Plant regeneration from protoplast derived calli in rice (Oryza sativa L.) using dicamba

Plant Science, 63 (1989) 187-- 198 Elsevier Scientific Publishers Ireland Ltd.

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PLANT REGENERATION FROM PROTOPLAST DERIVED CALLI IN RICE (ORYZA S A T I V A L.) USING DICAMBA*

B. JENES** and J. PAUK Cell and Tissue Culture Group, Wheat Breeding Departmen~ Cereal Research Institute, Szege~ P. O.B. 391 H-6701 (Hungary) (Received October 20th, 1988) (Revision received March 28th, 1989) (Accepted April 12th, 1989)

Thirty seven diploid and 7 haploid rice callus cultures were induced from the World Rice Collection maintained in Hungary. Cell suspension cultures were started from these calll in LS-2,5 liquid medium and subsequently transferred into amino acid (AA) medium. After 1 year of culturing 20 out of the 44 genotypes were found suitable for protoplast isolation. So far protoplast derived calli of genotypes have been obtained by culturing in RY-2 protoplast medium, and protoplast derived green plants of 3 genotypes have been regenerated through a two-step regeneration procedure. The protoplast derived plants are grown in pots under greenhouse conditions. Experiments are being carried out with the other genotypes developing the plant-protoplnst-plantsystem into a general method which is not dependent on the genotype. Key words: rice (Oryza sativa L.); cell suspension; protoplnst isolation and culture; plant regeneration

Introduction • To increase the efficiency of plant breeding is the most important task for a breeder. Nowadays the results of plant biotechnology seem to be promising to increase the plant breeding efficiency, integrating the latest achievments of cell and tissue culture into the traditional breeding methods. Embryo rescue culture, micropropagation or in vitro androgenesis can support effectually the plant breeders in spite of the lack of direct genetic manipulation. Most of the genetic transformation methods require a reproducible cell-plant system. The direct DNA uptake in the isolated protoplasts is an established

*Research work supported by OMFB, Budapest No. 132941 88. **On leave from Agricultural Biotechnology Center, G6d611~,Hungary, H-2101. -'~ Abbreviations: NAA, naphthylaeetic acid; LS, LinsmaierSkoog; Dicamba, 3,6-diehloro-o-anisicacid; 2,4-D, 2,4-dichloropbenoxyacetic acid; IAA, indol-3-acetic acid; BA, 6benzylaminopurine;AA, amino acid.

method of plant cell genetic transformation

[1,2]. In monocots the significant advance was marked by the team of Vasfl. Plants have been regenerated from protoplasts isolated from embryogenic cell suspensions of Pennisetum americanum, Pennisetum purpureum, Panicum mazimum and Saccharum officinarum [ 3 - 6]. Fujimura's research group in 1985 was the first [7] to successfully develop the protoplastplant system in rice using two Japanese genotypes. In 1986 Yamada et al. [8] emphasized the importance of calf-serum in rice protoplast culture medium and for the first time his group worked on a large variety scale (25 cultivars). After some preliminary results achieved by Coulibaly and Demarly [9], Thompson et al. [10] and Abdullah et al. [11] reported complete rice suspension culture and protoplast isolation procedure. Japanese researchers obtained over 100 regenerants from rice protoplasts [12,13] in four Japanese cultivars and attempted to apply this method for developing new variants through somaclonal (protoclonal) variation.

0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Great efforts have been made in many laboratories with other species of the Crraminea and considerable progress has recently been reported in other important cereals. In maize the first protoplast-derived plant was regenerated from embryogenic suspension culture [14] followed by successful genetic transformation experiments [15]. The protoplast originated plantlet of wheat was obtained from anther-derived suspension cultures of a commercial wheat variety [16]. In Triticale, protoplast-derived cell suspension and callus culture was obtained from isolated protoplasts [17]. In barley albino plantlets have recently been regenerated from protoplasts isolated from morphogenic cell suspension cultures [18]. According to the above data the protoplastplant system is going to be solved in the most important cereal species in the near future. Despite the results achieved in the last years, there is no method as yet which is not dependent strongly on the genotype. This paper summarizes our efforts to extend the genotype background of protoplast work on new Hungarian lines and other cultivars from abroad, which had never been investigated before in published studies. Materials and Methods

