Plant Cell Reports

Plant Cell Reports (1986) 5:89-92

© Springer-Verlag1986

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice) J a n u s z Z i m n y 1 and H o r s t L 6 r z 2 BotanicalGarden of the Polish Academy of Sciences, Warsaw, Poland 2 Max-Planck-Institut ft~rZt~chtungsforschung, D-5000 KOln 30, FRG Received December 24, 1985 / Revised version received February 17, 1986 - Communicated by I. Potrykus

ABSTRACT Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At i and 4 mg/l Dicamba and i mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation. ABBREVIAT

IONS

BM Basic medium 2.4 -D 2,4-dichlorophenoxyacetic acid Dicamba 3,6-dichloro-2-methoxy benzoic acid Picloram 4-amino-3,5,6-trichloropicolinic acid INTRODUCTION Plant regeneration from protoplasts is considered to be an important aspect for genetic manipulation of plants (L6rz et al. 1985). The material to be used should be morphogenic, allowing plant regeneration from transformed protoplasts. For this reason we are interested in cultures of meristematically active tissue. There exist two ways of in vitro plant regeneration: somatic embryogenesis and organogenesis, both of which are observed in the Gramineae. It has been shown possible to initiate somatic embryogenesis from irmmature and mature rice embryos (Heyser et al. 1983) and young leaves (Bhattacharya and Sen 1980; Wernicke et al. 1981) and plant regeneration by organogenesis has been described for several tissues and organs (reviewed by Dale 1983). Morphogenesis has been obtained also in callus cultures derived from seminal roots (Kawata and Ishihara 1968) and in callus derived from the "place of root system" (Nishi et al. 1968). In both cases, however, regeneration of plants was a rare event. So far, however, no efficient and reliable protoplast to plant regeneration system for any cereal species has been developed (reviewed by Ozias-Akins and L6rz 1984). The aim of this paper is to present a method for the initiation of rice root tip derived suspended

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to: H. L6rz

callus and the subsequent regeneration of plants from this tissue. These explants provide an easily accessible and well defined source of meristematic tissue and a good source of protoplasts with a possible capacity for plant regeneration. Cell suspension cultures obtained from the callus mentioned above are also described. MATERIALS

AND

METHODS

Callus initiation Kernels of japonica type rice, cv. Thaipei, were surface sterilized in 70% ethanol for i min and i% Na0CI for 20 min, then washed 3 times in sterile water. They were germinated in sterile water in 125 ml Erlenmeyer flasks (27°C, dark). Root tips (5-10 mm) were excised from 10 day old seedlings (Fig. la) and cultivated in BM (basic medium = Murashige-Skoog medium, 1962, thiamine 0.4 mg/l) supplemented with 2 mg/l 2.4-D. Erlenmeyer flasks (125 ml) containing ten root tips were shaken at 140 rpm and maintained at 27°C in darkness. Callus proliferation became visible between i0 and 30 days after culture initiation. The first subcultures were made after 4-5 weeks and thereafter every 10 days replacing one third of original 15 ml culture medium by fresh medium. Organogenesis and plant regeneration After 7, 16 and 24 weeks, calli derived from suspended root tip cultures were used in plant regeneration experiments. These were carried out by transferring callus pieces (2-3 mm) onto solid BM (0.7% Merck agar) containing 2.4-D, Dicamba or Pieloram (Table l).In the first experiments callus was cultured on the auxin containing medium for 7 weeks and thereafter transferred to regeneration medium (BM + i mg/l kinetin + 0.7% agar). In the third set of experiments after 10 days on the auxin medium containing i mg/l Picloram, calli which exhibited embryogenic structures were transferred to BM with 0.1 mg/l 2.4-D and 12 days later onto hormone-free BM. For organogenesis and regeneration, cultures were kept at 27°C and 16 h light period (ca. 200 lux, General Electric F96PG/7-power groove cool white). Regenerated plantlets were cultured in glass vessels on solid half-strength BM until they were 15 cm high. Afterwards plantlets were potted in a mixture of peat and sand and kept for the first seven days under high humidity by covering with plastic foil. They were then cultivated in the green-house at 26°C,

