdot}L^{-1}$)를 달리하여 처리한 결과, $1.0mg{\cdot}L^{-1}$ IBA, $15{\sim}20g{\cdot}L^{-1}$ sucrose가 첨가된 1/2 MS 배지에서 발근율(100%), 뿌리 수(10.45 ~ 13.60개/절편체), 뿌리 길이(7.41 ~ 8.33 cm) 및 신초 길이(4.93 ~ 5.38 cm)가 양호하였다. 15종의 SSR primer를 이용하여 증식된 20개의 소식물체를 분석한 결과, 총 30개의 대립유전자가 검출되었으며 모두 동일한 밴드 패턴을 보여 온실에서 자란 M.26 식물체와 유사하여 유전적으로 안정한 것으로 판단되었다.

Keywords

References

  1. Amberger PV, Shoemaker RC, Palmer RG (1992) Inheritance of two independent isozyme variants in soybean plants derived from tissue culture. Theor Appl Genet 84:600-607
  2. Bahmani R, Karami O, Gholami M (2009) Influence of carbon sources and their concentrations on rooting and hyperhydricity of apple rootstock MM.106. World Appl Sci J 6:1513-1517
  3. Calamar A, De Klerk GJ (2002) Effect of sucrose on adventitious root regeneration in apple. Plant Cell Tissue Org Cult 70:207-212 https://doi.org/10.1023/A:1016356123367
  4. Castillo A, Cabrera D, Rodriguez P, Zoppolo R, Robinson T (2015) In vitro micropropagation of CG41 apple rootstock. Acta Hort 1083:569-576
  5. Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2005) Hyperhydricity in apple: ultrastuctural and physiological aspects. Tree Physiol 26:377-388
  6. Cho KH, Heo S, Kim JH, Shin IS, Kim SE, Kim DH, Han SE, Kim HR (2010) Analysis of genetic diversity of apple cultivars using RAPD and SSR markers. Kor J Breed Sci 42:525-533
  7. Ciccotti AM, Bisognin C, Battocletti I, Salvadori A, Herdemertens M, Jarausch W (2008) Micropropagation of apple proliferationresistant apomictic Malus sieboldii genotypes. Agron Res 6:445-458
  8. de Klerk G-J, Pramanik D (2017) Trichloroacetate, an inhibitor of wax biosynthesis, prevents the development of hyperhydricity in Arabidopsis seedlings. Plant Cell Tiss Org Cult 131:89-95 https://doi.org/10.1007/s11240-017-1264-x
  9. Devarumath RM, Nandy S, Rani V, Marimuthu S, Muraleedharan N, Raina SN (2002) RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type). Plant Cell Rep 21:166-173 https://doi.org/10.1007/s00299-002-0496-2
  10. Dobranszki J, Teixeira da Silva JA (2010) Micropropagation of apple- a review. Biotechnol Adv 28:462-488 https://doi.org/10.1016/j.biotechadv.2010.02.008
  11. Geng F, Moran R, Day M, Halteman W, Zhang D (2016) Increasing in vitro shoot elongation and proliferation of 'G.30' and 'G.41' apple by chilling explants and plant growth regulators. HortScience 51:899-904 https://doi.org/10.21273/HORTSCI.51.7.899
  12. Ghanbari A (2014) Impacts of plant growth regulators and culture media on in vitro propagation of three apple (Malus domestica Borkh.) rootstocks. Iran J Gent Plant Breed 3:11-20
  13. Gimenez C, Garcia ED, Enrech NXD, Blanca I (2001) Somaclonal variation of banana: cytogenetic and molecular characterization of somaclonal variant CIEN BTA-03. In Vitro Cell Dev Biol Plant 37:217-222 https://doi.org/10.1007/s11627-001-0038-6
  14. Ivanova M, Van Staden J (2011) Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell Tiss Org Cult 104:13-21 https://doi.org/10.1007/s11240-010-9794-5
  15. Jain SM (2001) Tissue culture-derived variation in crop improvement. Euphytica 118:153-166 https://doi.org/10.1023/A:1004124519479
  16. James DJ, Thurnbon IJ (1981) Shoot and root initiation in vitro in the apple rootstock M.9 and the promotive effects of phloroglucinol. J Hortic Sci 56-15-20 https://doi.org/10.1080/00221589.1981.11514960
  17. Jones OP (1967) Effect of benzyladenine on isolated apple shoots. Nature 215:1514-1515 https://doi.org/10.1038/2151514a0
  18. Jun JH, Chung KH, Jeong SB, Hong KH, Kang SJ (2001) Rapid multiplication of M.9 apple rootstocks in vitro. Kor J Hort Technol 19:34-38
  19. Kadota M, Niimi Y (2003) Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots. Plant Cell Tiss Org Cult 72:261-265 https://doi.org/10.1023/A:1022378511659
