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

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.

In-vitro Flowering and in-vivo Sex Expression of Micropropagated Parthenocarpic Gynoecious Cucumber

A micropropagation protocol for parthenocarpic gynoecious cucumber reduces the burden of producing the seeds for each generation and their maintenance in-vivo. Thus an experiment was conducted in order to regenerate the plants in-vitro to check their performance after micropropagation. The micropropagation resulted in maximum shoot initiation (100%) from seedling excised cotyledonary explants with half strength MS medium supplemented with 0.5 mg/l IAA and 2 mg/l BAP along with half strength MS medium supplemented with 0.25 mg/l IAA for rooting and from stem nodal explants with Full MS + 1.5 mg/l IAA + 2 mg/l BAP media whereas half strength MS media without any hormones resulted in rooting and in both cases there were in-vitro flowers and change in their sex expression while grown in in-vivo conditions. On an average 61.11 and 48.15 percent survival was recorded from the plants regenerated through cotyledonary explants and stem nodal explants respectively. Out of five survived plants from regenerated parthenocarpic genotype CS 131 three showed monoecious sex expression and two exhibited gynoecious (parthenocarpic) sex expression. Mixed response of sex expression was evident in the regenerated parthenocarpic and gynoecious genotypes.

Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42–45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

AUXIN PULSE IN THE INDUCTION OF SOMATIC EMBRYOS OF Eucalyptus

ABSTRACT The objective of this study was to evaluate the effect of auxin pulse intervals on the induction of somatic embryos of Eucalyptus grandis x E. urophylla and to describe the embryogenic behavior of callus under the effect of auxinic stress. Cotyledons were inoculated in culture medium containing 207.07 µM picloram, a treatment considered as auxin pulse. Explants that were in the auxin pulse treatment were transferred to semisolid or liquid medium containing 20.71 µM picloram after one, two, four or eight days of auxin pulse. In a second experiment, explants that were on auxin pulse treatment were transferred to semi-solid medium containing 20.71 µM picloram after one, two or three days of auxin pulse. Auxiliary picloram pulse treatments (207.02 µM) can be used as an initial source of stress for the acquisition of embryogenic competence. The oxidation of cotyledonary explants may be considered as an indication of the formation of embryogenic calli. The presence of pectins in peripheral regions of somatic pro-embryos can be considered as a marker of somatic embryogenesis in cotyledonary explants of Eucalyptus grandis x E. urophylla.