Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Abstract

Abstract.Results on the enhancement of the frequency of protoplasts with micronuclei, and on the isolation and enrichment of smaller sub-diploid microprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reported. Suspension cells were treated with the spindle toxin amiprophos-methyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wall-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increased by a factor of 2-6. A shorter period (3 h) of incubation with a higher concentration of enzymes as well as a longer period (16 h) of incubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, synchronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, significantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono-and micronucleated) obtained after enzyme incubation was fractionated throuh a continuous iso-osmotic gradient of Percoll, using high-speed centrifugation. This resulted in one large and a few small bands, which contained a heterogeneous population of microprotoplasts, protoplasts and 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 microprotoplasts and protoplasts of the bands, discontinuous Percoll gradients and sequential filtration through nylon sieves of decreasing pore size (48--20-15-10-5 [am) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (~80%) small subdiploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as revealed by miAbbreviations: APH =aphidicolin; APM = amiprophos-methyl; Au = arbitrary units; CB = cytochalasin B; DAPI = 4,6-diamidino-2-phenylindole; FDA = fluorescein diacetate; HU = hydroxy urea Correspondence to: K.S. Ramulu; FAX: 31 (8370) 18094 crodensitometric and flow-cytometric analyses. The application of this technique for partial genome and limited gene transfer is discussed.

Preparation of Microprotoplasts for Partial Genome Transfer via Microprotoplast Fusion in Liliaceous Ornamental Plants

“…Although other spindle toxins, amiprophos-methyl and butamiphos, had been used for inducing efficient micronucleation in suspension-cultured cells of Nicotiana plumbaginifolia 11 , Solanum tuberosum 12,13,24 and Helianthus giganteus 2 , their efficiency on the induction of micronucleation in Hemerocallis hybrida was limited 21 .…”

A study of the process of synchronisation and micronucleation in Beta vulgaris and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry

“…Although a sequential treatment with APH-CA (4.5 mM) resulted in the highest MI (8.5 ± 0.5%), a lower frequency of MN-cells was observed (5.5 ± 1%). Cytological observations and CLSM showed that the process of micro-nucleation and evolution of micro-nuclei followed the same course of events as observed in N. plumbaginifolia (Ramulu et al 1993): i.e. mitotic arrest of dividing cells, chromosome spreading without centromere division as a consequence of APM or CA treatment, formation of nuclear walls around regrouping and decondensing chromosomes, formation of micro-nuclei and finally the restitution of intact nuclei by micro-nuclear fusions (Fig.…”

“…For induction of micronuclei, the suspension cells were subsequently treated for 24 h with APM (amiprophos-methyl; Tokunol M; 0-methyl-0-0-(4-methyl-6-nitrophenyl)-N-isopropyl-phosphoro thiamidate from Bayer Mobay Corporation, Agricultural Chemicals Division, Kansas City, MO., USA) or CA (Cremart; Butamiphos; Q-ethyl-Q-(3-methyl-6-nitro-phenyl) N-sec-butylphosphorothioamidate from Sumitome Chemical Comp., Osaka, Japan) at 15-30 mM or 2.25-36 mM respectively. These concentrations were chosen on the basis of our previous work with Nicotiana plumbaginifolia and potato (Ramulu et al 1993(Ramulu et al , 1994.…”

“…To disrupt protoplasts and to obtain MPPs, the dense solution mixture of mono-and micro-nucleated protoplasts was loaded onto a continuous iso-osmotic Percoll gradient Ramulu et al 1993) and was centrifuged at 10 5 g. for 30 min to 1 h. After gradient centrifugation, the bands were collected and diluted 4-to 5-fold in W5 solution. The mixture with protoplasts of different sizes was filtered through a series of nylon sieves with 15, 8 and 5 mm pore size, and was centrifuged at 40 and/or 160 g for 15 min to collect the MPPs.…”

“…Hydroxy urea and APH were obtained from Sigma Chemical Company, St Louis, MO, USA. The treatments with HU and APH were carried out as described by Ramulu et al (1993). Afterwards, the cultures were washed repeatedly with culture medium.…”