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Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

  • Zhen-Fang Yin
  • Bing Zhao
  • Wen-Lu Bi
  • Long Chen
  • Qiao-Chun Wang
Micropropagation

Abstract

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH 5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23 ± 2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.

Keywords

Genetic marker Genetic stability Leaf segment Lilium Shoot regeneration 

Notes

Acknowledgment

This study was financially supported by the Presidential Foundation of Northwest A&F University.

References

  1. Armitage AM (1997) Herbaceous perennial plants: a treatise on their identification, culture and garden attributes, 2nd edn. Stipes, ChampaignGoogle Scholar
  2. Bacchetta L, Remotti PC, Bernardini C, Saccardo F (2003) Adventitious shoot regeneration from leaf explants and stem nodes of Lilium. Plant Cell Tissue Organ Cult 74:37–44CrossRefGoogle Scholar
  3. Bahr LR, Compton ME (2004) Competence for in vitro bulblet regeneration among eight Lilium genotypes. HortSci 39:127–129Google Scholar
  4. Bornet B, Branchard M (2001) Nonanchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Mol Biol Rep 19:209–215CrossRefGoogle Scholar
  5. Dobránszki J, Teixeira da Silva JA (2010) Micropropagation of apple—a review. Biotechnol Adv 28:462–488PubMedCrossRefGoogle Scholar
  6. Feng JC, Yu XM, Shang XL (2010) Factors influencing efficiency of shoot regeneration in Ziziphus jujuba Mill. ‘Huizao’. Plant Cell Tissue Organ Cult 101:111–117CrossRefGoogle Scholar
  7. George EF, Davies W (2008) Effects of the physical environment. In: George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture, 3rd ed. Springer, pp 355–401Google Scholar
  8. Godo T, Mii M (2001) In vitro germplasm preservation of lily species utilizing callus cultures at low temperature. Acta Horticult 560:153–155Google Scholar
  9. Kato Y, Yoshinori Y (1977) Plantlet formation and differentiation of epidermal tissue in green callus culture from exised leaves of Lilium. Phytomorphology 27:390–396Google Scholar
  10. Kim MS, Jeon JH, Youm JW, Kim JH, Lee BC, Kang WJ, Kim HS, Joung H (2005) Efficient plantlet regeneration via callus formation from leaf segment of Lilium oriental hybrid ‘Casa blanca’. J Plant Biotech 7:129–134Google Scholar
  11. Liu XM, Yang GC (2012) Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation. In Vitro Cell Dev Biol Plant 48:172–179CrossRefGoogle Scholar
  12. Lopez-Carbonell M, Alegre L, Prinsen E, van Onckelen H (1992) Diurnal fluctuations of endogenous IAA content in aralia leaves. Biol Plant 34:223–227CrossRefGoogle Scholar
  13. Mandal AKA, Gupta SD (2001) Direct shoot organogenesis and plant regeneration in safflower. In Vitro Cell Dev Biol Plant 37:50–54Google Scholar
  14. Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496PubMedCrossRefGoogle Scholar
  15. Matsumoto T, Sakai A, Yamada K (1995) Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Cult 41:237–241CrossRefGoogle Scholar
  16. McRae EA (1998) Lilies: A guide for growers and collectors. Timber Press, PortlandGoogle Scholar
  17. Mitsukuri EK, Mori G, Johkan M, Shimada Y, Mishiba K, Morikawa T, Oda M (2009) Effects of explant position and dark treatment on bud formation in floret culture of Ponerorchis graminifolia Rchb.f. Sci Hortic 121:243–247CrossRefGoogle Scholar
  18. Modgil M, Mahajan K, Chakrabarti SK, Sharma DR, Sobti RC (2005) Molecular analysis of genetic stability in micropropagated apple rootstock MM106. Sci Hortic 104:151–160CrossRefGoogle Scholar
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  20. Nhut DT (1998) Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture. Plant Cell Rep 117:913–916CrossRefGoogle Scholar
  21. Nhut DT (2003) The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes. Plant Growth Regul 40:179–184CrossRefGoogle Scholar
  22. Nhut DT, Huong NTD, Le VB, Teixeira da Silva JA, Fukai S, Tanaka M (2002) The changes in shoot regeneration potential of protocorm-like bodies derived from Lilium longiflorum young stem explants exposed to medium volume, pH, light intensity and sucrose concentration pretreatment. J Hortic Sci Biotechnol 77:79–82Google Scholar
  23. Nhut DT, Le BV, Teixeira da Silva AT, Aswath CR (2001a) Thin cell layer culture system in Lilium: regeneration and transformation. In Vitro Cell Dev Biol Plant 37:516–523CrossRefGoogle Scholar
