Planta

Planta (1989)178:325-333

9 Springer-Verlag 1989

Plant regeneration from indica rice (Oryza sativa L.) protoplasts Lisa Lee *, Ronald E. Schroll**, Howard D. Grimes, and Thomas K. Hodges*** Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

Abstract. Rice ( O r y z a sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1.106 to 15" 106 viable protoplasts/l g flesh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0% ; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N 6* P r e s e n t a d d r e s s : Enichem Americas, Inc., Monmouth Junction, NJ 08852, USA ** P r e s e n t a d d r e s s : Coastal Plains Soil and Water Conservation, Florence, SC 29502, USA *** To whom correspondence should be addressed

2,4-D=2,4-dichlorophenoxyacetic acid; p c v = packed cell volume; BAP = N6-benzylaminopurine; F D A = fluorescein diacetate; F W = f r e s h weight; IAA=indole-3-acetic acid Abbreviations:

A A = M u l l e r and Grafe (1978); C P W = F r e a r s o n et al. (1973); Kao * = Kao (1977); LS = Linsmaier and Skoog (1965) ; MS=Murashige and Skoog (1962); N6=Chu etal. (1975); PCM = Ludwig et al. (1985) Media:

benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N6 or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about threefourths the height of seed-grown plants. Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-weekold primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions. Key words: Cell culture (plant regeneration) - Embryogenic cell and callus culture - Feeder layer - O r y z a (plant regeneration) - Protoplast (culture)

Introduction Regeneration of plants from protoplasts of japonica genotypes of rice have been obtained (Fujimura etal. 1985; Yamada etal. 1986; Abdullah etal. 1986; Toriyama et al. 1986; Kyozuka et al. 1987); however, there have been no reports of success at regenerating rice plants from protoplasts of the indica genotypes. It is especially important to regenerate the indica rice genotypes since these varieties are the principle food source in most of the tropical regions of the world. We have previously developed conditions for regenerating indica rice genotypes from primary callus of IR54 (Koetje et al. 1989). In the present study, we optimized conditions for obtaining high yields of viable protoplasts from primary callus

326 obtained from immature embryos and from suspension cells of IR54 (an indica genotype). Plant regeneration from rice protoplasts was obtained f r o m c e l l s u s p e n s i o n s as w e l l as f r o m p r o t o p l a s t s of the primary callus. Our procedure involves the u s e o f c e l l s o f a j a p o n i c a g e n o t y p e ( C a l r o s e 76) as a n u r s e c u l t u r e d u r i n g t h e i n i t i a l s t a g e s o f p r o t o plast growth, then selecting small colonies of emb r y o g e n i c c a l l u s , a n d t r a n s f e r r i n g t h i s c a l l u s t o regeneration medium containing a cytokinin which promoted shoot development.

Materials and methods Plant materials. Seeds of the indica rice (Oryza sativa L.) cv. [R54 were obtained from the International Rice Research Institute (Los Banos, Philippines). Calrose 76 seeds were obtained from Dr. Niel Rutgers, University of California, Davis, USA. Plants were grown in growth chambers as described in Koetje et al. (1989).

Induction of callus and suspension cultures. Immature embryos (5-7 d after panicle extrusion) or mature seeds were surfacesterilized and cultured on either MS (Murashige and Skoog 1962) o r N 6 ( C h u et al. 1975) media as described (Koetje et al. 1989) or on LS medium (Linsmaier and Skoog 1965) containing 3 % (w/v) sucrose, 2.5 rag/12,4-dichlorophenoxyacetic acid (2,4D), 250 m M tryptophan, and 0.4% (w/v) agarose (BRL; Life Technologies, Gaithersburg, Md., USA). Several different suspension-culture lines of IR54 were used in this study and they are designated as follows : IR54-1 (developed from immature embryo callus and the first line to exhibit growth of protoplasts into callus), IR54-2 (developed from callus obtained from mature seeds), IR54-3 (developed from protoplast-callus of IR54-1 line), IR54-4 and IR54-5 (younger lines developed from immature embryo callus). A suspension culture of IR52, developed from young inflorescences, was also used as a nurse culture. Suspension cell cultures (approx. I g fresh weight (FW)/ 40 ml liquid medium) were initiated by carefully teasing away embryogenic regions from three- to four-week-old primary callus and placing them into liquid medium. The liquid medium contained N6 basal salts and vitamins, 20 m M e-proline, 250 m M tryptophan, 2% sucrose, pH 5.8 prior to autoclaving, and either 4 mg/1 2,4-D for cell line IR54-I or 2 mg/l 2,4-D for the other IR54 cell lines. All suspension cells were cultured on a rotary shaker at 120 rpm in the dark at 25 ~ C. During the establishment of the initial cell suspensions, the medium was changed without removing any callus every ~ 3 d for two to four weeks; subsequently, the cell suspensions were subcultured twice each week using 5 ml packed-cell-volume (pcv) per 40 ml of medium for the next three months at which time the cultures had become established or stabilized. Thereafter, the suspensions were subcultured once each week with 5 ml pcv of cells to 40 ml of fresh medium.

