Standardisation of in vitro pollination and fertilization for generating genetic variability in Zingiber officinale (Rosc.)

Abstract

Investigations were carried out to standaridse in vitro pollination and fertilization technique for seed set in ginger, Zingiber officinale (Rosc.) at the Plant Tissue Culture Laboratory of Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during 1990-1993. Investigations to improve flowering showed that early planting (March- April) is an important factor, which induce flowering in ginger, when the crop is grown from rhizome bits of standard size i.e., 15 g. Cultivar difference was also found to exist with respect to flowering. But increasing the size of seed bit from 15 g to 200 g resulted in early flowering of all the seven cultivars. Maintaining the plants as biennial registered still earlier and more profuse flowering of all the cultivars. In biennial plants flowering started as early as in 1st July, while it was only on 17th September in annual plants from 15 g. In annuals the flowering season was for two months, while it was extended for four months in biennials. The application of growth regulators did not significantly influence the flowering of different ginger cultivars. The study of floral biology of ginger flowers showed that, an inflorescence of ginger took 30-32 days from initiation to blooming. The number of flowers per inflorescence ranged from 20 to 31 and the blooming period ranged from 15 to 22 days. The anthesis started by 3.00 pm and continued upto 4.30 pm. Anther dehiscence did not occur simultaneously with flower opening and took place only after ½ to 11/2 h after anthesis. The ginger flowers are characterized by a spiny stigma and a long style of mean length of 3.46 cm. The ovoid ovary measured a mean length of 2.71 mm and diameter of 2.59 mm and recorded a mean ovule number of 24.43. The ovules recorded a mean length of 535.13 μm and breadth of 323.41 μm at the middle. The pollen fertility with acetocarmine stain in the studied cultivars ranged from 14.15 per cent in Nadia to 35.28 per cent in SG-66. Attempt to develop a medium which will support pollen germination and tube growth resulted in the identification of ME3 medium with osmoticum as 12 per cent PEG. The maintenance of the medium at pH reactions of 4.0 to 8.0 not significantly influence pollen germination and tube growth. The maximum pollen tube length registered was 1042.17 μm. The pollen germination started within 3 h of incubation and continued till 24 h. The histological examination of ovules of flowers on the day of anthesis revealed the presence of viable egg cell. The flower buds for in vitro pollination were collected and surface sterilized prior to anthesis (3.00 pm). The bacteria destroying the cultures were identified as Psuedomonas solanacearum. Dipping the unopened flower buds in streptocyclin 500 mg 1-1 for 1 h followed by wiping with 70 per cent alcohol and rinsing with mercuric chloride 0.1 per cent for 3 min gave satisfactory microbial sterilization of flower buds for in vitro pollination. Initial experiments for culture establishment showed that ginger ovary would develop under in vitro condition in ½ MS, SH or Nitsch medium when supplemented with growth regulators and CW. There was no ovary development with out hormones. Among the various methods of pollination tried, ovules developed in placental pollination, modified placental pollination and ovular or test tube fertilization. In all the three cases, pollen grains along with ME3 medium were applied over the ovules. The observation of ovules after placental pollination under fluorescence microscopy revealed that pollen germination starts within 3 h of pollination and pollen tube growth is sufficient to fertilize the ovule. Histological examination of ovules 4 DAP showed eight celled pro-embryo and 40 DAP showed well developed embryo and endosperm rich in starch and oil grains. The aforesaid successful pollination techniques are suitable for selfing and crossing in ginger under in vitro condition. Among the successful methods of in vitro pollination tried placental pollination is the best as it registered maximum number of seeds per culture with minimum effort. The mean seed set per culture in this method in four favourable media combinations was 61.56 per cent. The mean number of well developed seeds per culture was 6.87 at 80 DAP. The flower buds collected on the day of anthesis and one day after anthesis were suitable for in vitro pollination. Studies were made to standardize media supplements for enhancing ovule development. The ovules developed at all levels of sucrose concentration 3.0, 6.0, 9.0 and 12.0 per cent levels. Considering the increased seed set, 6.0 to 8.0 per cent is the optimum. The auxins as well as cytokinins alone induced ovule development but combinations proved to be better. The combination of NAA 0.5 to 1 mg 1-1 with varying concentration of BAP from 2 to 10 mg 1-1 had shown positive effect. The effect of BAP in the ovule development could be replaced by KIN (2.0, 5.0 mg1-1) or 2iP (2.5 mg1-1). Similarly the effect of NAA (0.5 to 2.0 mg1-1) could be replaced by 2,4-D (0.05 to 1.0 mg1-1) or IAA (0.05 to 0.2 mg1-1). The GA levels (2.0, 4.0, 6.0 and 10.0mg1-1) did not favour ovary and ovule development. The IAA precursor, typtophan (0.1,0.5 and 1.0 mg1-1) also behaved in the same manner. Other supplements like CW (10 to 12 % v/v) and CH (200 to 500 mg1-1) enhanced ovule development along with cytokinin and auxin. The extract from 15 days old inflorescence of ginger at a concentration of 0.3 to 3.0 mg1-1 promoted ovule development with hormones. The aminoacid L-glutamine (25 to 500 mg1-1) did not inhibit ovule development while YE (250 to 1000 mg1-1) inhibited ovule development. The solid as well as liquid form of favourable media combinations supported ovule development after in vitro pollination. The ovary and ovules developed at 260C as well as 280C but the lower temperature was best for the visual assessment of ovary and ovule appearance. With respect to light intensities, they developed in dark, diffused light and light intensities of 500 and 1000 lux. The in vitro produced seeds and fruits of ginger grew rapidly at the initial stage of 20 Dap and later growth was slow and comparatively little. The colour of the seeds at the initial stage was creamy white and in the course of development about 14.95 per cent of cultures develop, purple red colour within a period of 30.65 days. The whole seeds of a culture turned black within a period of 60 to 90 DAP. In the culture condition about 40 per cent of ovaries developed orange colour of ripening within a period of 35 to 65 days. They turned black by 90 DAP. The fruit of ginger is a thick walled three valved capsule with small black arillate seeds. Eighty days after pollination they recorded a mean diameter of 6.5 mm and a maximum of 9.5 mm. The ginger seeds recorded a mean length of 2.20 mm and breadth of 1.60 mm 80 DAP. The arillate seeds showed two seed coats, the outer being thick and the inner one being thin. The seed coat encloses a cavity and in the cavity endosperm with embedded embryo is seen. The in vitro produced seeds of ginger germinated when 80 days old seeds were incubated initially in the medium of ½ MS with 2,4-D 8 mg 1-1 for two months and then in hormone combination of BAP 9 mg1-1 and 2,4-D 0.1 mg1-1.