In-vitro Propagation of Pomegranate (Punica Granatum L.) and Their Genetic Fidelity Assessment Using Morpho-physio-biochemical and Molecular Tools

Abstract

This investigation on the “In vitro propagation of Pomegranate (Punica granatum L.) and their genetic fidelity assessment using morpho-physio-biochemical and molecular tools” was carried out in the College of Horticulture. Punica granatum L. is an important fruit crop belonging to the family Punicaceae. India is the world’s leading country in pomegranate production in the world. Pomegranate is a good source of protein carbohydrate, minerals, antioxidants, vitamins C, B and A. The fruit has been useful in controlling heart disease, prostate cancer, diarrhoea, hyperacidity, tuberculosis, leprosy, abdominal pain, fever and many other complaints. Pomegranate is successfully propagated through hardwood and softwood cuttings. The plant is highly cross-pollinated due to heterostyly, which leads to wide variations in tree and fruit characteristics raised through the seeds. However, the supply of true to type planting material is scarce due to certain limitations like low multiplication rate of planting material, the requirement of a large quantity of propagating material due to narrow planting geometry, low success rate of hardwood and softwood cuttings, such limitations led to the non-availability of quality planting material for its plantation. The tissue culture technique has been exploited in pomegranate to meet the current demand. Tissue culture provides an alternative solution for many genetically similar, phyto-sanitary, and physiologically high-quality planting materials within a limited period. An attempt for regeneration protocol and acclimatization of in vitro regenerated plantlets with assured genetic constitution was carried out. Different combinations of PGRs were evaluated for various shooting, rooting and callus parameters. The earliest shoot initiation in 21.60±2.63 days was recorded under SNP 0.5 mg/l and BAP 1.0 mg/l in MS media and found best among the treatment used in the study. Maximum shoot regeneration (86.67±5.77%) was recorded under SNP1.0 mg/l + BAP 1.5 mg/l + KN 1.5mg/l, maximum number of shoots (4.53±0.55) under the combination SNP 1.5 mg/l + BAP 1.5 mg/l + KN 1.5mg/l and the longest shoot (4.1±0.56cm) was recorded under the treatment SNP1.5 mg/l + BAP 1.5 mg/l + KN 1.5 mg/l. The In vitro-regenerated shoots were transferred to the rooting media and earliest root induction (12.10±0.46 days) was observed under NAA 2.0 mg/l SNP 0.2 mg/l combination. The root induction percentage in MS media enriched with (NAA 1.5 mg/l SNP 0.1mg/l), (NAA 1.5 mg/l SNP 0.2 mg/l), (NAA 1.5 mg/l SNP 0.3 mg/l) and (IAA 2.0 mg/l SNP 0.1mg/l) was found non-significant where 100% root initiation was recorded. The maximum number of roots (6.40±0.72) was observed under (NAA 2.0 mg/l SNP 0.3mg/l). Callus formation study in MS media supplemented with 2,4-D and BAP was conducted and maximum callus weight 420.37±45.71mg was recorded under (1.5mg/l 2,4-D + 1.5mg/l BAP). 83.33±5.77% callus induction was highest which was recorded under the treatment (2.0 mg/l 2,4-D + 2.0 mg/l BAP) and Maximum surface area (193.33±14.95mm2) of callus was recorded under (2.0 mg/l 2,4-D + 2.0 mg/l BAP). A large number of plants can be produced under in vitro aseptic conditions, but there is always a danger of producing Soma clonal variants by tissue culture technology. Thus, the genetic fidelity of micro-propagated plantlets were evaluated using SSR and ISSR markers and most of the in vitro and mother plants were highly similar in their genetic constituents. Biochemical analysis using leaves of the mother plant by methanol extract showed highest phenolic content, flavonoid content and tannin content than in in vitro raised plants. And evaluation of antioxidant enzymes in PGRs used media and non PGRs used media (control) found that the antioxidant enzymes increasing in PGRs media than the non PGRs media, antioxidant defence of micro-propagated plants determines by the activities of antioxidant enzymes (Superoxide dismutase, catalase and guaiacol peroxidase) resulted in higher shoot formation and increasing of shoot number per explant.