In Vitro Regeneration of Pomegranate and Genetic Integrity Assessment of Derived Materials

Abstract

Pomegranate is an important plant with nutritional, economic and health benefits. In recent years, many people have become aware of health benefits of pomegranate but the existing propagation methods cannot meet the demand for suitable planting materials needed for commercial production. Micropropagation can lead to mass production of plantlets and callus-mediated in vitro regeneration can open avenues for application of genetic engineering to improve this crop. The aim of this study was to develop an appropriate in vitro regeneration protocol for pomegranate and assess the genetic integrity of derived materials. Cytokinins (BAP, KN, TDZ) were tested for shoot induction from nodal explants while auxins (NAA, IBA and IAA) were tested for root induction from in vitro regenerated shoots. NAA in combination with BAP were assessed for their ability to induce callus from cotyledon and leaf explants. For callus induction, there were two experiments; one experiment had five levels of NAA with a fixed level of BAP. The other experiment had five levels of BAP with a fixed level of NAA. Genetic variability between mother plant, derived callus and plantlets was assessed using SSR markers. Highest number of shoots and leaves from regenerated shoots were obtained on MS media supplemented with 6.9μM KN; an average of two shoots and 12.00 ± 1.15 leaves/ explant. The highest number of roots was achieved on half strength MS media supplemented with 4.9μM IBA; an average of 7.00 ± 1.00 roots/shoot. The longest root was obtained on media supplemented with 5.3μM IAA; an average of 15.00 ± 1.00 mm. Both IAA at 5.3μM and IBA at 4.9μM were suitable for root induction. Callus was induced on MS media supplemented with combinations of (2.2-11.0μM BAP) and (2.8-13.2μM NAA). xii Hence cotyledons and young leaves are suitable explants for callus induction in pomegranates. Eight SSR markers were used in assessment of variability in tissue culture derived pomegranate. Genetic variation with a similarity coefficient of 0.46-0.92 was found in the derived materials. This variation could be attributed to the seedling materials used in the study. Therefore tissue culture can be used in rapid multiplication of pomegranate propagation materials. However there is a need to optimise conditions for detecting somaclonal variation in derived materials. Combination of BAP (2.2-11.0μM) and NAA (2.8-13.2μM) can be used in pomegranate callus formation.