Somatic embryogenesis and plant regeneration from inflorescence segments of Pennisetum purpureum schum. (Napier or elephant grass)

Plant Science Letters, 25 (1982) 147--154

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Elsevier/North-Holland Scientific Publishers Ltd.

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM INFLORESCENCE SEGMENTS OF PENNISETUM PURPUREUM SCHUM. (NAPIER OR ELEPHANT GRASS)

DA-YUAN WANG and INDRA K. VASIL*

Department o f Botany, University of Florida, Gainesville, FL 32611 (U.S.A.) (Received September 18th, 1981) (Revision received November 16th, 1981) (Accepted November 17th, 1981)

SUMMARY

A white and compact embryogenic tissue was obtained from young inflorescence segments of Pennisetum purpureum (Napier or Elephant Grass) cultured on Murashige and Skoog's (MS) and N6 medium containing various concentrations and combinations of 2,4
INTRODUCTION

The potential uses of plant cell and tissue culture techniques in the improvement of cereals and grasses have been enumerated and discussed extensively [ 1--4]. Although regeneration of plants has been reported from tissue cultures of most of the important species of cereals and grasses, a number of difficult problems persist, preventing the realization of the full potential of this important technology. For example, plant regeneration in vitro is sporadic and transient, with the tissues often losing their morphogenetic competence after a few subcultures. Furthermore, only a few selected genotypes of each species respond optimally in vitro. It has also been suggested that regeneration from cultured cereal tissues represents the Abbreviations: BAP = 6-benzylaminopurine, CM = coconut milk; 2,4-D = 2,4-dicholorophenoxyacetic acid; GA = gibberellic acid, MS = Murashige and Skoog's medium; NAA = naphthaleneacetic acid. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.

148 derepression of presumptive shoot primordia which proliferate adventitiously [5]. However, somatic embryogenesis recently has been induced in tissue cultures derived from a variety of explants from several species of grasses [6]. The significance of somatic embryogenesis lies in the fact that like their zygotic counterparts, the somatic embryos also arise from single cells. This would exclude the possibility of chimerism at the initial stages of plant formation. Plants regenerated from somatic embryos have consistently been shown to have the normal complement of chromosomes [7--14]. In an earlier report we described the formation of somatic embryos and plants from young leaf explants of Pennisetum purpureum [13]. We now describe extensive somatic embryogenesis and plant regeneration from cultured segments of young inflorescence in the same species, which is an important source of forage, does not set many seeds in nature and is principally propagated vegetatively. MATERIALS AND METHODS Young, unemerged inflorescences (1--3 cm in length) of Pennisetum p u r p u r e u m Schum. (Elephant or Napier Grass)were obtained from field grown material (selections PP12 and PP13 from Merkeron Menlo) provided by Dr. Stanley C. Schank. After stripping the outermost leaves the material was rinsed in 70% ethanol for 15 s, sterilised with 25% Clorox for 4 m i n and washed 3 times with sterile distilled water. The inflorescences were dissected out, cut into 1--3 mm segments and placed in a Falcon Petri dish (9 × 55 ram) on 0.8% agar medium containing 3% sucrose and different concentrations and combinations o f 2,4-D, BAP, NAA and coconut milk (CM). The two basic nutrient media used were MS [15] and N6 [16]. The pH of the medium was adjusted to 5.8 before autoclaving. All cultures were incubated at 27°C in a growth chamber under 16 h of diffused light. Tissue for histological observations was fixed in formalin~cetic-alcohol, embedded in paraplast, sectioned at 8--10 ~m and stained with safranin-fast green. For chromosome counts, freshly excised root tips were pretreated with 0.02% colchicine for 3 h and fixed in 3 : 1 ethanol/acetic acid. After hydrolysis in 1 N HC1 for 10 rain, the roots were stained with ~qchiff's reagent. Specimens for scanning electron microscopy were prefixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room temperature, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, critical point dried and coated with gold. A Hitachi S-450 Scanning Electron Microscope was used for the examination and photography of the specimens. RESULTS AND DISCUSSION At the time of excision and culture, the inflorescence contained many spikelets and spikelet primordia (Fig. 1). Individual floral primordia were

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recognizable, sometimes with the outermost whorl of accessory floral organs at an early stage of their development. No stamen or carpel primordia were seen. The inflorescence rachis, in cross section, showed a large number of vascular bundles distributed largely in the peripheral portions of the parenchymatous ground tissue. The peripheral parts of the rachis contained small and richly cytoplasmic cells as compared to the central region which was comprised of large and vacuolated cells. Scores of meristematic centres were formed in the rachis of 7-day-old cultures. Although the exact fate of these meristematic loci could not be determined, they did not appear to be involved in the subsequent formation of the embryogenic callus tissue. These loci of rapid cell division were located throughout the parenchymatous ground tissue, but were especially conspicuous and abundant among the cells immediately surrounding the vascular bundles. The parenchymatous cells became increasingly cytoplasmic, developed a conspicuously thick wall and underwent several internal segmenting divisions to form discrete groups of very small and richly cytoplasmic cells with prominent nuclei. No increase in cell size, nor any external signs of callus formation were noticeable, and no organised structures were formed from such meristematic loci. Two weeks after culture two distinct types of callus were formed on the inflorescence explants; one, soft, friable, translucent and non-morphogenic; the other, white, compact and embryogenic (Fig. 2). On N6 medium containing 1 mg/1 2,4-D, 0.5 rag/1BAP and 1 rag/1 NAA, about 50% of the inflorescence segments produced embryonic callus. In 2--3-week-old cultures cambium like zones were formed from the surface and sub-surface layers of the rachis and, subsequently, gave rise primarily to the white, compact, organised and embryogenic callus tissue. Similar callus was also

