Plant Cell Reports

Plant Cell Reports (1990) 9:69-72

9 Springer-Verlag1990

Improved plant regeneration from shed microspore culture in barley (Hordeum vulgave L.) cv. igri A. Ziauddin, E. Simion, and K. J. Kasha Department of Crop Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada Received December 5, 1989/Revised version received April 10, 1990 - Communicated by E. D. Earle

Abstract This report describes rapid regeneration of green plants from microspores of the barley cultivar Igri. Use of 0.3 M mannito[ during maceration and isolation was essential for response from mechanically isolated microspores of barley cv. ] g r i grown under our conditions. A shed microspore culture system proved to be simple and gave a fast response; plants were obtained as early as 25 days after the material was taken from the donor plant. A 28-day cold-pretreatment of spikes can also be replaced with a 3-4 day pretreatment of anthers in mannitol. Shed microspores from 100 anthers produced an average of 292 plants with 91% of them green. Approximately 80% of the regenerated plants were spontaneously doubled-haploids.

Abbreviations: IAA: FHG:

MS:

Indole-3-acetic acid Hunter's media (1988) Murashige and Skoog

INTRODUCTION The use of hapLoids in barley breeding programs is wet[ established (Kasha and Reinbergs, 1979). Since the microspores are both single-celled and haploid, they also could become an attractive target for in vitro manipulation such as transformation. However, one limitation for cereals may be the low efficiency of embryo formation and regeneration of green plants from microspore cultures (Lorz et al. 1988). Sunderland and Roberts (1977) introduced a technique in Nicotiana tabacum whereby the microspores, shed from anthers after 2-3 days of culture, produced high yields of pollen embryos when recultured without the anthers. Shed microspore culture has an advantage over mechanically isolated microspore culture because it avoids comptex isolation procedures and possible damage to the microspores. Earlier work with barley shed cultures focused on cold-pretreatments of anthers, conditioning effect of anthers in the medium and the production of large numbers of calli using the cultivar Sabarlis (Xu et al., 1981; Sunderland and Xu, 1982). Embryo production or plant regeneration in these shed cultures was not mentioned. K6hler and Wenzel (1985)

Offprint requests to: A. Ziauddin

reported obtaining 1.2 plants per 100 anthers from shed microspore cultures of the cv. Dissa. However, by including a cold-pretreatment of spikes and changing the media, the frequency of plant regeneration was increased to 14 green plants/100 anthers for Dissa and 17 green plants/100 anthers for the cv. Igri (Datta and Wenze[, 1988). In both these reports, the responding microspores formed callus before regenerating into plants. Media changes over a period of time (Clapham, 1973; Foroughi-Wehr eta[. 1976; Olsen, 1987; Hunter, 1987; Sorvari and Schieder, 1987) have set the stage for improvements in barley anther/microspore culture response in some genotypes. Recent reports with the cv. Igri indicate that it has been very responsive in anther culture on solid media (Olsen, 1987; Hunter, 1988), float anther culture (Datta and Potrykus, 1989) and mechanically isolated microspore culture (Hunter, 1988). Our initial attempts to repeat the mechanical isolation results of Hunter (1988) failed. However, it is often difficult to repeat procedures used in other labs, presumably due to strong influence of plant growth conditions (Kasha et al. 1989). In this paper, we report modifications to Hunter's (1988) procedures and demonstrate an improved production of embryos and green plants from shed microspore culture with the cv. Igri.

MATERIALS AND'METHODS Donor plants. Seeds of barley (Hordeum vulgate L.) cv. Igri were germinated in turface at 20~ 75% RH and a 16h day (200 #E/m2/sec). After 7-10 days the seedlings were transferred to a vernalization cabinet at 4~ 60% RH and a 8h day (40 #E/m2/sec). After 8 weeks, the plants were transferred to 15 cm pots containing a 3:2:1 mixture of Guelph clay-loam, turface and peat moss and placed in a growth cabinet with 12~ day/10~ night for 16h of Grow-lux fluorescent plus incandescent 50W bulbs (100 #E/m2/sec). The plants were fertilized twice a week using water soluble 20-20-20 and 28-14-14, alternatively. The duration of the growth period was about T weeks. The spikes were harvested when the pollen was in the mid to late uninucleate stage (Wheatley et al. 1986).

