Isolation, Culture and Regeneration of Lettuce Leaf Mesophyll Protoplasts

Plant regeneration from mesophyll protoplasts of Lactuca perennis

“…Sustained division of L. perennis protoplasts was only possible if an initial 7d dark culture phase was routinely adopted, consistent with other Lactuca protoplast systems, namely L. sativa and L. saligna, studied previously in our laboratory (Brown et al 1987). It is interesting to note that other workers have not reported the requirement for a dark period during the culture of protoplasts of L. sativa (Engler & Grogan 1982/1983Chupeau et al 1989). However, Chupeau et al (1989) emphasised the need to maintain plants used for protoplast isolation in a growth room to minimise both water stress and large environmental fluctuations, to use an iso-osmotic mixture of glucose, sorbitol and glycine in the enzyme mixture and to include a high level of manganese in the protoplast culture medium.…”

“…In cultivated lettuce, plant regeneration procedures have been reported for protoplasts isolated from seedling cotyledons and from expanded leaves of seed-derived plants (Engler & Grogan 1982/1983Nishio et al 1987;Chupeau et al 1989). In one of the latter studies, electroporationmediated DNA uptake into isolated protoplasts was employed in transient expression studies of the /3-glucuronidase gene, while uptake of the neomycin phosphotransferase II gene resulted in kanamycin resistant protoplast-derived tissues from which stably transformed plants were regenerated (Chupeau et al 1989).…”

“…However, Chupeau et al (1989) emphasised the need to maintain plants used for protoplast isolation in a growth room to minimise both water stress and large environmental fluctuations, to use an iso-osmotic mixture of glucose, sorbitol and glycine in the enzyme mixture and to include a high level of manganese in the protoplast culture medium. Likewise, Engler & Grogan (1982/1983 grew their plants in a growth chamber prior to protoplast isolation.…”

“…Sea Plaque agarose was employed as the gelling agent for the medium used to culture freshly isolated protoplasts, since its low gelling temperature of about 30°C was reported to be less detrimental to the viability of lettuce protoplasts compared with higher gelling temperature agents, such as Nobel agar (Engler & Grogan 1982/1983. The use of Type I agarose, rather than Sea Plaque agarose as a gelling agent for raMS2 medium, resulted in optimum growth of protoplast-derived tissues of L. perennis.…”

Lettuce (Lactuca sativa L.)

“…The information that follows is taken from the two reports to date that describe these conditions (Berry et al 1982;Engler and Grogan 1983). The information that follows is taken from the two reports to date that describe these conditions (Berry et al 1982;Engler and Grogan 1983).…”