University of Nottingham

Effect of silver nitrate and culture vessel on growth and quality of Type II callus of the A188 genotype
--Southgate, EM; Power, JB; Davey, MR

Using the point counting method of Mottley and Keen (Plant Cell Rep. 6: 389-392, 1987), the effects of both culture vessel and silver nitrate on white, friable Type II callus growth (percentage increase in size) of inbred A188 were assessed. In addition, the effect on callus quality (in terms of root production, yellowing or non-Type II characteristics) was ascertained. Deep, vented 9 cm tissue culture dishes (Corning, Bibby-Sterilin) and 9 cm petri dishes (Bibby-Sterilin) were compared and, in order to investigate ethylene accumulation in the two types of culture vessels, half of the callus maintenance medium was supplemented with 10 mgl-1 AgNO3. The callus maintenance medium consisted of Chu N6 basal salts (Chu et al. Sci. Sinica 18: 659-668, 1975) supplemented with 25mM proline, 1 mgl-1 thiamine-HCl, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 20 gl-1 sucrose and 1.5 mgl-1 2,4-D at pH 5.8. Twenty-five calli (approx. 100 mg pieces; 5 calli per dish/5 replicates per treatment) were placed onto maintenance medium in the two types of culture vessel, with or without silver nitrate. For each treatment, the mean percentage increase in size was calculated over a 14 d period. In addition, the proportion of calli exhibiting yellowed regions (>10% coverage of callus), roots and non-Type II characteristics (>10% coverage of callus) after 14 d of culture was recorded. The use of silver nitrate in the culture medium of A188 Type II callus significantly (p<0.05) enhanced growth when using both types of culture vessel and this was most noticeable when the culture vessel was smaller and not vented (petri dishes). The assessment of callus quality (in terms of minimising yellowed or non-Type II callus and root growth), demonstrated that the presence of Type II callus was not affected by AgNO3 supplementation of the medium or the culture vessel. However, in the absence of AgNO3, the use of vented tissue culture dishes reduced the number of calli exhibiting rhizogenesis.

Table 1. Effect of AgNO3 and culture vessel on growth and quality of A188 Type II callus over a 14 d culture period.
A188 Type II calli exhibiting response (%)2
Culture vessel1 AgNO3 Mean increase in size (%)3 Type II callus Yellowed callus Root production Non-Type II callus
TC + 134.5 (11.2) a 97.5 (2.5) 52.5 (9.2) 20.0 (6.5) 12.5 (3.7)
" - 104.0 (9.7) bc 97.5 (2.5) 47.5 (10.0) 5.0 (5.0) 10.0 (5.3)
P + 129.4 (19.6) ab 97.1 (2.9) 51.4 (12.9) 17.1 (5.2) 14.3 (8.4)
" - 80.5 (7.4) c 95.0 (3.3) 60.0 (10.7) 20.0 (8.5) 27.5 (11.9)
1TC = tissue culture dish, P = petri dish.
2Percentage of calli (5 replicated dishes of 5 explants) exhibiting response. Data are a mean of 25 replicates ± SE. 3Percentage values followed by the same suffix are not significantly different at p<0.05 (determined by the Kruskal-Wallis and Mood's Median test for non-parametric data).

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