90 i l l u m i n a t e d for 12 h in a r t i f i c i a l light (Osram HQI-T, 400 W / D H P O W E R STARS) and grown to maturity. Cell s u s p e n s i o n

culture

For i n i t i a t i o n of cell suspensions, 5 m o n t h o l d root tip d e r i v e d cultures w e r e used. 15 ml s u s p e n s i o n w e r e k e p t in 125 ml E r l e n m e y e r flasks (BM + 2 m g / l 2.4-D) on a g y r a t o r y shaker (140 rpm, 27°C, dark) and subcult u r e d every 4 days by adding 5 ml fresh m e d i u m and s u b s e q u e n t l y r e m o v i n g 5 m l of supernatant. This p r o c e dure gave rise, w i t h i n 2 months, to a s u s p e n s i o n cont a i n i n g callus clumps and small cell c l u s t e r s of 5-10 cells. M e c h a n i c a l s e p a r a t i o n b y s i e v i n g was a p p l i e d several times to o b t a i n a m o r e h o m o g e n e o u s suspension. O n l y the f r a c t i o n p a s s i n g t h r o u g h a 250 Zm stainless steel sieve a n d r e t a i n e d by a 50 ~m sieve was subseq u e n t l y s u b e u l t u r e d in small v o l u m e s (5 m l in 25 m l E r l e n m e y e r flasks or 2 m l in m u l t i w e l l plates, Greinet no. 657102). These w e r e s u b c u l t u r e d every second day u s i n g B M w i t h 2 mg/1 2.4-D. RESULTS Callus

induction

and p r o l i f e r a t i o n

E m b r y o - l i k e structures w e r e p r o d u c e d w h e n 24 w e e k s o l d callus was c u l t u r e d for i0 days on s o l i d i f i e d BM w i t h i mg/1 Pieloram. It was a dry, c o m p a c t callus w i t h small c a v i t i e s t y p i c a l for early stage e m b r y o g e nesis. Some g r e e n spots w e r e v i s i b l e in these calli. The n u m b e r of these i n c r e a s e d after t r a n s f e r to BM w i t h 0.I mg/1 2.4-D though no c o m p l e t e shoot f o r m a t i o n was o b s e r v e d f r o m t h e s e ' s t r u c t u r e s . C u l t u r e s r e m a i n e d for i0 days on this medium. A f t e r w a r d s they w e r e t r a n s f e r r e d to m e d i u m c o n t a i n i n g i mg/l kinetin. Under these c o n d i t i o n s c o l e o p t i l e s w e r e formed, T h e y b e c a m e green, except for t h e i r skirts w h i c h were w h i t e and showed a t e n d e n c y to form callus. On m e d i u m w i t h o u t hormones, roots a n d g r e e n shoots d e v e l o p e d from the eoleoptile base leading to g r o u p s of 2-5 plantlets. P l a n t l e t s w e r e s e p a r a t e d and g r o w n further in glass vessels. Twelve of those p l a n t s w e r e g r o w n to m a t u r i ty and set seeds (Fig. ig). F r o m 7 and 16 weeks o l d s u s p e n d e d calli, c u l t u r e d i n i t i a l l y for 7 weeks on solid m e d i u m c o n t a i n i n g 7 mg/l Dicamba, w h i t e e m b r y o g e n i c callus was obtained. It was t r a n s f e r r e d onto BM w i t h 0.i mg/1 2.4-D to p r o m o t e embryo development. C o m p l e t e embryo formation, however, w a s n o t observed, b u t p l a n t l e t s showing a normal root s y s t e m d i f f e r e n t i a t e d f r o m these ealli after t r a n s f e r on BM c o n t a i n i n g i mg/l kinetin. The first leaves of these p l a n t l e t s were slightly green, though, the s u b s e q u e n t leaves w e r e white. T h e s e albino p l a n t s r e a c h e d a h e i g h t of a p p r o x i m a t e l y 15 cm b e f o r e dying.

R o o t tip m e r i s t e m s a n d root lateral m e r i s t e m s p r o d u c ed callus w i t h i n 2 w e e k s of culture in liquid B M supp l e m e n t e d w i t h 2 m g / l 2.4-D (Fig. ib). The r e s t of the root tissue d e c a y e d and did n o t show any p r o l i f e ration. W i t h i n 5 weeks o~ g r o w t h an average 4 g of callus (average size 2-7 m/n diameter) w a s o b t a i n e d from ten r o o t tips. These calli f r e q u e n t l y p r o d u c e d r o o t - l i k e structures. Whitish, n o d u l a r structures dev e l o p e d from callus which o t h e r w i s e t u r n e d m o s t l y b r o w n after some weeks in culture. A f t e r m e c h a n i c a l separation the n o d u l a r callus was u s e d for i n d u c t i o n of m o r p h o g e n e s i s .

