Cotton Protoplast Culture Using the Feeder Layer Technique

H.C. Peeters and R. Swennen


 
ABSTRACT

Plant regeneration from protoplasts is still a limiting factor in cotton to allow its improvement via techniques such as somatic hybridization, resulting in chloroplast, mitochondrial or nuclear genome transfer, and direct DNA transfer into protoplasts. Embryogenic cell suspensions of Gossypium hirsutum cv. Coker 312 were used as protoplast donor and nurse cells. These cell suspensions were initiated from callus cultures induced from hypocotyl explants (Trolinder and Goodin, 1987). Viable protoplasts were obtained following digestion in an enzyme solution for 18h on an orbital shaker (50rpms) at 25 C in darkness. The protoplasts, collected by sieving and filtering, were placed on filters over a feeder layer of nurse cells (Prioli and S”ndahl, 1989). The nurse cells were spread onto callus inducing medium containing auxins and cytokinins. The protoplasts formed very few or no colonies without the feeder layer, but in its presence, plating efficiency up to 8% was observed. Embryogenic calli, from which plantlets were regenerated, were recovered from the filters only in the presence of nurse cells. Further experiments demonstrated that the presence of plant growth substances in the nutrient medium was not necessary. The plating efficiency was 4.6% in hormone-free nutrient medium and 6.4% in the presence of plant growth regulators. Conditioned medium, prepared by mixing a cell suspension filtrate with one third volume of fresh nutrient medium, and autoclaved nurse cells had also promotive effects and resulted in a plating efficiency of 4% and 1.1%, respectively.

Germinating embryos were transferred to flasks saturated with Stewart and Hsu's medium (1977). Plantlets with at least two true leaves have been transferred to the greenhouse.



Reprinted from 1993 Proceedings Beltwide Cotton Conferences pg. 611
©National Cotton Council, Memphis TN

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Document last modified Sunday, Dec 6 1998