Somatic embryogenesis in sugarcane - Springer Link

Protoplasma 118, i 6 9 - 180 (1983)

PROTOPLASMA 9 by Springer-Verlag 1983

Somatic Embryogenesis in Sugarcane (Saccharum officinarumL.) I. The Morphology and Physiology of Callus Formation and the Ontogeny of Somatic Embryos WAI-JANE H o a n d INDRA K. VAS1L* Department of Botany, University of Florida, Gainesville, Florida Received October 14, 1982 Accepted in revised form March 16, 1983

Summary

1. Introduction

Embryogenic callus was induced on segments of young leaves of sugarcane (Saccharum offi'cinarum L.) cultured on Murashige and Skoog's medium supplemented with 0.5-3.0 mg/2,4-D, 5% coconut milk and 3-8% sucrose. The fourth and fifth leaves, especially their midrib and sheath regions within 5 cm from the leaf base, were most suitable for the induction of embryogenic callus. Many embryoids (= somatic embryos) were formed when the callus was transferred to low 2,4-D media (0.25-0.5 mg/1), or was allowed to remain on the high 2,4-D medium for a prolonged period. Plantlets obtained from the germination of embryoids were transferred to soil and grown to maturity. The embryogenic callus was formed by divisions in mesophyll cells situated primarily in the abaxial half of the leaf, and also from cells of the vascular parenchyma. The embryoids developed by internal segmenting divisions in single richly cytoplasmic cells located at the periphery of the embryogenic callus and showed the typical organization of grass embryos.

S u g a r c a n e (Saccharum officinarum L.), a m e m b e r o f the Gramineae, is a c r o p o f m a j o r i m p o r t a n c e p r o v i d i n g a b o u t 65% o f the sugar p r o d u c e d in the W o r l d . Tissue culture m e t h o d s have been used extensively to study its physiology, a n d aid in the p r o p a g a t i o n , m u t a t i o n a n d breeding o f s u g a r c a n e since 1961 (NIcI~EL~ 1967), a n d also for the e l i m i n a t i o n o f viruses (MoR~ 1971, L E v 1972, HENDRE et al. 1975). P l a n t r e g e n e r a t i o n f r o m tissue cultures o f s u g a r c a n e has been successfully a p p l i e d to b r e e d i n g a n d p r o p a g a t i o n p r o g r a m s b y m a n y w o r k e r s (HEINZ et al. 1977, PLOPER a n d VAZQUES

Keywords." Saccharum officinarum L.; Grasses; Somatic embryogenesis; Sugarcane; Tissue Culture.

Abbreviations: abscisic acid = ABA, 8-azaguanine = AZG, benzylaminopurine = BAP, chlorocholine chloride = CCC, casein hydrolysate = CH, coconut milk = CM, 2,4-dichlorophenoxyacetic acid = 2,4-D, gibberellic acid = GA, MURASHIGE and SKOOG'S medium = MS, naphthaleneacetic acid = NAA, 4-amino-3, 5, 6-trichloropicolinic acid = Picloram, yeast extract = YE. * Correspondence and Reprints: Department of Botany, University of Florida, Gainesville, FL32611, U.S.A. Florida Agriculturc Experiment Station Journal Series No. 4189.

DE RAMAkLO 1976, NADAR and HElNZ 1977, PAVAN et al. 1977, KALAW et al. 1977, KO6A a n d K v D o 1977, PLOPER a n d MARIOTTI 1978, CHAGVARDIEFF et al. 1981). T h e p l a n t s are said to develop b y the o r g a n i z a t i o n o f s h o o t meristems in callus cultures. H o w e v e r , no detailed histological studies have been carried o u t to u n d e r s t a n d the origin of the callus tissues or the plantlets. Recently, p l a n t s have been regenerated f r o m tissue, cell a n d p r o t o p l a s t cultures of several species o f t h e Gramineae, by the process o f s o m a t i c e m b r y o g e n e s i s (VAsIL 1982a, b, 1983, VASIL et al. 1982). In this c o m m u n i c a t i o n we describe the m o r p h o l o g y a n d p h y s i o l o g y o f callus f o r m a t i o n a n d the r e g e n e r a t i o n o f p l a n t s via the f o r m a t i o n o f s o m a t i c e m b r y o s f r o m cultured segments o f i m m a t u r e leaves o f s u g a r c a n e (Saccharum officinarum L.). This study c o m p l e m e n t s o u r r e p o r t on the f o r m a t i o n o f s o m a t i c e m b r y o s a n d

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WAI-JANEHo and INDRAK. VASIL: Somatic Embryogenesis in Sugarcane (Saccharumofficinarum L.). I.

p l a n t r e g e n e r a t i o n f r o m cell s u s p e n s i o n c u l t u r e s o f s u g a r c a n e ( H o a n d VASIL 1983).

