Plant Cell Reports

Plant Cell Reports (1991) 1 0 : 1 - 6

9 Springer-Verlag1991

Regeneration of fertile plants from protoplasts derived from embryogemc cell suspensions of barley (Hordeum vulgate L.) A. J/ihne

1,2,

p . A . Lazzeri 1, and H. Ltrz 1

1 Institut ftir Allgemeine Botanik, AMP II, Universit/it Hamburg, Ohnhorststral3e 18, W-2000 Hamburg 52, F R G 2 Resistenzlabor der Saaten-Union, Hovedisser Strage 92, W-4817 Leopoldshthe, F R G Received February 10, 1991/Revised version received March 14, 1991 - Communicated by I. Potrykus

Abstract. We report regeneration of f e r t i l e plants from barley (Hordeumvutgare L. cv. Igri) protoplasts isolated f r o m regenerable suspension cultures initiated f r o m anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered. Abbreviations : 2,4-D: 2,4-dichlorophenoxyacetic acid, 6-BAP: 6-benzylaminopurine, PP: Protoplasts Introduction For a tong time i t has seemed that cereal species are recalcitrant in cell-suspension and protoplast culture. However, the last few years have shown that it is indeed possible to regenerate fertile plants from protoplasts of cereals. In all cases where plants have been regenerated from protoplasts a prerequisite was the establishment of embryogenic cell suspensions, as reported for maize (Prioli and SShndahl 1989, Shitlito et at. 1989), rice (Fujimura et al. 1985, Kyozuka et al. 1988) wheat (Vasit et al.

1990) and barley (LQhrs and L6rz 1988, Lazzeri and LSrz 1990, Yan et al. 1990). Unfortunately it is generally observed that cereal suspensions tend to become less regenerable with age (Shillito et al. 1989, LQhrs and LSrz 1988), therefore it is important

Offprint requests to: H. Ltrz

to develop methods f o r fast and reproducible suspension establishment. In barley, callus derived from immature embryos is the most frequently used material for production of morphogenic suspension cultures (L~hrs and L6rz 1988, Lazzeri and L6rz 1990), but with this system the establishment of suspensions generally takes several months. Our previous experiments have shown that suspension production from anther cultures is faster and more efficient (J~hne et at. 1991). The culture of barley protoplasts isolated from various tissues has been described previously. Koblitz (1976) f i r s t reported colony formation from protoplasts isolated from friable callus cultures and since t h e n there have been several reports of protoplast isolation from suspension cells. Lehrs and L6rz (1988) f i r s t regenerated albino ptantlets from suspension protoplasts. Two years Later Lazzeri and L6rz (1990) succeeded in recovering green plants from protoplasts, but t h e s e plants did not survive transfer to soil. Yan et at. (1990) initiated suspensions directly f r o m immature e~ryos and isolated protoplasts f r o m which plants could be regenerated. These plants could be transferred to soil, but no mention of their f e r t i l i t y status was made.

Material

and M e t h o d s

Cell Suspension C u l t u r e s . For the i n i t i a t i o n of c e l t suspensions 4-week-old a n t h e r - d e r i v e d embryogenic c a l l u s of the c u l t i v a r I g r i was used, as described p r e v i o u s l y (J~hne et a t . 1991). I s o l a t i o n and Culture of ~ r o t o p l a s t s . Cell suspension lines were tested twice a month f o r t h e i r a b i l i t y to release p r o t o p t a s t s . P r o t o p l a s t s were i s o l a t e d and

