PhD

Investigations on “Rapid propagation and conservation of selected leguminous medicinal plants using in vitro techniques” were carried out at the Department of Plantation Crops and Spices and at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2001-2003. The cryopreservation experiments were conducted at the Division of Biotechnology, Tropical Botanical Garden and Research Institute, Palode during 2003-2004. Leguminous medicinal plants viz., Clitoria ternatea L., Mucuna pruriens Baker non DC. and Indigofera tinctoria L. having immense medicinal value and of which superior accessions were available in the Department of Plantation Crops and Spices were selected for the study. Standardisation of in vitro techniques for rapid propagation of these medicinal plants and their conservation via slow growth and cryopreservation were attempted in the present study.

Enhanced release of axillary buds from nodal explants, with maximum shoot proliferation of 6.33 and 5.17 was obtained in MS medium supplemented with BA 0.5 mg l-1 + Kn 0.5 mg l-1 in C. ternatea and M. pruriens respectively. Nodal explants of I. tinctoria recorded maximum shoot proliferation of 4.67 in MS medium with BA 1.0 mgl-1 + IAA 0.1 mg l-1. Leaves and cotyledons, when used as explants did not give rise to any shoots.

In order to initiate somatic (indirect) organogenesis, maximum callus index (267 to 367) could be produced in MS medium with NAA 2.0 mg l-1 + BA 0.5 mg l-1, NAA 1.0 mg l-1 + BA 0.5 m gl-1 and 2,4-D 2.0 mg l-1 + BA 0.5 mg l- in stem, leaf and cotyledon explants respectively in C. ternatea. 2,4-D 2.0 m gl-1 + BA 0.1 mg l-1, NAA 2.0 mg l-1 + BA 0.5 mg l-1 and 2,4-D 2.0 mg l-1 + BA 0.5 mg l-1 in MS medium recorded maximum callus index (199.99 to 283.00) in stem, leaf and cotyledon explants respectively in M. pruriens. 2,4-D 2.0 mg l-1 + BA 0.5 mg l-1 and 2,4-D 2.0 mg l-1+ BA 0.1 mg l-1 in MS medium recorded highest callus index (155.34 to 250.00) from stem and leaf explants respectively in I. tinctoria.
In C. ternatea, BA 2.0 mg l-1 + Kn 1.0 mg l-1 + NAA 1.0 mg l-1 + adenine sulphate 20 mg l-1, BA 2.0 mg l-1 + NAA 0.5 mg l-1 and BA 2.0 mg l-1 + Kn 1.0 mg l-1 + NAA 0.5 mg l-1 in MS medium were identified as the best media for shoot regeneration from stem, leaf and cotyledon derived callus respectively. In M. pruriens, Kn 1.0 mg l-1 + NAA 0.5 mg l-1 in MS medium was identified as the best medium for shoot regeneration from stem derived callus and BA 2.0 mg l-1 + NAA 0.5 mg l-1 in MS medium for shoot regeneration from leaf and cotyledon derived callus. BA 2.0 mg l -1 + NAA 0.5 mg l-1 in MS medium was identified as the best media for shoot regeneration from stem derived callus.

Somatic embryogenesis could be induced in Clitoria ternatea using yellow granular and friable leaf derived callus (from callus induction medium 2,4-D 0.5 mg l-1 + BA 0.1 mgl-1in MS medium) on culturing callus on to MS medium supplemented with BA 1.0 mg l-1, initiated globular, heart and torpedo shaped embryos. Embryos, when separated and cultured on GA supplemented medium or hormone free MS medium, torpedo shaped embryos developed into plantlets with root and shoot initials. Elongation of plantlets was achieved in hormone free MS medium. The fully developed plantlets could be acclimatized and transplanted to pots under ambient conditions with 40 per cent survival rate.

Rooting of in vitro raised shoots was achieved by culturing them on to NAA 1.0 mg l-1, IBA 2.5 mg l-1 and IAA 1.5 mg l-1 in MS medium in C. ternatea, M. pruriens and I. tinctoria, respectively.

In in vitro conservation, half MS with IBA 0.5 mg l-1 was identified as the suitable basal culture media for slow growth in all the three plant species under study. Cultures could be maintained without subculture for 28 weeks. Those cultures which gave more than 50 per cent survival and regeneration were again subcultured on to fresh slow growth medium to reassess the efficacy of the medium to induce slow growth. PVP 6.0 per cent, mannitol 1.0 and 0.5 per cent were identified as the best among the various additives tried to induce slow growth in C. ternatea, M. pruriens and I. tinctoria respectively, on storage up to 40 weeks in terms of cent per cent survival and regeneration and plant growth inhibition.

For cryopreservation, germination of seeds was found significantly influenced by its moisture content. Lower moisture level of 7.03, 5.24 and 5.35 per cent recorded a higher germination per cent of 90.44, 83.91 and 83.36 per cent respectively in C. ternatea, M. pruriens and I. tinctoria. Varying periods of storage in liquid nitrogen did not affect the germination of seeds in C. ternatea, M. pruriens and I. tinctoria.

Due to the large size of seeds in M. pruriens, zygotic embryos were found more suitable for cryopreservation. The highest survival (90.76 per cent) and plant regeneration (80.05 per cent) were recorded at a moisture level of 14.17 per cent, 150 min after desiccation.

In C. ternatea and I. tinctoria, 4 h desiccation was found to be most effective for cryopreservation of encapsulated axillary buds by encapsulation- dehydration method, while in M. pruriens none of the desiccated buds survived freezing in liquid nitrogen. On rapid freezing in liquid nitrogen with the addition of cryoprotectant, only meristems of M. pruriens survived. Cryoprotectant combination of sucrose 0.7 M + DMSO 5 per cent was found effective and none of the three species survived in other cryoprotectant treatments.

Germinated somatic embryos on encapsulation-dehydration gave maximum survival of 43.33 per cent at 5 h desiccation.

To assess the genetic fidelity of plantlets regenerated from cryopreserved materials, the RAPD patterns were compared with those of the non-cryopreserved plants. Six primers were selected for each plant species. No difference was observed in the banding pattern of cryopreserved and non-cryopreserved samples in all the three plant species under study, which confirmed the genetic stability of cryopreserved materials.