INTRODUCTION
In the last years, much effort has been made to develop genetic manipulation methods for monocots. The first transgenic cereal plants were produced via the uptake of DNA by protoplasts and the regeneration of plants from protoplasts derived calluses [10, 12].
However, protoplasts capable of division are usually only obtained from cell suspensions which in most cereals are difficult to establish and time-consuming, they need constant maintenance and rapidly lose regenerative competence. Moreover, fast growing cereal cell cultures accumulate genetic aberrations over time [7].
There has long been interest in isolating protoplasts directly from cereal tissues competent for plant regeneration to develop transformation systems which avoid the problems associated with cereal cell suspension cultures [10]. Recently, plants have been regenerated from leaf mesophyll and scutellum protoplasts of rice [4, 3].
A routine procedure for the isolation and culture of protoplasts capable of sustained division and fertile plant regeneration, isolated directly from scutellar tissues is presented. This system has potential as the basis for a protoplast-mediated transformation method for barley.
MATERIALS AND METHODS
Preparation of explants: Scutellar tissues from 1.4-1.6 mm embryos of barley cvs. Clipper, Dissa and Derkado were pre-cultured for 3 days on a modified L1 basal medium [9], containing 5.0 mM NH\4\NO\3 and 148.4 mM KNO\3, supplemented with C5 carbohydrate mixture (maltose, glucose and fructose each at 28 mM) and 9.0 µM 2.4-D (L8D2C5); they were then transferred onto the same basal medium containing sorbitol (88 mM) as the sole carbohydrate source and incubated in the dark for 16 hours at 4°C (two-stage pre-culture).
Protoplast isolation and culture: Protoplast isolation was performed according to a standard procedure used for barley cell suspensions [9], after vacuum infiltration of scutellar tissues with cell wall digesting enzymes (1% Cellulase Onozuka RS [Yakult, Tokyo] and 0.05% Pectolyase Y 23 [Seishin, Tokyo]). Protoplasts were embedded (final density of 1.2-1.3 x 10^6 /ml) in Na-alginate according to a method described previously [6]. Alginate "puddles" were then transferred to liquid protoplast culture medium using a feeder culture system as described by Funatsuki [2]. The liquid culture medium used was modified L3 medium [1], containing 5.0 mM ammonium nitrate, 500 mM maltose and 4.5 µM 2.4-D (L8D1). Protoplasts were co-cultivated with feeder lines and incubated in the dark. Two weeks after protoplast isolation, feeder cells were removed and the protoplast culture medium was diluted, every 7 days, with a standard suspension medium, until calli reached 2-3 mm diameter (5-8 weeks after protoplast isolation).
Embryogenesis induction and plant regeneration: Macroscopic calli were picked from Na-alginate, washed and subcultured onto embryogenesis induction medium, based on L1, but containing 3.1 mM NH\4\NO\3, 11.3 µM Dicamba and C5 carbohydrate mixture (L7Dc2.5C5). Embryogenic explants were further subcultured, after 4-5 weeks, for shoot induction and plant regeneration (3-4 months after protoplasts isolation). Regenerants were grown to maturity in pots and fertile plants were recovered.
RESULTS AND DISCUSSION
The most important factors in the development of the culture system were the pre-culture of donor scutella (on media of specific carbohydrate composition and with cold starvation treatment) and the co-cultivation of alginate-embedded protoplasts with nurse cells.
Optimization of the composition of the protoplast culture medium (such as the nitrogen content, the carbohydrate composition and the 2.4-D level), embedding protoplasts in Na-alginate and the co-cultivation of the protoplasts with nurse cells led to high protoplast plating efficiencies (Table 1).
Table 1 - Effect of feeder lines on protoplast plating efficiency.
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cell lines cultivars
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Clipper Dissa Derkado
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HTA 42A 2.36 ± 0.49 2.36 ± 0.06 2.37 ± 0.67
DLE3 6.37 ± 0.16 0.0 0.0
no feeder 0.0 0.0 NT
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HTA 42A (Tritordeum suspension line); DLE3 (cv. Dissa suspension
line).
The advantage of this new protoplast protocol is that it is relatively
simple compared with others previously reported such as egg cell
protoplasts [5] and microspore-derived protoplasts [11]. Additionally, this
method avoids the establishment of primary callus [8] and/ or suspensions
cultures. Our system has been used successfully not just in model
responsive genotypes, such as cvs. Dissa and Clipper, but also in an
agronomically important cultivar for the malting industry (cv. Derkado).
To develop a transformation system based on the scutellum protoplast protocol, direct gene transfer into scutellar protoplasts through PEG-mediated uptake has been tested, using a plasmid containing the GUS (uidA) and NPTII genes. Transient-GUS expression was obtained and calli have now been recovered from transformation-treated protoplasts grown under selection pressure. If this approach yields transgenic plants it may provide an alternative to particle bombardment for barley transformation.
References :
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