cultured according to a protocol of Lazzeri et al. (1991). Three days a ft e r subculture 2 to 4 grams of suspension c e l l s (fresh weight) were incubated with filter-sterilised enzyme solution (10 ml enzyme solution/1 g suspension cells). The enzyme solution consisted of I % Cellulase Onozuka RS (Yakult, Tokyo), 0,5 % Macerozyme RIO (Serva) and 0,05 % Pectotyase Y23 (Seishin, Tokyo) dissolved in washing solution (LW) at a pH of 5.7 and an osmotarity of 700-725 mOsm. The LW solution contains the macro and micro salts and amino acids of L1 medium and 0.6 M mannitol. Cells were incubated on a rotary shaker at 40 rpm at 25 ~ for 2 to 3 hours. After cell walt digestion the protoplast-enzyme mixture was rinsed through 100, 50 and 25 #m diameter sieves with LW solution (pH 5.7, 700 - 725 mOsm). Protoplasts were collected by centrifugation at 50 x g for 4 minutes. The protoplast preparation was washed twice in LW solution. After purification, protoplast pellets were suspended in 2 ml atiquots of L1 medium containing 2 mg/t 2,4D, 180 g/l maltose and 15 g/l SeaPlaque agarose (medium osmotarity adjusted to 700 mOsm with mannitot). Suspended protoplasts were pipetted into 60 mm diameter plastic petri dishes (Greiner, tissue culture quality). Cultures were incubated at 25 ~ under continuous low light. Plating efficiencies were determined after 14 days by counting viable microcalli. After 21 days of culture the agarose of the protoplast cultures was cut into segments (16 segments/dish) and 0.5 mt liquid LID2 medium was added to the surface. Plant Regeneration. For further culture, segments of agarose containing 5 to 6 week-old protocolonies were transferred to the surface of solid culture medium containing I mg/l 2,4-D and I mg/l 6-mAP. Two media which proved to be suitable to induce embryogenesis from suspension cells were compared: MSmDIBI and L3DIBI (J~hne et al. 1991). Protoplast-derived calli were subcultured monthly and selected for e~ryogenic tissues. Embryogenic c a l l i which appeared after two months of culture were transferred to the l i g h t (16 h, approx. 6000 Lux). Green shoots which arose from the protocolonies a f t e r 4 to 5 months were

transferred to hormone-free regeneration medium (L3) which contained 25 g/I of maltose. After 4 to 6 weeks of growth, plants with vigorous root systems were transferred to pots containing a peat/soil mix and were grown at 18 ~ with 16 h light, in a phytotron for about two months. Regenerants were vernalized for six weeks at 4 ~ with 10 h light (4000 Lux). After vernalization, plants were grown for a further two months in the phytotron. Shortly before tillering plants were transferred to the greenhouse where they were grown to maturity.

Results Cell Suspension Cultures. For the i n i t i a t i o n of embryogenic c e l l suspension cultures one-month-old anther-derived embryogenic c a l l i (Fig. 1A) were used as inoculation material, as described previously (JQhne et a l. 1991). Young cultures contained large and compact aggregates. After eight weeks of culture in macroplates suspensions with f i n e r aggregates were selected for culture in plastic vessels. After the third month of culture suspensions were still rather heterogeneous, but with selective suloculturing it was possible to increase the frequency of small, highlycytoplasmic cell aggregates (Fig. IB). For up to four months of culture suspension aggregates were plated directly on regeneration medium to test their morphogenic capacity. Fertile plants could be regenerated from these suspensions at high frequencies. Suspensions maintained their ability to regenerate fertile plants for more than six months, but with advancing age their regeneration capacity declined and the proportion of albino plantlets increased. For suspensions older than four months it was found to be necessary to plate the cells first on an induction medium before transfer to regeneration medium. Protoplast Isolation and Culture. For protoplast isolation suspensions comprising mainly small aggregates of highly cytoplasmic celts were selected.

I t was observed that freshly i n i t i a t e d suspensions were not suitable for protoplast is olat ions . Young suspensions always yielded very few protoplasts that divided poorly (Tab. 1). With regular subculture the y i e l d of protoptasts increased. V i a b i l i t y of freshlyisolated protoplasts (Fig. 1C) was determined by fluorescein diacetate. More than 90 % of protoplasts stained with this dye were highly fluorescent under UV l i g h t , indicating a high level of v i a b i l i t y . Protoplasts isolated from embryogenic suspension cultures were able to divide when plated in protoplast culture medium s o l i d i f i e d with 1.5 % Sea Plaque agarose. The protoplasts did not divide when they w e r e cultured in l i q u i d medium. The f i r s t protoplast divisions occurred a f t e r 5 to 9 days (Fig. 1D) and celt colonies were observed a f t e r !0 to 14 days of culture (Fig. 1E). After 14 days plating e f f i c i e n c y was determined. Plating e f f i c i e n c i e s were found to depend on p la t in g density (Tab. 2); tow plating densities resulted in very tow frequencies of protoplast d i v i s i o n , the best p la t in g e f f i c i e n c i e s were obtained when densities between 8 x 105 and 1 x 106 protoptasts/ml were used. On the basis of these results, a plat ing density of about 8 x 105 protoplasts/ml was adopted as standard.

Colonies derived from protoplasts resembted the aggregates present in the donor suspension, being compact with smooth surfaces. Around 4 weeks after protoptast isolation colonies had a diameter of I - 3 mm (Fig. IF). At this stage they were transferred to s o l i d c u l t u r e media.