Plant material Forty four genotypes of rice (O~yza sativa L.) were obtained from the World Collection of Rice Cultivars of the Research Institute for Irrigation, Szarvas, Hungary. These 44 genotypes are listed according to the classification of types in Table I. Donor tissues were collected from the rice nursery in August and September 1987. Donor plants were kept under Hungarian agronomical and climatic conditions. Callus induction and cell cultures Immature seeds of 37 rice genotypes were surface-sterilized using 20/0 NaOCI with Tween 80 for 20 min followed by rinsing three times in sterile distilled water.

Table I.

Rice genotypes used in the investigations.

Code No.

Variety or line

Origin (country)

China 1039 LA 110 SR 7466-18-1-2-3-4 AT-77-1 KK 9-6~ RP 1897-3790,~0-1 JKU/K/449-U8-2 LKU /K/ 450-172-10 Kashmir Basmati M-9

India USA Korea Sri Lanka Nepal India India India Pakistan USA

H-250 H-254 H-257 H-258 H-592 H-616 H~46 H-729 H-732 H-777 H~76 H-937 H-942 H]~7 HB-42 HB-44 HB-45 HB-46 IR 346 C11711 Volano Reimei Wasetoramachi 1 Stejaree 45 Cheolweon 31 Wasetoramachi 2 Partos 308 Miara

Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Hungary Korea China Italy Japan Japan USSR Korea Japan Rumania Italy

Indica t~,pes 21" 46 50 132 155 159 292 295 301 317

Japonica types 250 254 257 258 592 616 646 729 • 732• 777" 876" 937 942 B-7 B-42 B-44 t3-45 B-46 66 71 99 287 290 304 311 712 908* 918'

Unclassified types 74 875 970 981 1000b 1001 b

IR 1552 Anthocian hull Red rice i Red rice 2 A 8315 A 7927

IRRI Rumania Hungary Hungary China China

*Cultured on haploid level. bRice X Sorghum hybrides from Chen Shanbao et al. [27,28].

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Caryopses were shaken during sterilization. Immature embryos 1 2 - 1 4 days old were aseptically removed. These structures were cultured on LS-2.5 agar medium [10]. In most of the genotypes embryogenic and compact caUi developed from the dissected embryos within 3 weeks. Haploid calli of 7 genotypes were induced from uninucleated microspores on J-19 medium [19], in anther culture [20] after 10 days of cold treatment at 10°C. In 6 weeks microsporederived embryoids appeared on the surface of the anthers. Half of the embryoids were used for haploid plant regeneration, the other half was used for establishing cell suspensions. The 6-week-old, rapidly growing diploid and haploid calli were transferred into liquid LS 2.5 medium at 1:5 (inoculum/fresh medium) ratio in 100 ml Erlenmeyer flasks on gyratory shaker at 120 rev./min subcultured in 3-day intervals, because of the gell phase produced by the senescent cells. After 6 - 1 0 weeks, depending on the genotype, the suspensions consisted of large aggregates with diameter of 1 --2 mm and some small clusters containing 200--300 cells. These smaller aggregates were placed into amino acid (AA) liquid medium [10]. In another 6 weeks in the suspension aggregates consisting of 2 0 - 6 0 cells appeared which were found to be suitable for protoplast isolation. The plant regeneration capacity of the suspension cultures were tested on MSD 4 agar medium [10] with modified hormone components (without NAA) and the regenerants were grown on N ~ medium [ 2 1 ] without hormone. Plating efficiency of cell suspensions was calculated by counting colonies plated on MSD 4 agar medium growing on several square centimeters of one Petri dish after 3 weeks using a stereo microscope.

