In this thesis the interactive effects are described of tissue, medium, and other environmental factors on the process of adventitious bud formation in vitro
from bulb-scale explants of Lilium speciosum
Thunb. Besides, results are presented of experiments designed to test the hypothesis that the effects of some of the factors can be explained by their action on one physiological process. Some important results from the investigations are stated below:
Chapter I presents effects of the auxin, 1-naphthylacetic acid (NAA), and of the cytokinins, N 6
-benzyladenine (BA) and N 6
-The two cytokinins did not influence the number of adventitiously formed plantlets per explant, but severely reduced the bulblet growth of these plantlets.
-Low concentrations (≤0.5 μM) of NAA led to an increase in the number of plantlets per explant. Higher concentrations were less effective.
-Explants of cold-stored bulbs (at O°C) were more sensitive to the action of NAA than those of uncooled bulbs.
-The regeneration process on bulb-scale explants showed basipetal polarity. Addition of NAA to the nutrient medium suppressed this polarity.
-The optimum response to NAA of 'basal' plantlets was found at a lower concentration than that for 'non-basal' plantlets.
Effects of the factors wounding, TIBA, and temperature on plantlet number and polarity are described in Chapter II.
-Additional wounding of the explants by removal of the abaxial epidermis led to an increase in the number of plantlets per explant. This effect, observed when explants were cultured in the presence of 0.5 μM NAA, could be ascribed to the act of wounding itself and not to the absence of the abaxial epidermis or to altered diffusion conditions between the explant and the medium.
-Addition of law concentrations (≤2 μM) of 2,3,5-triiodobenzoic acid (TIBA) to the medium, in the presence of 0.5 μM NAA, resulted in an increase in the number of plantlets per explant. Higher concentrations were inhibitory.
-Within the range of 15°-25°C, a higher temperature during the culture period led to a linear increase in the number of plantlets per explant.
-Additional wounding, TIBA, and higher temperature suppressed the basipetal polarity of the regeneration sites in the explants. -The similarities in the effects of NAA, wounding, TIBA, and temperature, led to the hypothesis that interactions exist between the effects of these factors.
In Chapter III these interactions between the effects of NAA, wounding, TIBA, and temperature, are analyzed.-wounding and NAA:
additionally wounded explants regenerated more plantlets than normal ones, and were more sensitive to the action of NAA.-NAA and TIBA:
the stimulatory effect of TIBA on the number of plantlets, observed in the presence of 0.5 μM NAA, was absent or much smaller in the absence of the auxin. In the presence of 0.5 μM NAA, TIBA exerted its optimal effect at a higher concentration than without NAA.-wounding and TIBA:
additionally wounded explants were more sensitive to the action of TIBA than normal explants. -temperature, wounding, and NAA:
within the range of 15°-25°C, a higher culture temperature caused a linear increase in plantlet number. The slope of the line depended on the degree of wounding of the explants and on the presence of 0.5 μM NAA. When extrapolated, the lines intersected the abscissa (~number of plantlets per explant=0)at c. 3°C.
-The effects of each of the factors NAA, TIBA, wounding, and temperature, on the polarity of the regeneration sites in the explants, depended on the other ones.
-The endogenous concentration of free IAA in bulb-scale tissue of Lilium speciosum
Thunb. was established at 1.1 ± 0.1 ngg -1
-The hypothesis was made that the factors NAA, wounding, and temperature, acted, at least partly, by affecting one central physiological process, possibly that of ethylene biosynthesis.
In Chapter IV effects of the factors NAA, wounding, temperature, and aminoethoxyvinyl glycine (AVG) are described on the production of ethylene and ethane by bulb-scale explants during the course of the culture period.
-Ethylene and ethane were produced by the explants, the amounts depending on condition and stage of the culture.
-The first 1-2 weeks of the culture period, i.e., the stage of cell divisions, were characterized by an increase in the production of ethylene. The subsequent decrease in ethylene production by the explants corresponded with a phase of bud differentiation.
-During days 2-4 of the culture period, an exponential increase in ethylene production by the explants was established for all culture conditions, concomitantly with an exponential decrease of the ethane production. For all culture conditions, a negative linear relationship was found between the logarithm of the ethane production and the ethylene production.
-Various correlations were established between the ultimate number of plantlets per explant and the ethylene and ethane productions by the explants. A highly significant (p<0.0005) relationship was found for all culture conditions between the ultimate number of plantlets per explant and the slope of the ethane/ethylene regression lines.
Chapter V describes effects of AVG, 1-aminocyclopropane-1-carboxylic acid (ACC), and ethylene.
Addition of 0.3-30 μM AVG to the nutrient medium caused a concentration dependent decrease of the number of plantlets per explant and reinforced the basipetal polarity of the process.
-On the contrary, addition of 10-50 μM ACC to the medium led to an increase in the number of regenerated plantlets and suppressed the basipetal polarity. 50 μM ACC nullified the inhibitory effect of 30 μM AVG,
-In the presence of 0.5 μM NAA, ethylene (1-10 ppm in air), supplied during the first 3 or 7 days of the culture period, caused an increase in the number of plantlets per explant and suppressed the polarity of the process.
At the end of Chapter V, some conclusions are drawn:
1)Ethylene biosynthesis plays a key role in the process of adventitious bud formation from bulb-scale explants of Lilium speciosum
2)The effects of TIBA, wounding, temperature, and ethylene depend on the presence of 0.5 μM NAA in the nutrient medium. 3)The process of adventitious bud formation from bulb-scale explants of Lilium speciosum
is regulated by auxin and its basipetal transport, as influenced by, e.g., TIBA and/or ethylene.
A model sysytem (see Chapter V, Fig. 3) is proposed, which accounts for the observed effects. It designates auxin and its translocation as the regulating factors for bud initiation in bulb-scale explants, when the inevitable membrane damage is alleviated by the auxin-dependent ethylene-synthesizing system.