Fig. 1. Inflorescence at the time of culture. (x30). Fig. 2. White and compact embryogenic callus (ec) and friable non-embryogenic callus (ne) 6 weeks after initial culture (xl0).

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formed on associated spikelet and/or floral axes. In many instances the original parts of the spLkelets and the floral primordia were left intact and could be recognised several weeks after the initiation of culture and callus formation; the floral primordia did not undergo any further development. Embryogenic callus tissue was obtained on both MS and N6 media supplemented with 2,4-D, BAP and NAA. High concentrations of 2,4-D slowed and inhibited embryogenesis. The highest frequencies of embryogenic callus formation (more than 70%) were obtained in N6 medium with 1 rag/1 2,4-D, 0.5 mg/1 BAP and 1 mg/1 NAA, or in the MS medium with 2 rag/1 2,4-D and 5 rag/1 BAP. The N6 medium produced more typical embryoids and in a shorter time than those obtained in the MS medium. Higher concentrations of 2,4-D also inhibited the precocious germination of embryoids. The compact and organised callus tissues maintained their embryogenic potential for more than one year and through 18--20 subcultures on MS medium either lacking in plant growth substances or with 1 mg/1 GA to or 2.5 mg/1 2,4-D and 5% CM. On the former, the embryogenic callus proliferated rapidly and had to be subcultured every 15 days to prevent the formation of new embryoids and their immediate germination to form plantlets. The medium with CM did not support active callus growth and embryoids were not formed until 3--4 weeks after subculture.

Fig. 3. Plantlet formed from embryoid after transfer to MS medium without hormones. Fig. 4. Plantlets potted in soil. Fig. 5. R o o t tip chromosomes from regenerated plant, 2n -- 4x = 28. (X2500).

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The organised callus containing mature embryoids was transferred to MS medium either lacking in plant growth substances or with 1 rag/1 GA to induce the germination of embryoids and formation of plantlets. The frequency of plantlet formation after 3 months of subculture was more than 60%. The regenerated plants were successfully transplanted in soil (Figs. 3 and 4) and grown to maturity. An examination of root tips from the regenerated plants showed them to be normal tetraploids with 2n = 4x - 28 chromosomes (Fig. 5). The embryoids formed in vitro showed characteristic features of grass embryos including a well
Figs. 6 and 7. Mature embryoids formed in vitro (co, coleoptile; cr, coleorrhiza; sc, scutellum; sh, s h o o t meristem). Note presence of two shoot meristems within a single coleoptite. (X70, XS0).

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ii i

. . . . . . . . .

Figs. 8 and 9. Precocious germination of embryoids, formation of leafy scutellum with trichomes and multiple shoot meristems. In Fig. 8 four generations of embryoids (i--iv) can be seen. The oldest embryoid has germinated precociously, its scutellum has split into several parts, each with trichomes and multiple shoot meristems have been formed at the base of the scutellum. (×40, ×100). tion, b u t no somatic embryogenesis, from only a b o u t 5% o f the cultured inflorescence segments ofPennisetum purpureum. Their cultures also suffered from the rapid loss o f regeneration potential. Somatic embryogenesis is a preferred m e t h o d of plant propagation in vitro because it allows rapid p r o d u c t i o n o f a large n u m b e r o f plants within a short period of time. Plants derived through embryogenesis are unicellular in origin and, thus, are likely to be m o r e suited for genetic, breeding and m u t a t i o n research. Conversely, plants regenerated through t h e oz~xdz~tion of s h o o t meristems, which are multicelhflar in origin, can be cbimeral in nature [18--22] and, therefore, n o t useful for breeding or for the maintenance o f genetic stocks. Furthermore, no polyploids or aneuploids were f o u n d amongst t h e plants regenerated through somatic embryo~vnesis from tissue cultures o f a n u m b e r o f cereal and grass species [7--14]. Although no detectable genotypic o r phenotypicvariations were f o u n d in these plants, a rigorous cytological examination o f the plAnta, especially meiotic chromosome anatysis, needs t o b e undertaken to determine w h e t h e r chromosome translocations, deletions, inversions, etc., have taken place. ACKNOWLEDGEMENTS D-Y.W. is on leave of absence from the Institute of C i t ~ s , Chinese