70 Nechanica[ty i s o t a t e d m i c r o s p o r e c u t t u r e . The d e t a i l e d p r o t o c o l described by Hunter (1987, 1988), i n c l u d i n g the use of 28 day c o l d - p r e t r e a t e d spikes, was repeated. Subsequently, a m o d i f i c a t i o n to the technique found to be e s s e n t i a l was the use of 0.3 M mannitol r a t h e r than media f o r i s o l a t i o n and c e n t r i f u g a t i o n of microspores.

Shed Nicrospore Culture. The leaf sheaths with enclosed spikes were surface s t e r i l i z e d by spraying with 70% ethanol. Anthers from fresh spikes were c u l t u r e d in 5 cm diameter p e t r i dishes containing s t e r i l e 0.3 M mannitol at a d e n s i t y of 20 anthers per ml of mannitol. A t o t a l of 240 anthers were used in t h i s experiment. The P e t r i p l a t e s were sealed with p a r a f i l m , covered with f o i l and placed in an incubator at 25 to 28~ A f t e r 3 days, the mannitol s o l u t i o n containing the microspores shed from the 240 anthers was c o l l e c t e d . A number of shed microspores adhered to the p e t r i p l a t e s ; t h e r e f o r e the p l a t e s were g e n t l y shaken to get the microspores i n t o the mannitol s o l u t i o n and i f needed, e x t r a mannitol was used f o r r i n s i n g . To prevent unnecessary loss of microspores in p i p e t t e s and c e n t r i f u g e tubes, the microspore-mannitol suspension from d i f f e r e n t p l a t e s was pooled before c e n t r i f u g a t i o n at 500 r.p.m, f o r 5 minutes. The mannitol was discarded and the microspores were resuspended in 0.6 ml of ovaryc o n d i t i o n e d (K6hler and Wenze[, 1985) l i q u i d FHG medium (Hunter 1988) c o n t a i n i n g 1 mg/l IAA and 0.2 mg/l kinetin. A l l l i q u i d media were f i l t e r (Nalgene) sterilized, and o v a r y - c o n d i t i o n i n g was with Igri cultivar. FHG medium is the same as Olsen's (1987) anther c u l t u r e medium except f o r the replacement of sucrose with maltose (6.2%) and the absence of F i c o l [ 400 (see Kasha et a l . 1989). Approximately 0.2 ml of the suspension (1.8 x 105 microspores) was placed in d r o p l e t form on top of 3 ml of s o l i d i f i e d (0.8% Sea Plaque agarose) FHG medium. A t o t a l of three r e p l i c a t e s were set up and the p l a t e s were wrapped in p a r a f i l m , incubated in the dark at 25~ f o r 21 to 28 days. Observations on the development of microspores were made r o u t i n e l y with an i n v e r t e d microscope ( Z e i s s ) . I f the p l a t e s were d r y i n g out, one or two (.05ml) drops of fresh FHG l i q u i d medium were added. Regeneration of p t a n t t e t s . Between 21 to 28 days a f t e r culture initiation, the foil was removed and the c u l t u r e s were t r a n s f e r r e d to an incubator (22~ with low l i g h t (30-40 #E/m2/sec). Embryos and c a l l i that were 1.5 mm or l a r g e r in diameter were removed weekly and t r a n s f e r r e d to the regeneration medium (FHG solidified with 0.8% Sea Plaque agarose). This permitted further development of the smaller structures in the original plates. Once plantlets reached 2 to 4 cm in height they were transferred to vials or flasks with a solidified (0.8% agar) hormone-free MS medium (Murashige and Skoog, 1962) containing 2% sucrose as the sugar source. Plants were later transferred to small pots containing a mixture of peat moss and soil, placed in a growth room and covered for a week with clear plastic cups to maintain high humidity.

