Plant Cell Reports (1985) 4 : 1 1 5 - 1 1 7

Plant Cell Reports © Springer-Verlag 1985

Plant regeneration from tissue cultures initiated from immature inflorescences of a grass, Echinochloa colonum (L.) Link A. K. Tyagi *, S. Bharal **, A. Rashid, and N. Maheshwari U n i t for Plant Cell a n d M o l e c u l a r Biology a n d D e p a r t m e n t o f Botany, U n i v e r s i t y o f Delhi, Delhi-110007, I n d i a Received A u g u s t 14, 1984 / R e v i s e d version received F e b r u a r y 27, 1985 - C o m m u n i c a t e d by J. W i d h o l m

ABSTRACT Organised structures develop on a white and compact callus initiated from small segments of immature inflorescences of Echinochloa c o l o n u m cultured on M u r a s h i g e a n d Skoog's (MS) m e d i u m supplemented with 5.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) a n d 10% coconut milk. These develop into plantlets u p o n subculture onto M S medium containing 0 or 0.2 mg/l 2,4-D. T w e l v e out of 17 plantlets regenerated grew well on transfer to soil and eleven plants p r o d u c e d seeds.

INTRODUCTION

Krumbiegel-Schroeren et al. 1984). It is,therefore, possible that totipotent tissue cultures obtained from the immature inflorescence m a y find application in rapid clonal propagation of desirable geonotypes and a source material for genetic manipulation and improvement.

O n e of t h e i m p o r t a n t a r e a s of r e s e a r c h for plant scientists is the introduction of n e w p l a n t s from the wild into cultivation. Echinochloa, a grass, is a promising s p e c i e s w h i c h if a d a p t e d to c u l t i vation might be an important forage and grain crop for arid and semi-arid regions, as a single watering suffices from germination to harvest (Galston et al. 1980). This report describes for the first time regeneration of p l a n t s from tissue

Strategies to r e g e n e r a t e plants from tissue cultures of c e r e a l s and grasses have been evolving steadily during the past decade. Successes in obtaining plants in tissue cultures, particularly those initiated from immature embryos, young inflorescences and from leaves (Wernicke and

cultures initiated from of Echinochloa colonum.

Brettell 1980; Lu and Vasil 1981; Hanning and Conger 1982; Conger et al. 1983) a n d recently somatic embryos from suspension cultures of an orchard grass (Gray et al. 1984), have opened new opportunities for the application of in vitro techniques to improve cereals a n d grasses (G e n g e n b a c h et al. 1977; Schaeffer a n d Sharpe 1981; Hibberd and Green 1982; Sun et al. 1983; see also Larkin and Scowcroft 1981; Maliga 1984). Plant regeneration in tissue cultures initiated from immature inflorescences has already been reported in cereals and grasses via organogenesis (Gosch-Wackerle et al. 1979; Lo et al. 1980; Bajaj and Dhanju 1981; Nakamura et al. 1981; Chen et al. 1982; Nakamura and Keller 1982; Dale a n d Dalton 1983; M a d d o c k et al. 1983) as well as by e m b r yogenesis (Brettell et al. 1980; Dale et al. 1981; Vasil and Vasil 1981; L u and Vasil 1982; Ozias-Akins and Vasil 1982; W a n g a n d Vasil 1982; Conger a n d M c D o n n e l 1983;

Immature inflorescences of Echinochloa colonum (L.) Link were obtained from field g r o w n plants. After r e m o v i n g the outermost leaves, the material was surface-sterilized with chlorine water for 5 minutes a n d subsequently rinsed with sterilized distilled water three three-times. For preparing chlorine water, 50 ml of HCI (38%) w a s p o u r e d over 15 g of potassium p e r m a n g a n a t e taken in a filtering flask. Chlorine was a11owed to b u b b l e into 500 ml of distilled water. The inflorescences taken from fifteen plants -- ranging from 1-2.5 c m in size -- w e r e dissected and cut into 3-5 m m long segments. From one plant more than one inflorescence was taken. These explants were inoculated in culture tubes on the surface of agar-gelled M S m e d i u m (Murashige a n d Skoog 1962) containing 5.57 mg/1 Na_EDTA as iron source, 20 g/l sucrose, and 5 Zmg/l 2,4-D + 10% CM, as growth regulators. The medium was gelled with 8.0 g/l Difco Bacto-agar and the pH adjusted to 5.7 with 0.IN N a O H or HCI before autoclaving. Tissue cultures developing from the initial explants w e r e used for sub-culture. To obtain plantlets the growth regulators were either omitted or only 0.2 mg/l 2,4-D was used. Tissue culture initiatio~ w a s conducted in diffuse light of about 1.9 W / m - while plantlet regeneration was carried out at higher light intensities of about 5.27 W / m .

