Molecular diversity among in vitro and field grown grapevine cultivars (Vitis vinifera L.) from Pakistan

Abstract

In the current research work, conditions were optimized for establishing an effective protocol for pretreatment of explants, induction of shoots from nodal explants and their root formation from different cultivars of Vitis vinifera L. (Red Globe, Autumn Royal, Crimson, Thompson, Sundarkhani, Perlette and King‟s Ruby) using MS medium suplemented with different plant growth regulators. In the present study, for controlling the browning of the cultured explants due to the presence of phenolic compounds, a combination of 150 mg/L Citric acid and 100 mg/L Ascorbic acid proved effective for all the grapevine cultivars used. For shoot induction from nodal explants, effects of different hormones (BAP, KN and TDZ) and their combination, supplemented in MS medium was tried. Along with the shoot induction percentage, the morphology of the newly grown in vitro shoots (number and length) was also studied. Among the different cytokinins, best response was shown by BAP at 0.5 mg/L concentration in cultivars, Red Globe, Crimson, Perlette and King‟s Ruby while in others a higher concentration was favourable for shoot induction. Moreover, shoot length was also directly proportional to hormone concentration. Conditions were also established for root induction from in vitro grown shoots using half-strength MS medium fortified with different concentrations of IBA. The best root induction percentage was recorded at 2.0 mg/L in all of the cultivars. For acclimatization, rooted in vitro plantlets were shifted in sterile sand: soil (1:1) for hardening inside the culture room which was then shifted to the soil after one month. In the present study, antioxidant and antibacterial activity of leaf extracts of grapevine cultivars was also studied. For antioxidant activity, different scavenging assays including 1, 1 diphenyl- 2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) and Ferric reducing antioxidant power (FRAP) assay were tried with Ascorbic acid as standard. The results of inhibition in DPPH radical scavenging assay showed increasing order with an increase in the concentration of the plant extract of grape cultivars. Evaluation of antioxidant activity of methanolic grapevine leave extracts using the hydrogen peroxide (H2O2) scavenging activity showed an increase in absorbance with an increase in the concentration of leaf extracts in all the cultivars. Reductive capabilities exhibited by leaf extracts, evaluated by potassium ferricyanide reduction method or reducing power assay showed RP values of plant polyphenols. In general, strong and positive presence of antioxidants was observed in all cultivars studied. The antibacterial activity of various concentrations (25,50 and 100mg/mL) of the leaf extract of all grapevine cultivars was evaluated using six strains of bacteria (Proteus sp., Streptococcus viridans, Escherichia coli, Pseudomonas sp., Methicillin resistant Staphylococcus aureus (MRSA) and Clostridium septicum) using agar well diffusion method. The results showed that Escherichia coli was the most resistant bacterial strain and Proteus sp. was the least resistant bacterial strain against the leaf extract of cv. Autumn Royal among all the cultivars studied. It was observed that all the concentrations of plant extracts of all the cultivars exhibited a varying degree of antimicrobial activity by suppressing the growth of all the six bacterial strains tested. In the present research work, the clonal homogeneity of six months old tissue culture raised plants of all grapevine cultivars was checked with mother plants using Inter Simple Sequence Repeat (ISSR) markers. Morphological assessment of these in vitro raised plants maintained in greenhouse did not show any differences with mother plants. However, to test the genetic homogeneity of these plants, 6 ISSR primers [(GTG)5, (GACA)4, (GGAT)4, (GATA)4, (CA)8 and (CAA)5] were screened, out of which 4 primers [(GACA)4, (GATA)4, (CA)8 and (CAA)5] produced scorable and repeatable bands with all the cultivars except King‟s Ruby which responded only to 2 primers [(GATA)4 and (CA)8]. These 4 ISSR primers generated 74 distinct band classes with a total of 370 scorable bands. All primers showed uniform banding pattern in all plants of each cultivar to their mother plant. The allele sizes ranged from 150 to 2000 bp. The results of DNA based marker system in the present study revealed the genetic uniformity among the in vitro raised plants and demonstrated the reliability of in vitro propagation system used for grapevine cultivars. To assess the genetic diversity among all the cultivars of grapevine, molecular analysis of all the cultivars was performed using microsatellite fingerprinting method. In this study, 6 ISSR markers were also used for characterization of all seven grapevine cultivars and the size of amplification products ranged from 300 to 3000bp. The total number of scored bands was 84 and varied from 11 to 36 per primer with an average of 3 bands per template. Cluster analysis showed variations ranging from 0% to 86% among the cultivars except for King‟s Ruby which showed no banding pattern with three ISSR markers i-e, (GTG)5, (GACA)4 and (CAA)5 and Crimson which showed no motifs with (GTG)5. However, the cultivars were vegetatively propagated which discounted mutation as the source of differences among them during their data analysis. This work also shows that evaluating the genetic diversity of grapevine germplasm collections using ISSRs is very competent for elementary and applied research. It can be helpful for protecting germplasm resources and practicing breeding programs