Protoplast isolation from suspension One gram inoculum from the suspension was placed in a 5-cm diameter Petri dish in 1 ml AA medium and 1 ml of the enzyme solution was added. Protoplasts were released using the following enzyme mixture after Dudits' personal communication: Onozuka RS Cellulase

6%, Pectinase 2%, Driselase 2%, MES 585 mg/1, N a H ~ O , 100 mg/l, CaCIs X 2H20 1.0 g/l, mannitol 0~SM, ~sorbitol 0.35 M at pH 5.6. Protoplasts were released during 3--3.5-h digestion at 28 °C on gyratory shaker at 30 rev./ rain. The released protoplasts were washed three times in YM washing solution [8] and centrifuged at 1000 rev./min for 5 rain after each washing. The protoplast suspension was sieved through a 40 ~m stainless steel mesh to remove debris.

Culture of trrotoplasts and plant regeneration The isolated protoplasts were cultured at a density of 5 x 105/ml in 4 ml RY-2 liquid medium [8] and in agarose of the same medium [22] at 0.4 M, 0.425 M, 0.45 M, 0.5 M osmotic concentration with Sigma Glucose at 28°C in darkness for 10 days. The 0.6% agarose bed was adjusted e.g. by mixing double concentration (1.2O/o) Serva agarose medium (0.4 M) with 0.45 M liquid medium at ratio 1 : 1. After the first 1 - 2 weeks some drops of AA medium were added to the protoplasts into the liquid cultures. The protoplast derived small clusters embedded in agarose blocks were floated in 2 ml AA medium at 26 °C, exposed to 16/8 h dark/light periods on a gyratory shaker at low speed {30 rev./min) for about 30 days. After the 40th day the protoplast derived clusters, 1--2 mm in size, were plated on modified LS medium complemented with 2 mg/l Serva Dicamba (LS-2Di medium) containing 30/0 sucrose for 18--20 days. The yellow-brown compact and globular-structured protoplast derived calli, 4--5 mm in diameter, were transferred onto the modified R 3 and R 7 wheat regeneration media [23] containing 2 mg/l BA, 2 mg/l IAA (both) and 5 g/l myo-inositol (R3), 2 g/1 myc~inositol, casein hydrolysate 1 mg/l. Prolin 10 mg/l (in R7). The cultures were kept under light of 2000 lux. After a 3 - 4 - w e e k period shoot primordia 2 - 3 mm long without any roots were visible on the surface of protoplast-derived calli. These calli with shoots of about I cm were transferred onto N-6 medium complemented with 2 mg/l IAA to facilitate regeneration of normal roots.

190

Fig. 1. Developmentof rice suspension culture after 4 months of culturing: (A) rough suspension of 258 genotype in early stage; (B)mediumsize aggregates of 777 genotype;(C)fine and well growingsuspensionof BA2genotype.

After 2 weeks the well developed plantlets with roots were transferred into 250-m1 Erlenmeyer flasks on N-6 medium without hormones. The healthy and green plants were placed into pots and kept in the greenhouse for acclimatization (covered by PVC bag) and growing. Results

Development of suspension cultures Dissected immature rice embryos of indica and japonica types and anthers containing rice microspores of both types were used as inoculures for inducing diploid and haploid callus cultures. The somatic derived calli proliferated well on LS 2.5 agar medium. The anther culture derived calli were obtained on J-19 agar medium and subcultured in 2--3-week intervals. After starting suspension cultures from these calli in liquid LS 2.5 medium

differences were observed among the genotypes in the time needed to develop a finely dispersed cell suspension culture containing small cell clusters. After 1-year experiments 20 japonica type rice genotypes were found to be suitable for culturing in fine cell suspension as a source for releasing protoplasts. Among these japonica genotypes the shortest time was 3 months from callus induction to the first protoplast isolation from cell suspension of the Hungarian line 'B-42'. The stages of the development of suspension cultures after 3 months of subculturing can be seen on Fig. 1. The longest time needed to get optimal fine suspension was 12 months with Wasetoramachi, a Japanese cultivar among the 20 selected cultivars (Table V). After 12 months, 24 genotypes could only produce rough suspension consisting of aggregates with minimum 2 mm diameter. To produce a fine suspension the optimal

