Genetic diversity analysis and in vitro seedling production of mangosteen (Garcinia mangostana L.) accessions

Abstract

The study entitled “Genetic diversity analysis and in vitro seedling production of mangosteen (Garcinia mangostana L.) accessions” was conducted at the Department of Fruit Science and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, during the period 2023–2025. The investigation aimed to assess the genetic diversity of mangosteen accessions using molecular markers and in vitro seedling production to enhance seedling vigour. Thirty mangosteen genotypes cultivated in Thrissur and Wayanad districts of Kerala were subjected to molecular characterization. Genomic DNA was isolated from young leaves using the CTAB extraction method (Doyle and Doyle, 1987) with modifications proposed by Sulassih and Santosa (2020) incorporating polyvinylpyrrolidone (PVP) to eliminate polyphenolic impurities. The DNA samples showed UV absorbance ratios (A260/A280) between 1.79 and 2.07 indicating their purity. Ten Inter Simple Sequence Repeat (ISSR) primers, previously reported in mangosteen, were used for amplification. These primers produced a total of 71 loci across the thirty genotypes of which 63 were polymorphic resulting in a mean polymorphism percentage of 85.87%. The number of loci per primer varied from 3 (UBC 860) TO 11 (UBC 840) indicating considerable variation in amplification efficiency. The Polymorphic Information Content (PIC) values ranged between 0.04 (UBC 860) and 0.39 (UBC 825) with an average of 0.28, indicating moderate informativeness. Cluster analysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on Jaccard’s similarity coefficients revealed three major clusters at a similarity coefficient of 0.66 corresponding to 34% dissimilarity. Cluster I comprised a single accession (Acc. 017) that was distinctly separated indicating a high degree of divergence. Cluster II containing twenty six accessions was further divided into subclusters II A and II B at 0.68 similarity, while Cluster III included three accessions (Acc.004, Acc.048, and Acc.126). Principal Coordinate Analysis (PCA) further supported the clustering pattern, indicating the moderate genetic differentiation among the genotypes. Investigations were also carried out for in vitro seedling production of mangosteen. Mature seeds of mangosteen were aseptically cultured on Murashige and Skoog (MS) and 85 half-strength MS media containing different concentrations of sucrose (1%, 2%, and 3%) and gibberellic acid (GA₃) (0, 0.2, 0.5, and 1 ppm). Twelve treatment combinations were evaluated for their influence on germination, subsequent seedling vigour and growth. Significant variation was observed among treatments for all parameters studied. The mean number of days required for germination ranged from 12.33 to 22.67 days. The earliest germination was observed in treatment T₁₂ (½ MS + 2% sucrose + GA₃ 1 ppm). Similarly the germination percentage also varied significantly, with T₁₂ showing the highest value (95.24%) followed by T₁₀ (½ MS + 2% sucrose + GA₃ 0.2 ppm). Seedling vigour indices (SVI-I and SVI-II) reflected a similar trend with T₁₂ recording the highest values (1383.81 and 44.48, respectively), indicating the positive influence of reduced salt concentration and GA₃ supplementation on germination and early growth. Growth parameters recorded at the time of plant out revealed significant differences among treatments. The longest shoots (5.73 cm), roots (3.87 cm) and leaves (4.77 cm) were recorded in T₁₂. After four weeks of hardening, seedlings from T₁₂ maintained superior performance with the highest shoot length (7.10 cm), root length (7.17 cm), and leaf length (6.17 cm). The number of leaves was the highest in T₈ (MS + 2% sucrose + GA₃ 0.5 ppm) and T12 (½ MS Media (2% sucrose) + GA 1 ppm). Survival percentage did not differ significantly among treatments; however, T₁₂ and T₆ recorded the highest survival percentage (95.24), indicating the robustness of the developed seedlings. These results demonstrated that half-strength MS medium supplemented with 2% sucrose and 1 ppm GA₃ was optimal for inducing early germination, improving root and shoot elongation, and enhancing seedling vigour. The present study confirms the existence of genetic diversity among mangosteen accessions in Kerala despite their apomictic nature. The moderate polymorphism revealed by ISSR markers indicates underlying genetic differentiation likely resulting from somatic mutations or environmental adaptation. The optimized in vitro germination media (½ MS medium supplemented with 2% sucrose and 1 ppm GA₃) proved effective in enhancing germination, seedling vigour, and survival percentage.