1.1 Shoot formation on selective media
At the beginning, the frequency of transformation was assessed by shoot formation on 4-day-old explants in SM (Fig. 2). After co-culturing and during 5 to 6 weeks of processing, DO occurred efficiently (65%) from 4-day-old cotyledon explants of ‘Khatooni’.
1.2 PCR analysis
Kanamycin selection was not completely efficient because PCR analysis showed several “escapes” and no amplified DNA fragment for the nptII gene in regenerants. The PCR system amplified the selective marker in 8.4% of the regenerants and was established in the presence of 786 bp fragments of nptII in 11 transgenic lines regenerated from SM (Fig. 3-a). There was no Agrobacterium contamination in nptII PCR since the PCR of the Vir gene did not indicate any amplification in nptII positive lines (Fig. 3-b).
1.3 Genetic fidelity
Genetic stability of the positive PCR plantlets were analyzed by ISSR markers. Ten ISSR primers were used in this study, and all ten are common in research applications (Haddad et al., 2017). A total 112 well-resolved bands were observed in electrophoresis gel (ranging between 7 to 13 and an average of 11 bands per primer). The size of amplified fragments ranged between 230 bp to 2.0 kb. All were same among the analyzed regenerants. These results (Fig. 4) indicated that all amplified profiles the transplants are true-to-type plants mother.
1.4 Flow cytometry
The FMC pattern of all transgenic and mother plants was homogeneous in channel 200 nuclei (Fig. 5), which resulting no differences between the transgenic lines and standard plants (Yang et al., 2008; Carra et al., 2012). These observations indicating that polyploidization did not occur in transformation system.
2. Discussion
No study has so far assessed somaclonal variation in muskmelon transgenic lines by molecular markers. For the first time, it was assessed in this study where no somaclonal differnces was observed among the transformed lines and standard line, according to ISSR markers. The recent results show that the chromosomal composition here is very stable compared to other reports. Ren et al. (2012) asserted that the type of bacterial strain, infection period and density of the Agrobacterium growth media affect regeneration, transformation rate and genetic inconsistency in melon. Nuñez-Palenius et al. (2006) and Ren et al. (2013) found that 80% to 100% of the transgenic plants were tetraploid. Chovelon et al. (2011) proved that genetic instability in transformed plants produced from cotyledons (53%) was higher than those obtained from melon leaf explants (12.2%). This could be due to a greater effect of Agrobacterium on younger cells of the cytodenic exudates.
Guis et al. (2000) had observed more tetraploidy in younger cotyledons. In this research the ISSR markers and FMC were utilized to compare chromosomal composition and genetic differences between transformed muskmelons and their mother plants. The transformation efficiency and genetic instability of melon tissues are generally influenced by several agents. Different results have been reported from in vitro differentiation following Agrobacterium transformation.
Our experiment suggests that shoot formation of co-cultivated explants decreases as compared to non-inoculated explants. The meristematic tissues of recalcitrant species like muskmelon destruct after the infection and co-cultivation of explants with Agrobacterium suspension which could be due to the production of ethylene in the culture medium. Khanna et al. (2007) showed how ethylene affects many growth processes and reduces the frequency of melon regeneration and transformation.
Ntui et al. (2010) reduced ethylene production by explants after Agrobacterium infection, even as the ACC gene expression reduced and increased the transformation rate. A cytological study by Dan et al. (2010) on melon epidermal and sub-epidermal cells revealed a lower regeneration rate of the infected explants which can be related to the disability of cells in causing regeneration. Also, this may be due to the genetic instability of the infected meristematic parts.
Different results were also published about groundnut regeneration after Agrobacterium infection (Venkatachalam et al., 1998) whereas Guis et al. (2000) observed no difference in the regeneration frequency between infected and uninfected melon explants. Some studies have shown negative effects of Agrobacterium inoculation on regeneration. Deterrence or improvement of the proliferation in SM is possibly a manifestation of the role of some antibiotics in media culture (Bordas et al., 1997; Choi et al., 2012; Zhang et al., 2014). Antibiotics that were used in this study include cefotaxime, rifampicin and kanamycin which can be influential in regeneration. Valles and Lasa (1994) demonstrated that the frequency of shoot regeneration on an antibiotic-free medium was more than regeneration on media containing antibiotics. The frequency of regeneration has been affected positively by cefotaxime when specific concentrations are used in the culture media for some plant species such as durum wheat (Borrelli et al., 1992) and barley (Mathias and Mukasa, 1987). Cefotaxime is often supplemented in the organogenesis media after co-culture to prevent Agrobacterium growth (Yu et al., 2001).
This antibiotic is placed in the b-lactam class and is similar to plant PGRs. It has the least toxicity on in vitro plant culture, whereas some b-lactam antibiotics such as carbenicillin have shown inhibitory effects at higher concentrations and have reduced regeneration frequency (Grzebelus and Skop 2014). Phillips et al. (1981) affirmed that Rifampicin can effectively eliminate bacterial contamination but stated negligible effects on explant growth and regeneration.
Kanamycin has negative effects on the frequency of shoot and root formation in vitro regarding some melon subspecies (Bordas et al., 1997; Ren et al., 2012; Choi et al., 2012). Accordingly, we expected an increase in the regeneration of ploidy levels, whereas the ploidy level did not change after inoculation. In this study, a high genetic constancy (100%) of transformed muskmelon (‘Khatooni’) was successfully achieved using NOA+BAP PGRs.