PlantCell Reports

Plant Cell R e p o r t s (1995) 14:253-256

9 Springer-Verlag 1995

Efficient regeneration of fertile plants from protoplasts isolated from microspore cultures of barley (Hordeum vulgare L.) Marjatta Salmenkallio-Marttila and Veil Kauppinen V T T Biotechnology and F o o d Research, P.O. Box 1505, FIN-02044 VTT, Espoo, Finland Received 21 April 1994/Revised version received 1 September 1994 - C o m m u n i c a t e d by H. L6rz

Summary. Cultures of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar of malting barley) were used for isolation of protoplasts. The protoplasts were cultured embedded in agarose. The plating efficiency varied from 0.002% to 0.015%. Several hundred green plants were regenerated from the cultures. Plantlets regenerated from protoplasts were potted in soil within 4-5 months of collecting the spikes for microspore culture and the first plants are now setting seed.

Introduction Although representatives of most of the world's crop plants have been regenerated from protoplasts, the regeneration of fertile plants from protoplasts still remains a problem (Lazzeri et al. 1990, Roest & Gilissen 1993). Regeneration methods often apply only to particular varieties or even genotypes. A method optimized for one plant line may totally fail with another closely related one. Cells or tissues that in themselves are regenerable are the most likely to yield protoplast cultures capable of regenerating new plants. Therefore most regeneration methods are based on the production of embryogenic suspension cultures. Methods for protoplast regeneration have also been developed for the major cereals rice (Fujimura et al. 1985), maize (Rhodes et al. 1988a), wheat (Vasil et al. 1990), sorghum (Wei & Xu 1990) and barley (Yan et al. 1990, JS.hne et al. 1991). Key steps in these methods are the production of suspension cultures that provide a good source of protoplasts while still retaining their regeneration capacity. In common with other grarnineous species the establishment of regenerable cell suspensions of barley has proved to be difficult. Friable embryogenic callus that is suitable for the initiation of suspension cultures is difficult to obtain in the case of barley. Correspondence to: M. Salmenkallio-Marttila

Moreover, the morphogenic capacity of suspension cultures decreases during long periods of culture (Lfihrs & LSrz 1988, Datta et al. 1992, J~hne et al. 1991). Barley plants have been regenerated from protoplasts derived from embryogenic suspension cultures (Yan et al. 1990, J~ane et al. 1991, Funatsuki et al 1992). However, with respect to the use of the protoplast system for transformation, the relatively unstable plant regeneration reported in these studies remains a problem. In particular, young cell suspensions which retain high regeneration capacity typically produce low yields of fragile protoplasts which divide poorly. The aim of the present study was to develop an improved protocol for the regeneration of fertile plants from isolated barley protoplasts. The inability to regenerate high numbers of fertile plants has been considered to result from the time period needed to obtain homogenous suspension cultures from which dividing protoplasts can be isolated (J~ihne et al. 1991). In comparison with embryogenic suspension cultures, microspore cultures can be produced rapidly and under optimized conditions they show reproducibly high frequencies of fertile plant production. Therefore in the present study microspore cultures were used for the isolation of regenerable protoplasts.

Materials and methods Plant material. Barley grains (Hordeum vulgare L. cv. Kymppi, a tworowed spring barley) were obtained from Kesko Agronomic Station, Hauho. Plants were grown in a growth room (22/13~ day/night, 16 h light 4000/~mol m -~ s-l). The duration of the growth period was 6-8 weeks. The tillers were harvested when the sheath of the flag leaf had emerged 5-10 era, which correlates with the late uninueleate to early binueleate stage of microspore development. The tillers were wrapped in aluminlum foil and placed in beakers in a cold room at 7~ for 3-4 weeks.

