Induction of genetic variability in Musa sp. var. Nendran by in uitro methods

Abstract

Investigations were carried out at the Department of Pomology and Floriculture and Plant Tissue Culture Laboratory of the All India Co-ordinated Floriculture Improvement Project, College of Horticulture, Vellanikkara, Thrissur, during 1991-93 on the induction of variability in the banana variety Nendran (Musa AAB ‘Nendran’) by in vitro methods. Explants utilized for the study were shoot tip and eye bud for direct organogenesis through enhanced release of axillary buds and shoot tip, flower base, inflorescence axis, embryonic leaves and scalp for somatic organogenesis/embryogenesis. For culture establishment, axillary shoot initiation and in vitro rooting, different growth regulators, like NAA, 2, 4-D and 2, 4, 5-T (auxins) and BA and kinetin (cytokinins) were made use of. The plantlets produced in vitro were subjected to hardening treatments to ensure a better establishment of planted out plants and their growth parameters were studied. For shoot tip and eye bud explants, a combination of treatments involving, an initial dipping of explants in emisan (0.1 per cent) for 30 minutes followed by dipping in norfloxacin (0.1 per cent) for 30 minutes followed by dipping in norfloxacin (0.1 per cent) for 30 minutes and finally rinsing the explants in mercuric chloride (0.1 per cent) for 20 minutes was found to be best, but for flower base and inflorescence axis explants, emisan (0.1 per cent) treatment for 20 minutes and for embryonic leaves, dipping in alcohol for one second were in the best. Better and speedier establishment and growth of shoot tip and eye bud explants were observed on MS (semi-solid) medium containing NAA 2 ppm + BA 5 ppm. Addition of activated charcoal (500 mg per litre) to the medium, reduced media and explant discolouration due to polyphenol oxidation. When the performance of the shoot tip and eye bud explants was compared, eye bud explants took more time for culture establishment and growth. In shoot tip culture, on an average, each explant released 8.66 axillary shoots in the treatment involving MSb*+ NAA 2 ppm + BA 10 ppm. In the case eye bud, on an average, each explant released five axillary shoots. Continuous sub culturing was carried out at two week interval to assess the variation induced to cultured plants due to repeated subculturing. It was found that, the number of shoots produced per culture was not constant in all the subcultures. Still, the axillary shoots produced per explant per culture vessel increased at the mean rate of 5.90. BA alone at higher concentration (10 ppm) resulted in colloid (globular semi-hard, light green callus like structure) formation and subsequent regeneration. MSb*: MS medium containing half concentration of inorganic salts and full concentration of organic growth factors. For in vitro rooting, MSb medium containing NAA 10 ppm and AC 0.05 per cent was found to be effective for early root initiation and the maximum number of roots per shoot was produced at the treatment involving MSa* +NAA 5 PPM+ AC 0.05 per cent. Of the various explants, viz., shoot tip, inflorescence axis, flower base, embryonic leaves and scalp(in vitro) tried for initiating collus scalp and embryonic leaves recorded maximum response. Among the media tried for callus initiation, MSb media at liquid consistency was found to be more effective. Maximum callus index (266) was recorded for the treatment combination involving 2, 4-D 7 ppm and BA I ppm. For callus differentiation the treatments involving 2, 4-D and BA, BA alone and basal MS media resulted in rhizogenesis, and treatments involving 2, 4-D alone produced embryoid like structures from scalp callus. No shoot organogenesis was observed. Also treatments were conducted with changed levels of nitrate source in the media, but they did not give any favourable results. Embryoid like bipolar structures were recovered from scalp callus when they were transferred to media devoid of growth regulators. To study the variation, if any, induced due to derail subculturing, the shoots obtained from each subculture cycle (through enhanced release of axillary buds) were isolated and their MSa* : MS medium containing full concentration of inorganic salts and organic growth factors identify maintained. The shoots thus separated were rooted and planted out after subjecting them to a process of hardening. Somatic chromosome counts were made at the root tips of plantlets from 10 subcultures to confirm the ploidy. All the plants were triploids (2n = 33). The plantlets from different subcultures were planted out in sand, which was found to be the best medium. Observations made on growth parameters, at fifteen days interval, revealed that the plants from subcultures differed significantly with respect to the rate of growth in height and leaf area.