Genomic alterations in coding region of tissue culture plants of Coffea arabica obtained through somatic embryogenesis revealed by molecular markers
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In coffee, the micropropagation technique can be efficiently used in mass multiplication of superior F1 hybrids which is difficult using the conventional method. However, the in vitro regeneration protocol is genotype-specific. Therefore the development of an efficient regeneration protocol is critical to the success of large scale propagation of any coffee hybrids. In the present study, regeneration of two improved rust tolerant arabica hybrids S.4202 and S.4932 was achieved through somatic embryogenesis. The MS media supplemented with different concentrations of 2, 4-D, IAA and Kn were tested for callus induction using foliar explants. The optimum somatic embryogenesis in S.4202 and S.4932 was obtained when explants were incubated in callus induction media containing 0.4 to 0.6 mg/l 2, 4-D. The in vitro regenerated plants were planted in the field and were found to be phenotypically normal. The genetic fidelity of in vitro regenerated plants of both the genotypes along with their mother plant was tested using 20 SRAP and 12 SCoT markers. The average genetic similarity between the tissue culture-derived plants and mother plants of S.4202 was 0.977 using SRAP and 0.987 using SCoT markers. Similarly, the average genetic similarity between the mother plant and the in vitro derived plants of S.4932 was 0.966 and 0.993 using SRAP and SCoT markers respectively. The differential fragments amplified by SRAP primers in the regenerated plants were sequenced and in silico analysis of the fragments revealed the occurrence of somaclonal variation in the coding region with functional attribution to zinc finger protein.
Plant regeneration through somatic embryogenesis was achieved in two improved cultivars of Coffea arabica. The genetic fidelity of in vitro regenerated plants was tested using both SRAP and SCoT molecular markers. The differential fragments amplified in in vitro regenerated plants using SRAP primers were sequenced and in silico analysis revealed the genomic alterations in the coding region.
KeywordsCoffea arabica Plant regeneration SRAP SCoT Coding region Genome alteration
The authors thank Dr. Y. Raghuramulu, Director of Research Central Coffee Research Institute, Coffee Board, India, for providing laboratory facilities and encouragement. Funding support from Coffee Board, Govt. of India, is gratefully acknowledged.
MKM conceived the experiment and participated in the tissue culture of S.4932 genotype and wrote the manuscript. BM carried out the tissue culture experiments and field data collection and participated in manuscript preparation. PKJ performed the SRAP analysis and AKH performed the SCoT analysis. MKM edited the manuscript. All authors have gone through the manuscript and approved.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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