Plant regeneration from stem cortex protoplasts of Brassica napus

Abstract

Cell layer strips composed of the epidermis and 7-9 layers of subepidermal cells were isolated from the 3-4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [1 l] medium and subsequently incubated for 17-18h in a 0.4M mannitol solution containing 1% Macerozyme and 1% Cellulase 'Onozuka' R-10. Protoplast yield was 2-2.8 x 10 6 per 1.0 g of tissue. Protoplasts were cultured at 1 x 105/ml in three different media: S I [13], B [12] and L [8l. The first cell divisions occurred after 2-8 days of culture at frequencies of 20-54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1-2 mm in diameter were transferred onto B 5 12] medium with 3 mgl-~ zeatin, 1% sucrose, 0. ! M mannitol and 0.5 % agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.

Effects of a Ficoll-agarose system on early division and development of mesophyll protoplasts of winter oilseed rape (Brassica napus L.)

“…These division frequencies are similar to those reported for stem cortex protoplasts treated with Ficoll [8]. In the same study, however, it was reported that mesophyll protoplasts isolated from leaves of glasshouse-grown plants, and cultured using identical conditions, did not show any division activity.…”

“…But when monitored over a longer period of incubation in agarose, those cultures transferred at the earlier points (7-10 days) had overtaken the later transfers and were growing more rapidly and subsequent callus development was enhanced. The transfer time was earlier than in other reports [6,8] and was related to the early high division frequency obtained in this study.…”

“…Callus derived from the 2% treatment grew on more rapidly compared with that from other treatments, and on transfer to the regeneration medium described up to 50% of the calli had produced roots within 8-10 weeks of isolation. Lower survival rates reported under similar growth regulator regimes [8] may be associated with the longer periods of culture in liquid media in that study. Therefore in conclusion, the combination of an initial incubation in a medium containing 2% Ficoll followed by a transfer to agarose after 10 days would appear to produce efficient early regeneration with this winter variety ofB.…”

“…Few details have been published on work with winter varieties. Other work on Brassica napus protoplasts includes successful regeneration from hypocotyl protoplasts [5,6,7] and a recent paper utilising protoplasts from stem cortex tissue [8]. These reports indicate that there is wide scope for improvements and increased efficiency of regeneration methods.…”

“…Ficoll treatment and agarose have been used in a recent study on plant regeneration from stem cortex protoplasts isolated from B. napus cv. Westar [8] where a medium containing 2% Ficoll was considered optimal, but the effects of other Ficoll concentrations were not reported.…”