Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas).
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Date
2014-01
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Abstract
Aims: Sweet potato (Ipomoea batatas) cv. “Miyazakibeni” was used as material for shoot
apex culture, reverse transcription-polymerase chain reaction (RT-PCR) and clonal
propagation to establish an efficient and practical virus-free seedling supply system in
production of vegetatively reproductive plants.
Study Design: At first, efficient plant regeneration was achieved from shoot apex culture
of sweet potato. Secondly, RT-PCR method was used to detect the sweet potato feathery mottle virus (SPFMV) viral infection of tuber surface of edible sweet potato using the
RNAs from the plants obtained from shoot apex culture. Finally, the virus-free plants
verified by RT-PCR were propagated clonally by culture of suckers cut from stems of the
virus-free plants.
Place and Duration of Study: Faculty of Environmental and Horticultural Science,
Minami Kyushu University, between June 2008 and December 2012.
Methodology: The best efficiency for material sterilization was tested using different
concentrations (0.1% - 1.5%) of sodium hypochlorite solution (SHS) and the treated times
(5 min – 20 min). Theshoot apexes less than 0.3mm in size were cultured on Komamine
and Nomura (1998) (KN) medium and Murashige and Skoog (1962) (MS) medium. The
regenerated plants were used for RNA extraction and then, used for RT-PCR for detection
of SPFMV. Based on the result of RT-PCR, the suckers cut from stems of virus-free
plants were cultured and propagated clonally and routinely within a short period.
Results: The combination of 0.3% of SHS and 10 and/or 20 min gave the best result
(100%) of surviving rate for material sterilization. The culture of shoot apexes less than
0.3 mm in size gave plant regenerating rates of 82% and 65% on KN and MS medium,
respectively. The results of RT-PCR of RNAs from plants obtained from shoot apex
culture and plants of SPFMV infection showed that SPFMV virus was clearly removed by
shoot apex culture conducted in this study. For clonal propagation, 80-100% of suckers
cut from the stems of the virus-free plants detected grew into complete plants after 6
weeks of culture, indicating that the virus-free plants could be routinely propagated 5
times in number each time and repeatable by the short circle. The sweet potato produced
in field showed no symptom called as russet crack-like symptom (RC-LS) even after
cultivation two seasons.
Conclusion: Overall, an efficient and practical virus-free seedling supply system was
established in sweet potato by the three steps of 1) virus-free plant regeneration from
shoot apex culture, 2) quick detection of SPFMV using RNA of the regenerated plants by
RT-PCR, and 3) the verified virus-free plants were propagated clonally and routinely
within a short period using culture of suckers cut from the stems of virus-free plants.
Description
Keywords
Ipomoea batatas (L.) Lam, clonal propagation, reverse transcription-polymerase chain reaction, shoot apex culture, sucker culture
Citation
Chen Lanzhuang, Chenti Xu, Du Zhaosheng, Hamaguchi Takuro, Sugita Toru, Nagata Ryutarou, Guan Liming. Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas). British Biotechnology Journal. 2014 Jan; 4(1): 51-63.