RESULTS AND DISCUSSION

Our initial attempts to repeat the isolated microspore culture of barley cv. Igri as described by Hunter (1987, 1988) failed to give any response. As the microspores appeared to abort immediately, two aspects of mechanical

i s o l a t i o n were explored - one being the i s o l a t i o n in the FHG medium, and the second, the i s o l a t i o n technique itself. Varying the maceration procedure and c e n t r i f u g a t i o n speeds d i d not improve the response. However, by substitution of 0.3 M mannitol for the FHG medium during isolation, viable microspores were obtained and we were able to repeat Hunter's success with mechanical isolation of microspores. Mannitol was first used in the preculture of anthers for 3-4 days as a substitute for the 28 day coldpretreatment of spikes (R.B. Jorgenson, Pers. comm). When fresh anthers of !gri were precultured in 0.3 M mennitol, approximately two-thirds of the microspores (2208/anther) were shed into the mannitol during the first 3 days (Fig. la). When these shed microspores were recultured in the medium, they underwent rapid divisions and formed multicellular structures (Table I; Fig. Ib). Since the shed culture technique was very simple and response from microspores very rapid, efforts were concentrated on improving embryo induction from these cultures. The temperature at which donor plants are grown is important for obtaining a large number of shed microspores. Chen (Pers. comm.) obtained very few shed microspores of Igri when plants were grown at the higher temperature of 22~176 - day/night. Tabte 1:

General development of shed barley (cv Igri) microspores over the period of culture.

Time After Culture Initiation

Development

24h -3 days - Start to see microspores numbers. (Fig. la)

shed

in large

5-6 days

-A large percent of the microspore population is undergoing divisions (Fig. Ib)

7-I0 days

-

18 days

- A number of embryos have formed (Fig. Ic).

Rupture of exine to release multicellular structures.

21-25 days - Some spontaneous germination of embryos and p l a n t l e t formation in the o r i g i n a l media (Fig. ld). Begin harvesting embryos of appropriate size. 28 days

- Plantlet development on regeneration media. (Fig. le).

A combination of mannitol pretreatment with ovaryconditioned FHG media, provides a rapid system of embryo formation (Fig. Ic) and plant development from shed microspores. In cases where the embryos germinate spontaneously in the initial culture media (Fig. Id) plants can be regenerated as early as 25 days after the material was taken from the donor plant. Both Hunter (1988) with mechanically isolated microspores and Datta and Wenzel (1988) with shed microspores obtained plant regeneration 8-10 weeks after the material was taken from the donor plant. When the microspores were mechanically isolated after a mannito[ pretreatment of anthers, the response was good but embryo and plant development was slower than with the shed system (unpublished observations). However, comparison of t h e

?] two systems using identical densities of microspores is required. Since centrifugation is common to both systems it is possible that the mechanical isolation process itself may cause the slowdown of microspore response. Degrading enzymes or toxic substances may be released from the damaged anther walt tissues or celts and cause problems in spite of several washes of microspores.

The average value obtained for plant regeneration from shed microsperes was 292 plants per 100 anthers with 91% of them green (Table 2). We estimate that 0.12% of the microsperes that were shed produced green plants (Table 3). The plant regeneration numbers given are conservative. By r e p l e n i s h i n g with fresh media and h a r v e s t i n g embryos at r e g u l a r i n t e r v a l s , the number of p l a n t s regenerated could be h i g h e r .

Table 2: Number of plants produced from shed microspere culture in barley cv. Igri.

Table 3: Efficiency of the shed microspere culture system in barley cv. Igri.

Replication*

I 2 3 Total(240 anthers) # of plants 100 anthers

Green Plants # % 181 87.4 190 88.8 268 95.7

# 26 24 12

% 12.6 11.2 4.3

207 214 280

639

62

8.2

701

Efficiency = p l a n t s per anther = 2.66 x 100 = 0.12% microsperes shed per a n t h e r 2208

292

* 3 days a f t e r anthers put in m a n n i t o l .

