An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

Abstract

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basis layer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l-1 kanamycin or 5 mg l-1 paromomycin. Single resistant cells can be recovered from about 10,000 sensitive cells in one alginate layer. Injection of the neo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.

High efficiency transient and stable transformation by optimized DNA microinjection intoNicotiana tabacumprotoplasts

“…For protoplast isolation plants that had been growing for 6 weeks since the last subculture were used. Protoplast isolation was performed as described by Schnorf et al (1991).…”

“…(4) Single plant cells do not adhere firmly enough to the supporting matrix to anchor them for microinjection. Although stable transformation of an alga and different plant species has been achieved by DNA microinjection (Neuhaus and Spangenberg, 1990;Schnorf et al, 1991) other methods are generally applied for plant transformation, that are technically less difficult and more efficient in terms of generating transformed clones per unit time (Kung and Wu, 1993). However, gene transfer by microinjection has a number of unique advantages, which can be exploited for specific applications.…”

“…Protoplasts have been immobilized using holding capillaries (Crossway et al, 1986), adhesive substances (e.g. polylysine; Steinbiss and Stabel, 1983;Reich et al, 1986) or embedding in medium containing either agarose (Lawrence and Davies, 1985;Aly and Owens, 1987) or alginate (Schnorf et al, 1991). Injection solutions stained with Lucifer yellow or other fluorescent dyes were occasionally used to control the injection process visually (Steinbiss and Stabel, 1983;Aly and Owens, 1987).…”

“…Single cell culture systems have been developed that allow the propagation of individual injected protoplasts (Reich et al, 1986;Crossway et al, 1986). Following DNA microinjection into protoplasts high efficiency stable transformation has been reported (Reich et al, 1986;Crossway et al, 1986) and, using an effective protoplast embedding and culture system, stably transformed tobacco lines were produced (Schnorf et al, 1991). However, none of the protoplast microinjection systems established to date combined all the requirements for performing large-scale conclusive studies on the different parameters affecting the DNA delivery to target cells, the survival of injected cells and the stable integration of transferred genes.…”

“…Based on the methodology established by Schnorf et al (1991) we have developed an effective system for DNA microinjection into tobacco mesophyll protoplasts that can be used to optimize, step by step, the process leading to stable transformation. The system allows routine observation of the delivery of injection solution as well as of the fate of individual injected cells.…”