254 Microspore isolation and culture. Microspores were isolated and cultured in 108-medium, which is the /q6 basal medium (Chu et al. 1975) supplementedwith 1.1 m M glutamine, 2.8 m M m-inositol, 0.175 M maltose, 7.9 /.dVl 2,4-D and 1.2 p.M kinetin. For mierospore isolation ten spikes were cut into 3-4 pieces and macerated with a teflon rod in the 108-medium. The crude microspore preparation was then filtered through an 80 # m nylon sieve and the microspores were collected by centrifugation for 5 min at 300 x g. The microspores were resuspended in 9 ml of the same medium and the total number of microspores and the number of embryogenic microspores were counted in a Fuchs-Rosenthal haemocytometer. The microspores were cultured in 108-medium at a density of 0.8-1.0xl0 ~ ml -~ in 5 cm Petri dishes in the dark at 23~ The cultures were incubated stationary for the first 1.5 weeks and then in a rotary shaker (65 rpm, stroke radius 2.5 cm). Protoplast isolation and culture. Three- to four week old cultures of mierospores were used for protoplast isolation. Protoplasts were isolated and cultured according to the protocol of Lazzeri et aL (1991) for barley suspension cultures. 2-3 g o f microspore mass was incubated in 20 ml o f enzyme solution containing 1.0% cellulase Onozuka RS, 0.5 % Macerozyme R I 0 and 0.05 % peetolyase Y23 in washing solution (LW). After 2-3 h incubation the suspension was diluted with an equal volume of LW and filtered through 160 #m, 55 #m and 20 # m nylon sieves. The protoplasts were washed twice with LW (100 x g, 5 min) and suspended in protoplast culture medium at a density o f 2-3 x 105 ml-x. Two different media were used for protoplast culture, modified N6-medium (Chu et al. 1975) derived from the medium used for microspore culture (medium 108) and L1 medium used for barley protoplast culture by Lazzeri et al. (1991). The protoplast media were supplemented with 2.3 /.tM 2,4-D, 0.5 M maltose and 1.2% agarose (Sea-Plaque*, FMC Corporation). One ml aliquots of the protoplast suspension were plated on Millicell"-CM culture plate inserts (Millipore), which were placed in 9 cm Petri dishes containing 8 ml of nurse culture. The nurse cultures were prepared by suspending approximately 0.5 g of the same microspore mass as was used for protoplast isolation in 8 ml of protoplast medium without agarose. Cultures were incubated on a rotary shaker (65 rpm, stroke radius 2.5 em) at 23~ in the dark. After one week of culture the feeder cells were removed and 2 ml o f fresh 108 medium (0.14 M maltose, 9.0/.tM 2,4D) was added to the plates. After another week of culture the old medium was replaced with fresh 108 medium. After one more week the agarose pieces were transferred to L2 medium supplemented with 11.3 /,M 2,4-D and solidified with 0.3% gellan g u m (Gelrite", Scott laboratories). The plating efficiencies were determined as the number o f protoplasts producing microcalli in four weeks. Plant regeneration. The regeneration media II and III were the modified MS-media II and III respectively used by Olsen (1987) for barley anther culture. The medium 1I contained maltose instead o f sucrose (Hunter 1987) and the media were solidified with 0.3 % gellan gum. Embryogenic structures developing from the dividing protoplasts were transferred to regeneration medium II for further development. The plates were incubated with illumination (50 /,mol m-2s-l). Green plantlets were transferred to medium HI for further growth and potted in soil when about I0 cm tall.

Results and discussion Protoplast isolation and culture

The microspores isolated from 20 spikes produced approximately one gram of mass in three weeks. The protoplast yield varied between 5 and 15 x 106 per g fresh weight (Fig. la). Therefore one spike corresponded to approximately 0.5 x 106 protoplasts.

