ISOLATION OF SUB-DIPLOID MICROPROTOPLASTS FOR PARTIAL GENOME TRANSFERIN PLANTS - ENHANCEMENT OF MICRONUCLEATION AND ENRICHMENT OF MICROPROTOPLASTS WITH ONE OR A FEW CHROMOSOMES

Results on the enhancement of the frequency of protoplasts with micron uclei, and on the isolation and enrichment of smaller sub-diploid micr oprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reporte d. Suspension cells were treated with the spindle toxin amiprophos-met hyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wa ll-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increa sed by a factor of 2-6. A shorter period (3 h) of incubation with a hi gher concentration of enzymes as well as a longer period (16 h) of inc ubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, syn chronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, s ignificantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono- and m icronucleated) obtained after enzyme incubation was fractionated throu gh a continuous iso-osmotic gradient of Percoll, using high-speed cent rifugation. This resulted in one large and a few small bands, which co ntained a heterogeneous population of microprotoplasts, protoplasts an d cytoplasts. In contrast to the large band, the small bands contained a relatively higher frequency of small sub-diploid microprotoplasts. To separate the small sub-diploid microprotoplasts from the large micr oprotoplasts and protoplasts of the bands, discontinuous Percoll gradi ents and sequential filtration through nylon sieves of decreasing pore size (48 - 20 - 15 - 10 - 5 mum) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (almost-equal-to 80%) small sub-diploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as r evealed by microdensitometric and flow-cytometric analyses. The applic ation of this technique for partial genome and limited gene transfer i s discussed.