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The influence of some culture conditions on growth of plant tissues in vitro Florian, Svatopluk Fred

Abstract

The response of various plant tissues to different conditions of culture was compared. The tissues used were cambium-containing discs from carrot roots, undifferentiated carrot callus, bacteria-free sunflower tumorous (crown-gall) tissue, and segments of sunflower stems. The culture conditions compared, in combination, were agar versus liquid medium, shaken versus non-shaken liquid medium, and continuous light versus continuous dark. The response of the tissues to White's basal nutrient medium with added coconut milk (15 %) and indoleacetic acid (0.1 mg/l) and Hildebrandt's improved sunflower medium was also compared under these different culture conditions. Agitation of the liquid medium was accomplished through the use of a newly designed shaker, which consists basically of a horizontally oscillating bank of shelves. The tissues rested on the bottom of culture flasks (medicine bottles) on these shelves and were alternately exposed to medium and air as the liquid medium washed back and forth. Any horizontally oscillating platform could replace this shaker and almost any type and size of culture flask could be used. Probably any type of plant tissue could be cultured under these shaking conditions. It is not necessary that the tissues adhere to the walls of the culture vessels as in other agitation methods so far used in plant tissue culture. Growth (weight increase) of all tissues in shaken liquid medium (in both light and dark) was markedly superior (two to six times greater average weight in 42 days) to that of tissues on agar and in non-shaken liquid medium. The superiority of growth in shaken liquid medium is probably due to several factors; nutrients and gasses are supplied to the entire surface of the tissues, there is no drying and hardening of the tissue surfaces, resulting in a greatly increased surface area, harmful excretions can not collect at the tissue surface, and diffusion of nutrients is not hindered by adsorptive effects of agar particles. To compare the growth of these cultures with those of other workers using agitation methods is difficult due to the different sources of plant material, different sizes of tissues cultured, and different periods of culture used. In general the stimulatory results of shaking obtained appear to be at least as good as those obtained by Caplin and Steward with the much more elaborate and limited 'auxophyton '. There was no sign of eventual growth stoppage as obtained by White, using roller tubes. Light consistently stimulated tissues grown in liquid medium, particularly those in shaken liquid medium. The effect was especially marked on carrot callus and tumorous sunflower tissues grown in Hildebrandt's medium. It is suggested that light may play a role in the synthesis of growth factors supplied by coconut milk. Light had no significant effect on the growth of tissues on agar medium, indicating that the primary limiting factor in the growth of such tissues may be the rate of diffusion of nutrients from the agar. Carrot tissues showed better overall growth in the enriched White's medium while the sunflower tumorous tissue grew better in Hildebrandt's medium. The effect on carrot was probably primarily through indoleacetic acid and coconut milk. The response of sunflower tissue is difficult to evaluate at present. All carrot tissues developed chlorophyll throughout all of the experiments if cultured in light, while tumorous sunflower tissue remained white until placed in Hildebrandt's medium, when it turned light green. The significance of these differences is not known. One experiment showed that carrot discs derived from different carrots grew at significantly different average rates, indicating that discs to be compared should be derived from the same root. The plane in which the discs were cut did not seem to influence subsequent growth. 'Intra root' variation in disc growth necessitates replication.

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