Present *

address:

Botanisches Institut der Universit[t D{Jsseldorf, Universitatstrasse i, D-4000 D{lsseldorf 1 FRG.

** Gargi Road,

College, University of Delhi, N e w Delhi-l10049, India.

Siri

Fort

MATERIAL

AND

immature

inflorescences

METHODS

116 Light w a s provided by cool daylight fluorescent tubes (Philips, TL 40W/54 or TL 65-80W/54). Regenerated plants w e r e transplanted to soil in plastic pots, covered with beakers 2for the first 15 days, and g r o w n in about 1 0 W / m light provided by fluorescent tubes (as above) a n d tungsten bulbs (Philips Argenta, I00 W). The temperature of the culture room w a s 26_+2°C in light (6 a.m. to i0 p . m . ) a n d 24_+2°C during darkness. RESULTS

AND

DISCUSSION

Callus growth on immature was visible after 15 days

inflorescence explants of culture on M S +

5.0 mg/l 2,4-D + 10% CM. After 30 days, two types of callus b e c a m e distinguishable: (a) white and compact, (b) off-white and friable. The former resembled the morphogenic while the latter was like n o n - m o r p hogenic tissue described in the literature on cereals and grasses (see Vasil 1983). Subsequently, proliferating morphogenic tissue became nodular and produced organized structures (Fig. IA) w h e r e a s the n o n - m o r p h o g e n i c tissue produced hair-like structures ( Fig. IB, white arrows). Sometimes, both types of tissues were produced from the same explant as seen in Fig. IB. During three separate experiments, in w h i c h a total of 134 explants w e r e inoculated,

Fig. i. Regeneration of plants in tissue cultures initiated from immature inflorescences of Echinochloa colonum. A, Organized structures p r o d u c e d on compact a n d white callus from inflorescence explant cultured on M S + 2,4-D 5.0 mg/l + C M 10%. B, Organized structures (black arrows) a n d friable, off-white callus (white arrows) on the m e d i u m M S + 2,4-D 5.0 mg/l + C M 10%. Note several hair-like structures on friable callus. C, A n organized structure develops into plantlet u p o n subculture on M S m e d i u m alone. D, E, Several plantlets emerging from subcultured explant with organized structures on M S * 2,4-D 0.2 mg/l and on M S m e d i u m alone, respectively. F, A plantlet before transfer to soil. G, A plantlet with inflorescence about three m o n t h s after transfer to soil.

117 100 (74.6%, range 50-93%) produced organized structures. These organized structures did not develop further even after 80 d a y s of culture in the above medium, although the callus continued to g r o w o n t h e s a m e m e d i u m a f t e r subculture. However, when the morphogenic tissues with organized structures from the above explants were subcultured o n t o MS m e d i u m w i t h 0 or 0.2 mg/1 2,4-D, the organized structures developed into plantlets ( F i g s I C - E ) . T h e s t r a t e g y of l o w e r ing or omitting 2,4-D (or other auxins) from the growth medium has been widely used by many workers to regenerate plants from tissue cultures of grasses (see review, Green 1978; Brettell et al. 1980; Dale et al. 1981; Lu and Vasil 1982). In several cultures, off-white, friable callus also developed. These cultures produced many roots. The per cent of cultures forming shoots and plantlets on MS m e d i u m w i t h o u t 2,4-D was about 37% (35 c u l t u r e s o u t of 94 p r o d u c e d shoots and plantlets) and with 0.2 mg/1 2,4-D was about 32% (30 c u l t u r e s o u t of 94 p r o d u c e d shoots and plantlets). When the white compact and off-white, friable tissues w e r e subcultured separately in further experiments, plantlet formation w a s observed only from the white, c o m p a c t cultures. The organized structures germinate and grow into plants w h e n excised and plated separately . The efficiency of regeneration of plants from these structures w a s about 65%. T h e rnorp hogenic potential of the callus could be maintained for more than a year. In order to assess the capability of plants to grow in soil, five plantlets in the first experiment and 12 in the second with well-developed shoot-root systems (Fig. IF) were transferred to soil in plastic-pots after removing agar adhering to the plantlets under flowing tap water. Plantlets were covered with beakers for the first 15 days. T h r e e plants from the first e x p e r i m e n t and nine from the second survived and grew well and two from the first and nine from the second have produced seeds (Fig.lG). The overall success in regenerating plants is thus about 50 per cent. These results suggest that methods might be developed for in vitro propagation of Echinochloa colonum, which might be exploited for genetic manipulation of this species. If the organized structures observed in this study are somatic embryos, they may be we11-suited for future genetic manipulation studies.

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Thanks are due to the Department of Science and Technology, government of India, Delhi for financial support. We express sincere gratitude to Professor S.C. M a h e s h w a r i for his interest.

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