The m o s t s u i t a b l e h o r m o n a l s u p p l e m e n t of the m e d i u m appears to be i and 4 m g / l D i c a m b a for 7 w e e k s o l d callus f o l l o w e d b y i mg/1 k i n e t i n w h i l s t for 24 weeks o l d callus i mg/l P i c l o r a m f o l l o w e d by 0.i mg/1 2.4-D and i m g / 1 k i n e t i n was optimal. Using o t h e r c o m b i n a tions of auxins m e n t i o n e d in Table i it was p o s s i b l e to g r o w callus, b u t this tissue was u n a b l e to d i f f e r e n t i a t e into shoots or roots.

Plant r e g e n e r a t i o n

Cell s u s p e n s i o n

Seven and 16 weeks after culture i n i t i a t i o n callus was t r a n s f e r r e d onto solid B M c o n t a i n i n g d i f f e r e n t auxins (Table i). D u r i n g the culture these n o d u l a r calli often f o r m e d roots and t u r n e d brown. However, some of these calli r e m a i n e d light a n d o c c a s i o n a l l y showed green spots, w h i l s t some parts of the tissue showed a h i g h l y s t r u c t u r e d surface. This type of tissue was p l a t e d on B M c o n t a i n i n g i mg/l kinetin. A f t e r one w e e k w h i t i s h e o l e o p t i l e s d e v e l o p e d w h i c h later b e came green. F r o m the b a s e of these structures normal shoots a n d roots d e v e l o p e d (Fig. id,e,f).

Callus m a s s e s s u b c u l t u r e d every fourth day in liquid m e d i u m contained, after 2 months, a m i x t u r e of m e r i stematic clumps, callus pieces, and n o n - d i v i d i n g single cells, w h i c h u s u a l l y decayed. C e l l clusters, 50250 ~m in d i a m e t e r , w e r e m e c h a n i c a l l y s e p a r a t e d and c u l t u r e d in E r l e n m e y e r flasks on a g y r a t o r y shaker. As t h e y b e c a m e larger it was impossible to m a i n t a i n a h o mogeneous suspension. The s u s p e n s i o n s always c o n t a i n e d cell c l u s t e r s and p i e c e s of callus f r o m about 50 z m to 4 mm. However, w h e n a m e c h a n i c a l l y s e p a r a t e d fraction, 50-250 ~m, was c u l t u r e d for two w e e k s in m u l t i w e l l p l a t e s the cell culture produced highly cytoplasmic single cells and small g r o u p s of cells (Fig. 2).

TABLE

i: E f f e c t of auxins

on the i n d u c t i o n

culture

of m o r p h o g e n e s i s

........................ ................................... Time of 2.4-D mg/l D I C A M B A mg/l P I C L O R A M mg/1 root-tip culture 1 2 1 4 7 1 2 3 ............................................................ 7 weeks c c P P Pa ........................................................... 16 weeks c c c e Pa ........................................................... 24 weeks c c c e c P c c ...........................................................

c

not tested callus g r o w t h o n l y

P Pa

plant regeneration albino p l a n t s o n l y

9!

FIG.

i: Callus i n i t i a t i o n and p l a n t r e g e n e r a t i o n f r o m suspended root tip cultures a) Ten day o l d a s e p t i c a l l y g r o w n rice seedlings used for the i s o l a t i o n of root tips. b) Callus p r o l i f e r a t i o n 3 w e e k s a f t e r culture initiation. c) N o d u l a r structures and early stages of shoot d i f f e r e n t i a t i o n on callus c u l t u r e d for 7 w e e k s on s o l i d i f i e d BM w i t h 7 mg/1 D i c a m b a and for 2 weeks on BM c o n t a i n i n g I mg/l kinetin. d) D e v e l o p m e n t of c o l e o p t i l e s after 8 w e e k s of culture on s o l i d i f i e d a u x i n m e d i u m f o l l o w e d b y 2 w e e k s culture on k i n e t i n c o n t a i n i n g medium. e) Shoot regeneration. f) Root and shoot development. g) Fertile, r o o t - t i p culture d e r i v e d rice plant, cv. Thaipei.