0

0

D

0

i

t~

193

interval of subculturing was 3 - 4 days in LS 2.5 medium and 7 days in AA medium. After transferring the clusters from LS 2.5 into AA medium the suspensions contained small, 3 0 100 cells, aggregates. Regeneration ability of the suspension cultures was checked monthly and the plantlets were grown on N ~ medium. The regeneration ability decreased during the development of suspension culture, however a low but stable ability was retained by most of the fine suspensions. About 1--2% of the plated suspensionderived clusters could regenerate green plants. Table II summarizes the regeneration ability of the 11 -- 12-month-old suspension cultures. Haploid cultures, 729, 732, 777, 876, 908, 918 japonica rice genotypes, formed fine cell suspension in a shorter time ( 4 - 5 months) than most of the diploid cultures which needed generally 5 - 6 months.

Protoplast isolation During the experiments indica and unclassified type rice suspension proved to be unsuitable for efficient protoplast isolation, that is why the successful protoplast trials were carried out only in japonica types. Protoplasts were released by cell wall digestion in an enzyme mixture (Fig. 2). Digestion time of 3.5 h was optimal for obtaining a considerable amount of round shaped cytoplasmic protoplasts. In case of a shorter digestion time the number of released protoplasts was low, a Figs. 2--9.

longer digestion was harmful to many of the protoplasts because of the damage of cell membrane. During the isolation procedure the speed of centrifugation was raised up to 1800 rev./min without causing any damage of the protoplasts. When the speed was increased to 1800 rev./ rain, the centrifugation time was shortened to 2 min. The effect of suspension culture medium on the amount of released protoplasts was demonstrated by digestion of clusters cultured in LS 2.5 and AA media in the enzyme mixture. During the same incubation time (3.5 h) there were five times more protoplasts released from the suspension cultured in AA as compared to those cultured in LS 2.5. Isolated protoplasts were free of cell walls which was indicated by the observation that they readily fused with each other and abnormally huge protoplasts (arrow on Fig. 2) fusion products were visible among the normal ones.

Protoplast culture At the end of the isolation procedure the protoplasts were resuspended in RY-2 culturing medium parallel in liquid culture as well as embedded in agarose without nursing. The first cell divisions occurred 4 - 7 days after the isolation in both liquid culture and agarose, although in some cases, e.g. 908 genotype, first divisions were visible already on the second day (Fig. 3). Comparing the two methods for the

Protoplast-plant system in rice.

F i g .2. Freshly released protoplasts of B-42 genotype in the enzyme mixture. Spontaneous fusion product can be seen (arrow). Fig. 3. The first divisions of 777 genotype cultured in RY-2 medium on the 5th day after the isolation. Fig. 4. Protoplast-derived clumps of B-42 genotype on the 14th day following the isolation. Fig. 5.

Protoplast-derived colonies of 258 genotype embedded in agarose blocks on the 40th day after the isolation.

Fig. 6. Regeneration of shoot primordia on the globular-structured protoplast-derived callus of B-42 genotype on R7 medium. Fig. 7. Root regeneration on protoplast-derived callus and shoot primordium of B-42 genotype on N-6 medium complemented with 2 mg/1 IAA. Fig. 8.

Protoplast-derived plantlet of B-42 genotype on N-6 medium without hormones.

Fig. 9.

Protoplast-derived plants of B-42 genotype in pots under greenhouse condition.

11 11 11 12 12 12 12 12

B-42 B-45 II-46 258 732’ 777’ 376’ 908’

Qilture of haploid origin.

Age of suspension (month)

32 20 11 14 36 32 38 21

Liquid 91 31 29 33 88 76 82 68

Agarose

Colony formation after 5 weeks f%)

100 200 106 106 100 166 106 100

No. of colonies transferred on different medium 75 35 54 33 71 63 0 0

Morphogen structure on LS-2Di 50 33 46 61 12 20 0 0

Green spots on R.

% of colonies with

15 2 4 5 0 0 0 0

Shoot regeneration

7 0 2 3 0 0 0 0

Root reg. after shoot reg.

Characterization of protoplast culture and plant regeneration from protoplast derived callus of rice (Oryzo sat&z L.).

