The response of Cymbidium tissue cultures to 1-naphthylacetic acid

Abstract

A comparative study was conducted to determine the auxin requirements and cultural conditions for growth and development of Cymbidium cultivars; 259 (Chief Joseph 'Pathfinder'), 142 (Zuma Boyd 'Johns Pride'), RBL (Rothesay 'Black Label') and DHL (Delores Hoyt 'Limelight').

The tissues were cultured in erlenmeyer flasks containing Vacin and Went media in a growth cabinet that was maintained at 25°C and continuously illuminated. Measurements were made for fresh weight gain and proliferation count, (the number of protocorm like bodies).

In most cases 1-naphthylacetic acid (NAA) inhibited fresh weight gain although at low levels, (1.0mg 1⁻¹) growth was promoted in cultivars DHL and 142. Fresh weight inhibition by NAA and possible disruption of chlorophyll synthesis appeared to be linked in cultivars DHL and 142. In cultivar 259 NAA promoted proliferation count but at the expense of fresh weight gain. Kinetin at low levels, (0.1 and 1.0 mg 1⁻¹) in the presence of NAA inhibited fresh weight gain and proliferation count in shake culture but not in stationary culture. Liquid shake culture increased proliferation count compared with stationary culture at the expense of fresh weight gain and was not found to be essential for successful growth of the tissue in a liquid media.

Ethylene at low levels in flask atmospheres, (0.1 µl g⁻¹ h⁻¹) appeared to be a normal by- product of metabolism. At low levels of NAA, (1.0 mg 1⁻¹) ethylene evolution was promoted, (0.3 µl g⁻¹ h⁻¹). Shake culture stimulated ethylene evolution compared with stationary culture by a factor of ten. However, freshly cut tissue had no significant effect on ethylene production.

Accumulation of 14C NAA equivalents by tissue pieces was concentration and cultivar dependent. At 1.1mg 1⁻¹ NAA cultivars RBL and DHL accumulated 14 ng g⁻¹ 14C NAA equivalent respectively, after 48 h of incubation. Cultivar DHL at 16 mg 1⁻¹ NAA had, after one week of incubation, accumulated 1600 ng g⁻¹ 14C NAA equivalent in the tissue. The free NAA was readily metabolised to its conjugates which were tentatively identified as NAA-aspartate and NAA-glucose. Of the total activity applied only 22.5 percent remained as the free acid after 24 h of accumulation. Losses of the 14C label from the system were significant, (mean average; 11% week ⁻¹) and were possibly the result of decarboxylation. In cultivar DHL 14C equivalent was retained in the tissue over at least 4 weeks.

The high levels of ethylene evolution in response to shake culture tended to support the hypothesis that shake culture promoted meristematic activity (proliferation count) via auxin induced ethylene evolution. Kinetin may act to promote NAA uptake and in turn promote ethylene evolution.