Tab.

1: Isolation and Culture of Protoplasts from different Cell Suspension Lines of Cuttivar Igri

Line

Age (Weeks)

IL VI

4 8 12 16 20

IL IX

IL 1

IL3

Yield of PP/ Gram Celts

0.5-I.0xi06 1.0-2.0xi06 1.0-5.0xi06 1.0-5.0xi06

Plating Efficiency

0 0.I-0.2 1.0-3.0 ~ 1.0-3.0 %

+ +

4 8 12 16

0.5-I.0xi06 0.5-I.0xi06 1.0-3.0xi06

0 % 0 0 %

4 8 12 16 20

1.0-5.0xi06 1.5-2.5xi06

0.1-0.2 % not yet known 0.1-0.5 % not yet known

4 8 12 16 20

Tab.

1.0-1.5xi06 1.0-1.5xi06 1.0-4.0xi06

0.5-I.0 % not yet known 1.0-2.0 % not yet known 1.0-3.5 % not yet known

2= Influence of Plating Density on the Plating Efficiency of Protoplasts, Suspension Line: IL VI, Age of Suspension: 18 weeks

P l a t i n g Density No, of PP/ml)

Plating Efficiency

2 3 4 5 6 7

O~ 0 0 0.1 0.I 0.5 0.5

-

1

- 3

~

1

- 3

~

x x x x x x

Embryogenic Capacity

105 105 105 105 105 105 8 x 105 x 105 x 106

0.01 0.05 0.5 0.8 2 3

~ % % % % %

Numberof Replicates

5 5 8 8 10 18 18 18 25

Figure

1: Regeneration of Plants from Embryogenic Barley Suspension Cultures

A) Anther-derived embryogenic callus after one month of culture B) Suspension cultures in two different media C) Freshly isolated protoptasts, scale bar = 50 #m D) First divisions of barley protoplasts (after 6 days of culture, scale bar = 20 #m E) Colony formation from a barley protoplast after 14 days, scale bar = 20 #m F) Protoplast-derived colonies after 4 weeks of culture (Plating efficiency: 1.5 ~, plating density: 8 x 105 ) G) Protoplast-derived embryogenic callus H) Plants regenerated from protoplasts after vernalization ]) Fertile plants regenerated from protoplasts, in the greenhouse Plant Regeneration. For regeneration experiments we used c u l t u r e media p r e v i o u s l y t e s t e d f o r the c u l t u r e of suspension aggregates. We found two media s u i t a b l e f o r the induction of somatic embryogenesis: MSmD1B1 and L3D1B1. These media were also tested f o r c u l t u r e of p r o t o p l a s t - d e r i v e d c a l l i . A f t e r e i g h t weeks of c u l t u r e the formation of embryogenic s t r u c t u r e s could be observed ( F i g . 1G). Both media supported the induction of embryogenesis, but the MSmD1Bl-medium was s u p e r i o r to the L3D1Bl-medium. Embryogenic structures

were

selected

for

further

cutture

and

t r a n s f e r r e d i n t o the t i g h t . A f t e r f i v e months of s e l e c t i o n f o r embryogenic tissues green and a l b i n o shoots al~oeared, which were t r a n s f e r r e d t o hormonefree regeneration medium. In t o t a l 22 green p l a n t l e t s were regenerated. The p r o p o r t i o n of a l b i n o p l a n t l e t s was very high (18 a l b i n o : 1 green). A f t e r one month of c u l t u r e on regeneration medium rooted p l a n t l e t s were t r a n s f e r r e d to s o i l . From nine p l a n t l e t s which survived v e r n a l i z a t i o n ( F i g . 1H) s i x proved to be f e r t i l e ( F i g . 1 I ) . These s i x p l a n t s were g e n e r a l l y s i m i l a r in morphology to seed-grown p l a n t s , but tended to produce l a r g e r numbers of t i l l e r s . Root t i p chromosome counts showed these p l a n t s to be d i p l o i d . Three p r o t o p l a s t - d e r i v e d barley plants appeared abnormal. Two of them looked like haploid plants, but examination of their karyotypes revealed that one was diploid and one aneuploid. These two plants were both sterile. The third abnormal plant was tetraploid and set a few seed.