Protoplast isolation and culture. Several parameters were evaluated in order to obtain high yields of viable protoplasts from rice cells. The final procedure used was as follows, and this was the procedure for the optimization experiments except as indicated in the figure legends. About 0.75-1 g FW of primary callus or suspension cells were incubated in 10 ml enzyme solu-

L. Lee et al. : Regeneration of indica rice from protoplasts tion that contained 1% (w/v) cellulase "Onozuka'" RS (Yakult Honsha Co., Tokyo, Japan), 0.1% (w/v) Pectolyase Y-23 (Seishin Pharmaceutical Co., Tokyo, Japan), in CPW salts (Frearson et al. 1973), 0.4 M mannitol, and 5 m M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. The cells were digested for 4 h on a 40-rpm shaker in the dark at 25 ~ C. The mixture was filtered through a 30-gm nylon mesh (Tetko Co., Elmsford, N.Y., USA), washed with two volumes of the CPW medium, and centrifuged at 80.g for 15 min. The protoplasts were then suspended in 1 ml of CPW medium, layered onto 5 ml of 0.6 M sucrose, and centrifuged at 40.g for 10 min. The floating and purified protoplasts (containing less than 0.1% cells) were collected, washed with CPW medium, and suspended in a protoplast culture medium (Kao*) which was slightly modified from Kao (1977) by including 50 mg/1 arginine, 0.5 rag/1 2,4-D, and 7% (w/v) glucose. The osmolality of this medium was 459 m m o l . k g - 1 Fluorescein diacetate (FDA) (ICN Pharmaceuticals, Cleveland, Oh., USA) was used to determine the viability of the protoplasts (Widholm 1972). Cell contamination was determined by staining cell walls with Calcofluor White (Polysciences, Warrington, Pa., USA) (Galbraith 1981). Prior to protoplast isolation, the suspension cell cultures were transferred to either fresh N6 medium as described above, or to an MS or an amino acid (AA) medium (Muller and Grafe 1978) containing 2% sucrose and 2 mg/1 2,4-D and using 0.5 or 2.0 pcv per 40 ml of liquid. Protoplasts were cultured using a feeder-layer method originally developed for maize (Ludwig et al. 1985; Kamo et al. 1987) but with the following modifications. Protoplasts in Kao* medium (0.2 ml) were plated at a density of 5.10 s .ml-1 onto a 0.8-gm-pore Millipore filter (Millipore Co., Bedford, Ma., USA) which was placed onto feeder cells imbedded in agarose. The feeder-cell layer was prepared according to Kamo et al. (1987) and consisted of 1.5 ml pcv of 4-d-old suspension cells placed into 20 ml Kao* medium containing 0.5 mg/1 2,4-D with 0.8% (w/v) agarose (Sea Plaque L G T ; F M C Co., Rockland, Me., USA), and 5 ml of this mixture was transferred to a Petri plate (60 mm diameter, 15 mm high). The feeder cells were obtained from one of the following sources: a Calrose 76 cell suspension that was maintained in AA medium, two IR54 cell suspensions (IR54-4, IR54-5) that were maintained in N 6 medium, and an IR52 cell suspension (initiated from callus obtained from young inflorescences, S. Yang and T.K. Hodges, personal communication) that was maintained in LS medium. The Calrose 76 cell suspension was initiated with callus obtained from immature embryos, and it was maintained originally on an N6 medium with 2 mg/1 2,4-D. After 22 months, this culture was transferred to AA medium in order to improve protoplast yields, and it is currently maintained in the AA medium. The plated protoplast cultures were incubated in the dark at 25 ~ C. After three weeks, the plating efficiency was determined by counting the number of colonies that were at least 0.1 mm in diameter (with the aid of 8 x magnification of a dissecting microscope) and dividing the number of colonies by the number of protoplasts plated onto each filter. The filters were then transferred to 25 ml fresh LS basal medium containing 2% (w/v) sucrose, 0.5 mg/1 2,4-D, and 0.4% (w/v) agarose (BRL) in Petri plates (t00 mm diameter, 15 mm high) and cultured in the dark at 25 ~ C. Three weeks later, callus colonies 1-2 mm in diameter with embryo-like structures were transferred to regeneration medium containing either MS basal salts or N6 basal salts medium supplemented with 3% (w/v) sucrose, 0.4% agarose, 0.03 pM naphthalene-l-acetic acid, and various cytokinins at a concentration of 9 gM in Petri plates (100 mm, 25 mm). Five colonies (about 1-2 mm in diameter) were plated onto each plate. Plates were kept in the dark for one week and were then transferred to light chambers (75 p.mol, m - 2. s - 1

