Induction of genetic variability in ginger (zingiber officinale rosc.) through in vitro fertilization

Abstract

Investigations on ‘Induction of genetic variability in ginger (Zingiber officinale Rosc.) through in vitro fertilization’ were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2004-2005. Molecular characterization studies of the eight ginger cultivars -Z-0-78 (V1) Kodakara Local (V3), Kuruppampady (V4), Mahima (V5), Maran (V6), Rejatha (V7), Rio-de-Janeiro (V8) and Varadha (V9)- by RAPD using nine random primers revealed that the genetic variability among the cultivars very narrow in the range of 5 to 19 per cent. The cultivar Rio-de-Janeiro was distinct with maximum variability. The variability among the released varieties from IISR ranged from 5 to 10 per cent. The flowering season in ginger extended from August to November. The cultivars showed variability with respect to duration for initiation of flowering and flowering duration. The mean pollen fertility and viability among the ginger cultivars were 35.76 per cent and 8.11 per cent respectively. The autotetraploids had high pollen fertility and viability compared to diploids. The range of pollen viability in autotetraploids was from 12.03 to 15.68 (Z-0-78) and in diploids it was from 3.68 to 8.90 per cent. Among the diploids the released varieties Varadha, Mahima and Rejatha had more pollen fertility and viability. Attempts were made to refine the in vitro pollination technique with respect to basal media combination, growth regulators, vitamins and gelling agent. Studies conducted in the cross Varadha X Z-0-78 using placental pollination technique. The basal medium of half MS with 2X vitamins was superior to B5 medium. As gelling agent phytagel 0.18 per cent was superior to agar 0.65 per cent. Among the various plant growth regulator combinations tried the combination of picloram and BA was found to be best. The medium of half MS with 2X vitamins + 3 per cent sucrose + BA 2.5 mgl-1+ picloram 0.2 mgl-1 + 0.18 per cent phytagel was the best with respect to maximum ovule swelling, percentage of cultures with ovule development and percentage setting per culture. Seed set and seed development in various crosses involving autotetraploids and diploids were assessed. The parental combination between autotetraploids (Z-0-78 X Z-0-86) produced seeds with very good swelling in maximum number of cultures with maximum setting per culture. The parental combination Z-078 X Varadha and its reciprocal also favoured very good ovule swelling. The parental combination Z-0-86 X Varadha as well as Varadha X Mahima and their reciprocals produced seeds with good swelling. The size increase of the developing ovules after in vitro pollination was monitored from the day of pollination to 60 DAP. The size increase upto 15 DAP was rapid. At 60 DAP the ovules attained four-fold increase in size. The pollinated ovules cultured, in the course of maturation turned pink and later blackened. The seeds showed complete endosperm filling at 10 DAP. The soft and jelly like endosperm became hard by 20 DAP. Viability test with tetrazolium showed that the seeds were viable up to 50 DAP. Embryo was found to be seated towards the chalazal end and endosperm constitutes the major portion of the seed. Germination studies were conducted in a number of plant growth regulator combinations. The 60 days old seed derived from the parental combination of Kuruppampady X Z-0-78 showed radicle emergence in the medium of half MS + 3 per cent sucrose + 0.65 per cent agar + IBA 3.0 mg l-1. The seeds of parental combination Z-0-78 X Varadha germinated through somatic embryogenesis in the medium of B5 + 3 per cent sucrose + 0.18 per cent phytagel + 2,4-D 0.2 mg l-1 + BA 2.5 mg l-1 105 days after in vitro pollination. The germination was 10 per cent It was found that the seed coat was pushed apart by the callusing embryo and it developed globular somatic embryoids. Embryoids developed root and shoot poles in the same medium. The somatic embryoid is in the initial stage of development. Embryo culture studies with embryos excised from seeds of 15 to 60 DAP in various media combinations did not give positive response. Prolonged culturing may give positive results.