Research Square
Research Square
Preprint
3 years ago

Genetic Stability in Melon Transformation System: Assessment by Flow Cytometry and ISSR Markers

Mohammad Reza Raji, Mostafa Farajpour
Genetic instability in melon species sometimes occurs as a result of in vitro tissue culture and transformation systems. This study describes a new regeneration technique for agrobacterium-mediated co-culture of muskmelon explants (Cucumis melo L. c.v. 'Khatooni'). Here, no genetic instability was observed in positive PCR regenerants. 4-day-old cotyledonary explants had been infected with LBA4404 Agrobacterium suspensions. The co-cultivation occurred in the presence of 100mg/l rifampicin and 50mg/l kanamycin. The bacteria contained a binary vector pBI121 carrying nopaline synthase by the promoter-neomycin phosphotransferase gene. The regeneration succeeded 65% in selective MS medium containing N6-benzylaminopurine (600 µg/l), β-naphthoxyacetic acid (25 µg/l) and 50mg/l kanamycin in inoculated 4-day-old cotyledonary explants. According to the polymerase chain reaction analysis of neomycin phosphotransferase II gene, transformation was merely successful (8.4%), indicating a substantial miss on a large number of regenerated plants in the selective medium, as a consequence of PGR and antibiotic imbalances. Inter Single Sequence Repeat markers and flow cytometry analyses were used for evaluating the genetic stability and ploidy level of transplants, respectively. The integrated approach underlined that Agrobacterium inoculation and plant growth regulators were successfully combined in vitro to enable muskmelon transformation.

Publisher URL: https://www.researchsquare.com/article/rs-756109/latest

DOI: 10.21203/rs.3.rs-756109/v1

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