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 N 6 -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 ow 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 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.


Introduction
Muskmelon (Cucumis melo L.) is a common vegetable in several tropical and subtropical areas (Feyzian et al., 2009). Though belonging to the Cucurbitaceae family and the Reticulatus subspecies, muskmelon can sometimes be taken mistakenly for cantaloupes which are vegetables of the Cantalupensis subspecies (Malik et al., 2014). Pitrat (2008) reported that muskmelon originated in South Africa, but Schaefer et al. (2009) and Pitrat (2014) later claimed that muskmelon may have bio-historical roots in Asia. Muskmelon is increasingly becoming important in the drug and food industries because the fruits components (Kim et al., 2015). Melon seed oil can treat intestinal worms, reduce blood LDL levels (lowdensity lipoprotein cholesterol) and common blood clots. Also, it can prevent irregular heartbeats and reduce the likelihood of kidney and bladder stones ( variation is frequently observed in regeneration and transformation systems which can be attributed to genetic and somaclonal variation (Guis et al., 1998;Xing et al., 2010).
The culture conditions may affect genetic instability (Orzechowska et al. 2016). Wei et al. (2006) discovered three factors that can assist in increasing organogenesis: the kind of explant, combination and concentration of PGRs, and culture situation. Agrobacterium mediated transformation is the preferable procedure by which DNA fragments are inserted into herbaceous plants and genetic instability is altered. Exposing explants to co-cultivated Agrobacterium can arrest plant tissue growth for an amount of time, thereby reducing proliferation. Some antibiotics such as ampicillin, neomycin and carbenicillin are often employed in suspensions to alleviate this problem. Ren  Nonetheless, no study has applied this marker for the evaluation of transgenic melons. Since high levels of genetic instability are common in regeneration and transformation systems when studying melons, the purpose of this study was to introduce a new method for achieving transformation with high genetic sustainability in muskmelon. The genetic delity of transgenic melon was examined by ow cytometry and for the rst time by molecular markers.

Seed sterilization
Mature seeds were obtained from the muskmelon cultivar 'Khatooni'. The seeds were uncoated and surface sterilized according to Raji et al. (2018). So, the seeds were dried under laminar ow and on sterile paper.

Explant preparation
In following seeds were placed in jars lled with half-MS media (Murashige and Skoog 1962) and after germination, explants were prepared from 4 day-old seedlings. Then those were used to co-cultivation and shoot regeneration.

Transformation and co-cultivation
Further to transformation, 4-day-old cotyledons were cut into small pieces (2 by 5 mm) and then infected by the LBA4404 Agrobacterium tumefaciens strain containing a binary vector pBI121 that harbored the NoS promoter-nptll gene (Fig. 1).
The Agrobacterium suspension was grown in 10 ml Luria-Bertani liquid media including 50 mg l − 1 kanamycin and 50 mg l − 1 rifampicin for 12 h at 28°C in an incubator shaker operating at 200 rpm. The Agrobacterium was sedimented by centrifugation at 10000 rpm for 5 min.
The sediment was resolved in the MS medium containing 25 mg/l acetosyringone. Cells were adjusted at OD600 (5⋅108 cells/ml). The explants were suspended in the Agrobacterium dilution for 2-3 min and were relocated to be dried on lter paper. They were cultured on MS medium without plant growth regulators (PGRs) in an incubator at 28°C for two days of co-cultivation. The incubation occurred in a dark environment.

Organogenesis and acclimation of co-cultivated explants
The co-cultivated explants in free MS, organogenesis in selective medium (SM), including MS media containing BAP (600 µg/l) mixed with NOA (25 µg/l) and adaptation in greenhouse were done based on our previous report ???? et al (2020).
The SM also containing kanamycin (50 mg l − 1 ) and cefotaxime (300 mg l − 1 ). After two weeks of explant culture, proliferation record was started. The irrigation and fertilization occurred in hydroponic systems. DNA extraction DNA was extracted from mother and transgenic plants according to bahmankar et al () study by the modi ed CTAB method (Doyle, 1987). The quality (260/230 and 260/280 ratios) and concentrations of DNA were assessed by a NanoDrop Spectrophotometer (Thermo Scienti c -Waltham, Massachusetts).

Polymerase chain reaction
The presence of the NptII gene in regenerated lines was analysed by PCR using speci c forward (5'-GAACAAGATGGATTGCACGC-3') and reverse primers (

Data analysis
To con rm the transformed lines, 786 bp ampli ed fragments were considered for nptII genes, while no ampli cation of the bands in them should include a vir gene primer.
To consider genetic delity as per ISSR markers, the evaluations were focused only on the bands showing consistent ampli cation between 200 and 2,000 bp. PCR products were scored for the presence (1) or absence (0) of ampli ed bands. The polymorphism was measured base on allele frequencies and number of polymorphic loci/number of total loci.

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 e ciently (65%) from 4-day-old cotyledon explants of 'Khatooni'.

PCR analysis
Kanamycin selection was not completely e cient because PCR analysis showed several "escapes" and no ampli ed DNA fragment for the nptII gene in regenerants. The PCR system ampli ed 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 ampli cation in nptII positive lines (Fig. 3-b).

Genetic delity
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 wellresolved bands were observed in electrophoresis gel (ranging between 7 to 13 and an average of 11 bands per primer). The size of ampli ed fragments ranged between 230 bp to 2.0 kb. All were same among the analyzed regenerants. These results (Fig. 4) indicated that all ampli ed pro les the transplants are true-to-type plants mother.

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.

Discussion
No study has so far assessed somaclonal variation in muskmelon transgenic lines by molecular markers. For the rst 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 e ciency and genetic instability of melon tissues are generally in uenced 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 noninoculated 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. 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 in uential 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 speci c 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).   Genetic delity assessment of transformed lines L. (Khatooni cultivar) using ISSR markers, where in pro les were obtained with primer ISSR11+11b. The ampli cation bands of transgenic plants were similar to mother. Lane M: marker; lane MP: mother plant; lanes R1-11: transgenic plants.

Figure 5
Measurement of chromosomal contents of nuclei in transgenic melon by FMC. Pattern similar of ploidy levels of transgenic and mother plant (standard plant).