Standardisation of in vitro techniques for mass multiplication of aranthera and dendrobium

Abstract

Standardisation of in vitro techniques for mass multiplication of Aranthera and Dendrobium orchid varieties was attempted. The studies were carried out at the Plant Tissue Culture Laboratory, Department of Horticulture, College of Agriculture, Vellayani, during 1992-96. Attempts for the in vitro propagation via enhanced release of axillary buds, somatic organogenesis and somatic embryogenesis were made. One variety Annie Black of the monopodial orchid Aranthera and five varieties of the sympodial orchid Dendrobium were subjected to the initial response studies Sonia-17 (Dendrobium) and Annie Black (Aranthera) varieties were selected for detailed studies. Explants like shoot apices, leaf segments, root segments, keikis, inflorescence stalk were used. Mercuric chloride 0.1 per cent for ten minutes was identified as an effective surface sterilant. The lowest rate of microbial contamination was observed from January to May. The effect of culture medium (basal medium, mode of culture, strength of MS basal medium, major nutrient element, plant growth substances, casein hydrolysate, peptones, glutamine, sucrose, vitamins, yeast and malt extracts, fruit juices, coconut water, ethylene inhibitors) and culture conditions on in vitro shoot proliferation via enhanced release of axillary buds were studied.

Among the various explants tried culture establishment via enhanced release of axillary buds could be induced only from shoot apex explants of all the varieties. Culture establishment could be best induced in Vacin and Went basal medium. Sonia-17 recorded 90.0 per cent bud initiation in VW media supplemented with NAA 1.5 mg/l, BA 1.0 mg/l, Sucrose 30.0 g/l, Coconut water 15.0 ml/l, agar 6.0 g/l within 14 days. Sonia-17 was used for shoot proliferation studies. Maximum shoot proliferation (35.33 shoots per culture) from shoot apex explants of Sonia-17 could be induced in half-strength MS basal medium supplemented with BA 2.5 mg/l, NAA 1.0 mg/l, Sucrose 30.0 g/l, boiled and filtered coconut water 150.0 m/l and agar 6.0 g/l. Among the Dendrobium varieties only Sonia-17 responded to direct organogenesis. Of the various explants tried only the leaf base (from culture) could initiate direct organogenesis. The best treatment identified was half- strength MS basal medium (solid) supplemented with NAA 1.5 mg/l, BA 1.0 mg/l, sucrose 30.0 g/l, Coconut Water 150.0 ml/l, agar 6.0 g/l activated charcoal 1.0 g/l, under darkness. This treatment recorded 9.17 shoot of 4.92 cm length having 3.50 leaves per shoot. Maximum proliferation .of shoot (3.33 shoots/culture) obtained via direct organogenesis could be achieved in half-strength MS basal medium (solid) supplemented with BA 2.5 mg/l, NAA 1.0 mg/l, sucrose 30.0 g/l, agar 6.0 g/l and coconut water 150.0 ml/l. Direct organogenesis from the leaf base of Arantliera var. Annie Black could be initiated in half-strength MS basal medium supplemented with BA

3.0 mg/l, NAA 2.0 mg/l, sucrose 30.0 g/l, coconut water 150.0 ml/l, agar 6.0 g/l in the presence of light. Only one shoot per culture could be induced. Half-strength MS basal medium supplemented with BA 2.5 mg/l, NAA 0.1 mg/l, ascorbic acid + citric acid (150 ppm) supplemented with coconut water 150 ml/l, agar 6.0 g/l and sucrose 30.0 g/l was the treatment identified as best for shoot proliferation in Annie Black. Half-strength MS basal medium supplemented with BA 3.0 mg/l NAA 2.0 mg/l, Sucrose 30.0 g/l, agar 6.0 g/l and coconut water 150 ml/l could induce the development of protocorm-like Bodies (PLB's) on the shoot apex explants of Annie Black in 50.0 per cent of the cultures. Attempts to standardise in vitro propagation via callus-mediated somatic organogenesis and somatic embryogenesis were not successful. In vitro flowering in Sonia-17 was observed when half strength MS basal medium supplemented with BA 2.5 mg/l and sucrose 30.0 g/l, coconut water 150.0 ml/l and agar 6.0 g/l was left unsubcultured for 3-4 months. In vitro regeneration of roots in Sonia-17 could be best obtained in half-strength MS basal medium (solid) supplemented with NAA 1.0 mg/l, sucrose 30.0 g/l, agar 6.0 g/l in the presence of light. This treatment could produce 20.17 roots of 5.08 cm length. An increase of 2-8 roots/shoot could be observed after a period of one and a half-month.

Ex . vitro rooting studies were not successful. All the plantlets survived in the different containers used. Coconut husk was found to be the best medium/container. Hardening the in vitro plantlets in a green house with misting facility recorded cent per cent survival. Survival of plantlets irrigated with nutrient solution as well as hormone spray solution was poor. An increase in shoot length, leaf number, leaf length, leaf width, root number and root length by 2.72 cm, 1.06, 4.25 cm, 1.32 cm, 6.40 and 5.0 cm respectively could be observed after four months of planting out. The mean number of stomata per unit area, total chlorophyll, chlorophyll a were less in the leaves of in -vitro grown plantlets. Rate of water loss through leaves was greatest through the leaves of in vitro plantlets. The cost of production of a single orchid plantlet was found to be Rs. 3.68.