MSc

Investigations were carried out to study the response of gladiolus to rapid cloning through in vitro techniques at the Department of Pomology and Floriculture and Plant Tissue Culture Laboratory of All India Co-ordinated Floriculture Improvement Project, College of Horticulture, Vellanikkara, during 1992-94. The main objective was to identify the most suitable explant and media combination for in vitro cloning.
The explants used were corm axillary buds, cormel tips, inflorescence nodal segments (for enhanced release of axillary buds), inflorescence internodal segments, flower buds, flower bud bracts, root segments (for somatic organogenesis) and leaf segments (for somatic embryogenesis). The best season for the collection of corm axillary buds and cormel tips was from September to May. Surface sterilization of the explants could be effectively done with 0.1 or o.2 per cent mercuric chloride and the duration of treatment varied from I to25 minutes.
Culture establishment of the corm axillary bud, cormel tip explants were better in MS medium supplemented with BAP ranging from 1.0 mg 1 -1 to 4.0 mg 1-1. The concentration of BAP required varied according to the stage of development of corms and cormels. Higher levels of BAP was ideal during early stages of development of corm and cormels. Of the different media (White’s, SH and MS) tried, MS medium was found to be the best culture establishment (Stage 1) when supplemented with 3.0 mg 1-1 BAP.
Elongated shoots of Stage I were subjected to shoot proliferation (Stage 2). Multiple axillary bud production was very high when the MS medium was supplemented with BAP 1.0 mg 1-1 and NAA 0.5 mg 1-1 or BAP 2.0 mg 1-1 and NAA 0.5 mg 1-1. Callus production from the base of the elongated shoots were observed when the concentration of NAA increased in the medium.
Of the different cytokinins (BAP, kinetin and 2ip) tried, BAP was found to be the best in Stage 2. Frequent subculturing onto the MS medium containing BAP 2.0 mg 1-1 and NAA 0.5 mg 1-1 increased the production of multiple axillary buds. These when transferred to the MS medium devoid of growth regulators resulted in elongation of shoots.
The elongated shoots produced maximum number of roots in the MS medium containing 1.0 mg 1-1 IBA under the exclusion of light. However, early rooting was obtained in MS liquid medium devoid of growth regulators.
Plantlet survival was maximum when treated with 0.2 per cent Bavistin immediately after removing from the culture vessels, followed by treatment with 0.2 per cent mancozeb and norfloxacin at the time of transplanting and post planting treatment with 1/10 MS solution and drenching with triadimefon 20.0 mg 1-1 at three days interval inside improvised mist chamber.
Direct organogenesis could be obtained from immature inflorescence segments in modified MS medium supplemented with 15.0 mg 1-1 NAA and 3.0 mg 1-1 BAP.
Among the various explants tried for callus mediated organogenesis, callus index was the maximum (400) when immature inflorescence segments were inoculated to the modified MS medium supplemented with NAA 15.0 mg 1-1 in 16 h photoperiod and also in the medium supplemented with 15.0 mg 1-1 NAA + 2.0 mg 1-1 BAP and kept under exclusion of light. The callus derived from inflorescence segments differentiated into shoots in the MS medium supplemented with 3.0 mg 1-1 BAP and also in the medium supplemented with 1.0 mg 1-1 BAP and 0.5 mg 1-1 NAA. Callus also could be obtained from flower buds and flower bud bracts.
The callus derived from the corm axillary buds and cormel tip explants in Stage 2, differentiated in the basal MS medium devoid of growth regulators or supplemented with 20.0 ml 1-1 coconut water and also in the medium with 0.5 mg 1-1 BAP.
The root segments (both in vitro and in vivo) produced callus in MS medium supplemented with 1.0 mg 1-1 NAA and the differentiation was obtained in the medium containing 3.0 mg 1-1 BAP an 1.0mg 1-1 NAA.
Leaf segments failed to develop callus. However, the explants collected from the leaf covering the inflorescence boot leaf) when cultured in modified MS medium supplemented with 15.0 mg 1-1 NAA and 1.0 mg 1-1 BAP and incubated under darkness for three months developed somatic embryos.
In vitro corm production was noticed in the cultures, if planting out was delayed. Earliest and large sized corm induction was made possible in elongated shoots of gladiolus from Stage 2 in Ms medium containing 5.0 per cent sucrose, 0.5 mg 1-1 NAA and 5.0 mg 1-1 triadimefon kept under etiolated condition. The size of the in vitro produced corms enlarged from 0.2 cm to 2.3 cm in the MS liquid medium containing 5.0 per cent sucrose and 3.0 mg 1-1 triadimefon.