,-Ix

Discussion

by donor suspension celts frequently

We have developed methods for the regeneration of fertile plants from protoplasts of barley. A prerequisite in establishing this method was the selection of embryogenic suspension cultures suitable for the isolation of totipotent protoplasts. We have previously reported (JQhne et at. 1991) that the use of anther culture-derived tissues is an efficient approach for the establishment of embryogenic barley suspension cultures. [ t is possible to i n i t i a t e numbers of embryogenic suspensions from which f e r t i l e plants c a n be regenerated, but only a very small number of these suspensions prove to be suitable for the i s o l a t i o n of protoplasts. Most of the embryogenic suspension lines produce only hard and compact aggregates which do not give rise to protoplasts, even w h e n solutions with elevated enzyme concentrations were used (data not shown). In our experiments the critical step for successful protoplast isolation was the selection of suspension lines which produced small highly cytoplasmic aggregates with smooth surfaces which still maintained their embryogenic capacity. In order to have a continuous supply of regenerable protoptasts we routinely establish new embryogenic suspensions. Reports of maize protoptast cultures (Vasil and Vasil 1987, Kamo et al. 1987) have suggested that the morphogenic capacity of donor cell suspensions can be partially or completely lost in protoptast-derived calli. The results obtained for barley cultures demonstrate that protoplast-derived calli are able to maintain the morphogenic capacity of the donor suspensions. This has also been shown with suspensions established from embryogenic callus of immature embryos. These suspensions gave rise to protoplasts from which albino (Lehrs and L6rz 1988) and green plantlets (Lazzeri. and L6rz 1990) could be regenerated. However, the plant regeneration capacity of these suspensions was very low, therefore we consider the use of donor suspensions with high plant regeneration capacity critical for achieving reproducible plant regeneration from barley protoplasts. The plating efficiencies of the cultured protoplasts were found to be closely related to the quality and to the age of the donor suspension cultures. Our data also show that the plating densities have a great impact on the plating efficiencies (Tab. 2). Highest plating efficiencies were obtained when densities of 8 x 105 - I x 106 protoplasts/ml were used. This agrees with the reports of Yan et al. (1990) where good results were obtained when plating densities between 5 x 105 and I x 106 were used. In our experience plating efficiencies could be further increased to 10 % when protoplast cultures were fed

reported

for

(data not shown)

maize

protoptast

as

is

cultures

(Prioli and S6hndahl 1989, Shittito et at. 1989, Rhodes et at. 1988). For our experiments it was not routinely necessary to use a feeder system because without feeding we obtained enough calli for regeneration experiments, but for protoplast transformation it may prove helpful to use such feeder cultures. The regeneration of plants from protoplast cultures took a relatively long time compared to other reports (Shillito et at. 1989, Yamada et at. 1986), where plants could be transferred to soil approximately 3 months after isolation of the protoptasts. For our protoptast cultures it was necessary to subculture the calli regularly and to select carefully for embryogenic structures. The induction media which proved to be successful for suspension cultures also were suitable for induction of somatic embryogenesis in protoplast cultures. Although embryogenic structures appeared already after 2 months of culture these somatic embryos had a very poor germination ability. Further selective subculturing lead to the regeneration of many albino and some green plantlets. It is known that in gramineous species the frequency of albino regenerants generally increases with the length of time in culture (Kott and Kasha 1984, Vasil 1987). The green plantlets rooted easily after transfer to regeneration medium and could be transferred to soil after one month of culture, but only the most vigorous plants grown in soil continued growth after vernalization. Two of these plants were abnormal and exhibited chromosomal abberations, a third was phenotypically abnormal although it was diploid. At present it is not possible to control cultureinduced chromosomal abberation and loss of regeneration capacity, although the speed of the process may be influenced by factors such as levels of hormones in culture media (Ziauddin and Kasha 1990). One approach to the problem is the storage of competent cultures by cryopreservation (Shittito et al. 1989, Fretz and L6rz 1990) but ultimately methods for the continuous production of regenerable suspensions are needed. The possibility of regenerating plants from protoplasts makes the transformation of barley via direct gene transfer now feasible. The stable transformation of barley protoptasts f has recently been reported (Lazzeri et at. 1991) and these methods are now being applied to protoplasts from regenerable cell tines.

Acknowledgement s The authors would Like to thank Marion Gohra for her painstaking care of regenerant plants, Etisabeth RoBa for her excellent assistance in cell culture experiments and Xiao-Hui Wang for the root-tip chromosome preparations. We acknowledge the financial support of the Bundesministerium for Forschung und Technolgie, Bonn ("Forschungskooperation zwischen Industrie und Wissenschaft") during this work. This article is based on a doctoral study by Atwine J~hne in the Faculty of Biology, University of Hamburg.

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