Protoplasts failed to divide without the support of nurse cells. As the nurse cultures used were highly regenerable, culture well inserts were used to ensure the separation of protoplast cultures from the nurse cells (Fig. lb). The importance of nurse cultures for the regeneration of barley protoplasts has also been demonstrated in other studies (J~ihne et al. 1991, Funatsuki et al. 1992), in which feeder cultures consistently gave better plating efficiencies. The effect was strongest in the young lines (Funatsuki et al. 1992). Both colony formation and plant regeneration were improved by the use of feeder cultures. Two different media were tested for protoplast culture, modified N6 medium (Chu et al. 1975) derived from the medium used for microspore culture, and L1 medium used for barley protoplast culture by Lazzeri et al. (1991). N6 medium has been successfully used for the regeneration of rice protoplasts (Datta et al. 1992) but the barley protoplasts failed to divide in this medium. Sustained protoplast divisions were obtained when L1 medium was used. The medium has been effective in the culture of protoplasts isolated from suspension cultures of barley (Lazzeri et al. 1991, Jiihne et al. 1991, Funatsuki et al. 1992). Because the concentrations of macro salts in these two media are very similar, the influential factor is probably among the organic elements. High concentration of sugar is used to adjust the osmotic pressure of the protoplast culture medium. The type of sugar used is also important, because at the same osmolality maltose was superior to glucose for the culture of protoplasts isolated from barley microspores. The protoplasts divided poorly in medium containing glucose, the plating efficiencies were only a few percent of the ones obtained with maltose, and no plants could be regenerated. The first protoplasts divided after one week of culture and colonies were visible after 2-3 weeks (Fig. lc). Plating efficiencies, counted as microcalli formed from protoplasts after four weeks, varied between 0.002% and 0.015%. Protoplast-derived calli (Fig. ld, e) resembled very much in appearance the embryogenic calli derived from microspore cultures. Regeneration o f plants

The plating efficiencies obtained in our experiments, between 0.002% and 0.015%, were lower than those reported earlier for barley (Jiihne et al. 1991, Funatsuki et al. 1992, plating efficiencies even as high as 10%). However, we obtained repeatedly good plant production (Table 1). The production of green plants varied between 1 and 20 per lxl06 protoplasts, with a mean of 8.3. This is approximately one tenth of the regeneration capacity of the microspore cultures. The first plants transferred to soil are now setting seed (Fig. 1f). We isolated regenerable protoplasts from microspore

255

Fig. 1. Culture and regeneration of barley protoplasts, a) Freshly isolated protoplasts, scale bar 200 lam. b) Culture plate inserts containing the protoplast cultures were placed in Petri dishes with the nurse cells, e) Microcalli formed from protoplasts are visible after 3-4 weeks of culture, d) Embryos differentiating from calli derived from protoplasts, e) Plantlets regenerated from protoplasts, f) Fertile plants regenerated from protoplasts.

256 Table 1. Regeneration of barley protoplasts, summary of seven separate experiments. Total number of spikes

100

Total number of protoplasts

55.5x106

Total number of microcalli (plating efficiency)

5116 (0.01%)

Number of green and albino plants

459

584

Green plants per lxl06 protoplasts

8.3

Green plants per spike

4.6

cultures, not from embryogenic suspensions as in the earlier studies (Liihrs & LSrz 1988, JSJane et al. 1991, Funatsuki et al. 1992). Several months are required for the production of suspension cultures and very few of them are suitable for isolation of regenerable protoplasts. In these studies it was observed that freshly initiated suspensions were not suitable for protoplast isolation. Young suspensions yielded very few protoplasts, which divided poorly. With our protocol good plant production was obtained repeatedly and plantlets regenerated from protoplasts were potted in soil within 4-5 months of collecting spikes for microspore culture. Protoplasts are of great value for the genetic modification of plants. They can be transformed by direct gene transfer, i.e. uptake of DNA through the plasma membrane stimulated either chemically or electrically. Transgenic cereals have been obtained by direct gene transfer to protoplasts (Rhodes et al. 1988b, Toriyama et al. 1988, Zhang & Wu 1988, Zhang et al. 1988). The present study demonstrates the usefulness of microspore cultures for obtaining regenerable protoplasts of barley. The system will be applied to the production of transgenic barley plants.

Acknowledgements. The skillful technical assistance of Jaana Juvonen and Tuuli Teikari is gratefully acknowledged.

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Efficient regeneration of fertile plants from protoplasts isolated from microspore cultures of barley (Hordeum vulgare L.).

Cultures of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar of malting barley) were used for isolation of protoplasts...
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