91.2

266+_48

Albino

26+_8

Total # of Plants

* Each replication contained shed microsperes from approximately 80 anthers.

Fig. 1 -

Microsperes shed/anther =

2208 ~ 340

Green p l a n t s / a n t h e r = 2.66

The use of o v a r y c o n d i t i o n e d media has been found to give relative uniformity among replications (Table 2) and consistent results between experiments. When non-

Response from shed microspore c u l t u r e s . The number of days i n d i c a t e s time a f t e r anthers c u l t u r e d . a) Shed microspores a f t e r 3 days in m a n n i t o l . b) Microspores d i v i d i n g at day 5 in FHG medium. c) Typical embryo f o r m a t i o n at about day 18. (12 X). d) Spontaneous development of p l a n t l e t s i n the o r i g i n a l c u l t u r e medium (5 cm p l a t e s ) at day 21 e) Plant development on MS medium at day 28. Bars = 60#.

?2 conditioned media was used, there were initial divisions in the microspores, but these generally did not develop further into embryos or calli. Since the data for this study was collected, we have been able to repeat the goed microspore culture response from the shed cultures a number of times up to the embryo production stage typical of Fig. Id, but have not attempted to regenerate plants. In contrast to previous reports of success with microspores from cv. Igri (Hunter, 1987; 1988), our results are with material that has not been coldpretreated. The conditioning of media is probably more critical for microspores without a l o n g cold pretreatment since they lack the benefit of anther substances that may be released during that period. The actual mechanism by which the immature anthers dehisce is not known. A very brief nutrient starvation of the anthers (Heberle-Bors, 1989), resulting from culture in mannitol, might accelerate the rapid senescence of the anther wall and cause the anthers to dehisce readily. However, we have also observed a large number of microspores shed when fresh anthers are cultured in water. The microspores in water soon start to degenerate and abort within a few days. Thus, mannitol may provide a better osmotic balance for the microspores during the short preculture period. A small number of microspores are also shed when anthers are cultured directly in liquid media. However, our observations indicate that these microspores do not grow in the presence of anthers. The calli/embryos found floating in the liquid anther culture media are generally developed within the anther and then pushed out. Subsequent removal of the anthers in the initial stages (3-7 days after culture initiation) enables the shed microspores to start dividing and form multicellular structures but these sink to the bettomof the Petri plate and rarely regenerate (unpublished observations). Use of Ficoll in the media at the concentrations (100-200 mg/l) normally used for anther culture (Kao, 1981) has not been beneficial for shed microspores since a large percentage of microspores plasmolyze rapidly.

RisS; F.L. Olsen, Carlesberg Res. Inst., Denmark; B. .Foroughi-Wehr, GrOnbach, FRG and M. Bolik, Univ. Munich, FRG and are also acknowledged. We are indebted to the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture and Food for financial grant support.

Green plants regenerated from the shed microspore cultures have been found to consist of 80% spontaneously doubled haploids that are fertile. The rest of the population consisted of haploids, tetraploids and a few aneuploids. With such frequencies of diploids, a colchicine treatment would not be needed. The diploids also appear to be very uniform. In conclusion, reculturing the shed microspores in drops of liquid media on top of media that has been solidified (Hunter 1988) has enabled a high frequency of the dividing microspores from shed cultures from cv. Igri to continue their development and rapidly regenerate into plants. The importance of donor plant conditions (Kasha et at. 1989; Kuhlmann and ForoughiWehr, 1989) and use of carefully controlled conditions for experimentation continues to be of great importance. The simple procedure, starting from single microspores which undergo embryogenesis and form plants, could be suitable for haploid production, mutation and selection, and perhaps genetic transformation techniques.

K6hler F, Wenze[ G (1985) J Plant Physiol 121: 181-191.

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