L. Lee et al. : Regeneration of indica rice from protoplasts

327

I O0

~'6.0

N6AGAR 0.0285 80

5.0

MS- . 4.0

60

~

0.797 (63%)

AGAR

v

55 03 m 13,. 0

s

13. ~3

5~

40

2.0 2O

1.0

AGAROSE

0.0 0.0

N6- 1 0.104 (79%)

AGAROSE

(81%) i

0.2

0.4

0.6

0.8

0

photosynthetic photon fluence rate) with 16 h-photoperiods at 25 ~ C. Plantlets with shoots 5 cm or longer were then transferred into jars (100 mm high, 60 mm in diameter) containing 0.8% (w/v) agar with MS basal salts and 3% (w/v) sucrose to promote root development. Regenerated plants ( > 10 cm in length) were then transferred to pots containing soil and placed in a growth chamber (day temperature 30~ C, night temperature 24~ C, with a 10-h photoperiod) and grown as described for seed-grown plants (Koetje et al. 1989).

1

|

2

Viable Protoplasts (xl0 6 ) /gFW

Mannitol Conc. (M) Fig. 1. The effect of mannitol concentration on protoplast yield and viability in IR54 indica rice. Embryo-derived, primary callus was grown on MS medium supplemented with 2 rag/1 2,4-D for three weeks. Callus (approx. 1 g) was teased apart and placed into 10 ml of enzyme solution, pH 5.6, (see Materials and methods) at various mannitol concentrations for 4 h. Protoplasts were then isolated as described in Materials and methods, and viability was determined by FDA staining

|

Fig. 2. The effect of basal medium (MS or N6) and solidifying agent (agar or agarose) on protoplast yield and viability in IR54 indica rice. Embryo-derived, primary callus was grown on either MS-Agar (1.5%), MS-Agarose (0.4%), N6-Agar (1.5%), or N6-Agarose (0.4%) for three weeks. All four treatments included 2 mg/1 2,4-D. Callus (approx. 1 g) was teased apart, placed into 10 ml of enzyme solution, pH 5.6, containing 0.4 M mannitol, and incubated for 4 h. Protoplasts were then isolated as described in Materials and methods, and viability was determined by FDA staining. The numbers without parentheses refer to the protoplast yield; the numbers in parentheses indicate the viability (%) observed in each treatment

v

6.0

1 oo

5.5

90

5.0 80 •

Results

Protoplast isolationfrom primary callus and suspension-culture cells. Initial experiments on the isolation of protoplasts from primary callus grown on N 6 medium using our standard culture conditions (Koetje et al. 1989) resulted in yields of only 1.104 to 5-104 protoplasts/gFW, which was too low to carry out substantial experiments related to growth of the protoplasts. Therefore, experimental conditions were sought that would produce at least 106 viable protoplasts/gFW. Hydrolyzing enzymes (cellulase and pectinase) used for removing cell walls were suspended in three different media (MS, PCM, CPW) and the highest yields of viable protoplasts were obtained with CPW (data not presented). The osmotic potential of the medium containing these enzymes in CPW was adjusted with mannitol, and although this adjustment was extremely important, a broad concentration range (0.3-0.6 M) was suitable for stabilizing the protoplasts (Fig. 1). Higher yields of protoplasts of maize were obtained when callus was grown on MS medium (Imbrie-Milligan and Hodges 1986),