153 A c a d e m y o f A g r i c u l t u r a l Sciences, C h u n g k i n g , T h e Peoples R e p u b l i c o f C h i n a a n d is s u p p o r t e d b y t h e Chinese Ministry o f A g r i c u l t u r e a n d b y f u n d s p r o v i d e d t o I . K . V , b y t h e U n i v e r s i t y o f Florida. We t h a n k Dr. Chin-yi Lu f o r assisting in t h e histological e x a m i n a t i o n and scanning e l e c t r o n m i c r o s c o p y a n d Dr. H e n r y C. A l d r i c h f o r use o f t h e Biological U l t r a s t r u c t u r e L a b o r a t o r y . F l o r i d a A g r i c u l t u r e E x p e r i m e n t S t a t i o n J o u r n a l Series N o . 3 2 0 0 . REFERENCES 1 C.E, Green, In vitro plant regeneration in cereals and grasses, in: T.A. Thorpe (Ed.), Frontiers of Plant Tissue Culture 1978, University of Calgary, Canada, 1978, pp. 414-418. 2 P.J. King, I. Potrykus and E. Thomas, In vitro genetics of cereals: problems and perspectives, Physiol. Veg., 16 (1978) 381. 3 Y. Yamada, Tissue culture studies on cereals, in: J. Reinert and Y.P.S. Bajai (Eds.), Plant Cell, Tissue and Organ Culture, Springer-Verlag, Heidelberg, 1978, pp. 144--159. 4 I.K. Vasil, Plant cell culture and somatic .cell genetics of cereals and grasses, PI. Mol. Biol. Newsiett., 2 (1981) 9. 5 R.I.S. Brettell, W. Wernicke and E. Thomas, Embryogenesis from cultured immature inflorescences of Sorghum bicolor, Protoplasma, 104 (1980) 141. 6 I.K. Vasil, V. Vasil, C. Lu, Z. Haydu, D. Wang and P. Ozias-Akins, Somatic embryogenesis in cereals and grasses, in: E.A. Earle (Ed.), Regeneration and Genetic Variability, Praeger Press, New York, 1982, in press. 7 V. Vmdl and I.K. Vasil, Somatic embryogenesis and plant regeneration from tissue cultures o f Pennisetum americanum and P. americanum × P. purpureum hybrid, Am. J. Bot., 68 (1981a) 864. 8 V. Vasil and I.K. Vasil, Somatic embryogenesis and plant regeneration from suspension cultures of pearl millet (Pennisetum americanum), Ann. Bot., 47 (1981b) 669. 9 V. Vasil and I.K. Vasil, Characterization of an embryogenlc cell suspension culture derived from cultured inflorescences of Pennisetum americanum (pearl millet, Gramineae), Am. J. Bot., 69 (1982) in press. 10 C. Lu and I.K. Vasil, Somatic embryogenesis and plant regeneration from tissue cultures of Panicum maximum, Am. J. Bot., 69 (1982) 77. 11 C. Lu and I.K. Vasil, Somatic embryogenesis from freely suspended cells and cell groups of Panicum m a x i m u m Jacq, Ann. Bot., 48 (1981a) 543. 12 C. Lu and I.K. Vasil, Somatic embryogenesis and plant regeneration from leaf tissues of Panicum m a x i m u m Jacq, Theor. Appl. Genet., 59 (1981b) 275. 13 Z. Haydu and I.K. Vasil, Somatic embryogenesis and plant regeneration from leaf tissues of anthers o f P e n n i s e t u m purpureum Schum, Theor. Appl. Genet., 59 (1981) 269. 14 P. Ozias-Akins and I.K.Vasil, Plant regeneration from cultured immature embryos and inflorescences of Triticum aestivum L. (wheat): evidence for somatic embryogenesis, Protoplasma, (1982) in press. 15 T. Murashige and F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue culture, Physiol. Plant, 15 (1962) 473. 16 C.C. Chu, C.C. Wang, C.S. Sun, C. Hsu, K.C. Yin and C.Y. Chu, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica, 18 (1975) 659. 17 Y.P.S. Bajaj and M.S. Dhanju, Regeneration of plants from callus cultures of Napier gras~ (Pennisetum purpureum), Plant Sci. Lett., 20 ( 1981 ) 343. 18 M.D. Sacristan and G. Melchers, The karyological analysis of plants regenerated from tumorous and other callus cultures of tobacco, Mol. Gen. Genet., 105 (1969) 317.

154 19 H. Ogura, The cytological chimerasin original regenerates from tobacco ~iuue cultures and i:heir offsprings, Jap. J. Genet., 51 (1976) 161. 20 K. Sree Ramulu, M. Devereux, G. Ancora and U. Laneri, Chimerism in Lycopersicum peruvianum plants regenerated from in vitro cultures of anthers and stem internodes, Z. Pflanzenz~chtg., 76 (1976) 299. 21 A. Bennici and F. D'Amato, In vitro regeneration of durum wheat plants. 1. Chromosome numbers of regenerated plantlets, Z. Pflanzenz~chtg., 81 (19"/8) 305. 22 G. Mix, H.M. Wilson and B. Foroughi-Wehr, The cytological status of plants of Hordeum vulgate L. regenerated from microspore callus, Z. Pflanzenz/ichtg., 80 (1978) 89.