In vitro culture of tissues, cells and protoplasts of cotton (Gossypium bartbadense L.)

Elshihy, Osama Mohamed Abdel-hami (1986) In vitro culture of tissues, cells and protoplasts of cotton (Gossypium bartbadense L.). University of Southampton, Doctoral Thesis.

Abstract

Explants from different organs and tissues from six varieties of Gossypium barbadense were cultured in vitro under a range of different conditions. The concentration and type of growth regulators and the level of light intensity most suitable for callus production varied depending upon the explant source, but not upon the plant variety. The other nutritional and environmental requirements for callus production were the same for each type of explant. The amount of callus produced varied according to the origin of the explant. On subculture the growth regulator levels most suitable for continued callus production differed in all cases from those which had produced the most callus on the primary explant. But with the exception of mesocarp tissue, the yield of callus declined with each successive subculture. Alteration in the growth regulator levels stimulates callus growth after the 6th subculture but not to the same extent as in the first. Cultured immature embryos under appropriate conditions developed into seedlings instead of forming callus. In one experiment, four seedlings grown in this way formed dwarf plants which fruited early. This feature has been maintained through three generations. After a heat shock, a callus line habituated for auxins and cytokinins was recovered. The growth characteristics of this line were examined both in the presence and absence of growth regulators. Some interaction between the growth regulator requirements and the physical environmental condition was observed. A reversion to growth regulator dependence occurred when the line was cultured with growth regulators. Suspension cultures were initiated but satisfactory growth was only obtained with the habituated line. Conditions for the isolation of protoplasts were examined using six varieties of cotton. A range of tissues taken directly from the plant or from callus derived from different sources were used. Methods for the recovery of viable protoplasts were established. The protoplasts were cultured in a wide range of nutritional and environmental conditions. Division was obtained with protoplasts from cotyledon, cotyledon petiole callus, hypocotyl callus and habituated callus. Habituated protoplasts divided in the absence of growth regulators, whilst protoplasts from the other sources had a critical growth regulator requirement. In addition, cotyledon protoplasts required a critical level of mineral elements in the medium. After a few divisions, further colony development ceased unless the culture was diluted with fresh medium of the appropriate composition. Callus colonies were then obtained which could be transferred to agar-solidified medium. Extended subculture of callus derived from cotyledon protoplasts leads to the recovery of shoots and subsequently entire plants. (D72315/87)

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