  24. Nhut NT, Le BV, Fukai S, Tanaka M, Tran Thanh KTV (2001b) Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture. Plant Growth Regul 33:59–65CrossRefGoogle Scholar
  25. Niimi Y (1984) Bulblet productivity of explants from scales, leaves, stems and tepals of Lilium rubellum Baker. Sci Hortic 22:391–394CrossRefGoogle Scholar
  26. Ostry M, Hackett W, Michler C, Serres R, McCown B (1994) Influence of regeneration method and tissue source on the frequency of somatic variation in Populus to infection by Septoria musiva. Plant Sci 97:209–215CrossRefGoogle Scholar
  27. 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 20:1061–1066CrossRefGoogle Scholar
  28. Pereira AMS, Bertoni BW, Appezzato-da-Glória B, Araujo ARB, Januário AH, Lourenço MV, França SC (2000) Micropropagation of Pothomorphe umbellata via direct organogenesis from leaf explants. Plant Cell Tissue Organ Cult 60:47–53CrossRefGoogle Scholar
  29. Pérez-Tornero O, Egea J, Vanoostende A, Burgos L (2000) Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot. Plant Sci 158:61–70PubMedCrossRefGoogle Scholar
  30. Petri C, Scorza R (2010) Factors affecting adventitious regeneration from in vitro leaf explants of ‘Improved French’ plum, the most important dried plum cultivar in the USA. Ann Appl Biol 156:79–89CrossRefGoogle Scholar
  31. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238CrossRefGoogle Scholar
  32. Sakai WS (1973) Simple method for differential staining of paraffin embedded plant material using toluidine blue O. Biotech Histochem 48:247–249CrossRefGoogle Scholar
  33. Saker MM, Adawy SS, Mohamed AA, El-Itriby HA (2006) Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis. Biol Plant 50:198–204CrossRefGoogle Scholar
  34. Scowcroft WR (1984) Genetic variability in tissue culture: Impact on germplasm conservation and utilization. IBPGR, Rome, 1984Google Scholar
  35. Sharma S, Pamidimarri DVNS, Anand KGV, Reddy MP (2011) Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis. Ind Crop Prod 34:1003–1009CrossRefGoogle Scholar
  36. Swanberg A, Dai W (2008) Plant regeneration of periwinkle (Catharanthus roseus) via organogenesis. HortSci 43:832–836Google Scholar
  37. van Aartrijk J, Blom-Barnhoorn GJ, Van der Linde PCG (1990) Lilies. In: Ammirato PV, Evans DR, Sharp WR, Bajaj YPS (eds) Handbook of plant cell culture, vol. 5. Ornamental species. McGraw-Hill, New York, pp 535–576Google Scholar
  38. van Tuyl JM (1996) Interspecific hybridization of flower bulbs: a review. Acta Horticult 430:465–476Google Scholar
  39. Varshney AM, Lakshmikumaran M, Srivastava PS, Dhawan V (2001) Establishment of genetic fidelity of in vitro raised Lilium bulblets. In Vitro Cell Dev Biol Plant 37:227–231CrossRefGoogle Scholar
  40. Vos P, Hogers R, Bleeker M, Reijans M, Lee TDE, Hornes M, Frijters A, Pot J, Pelemen J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:407–414CrossRefGoogle Scholar
  41. Wang QC, Valkonen JPT (2009) Cryotherapy of shoot tips: novel pathogen eradication method. Trends Plant Sci 14:119–122PubMedCrossRefGoogle Scholar
  42. Wang H, Alburquerque N, Burgos L, Petri C (2011) Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: a feasible alternative for apricot genetic engineering. Sci Hortic 128:457–464CrossRefGoogle Scholar
  43. Xu LF, Ma FW, Liang D (2009) Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor). Sci Hortic 119:458–461CrossRefGoogle Scholar
  44. Yadav RC, Saleh MT, Grumet R (1996) High frequency shoot regeneration from leaf explants of muskmelon. Plant Cell Tissue Organ Cult 45:207–214CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2013

Authors and Affiliations

  • Zhen-Fang Yin
    • 1
    • 2
    • 3
  • Bing Zhao
    • 1
    • 2
    • 4
  • Wen-Lu Bi
    • 1
    • 2
    • 3
  • Long Chen
    • 1
    • 2
    • 3
  • Qiao-Chun Wang
    • 1
    • 2
    • 3
  1. 1.State Key Laboratory of Crop Stress Biology in Arid RegionNorthwest A&F UniversityYanglingChina
  2. 2.Key Laboratory of Genetic Improvement of Horticultural Crops of Northwest ChinaNorthwest A&F UniversityYanglingChina
  3. 3.College of HorticultureNorthwest A&F UniversityYanglingPeople’s Republic of China
  4. 4.College of ForestryNorthwest A&F UniversityYanglingPeople’s Republic of China

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