4.5

r

"i" ">"

70

40 3.5

60

~3.0 r

2 50

2.5

.) 0

2

,,., 4

6

.~ .~ . ~ , ~ , = .~ . t , ,. 40 8 10 12 14 16 18 20 22 24 Time (h)

Fig. 3. The effect of incubation time of the callus with the enzyme solution on protoplast yield and viability in indica rice. Embryo-derived, primary callus was grown on MS medium supplemented with 2 mg/1 2,4-D and 0.4% agarose. Callus (approx. 1 g) was teased apart, placed into 10 ml of enzyme solution, pH 5.6, containing 0.4 M mannitol, and incubated for 4 h. Protoplasts were then isolated as described in Materials and methods, and viability was determined by FDA staining

and this was also true for rice (Fig. 2). Also, note that the yield of viable protoplasts was much higher when the cells were grown on agarose as compared to agar. The length of time the callus tissue was digested with the cell-wall-hydrolyzing enzymes was critical for obtaining both high yields and high viability of the protoplasts (Fig. 3). Four

328 [] v

L. Lee et al. : Regeneration of indica rice from protoplasts

100

7.0

LL

r

6.0

b

5.o

9O

80 --=

4.0

e~

C/3

.m

>

~ . 3.0 o

o

n

7O

2.0 1.0

>

5.2

5.6

6.0

6.4

6.8

7.2

o~

60

pH of Enzyme Solution

Fig. 4. The effect of initial pH of the enzyme solution oll protoplast yield and viability in 1R54 indica rice. Embryo-derived, primary callus was grown on MS medium supplemented with 2 rag/1 2,4-D and 0.4% agarose. Callus (approx. 1 g) was teased apart and placed into 10 ml of enzyme solution containing 0.4 M mannitol and incubated for 4 h. The initial pH of the enzyme solution was varied by either K O H or HC1 titration. Protoplasts were then isolated as described in Materials and methods, and viability was determined by F D A staining

hours of digestion was chosen as the standard time for subsequent experiments. We also found that the pH of the enzyme solution was very critical (Fig. 4). A slight deviation from pH 6.0 caused marked reduction in both the yield and viability of protoplasts. Taken together, these experimental conditions increased the yields of viable protoplasts from the initial values of 1.104- 5.104 protoplasts/gFW of primary callus up to about 5. 106--7.106 viable protoplasts/gFW. The protoplast viabilities in subsequent experiments was generally greater than 90%. Protoplast yields from cells grown in N 6 liquid suspensions were initially quite low. However, when the above experimental conditions were applied to several different suspension culture lines, even when grown in N 6 medium (see next section), protoplast yields varied from 1.106 to 15.10 6 viable protoplasts/gFW. These yields were sufficiently high that large-scale protoplast-culturing studies could be undertaken.

Growth and regeneration of cell suspensions. Several IR54 cell suspensions were established using callus obtained from either immature embryos (IR54-1, IR54-4, IR54-5) or mature seeds (IR54-2). The suspensions were initiated from clusters of callus that contained embryo-like structures. The growth rates of the suspension cultures were slow, with doubling times of 3-7 d for different cultures. Regeneration of plants from IR54-1 and IR54-2 suspension cultures were evaluated when the cultures were ten

months old. Both cultures contained cell clusters of several sizes ranging from less than 10 cells per cluster to more than 100 cells per cluster. These cells were highly cytoplasmic and contained small vacuoles. Clusters of 1-2 mm in diameter were selected from the suspension cultures and subcultured onto agar-solidified MS medium containing 0.5 mg/1 2,4-D in Petri plates (five calli per plate). After three weeks the calli were transferred to fresh MS medium lacking 2,4-D. About one-third of the calli cultured produced plants with an average of six plants per callus piece.