Genotype

Table III.

0 0 5 5 0 0 0 0

Plantlet

7 0 0 1 0 0 0 0

Plant

No. of regener.

195 Treble IV.

Development of protoplast-derived cal]i on different culture media.

No. of calli transferred onto

% of calli formed morphogen struct, on

No. of plants regenerated from ealli grew on

LS-2.5

LS-2Di

LS-2.5

LS-2.5

LS-2Di

B-42

100

100

5

75

0

7

258

100

100

2

33

0

1

Genotype

efficiency of colony formation, the agarose-bead method proved to be three times more effective than the liquid medium method in case of most genotypes (Table III). The best osmotic effect was obtained at 1 : 1 mixture of 0.40 M and 0.45 M osmolarity of RY-2 culture media in agarose and at 0.45 M in liquid culture. The efficiency of colony formation (Figs. 4 and 5) in protoplasts isolated from various genotypes was different. So far four diploid, B42, B-45, B46, 258, and four haploid, 732, 777, 876, 908, genotypes were set into protoplast isolation trials and the efficiency of colony formation of the haploid lines was twice as high as that of diploids.

Plant regeneration from protoplast-derived callus Protoplast-derived caUi 4 0 - 5 0 days old 1 - 2 mm in size were put on pre-culture medium for regeneration on LS-2Di. After 3 weeks the calli cultured on LS-2Di medium were well growing with better efficiency than on the formerly used pre-culture medium LS-2.5. For example in case of B-42 75% of the calli were globular, compact and brown-yeilowish. Upon transferring these calli onto R3 and R7 media, green spots appeared on the surface of the globular structured, compact calli grew on LS-2Di in 10--20 days, but embryogenic structures were not visible on most of the calli grown on LS-2.5 (Table IV). In another 10 days green spots appeared on the surface of 50% of the calli (Fig. 6.) and 20% of these calli regenerated shoot primordia without any roots (Table III). All the regeneration events occurred without root regeneration (Fig. 7). In the next step the calli with shoots were placed onto N-6 medium containing 2 mg/1 IAA for regeneration of roots which followed in 10 days (Fig. 8).

LS-2Di

From B-42 there are 7 green plants in pots which are flowering (Fig. 9). Six of them have a normal phenotype, 1 plant has a different phenotype from the original one. One regenerated green plant and 5 plantlets of 258 genotype, and 5 plantlets of B-46 genotype have been obtained so far (Table V). Discussion In our trials we succeeded in regenerating and growing intact rice plants from suspensionderived protoplasts of japonica rice, similarly to the results reported previously in the literature [7,8,11,13,24]. The present study demonstrated that cell suspension technique, protoplast culture and plant regeneration methods can be extended to several different genotypes of rice (Oryza sativa L.). The establishment of suspension cultures from 44 genotypes following the procedure described in the literature was successful similar to Toriyama and Hinata [25], but we found considerable differences between the genotypes in the time needed to develop a fine cell suspension mainly between the japonica and the other type rices. We could produce finely dispersed cell suspension culture suitable for protoplast work only from japonica type rice but we failed to do it from the indica and unclassified types (Table V.). The best result was obtained in the case of the Hungarian japonica genotype B42. In order to use this method for breeding, further modifications are necessary. In our experiments we observed that some of the genotypes, B-42, 777, B-46, formed fine suspension cultures in both LS-2.5 and AA liquid media but most of the genotypes formed fine suspension only in AA medium. We used AA liquid medium for rice cell culturing in contrast

196

T a b l e V. culturing. Genotype

Six s t a g e s in t h e d e v e l o p m e n t of s u s p e n s i o n a n d p r o t o p l a s t culture in 44 rice g e n o t y p e s a f t e r 12 m o n t h s in vitro

(1)

(2)

(3)

(4)

(5)

(6)

Rough susp.

Fine susp.