  20. Ko S-M, Lee J-H, Oh M-M (2018) Development of nutrient solution for in vitro propagation of ‘M9’ apple rootstock plantlets. Hortic Sci Technol 36:202-214
  21. Lane WD, Mcdougald JM (1982) Shoot tissue culture of apple:comparative response of five cultivars to cytokinin and auxin. Can J Plant Sci 62:689-694 https://doi.org/10.4141/cjps82-100
  22. Li X, Xu M, Korban SS (2002) DNA methylation profiles differ between field- and in vitro-grown leaves of apple. J Plant Physiol 159:1229-1234 https://doi.org/10.1078/0176-1617-00899
  23. Liebhard R, Gianfranceschi L, Koller B, Ryder CD, Tarchini R, Van de Weg E, Gessler C (2002) Development and characterisation of 140 new microsatellites in apple (Malus ${\times}$ domestica Borkh.). Mol Breed 10: 217-241 https://doi.org/10.1023/A:1020525906332
  24. Liu M, Jiang F, Kong X, Tian J, Wu Z, Wu Z (2017) Effects of multiple factors on hyperhydricity of Allium sativum L. Sci Hortic 217:285-296 https://doi.org/10.1016/j.scienta.2017.02.010
  25. Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492-496 https://doi.org/10.1007/s00299-004-0870-3
  26. McMeans O, Skirvin RM, Otterbacher A, Mitiku G (1998) Assessment of tissue culture-derived 'Gala' and 'Royal Gala' apples (Malus ${\times}$ domestica Borkh.) for somaclonal variation. Euphytica 103:251-257 https://doi.org/10.1023/A:1018316422435
  27. Modgil M, Mahajan K, Chakrabarti SK, Sharma DR, Sobti RC (2005) Molecular analysis of genetic stability in micropropagated apple rootstock MM106. Sci Hortic 104:151-160 https://doi.org/10.1016/j.scienta.2004.07.009
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  29. Palombi MA, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev). Plant Cell Rep 20:1061-1066 https://doi.org/10.1007/s00299-001-0430-z
  30. Pathak H, Dhawan V (2012) ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793. In Vitro Cell Dev Biol Plant 48:137-143
  31. Picoli EAT, Otoni WC, Figueira ML, Carolino SMB, Almeida RS, Silva EAM, Carvalho CR, Fontes EPB (2001) Hyperhydricity in vitro eggplant regenerated plants: structural characteristics and involvement of BiP (Binding Protein). Plant Sci 160:857-868 https://doi.org/10.1016/S0168-9452(00)00463-5
  32. Prado MJ, Gonzalez MV, Romo S, Herrera MT (2007) Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation. Plant Cell Tiss Org Cult 88:1-10 https://doi.org/10.1007/s11240-006-9116-0
  33. Snir I, Erez A (1980) In vitro propagation of Malling Merton apple rootstocks. HortScience 15:597-598
  34. Sun QR, Sun HY, Bell RL, Li LG, Xin L, Tao JH, Li Q (2014) Optimisation of the media for in vitro shoot proliferation and root induction in three new cold-hardy and dwarfing or semi-dwarfing clonal apple rootstocks. J Hortic Sci Biotech 89:381-388 https://doi.org/10.1080/14620316.2014.11513096
  35. Van Nieuwkerk JP, Zimmerman RH, Fordham I (1986) Thidiazuron stimulation of apple shoot proliferation in vitro. HortScience 21:516-518
  36. Werner EM, Boe AA (1980) In vitro propagation Malling 7 apple rootstock. HortScience 15:509-510
  37. Yadav MK, Gaur AK, Garg GK (2003) Development of suitable protocol to overcome hyperhydricity in carnation during micropropagation. Plant Cell Tiss Org Cult 72:153-156 https://doi.org/10.1023/A:1022236920605
  38. Yepes LM, Aldwinckle HS (1994) Micropropagation of thirteen Malus cultivars and rootstocks, and effect of antibiotics on proliferation. Plant Growth Regul 15:55-67 https://doi.org/10.1007/BF00024677
  39. Yoon TM, Park HY, Sagong DH (2005) Effect of root pruning on tree growth and fruit quality of 'Fuji'/M.9 apple trees. Kor J Hort Sci Technol 23:275-281
  40. Ziv M (2005) Simple bioreactors for mass propagation of plants. Plant Cell Tiss Org Cult 81:277-285 https://doi.org/10.1007/s11240-004-6649-y