Protoplast culture. Protoplasts were obtained from the IR54-1 suspension line in high yields (using the optimized protocol for protoplast isolation from primary callus) when the cells were maintained on N6 medium or if they were subcultured into either AA or MS media for 6 d prior to protoplast isolation. The protoplast yields ranged from 1.106 to 3./06/gFW f o r cells cultured in N 6 medium, from / . / 0 6 tott5.106/gFW for cells on AA medium, and from 1.106 to 6" i06/gFW for cells on MS medium. Protoplast size ranged from 5 to 20 pm in diameter (Fig. 5 b). For different protoplast isolations, the final preparations obtained after flotation on a pad of sucrose contained either no contaminating cells (with walls) or less than 0.1% cells. Plating efficiencies for protoplasts of the IR54/ line were determined after three weeks of culture (Table 1) and in the presence or absence of different cell lines as feeders. With Calrose 76 as the feeder cells, protoplast plating efficiencies for IR54-1 ranged from 0.5 to 3.0% when the suspension cultures were of different ages, and when they had been cultured in different media for 6 d prior to protoplast isolation. The particular media and cell density at the time of the last subculture prior to protoplast isolation did not appear to make any consistent difference in plating efficiency when cells of Calrose 76 (Fig. 5 a) were the feeder (Table 1). In some experiments, the effectiveness of Calrose 76 as the nurse culture varied from flask to flask. The basis for this variation is currently being evaluated, but it does not appear to be related to rates of growth or changes in cluster sizes. When IR54-/ was cultured in AA medium, both IR54-4 and IR52 functioned initially as effective nurse cultures (plating efficiencies of 0.5%) but these nurse cultures did not sustain the growth of the IR54-1 protoplasts back into callus. In subsequent studies, however, the IR52 suspension culture (an indica genotype) was found to be capable of supporting IR54-1 protoplasts to grow into cal-

L. Lee et al. : Regeneration of indica rice from protoplasts

329

Fig. 5. a Clusters of Calrose 76 japonica-rice cell suspensions that were used for nurse cells in the protoplast culture studies. b Freshly isolated protoplasts from IR54-1 indica-rice suspension cells, e Callus obtained from protoplasts of IR54-1 grown on a filter for four weeks after transfer to LS medium (seven weeks after isolation), d Embryo-like structures on callus obtained from protoplasts of IR54-1 cell suspensions, e Protoplast-derived callus from IR54-1 cell suspensions with a shoot breaking through a coleoptile

lus and to generate plants. In separate experiments it was found that IR54-1 was ineffective as a feeder culture for its own protoplasts. It should be noted that protoplast growth back into callus of an indica genotype (IR54-1) was most effective when a japonica genotype (Calrose 76) was the feeder. These results illustrate the strict necessity for an appropriate nurse culture in order for protoplasts of the IR54-1 line to grow into callus. Calli derived from protoplasts were easily seen with the unaided eye on the filter about four weeks after isolation, and a lawn of callus was formed about seven weeks after isolation (Fig. 5c). Many

embryo-like structures were visible on the callus (Fig. 5 d). Calli containing these embryo-like structures were selected and transferred to regeneration media. Shoot-like structures appeared about two to three weeks after transferring the callus to regeneration medium and into light (Figs. 5 e, 7 a).

Regeneration of IR54-! plants from protoplast-derived-callus; importance of cytokinins. Ten (or fifteen for the MS-2 treatment) pieces of calli containing embryo-like structures (Fig. 5d) were selected and transferred to regeneration media containing different cytokinins (Fig. 6). The regenera-

330

L. Lee et al. : Regeneration of indica rice from protoplasts

Table 1. Effect of feeder (nurse) cells on the plating efficiency of rice protoplasts obtained from IR54-1 suspension cells when the culture was 7, 10, or 12 months old. Data are protoplast plating efficiency (%) Feeder cells

IR54-1 age (months)

None Catrose 76

IR54-4 IR52 IR54-5

IR54-1 suspension media 6 d prior to protoplast isolation N6_5a

AA-2

AA-0.5

MS-2

MS-0.5

7 7 10 12

0 2.5 2.6 1.4

0 0.5 0.5 1.7

0 3.0 0.9 1.6

0 3.0 0.7 1.3

0 1.3 0.8 1.3

7 7 7

Plant regeneration from indica rice (Oryza sativa L.) protoplasts.

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying pro...
2MB Sizes 0 Downloads 0 Views