Isolated protoplast

Protoplastderived caHus

Protoplastderived plantlet

Protoplastderived plant

1000" 21 b B-7c 257" 46 b 50 b 71 c 74" 9~ 132 b 165 b 159 b

292 b 295 b 287' 304 c 317 b 59~ 616 c 646 c 712` 875" 937 • 942" 981" 301 b 29~ 918 ~ 729• 311': 1001" 9"/0" 250. B.44 ° 254 ~

66 • 732"

777 ° 876 c 908 • BA5. B-46c 258 c B-42" "Unclassified t y p e rice. bIndica t y p e rice. cJaponica t y p e rice.

197

with the methods described, wheat [16], maize [14] and barley [18] cell suspension and protoplast cultures. The use of amino acid-based medium enhanced the establishment of fine cell suspension cultures in rice and has been found to be suitable for rice cell culturing as it was reported in several studies [10,11,24,25]. Appearance of cell colonies in suspension culture medium varied according to the culture media. In our trials similarly to the experiments of Toriyama and Hinata [25] the characteristic features of cells in suspension appeared to vary depending on the nitrogen source of the culture media. For this reason the inoculum obtained from suspension cultured in AA medium yielded five times more protoplasts than that cultured in LS-2.5 medium. In the enzyme mixture used for protoplast isolation the Onozuka RS Cellulase enzyme (Yakult Pharmaceutical Ltd. Japan) seems to be indispensable to release good quality protoplasts with high efficiency as it was also used in other author's experiments [7,8,10-12], although some researchers succeeded in protoplast isolation and plant regeneration without using RS Cellulase enzyme. The agarose bead technique was found to be beneficial for rice protoplast culture. A similar observation was made by Thompson et al. [1012]. This method was also useful for barley (Hordeum vulgate L.) protoplast culture [18]. Nevertheless, in our trials there was no difference in the time of the first division and in the development of protoplast-derived clusters during the first 14 days between the agaroseembedded cultures and the liquid cultures. After the 14th day the growth rate of protoplast~lerived clusters decreased in liquid culture. Plant regeneration from protoplast-derived suspension was carried out in two steps. In the first steps morphogenic-structured calli were obtained from the suspension plated on agar LS-2Di medium. Dicamba hormone was used for plant regeneration from rice suspension culture by Zimny and L6rz [26] and from barley

protoplast [18]. This is the first reported case of plant regeneration from rice (Oryza sativa L.) protoplasts with the application of Dicamba as auxin in the regeneration method. Green plantlets were obtained from 20% of the morphogenic-structured calli produced by the use of Dicamba in the second step. In this study we worked with both diploid and haploid cell cultures. The haploid protoplasts seem to be more valuable for somatic cell genetics such as DNA transfer, protoplast fusion in comparison with the diploids because the expression of incorporated genes could be easily detected in the haploid state [24]. We could produce diploid protoplast-derived plants from japonica type rice, but could not produce regenerated plants from protoplasts of indica and unclassified type rice because of the lack of suitable cell suspension. So far we could not regenerate plants from protoplasts of haploid cell culture. The time of progress is considerable in these experiments. Seven months after the initiation of this program the first protoplast-derived calli of B-42 were cultured and after <10 months the first regenerated green plant of B42 was growing in flask. Subsequently plantlets and plants were regenerated from two other genotypes (258, B-46). We are ready to offer these genotypes to other research groups if requested, since there are very few well regenerating genotypes reported in the literature which are suitable for genetic transformation or somatic hybridization. Further experiments are in progress according to Table V to elucidate the protoplast-plant regeneration system of all the 44 genotypes. We hope to increase the number of genotypes from which plants can be regenerated by means of the protoplast technique. Experiments are in progress using the described methods for genetic transformation through treatment of protoplasts with vector DNA. Acknowledgements The authors are grateful to Z. Barabas for

198

supplying the conditions in the Tissue Culture Laboratory of Wheat Breeding Department, I.K. Simon for providing the authors with plant material and for her useful advice, D. Dudits for his valuable advice, Mrs. A. Fej~rv~ri, Mrs. I. BartOk, Mrs. I. Pusztai and Mrs. J. Mesterh~zy for technical support and G. Tak~es for photographic assistance.

14

15

16

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