The cells and cell c l u s t e r s d i v i d e d g i v i n g rise to m u c h more h o m o g e n e o u s suspensions. To m a i n t a i n such suspensions it was i m p o r t a n t to subculture regularily, otherwise the s u s p e n s i o n b e c a m e very dense and some a g g r e g a t e s f o r m e d large callus pieces. Calli b i g g e r than 250 b m were s e p a r a t e d m e c h a n i c a l l y and u s e d for r e g e n e r a t i o n experiments. Shoot and root formation from these calli was p o s s i b l e and is p r e s e n t l y b e i n g studied in more detail. P r e l i m i n a r y e x p e r i m e n t s also

indicate that viable p r o t o p l a s t s can be i s o l a t e d rep r o d u c i b l y from these suspensions. DISCUSSION In this study we have r e g e n e r a t e d rice p l a n t s f r o m root tips via suspended callus d e m o n s t r a t i n g the tot i p o t e n c y of the tissue u s e d as explant. It was observed that the callus m u l t i p l i c a t i o n rate in liquid m e d i u m was faster than on solid medium. Root tips of

92

FIG. 2: Cell suspension culture a) Parabolic, highly cytoplasmic cells separating from callus clumps during initial stage of suspension culture. b,c) Cell division of single cells in an established suspension.

rice seem to have particular advantages in callus formation and morphogenetic capacity as demonstrated by the fact that repetition of this procedure when applied to rye (Secale cereale) or barley (Hordeum vulgare) has not so far been successful. Regeneration of albino plants appears to be dependent on the level of auxins in the regeneration medium. Albinos were obtained in two independent experiments namely when the induction medium contained a high concentration (7 mg /i) of Dicamba. Nishi et al. (1968) reported regeneration of plants after transferring primary callus from 2.4-D containing medium to one that was hormone free. In our study it was necessary to transfer the callus to auxin free but kinetin containing medium. However, in one set of experiments (24 weeks old callus cultured on solidified auxin media) kinetin inhibited plant development from the coleoptile stage onwards. For complete plant development hormone free medium was necessary. Somatic embryogenesis in Gramineae is commonly reported in the literature to arise from meristematically active tissue such as immature embryos and leaf bases. Although early stages of somatic embryogenesis have been observed, no complete plant regeneration via somatic embryogenesis was achieved. The fact that plant regeneration by shoot/root morphogenesis is possible, makes the material described above an alternative source of tissue for the establishment of cell suspensions and the isolation of potentially totipotent protoplasts. Previously immature embryos have been used to initiate cell suspensions which have been used subsequently to isolate viable and dividing protoplasts of rice (G~bel et al. 1985).

ACKNOWLEDGEMENTS One of us (J.Z.) gratefully acknowledges the rec@ipt of a Research Fellowship from Deutscher Akademischer Austausch-Dienst, Bonn, Fed. Rep. of Germany. We also thank our colleagues Elke G@bel and Peter Brown for discussions, criticism and encouragement.

REFERENCES Bhattacharya P, Sen SK (1980) Theor Appl Genet 58: 87-90 Dale P (1983) In: Potrykus I e t al (eds) Protoplasts 1983. Birkh~user, Basel, pp 31-41 G6bel E, Ozias-Akins P, LSrz H (1985) In: Aldersen PG, Withers LA (eds) Plant Tissue Culture and Its Agricultural Applications. Butterworth, Kent, pp 359-365 Beyser J ~ Dykes TA, DeMott KJ, Nabors MW (1983) Plant Sci Lett 29: 175-182 Kawata S, Ishihara A (1968) Proc Japan Acad 44: 549553 LSrz H, Baker B, Schell J (1985) Mol Gen Genet 199: 178-182 Murashige T, Skoog T (1962) Physiol Plant 15: 473497 Nishi T, Yamada Y, Takahashi E (1968) Nature 219: 508-509 Ozias-Akins P, L~rz H (1984) Trends in Biotechnology 2: 119-123 Wernicke W, Brettell R, Wakizuka T, Potrykus I (1981) Z Pflanzenphysiol 103: 361-365