Micropropagation of phalaenopsis

Abstract

Investigations were carried out at the Plant Tissue Culture Laboratory attached to the All India Co-ordinated Floriculture Improvement Project (AICFIP), College of Horticulture, Vellanikkara, during 1993-96 to standardise the micropropagation technique in Phalaenopsis.

Out of the different explants tried, response was shown by inflorescene stalk node, inflorescence stalk tip and pollinia collected from the field grown plants and apical bud, shoot node, basal portion, leaf and root of plantlets grown in vitro.

Maximum survival (40%) of nodal explants was recorded at the sterilant combination involving mercuric chloride (0.01%) for 30 min., streptomycin + pencillin (0.01%) for 90min., followed by final sterilization using mercuric chloride (0.10%) for 10 min. For flower bud, the combination involving emisan (1.00%) for 30 min. followed by alcohol (50%) for 1 min. recorded the maximum survival percentage (55).

The ½ MS liquid medium containing BA 5 ppm+ NAA 2ppm + 2, 4-D ppm + CW 15 percent recorded the minimum number of days for nodal swelling and bud development (6 and 14 days, respectively). Basal portion was found to be the best explants with respect to rate of increase in shoot number (8.00) and leaf number (7.67), followed by shoot node. The latter recorded the highest root number (7.67) at the end of 12 weeks of culturing. As to the position of explant, highest increase in the length of the buds was recorded for first node, followed by second, third and fourth.

Full strength MS and KC media were far inferior to ¼ MS, ½ MS, ¾ MS and VW media, for culturing. Maximum number of shoots (5.00) and leaves (7.67) were recorded for ¼ ms AND ½ ms media after 8 weeks of culturing.

Physical state of the medium, viz. liquid and semi –solid did not show any significant difference.

Sucrose at 1.5 percent level recorded the maximum number of shoots and leaves after 8 weeks of culturing. Thiamine – HCL increased the shoot and leaf number at 20ppm level at the end of 8 weeks, whereas the presence did not favour the production of roots.

The time taken for callusing in pollinia was minimum (2.0 days) at BA 5 ppm + NAA 2 ppm + 2, 4-D 2ppm in ½ MS medium containing 3 percent sucrose. When 90 day old pod was used, protocorm formation was observed in ½ MS medium containing BA 10ppm + NAA 1 ppm and KIN 5 ppm +2, 4-D 2ppm.

When the effect of NAA was considered root production was increased at the combination NAA 5 ppm + BA 10ppm + adenine 10 ppm.

With regard to the effect of BA, that at 25ppm in combination with adenine 10 ppm + NAA 1 ppm recorded maximum number of shoots and leaves.

Shoot and leaf number was maximum at ¼ MS medium containing BA 20 ppm+ 2, 4-D 2.5ppm, whereas root production was maximum at BA 20ppm + 2, 4-D 5 ppm. As to the combined effect of BA, NAA and 2,4-D in ¼ MS medium, the combination BA 5ppm + NAA 2 ppm + 2,4-D 2 ppm recorded the maximum number of shoots and leaves.

Different levels of KIN, was found to have no significant influence on the production of shoots, leaves and roots. When the combined effect f KIN, NAA and 2, 4-D was considered ¼ MS medium containing KIN 20ppm + NAA 2 ppm + 2, 4-D 2 ppm recorded maximum number of shoots and leaves.

The apical bud did not show any multiple shoot production during the 12 weeks culture period as influenced by coconut water, whereas first node at CW 10 percent, shoot node at CW 10 per cent, shoot node at CW 15 percent, and basal portion at CW 25 percent recorded the highest number of shoots.Maximum number of leaves was produced by apical bud at CW 20 percent , first node and shoot node at CW 10 percent, and basal portion at CW 25 percent.

Both tender and mature coconut water were equally effective. Fresh and upto 6 days old coconut water could also be used with similar effect.

Peptone at 1000 ppm was found to influence favourably the induction of multiple shoots from in vitro shoots.

Culturing the nodal explants in liquid media with filter paper bridge or keeping in the dark were found to reduce phenolic blackening. With the increase in the concentration of antioxidants, there was a proportionate reduction in media discolouration.

Activated charcoal and triadimefon added in the media were found to influence the root production from shoots. Length of the root was maximum at triadimefon 20 ppm.

Sucrose at 1.5 percent level recorded the minimum number of days for PLB development and the maximum number of PLB’s developed. Thiamine – HCL, coconut water, tomato juice and peptone did not significantly influence the time taken for PLB formation, but favoured the number of PLB’s developed.

With regard to PLB formation from shoot node, ½ MS medium containing BA 5 ppm + NAA 2 ppm + 2, 4-D 2 ppm recorded the minimum number of days for PLB development and maximum number of PLB’s. When PLB formation from in vitro leaf was considered, cent percent of the leaf cultures developed PLB’s at the combination BA 25 ppm + adenine 10 ppm + NAA 1 ppm and the time taken for PLB formation was minimum.

Cent percent of the cultures developed PLB’s from in vitro roots at BA 25 ppm + adenine 10 ppm + NAA 1 ppm and the time taken for PLB formation was minimum. Further growth of PLB’s and Plantlet development was the best in ¼ MS medium containing BA 15 ppm +NAA 1 ppm followed by adenine 8 ppm + BA 16 ppm. Regarding the effect of coconut water, PLB growth at CW 15 percent and plantlet development at CW 25 percent recorded the best results.

Light favoured plantlet development, multiple shoot formation and PLB formation from shoot node and in vitro root, whereas dark period favoured early development of PLB’s from in vitro root, whereas dark period favoured early development of PLB’s from in vitro leaf, callusing and PLB proliferation.

Healthy, large and robust plants were produced when plantlets were grown in 250 ml conical flask, followed by large test tubes.

When the plantlets grown previously in medium triadimefon 20 ppm were hardened by spreading over sterile charcoal pieces for two weeks, planted in coconut husk and were hung in the orchidarium with high humidity, cent percent survival was recorded even after 8 weeks of planting out.

The nutrient solution 30:10:10(0.50%) and 17:17:17 (0.10%) recorded the highest survival percentage after 12 weeks of planting out. The growth characters of the plants. Viz., plant height, leaf number, leaf length and width, root number and root length were found to be maximum for the plants sprayed with 17:17:17 at 0.10 percent level.

The survival percentage of plants showed a slight decrease with time and all the plant characters increased with time except the number of leaves.