2. Material and Methods Leaf explants were obtained from shoots of sugarcane clone 68-1067 (courtesy of Dr. James R. Miller, U.S.D.A. Sugarcane Station, Canal Point, FI) either grown in the greenhouse or in the field at Gainesville, F1. All of the outer mature leaves were removed. The remaining apical part of the shoot, which consisted of several pale-yellow furled leaves, was trimmed to 30 cm in length. The shoot was surface sterilized in 75% ethanol for one minute, disinfected in 20% Clorox solution for 20 minutes, and washed with four changes of sterile water. After removal of several outer layers of leaves, the innermost 5 6 tightly furled leaves were sequentially cut into 2ram-thick transverse segments, starting at the base of the leaves and up to 8 cm towards the tip. The sequential leaf segments, with a cut surface in contact with the culture medium, were placed in each Falcon Petri dish (100 x 15 mm) containing approximately 30 ml of the medium. Also, 2 ram-thick transverse segments were cut from the top 1 cm of young stems and cultured as described for leaf segments. M URASIJrGEand S ~:oo6's (1962) salt solution with 1.0 rag/1 thiamine, 100 mg/1 inositol and 30 gm/1 sucrose was jelled with 0.8% agar and adjusted to pH 5.8 before autoclaving. Different levels and combinations of 2,4-D (0-3.0 mg/1), NAA (0-1.0 mg/1), Picloram (0.16.0 rag/l), BAP (0-9.5 mg/1), CM (5%), YE (500-1,000 mg/1), CH (5001,000 mg/1), glut~mic acid (1 gm/1), arginine (30-50 mg/1), ABA (0.010.2 rag/l), AZG (0.01-0.2 rag/l), CCC (0. l(I.5 mg/1 and sucrose (1-8%) were tested to determine optimum levels and combinations for callus induction, growth and differentiation. The N 6 (CHuet al. 1975) and MS medium, without NH4 + or NO3-, were also tried. The MS medium containing no NH 4 + was developed by replacing NH4NO 3 with 20 or 60 mM KNO 3. Ten or 30 mM ammonium malate and 20mM KC1 were added to replace KNO~ in N O 3 - - f r e e MS medium. Ammonium malate was made by dissolving malic acid in water and increasing the pH to 5.0 with NH4OH. Data were obtained from 10 replicates for each treatment. All cultures were incubated in the dark at 27 ~ for callus induction and growth. The effect of various media on fresh weight increase of callus was determined by culturing 10 pieces of callus (total fresh weight 1 gin) in each Petri dish containing 30 ml of medium. A total of 5 dishes for each treatment were used. Fresh weights were recorded after one month. Leaf tissues were sampled after 0~ 1, 2, 4, 8, 16, 24~ and 32 days and fixed in formalin-acetic-alcohol (FAA), dehydrated in a tertiarybutyl alcohol series and embedded in Paraplast. Sequential paraffin sections were cut at 5 10,am on a rotary microtome, stained with safranin-fast green and mounted with Permount. FAA-fixed tissues were dehydrated also through a 70, 85, and 95~ ethyl alcohol series for 1-2 hours each, then infiltrated and embedded

in JB4 plastic (Polysciences). The polymerized and trimmed plastic blocks were sectioned with a glass knife mounted on a Sorvall MT2-B "Porter Blum" ultra-microtome to obtain 2-3 ~m sections. Dry sections were stained with periodic acid Schiff's method (McMANUS 1948), post-stained with 1% anilin blue (prepared in 3.5% v/v glacial acetic acid), washed with running water until the water was clear and air dried. Dry sections were mounted in Permount. For scanning electron microscopic studies, tissues were fixed for 2 hours at room temperature in 2% glutaraldehyde buffered to pH 7.2 with caeodylate, washed with the same buffer 3 times and post-fixed overnight in 1% OsO4. After dehydration through a graded ethanol series to absolute ethanol, the tissues were critical point dried and sputter coated with gold. Observations and photographs were made on a Hitachi S-450 scanning electron microscope at 20 kV.

3. Results

3.1. Formation of Embryogenic Callus and Somatic Embryos C a l l u s tissue first b e c a m e visible at t h e cut e n d o f e x p l a n t s 4 d a y s after c u l t u r e o n M S m e d i u m c o n t a i n i n g 0.5-3.0 r a g / 1 2 , 4 - D . It a l w a y s f o r m e d first n e a r a v e i n o r the c u t e n d s o f the l e a f (Figs. 1 a n d 2). V e r y little o r n o callus was f o r m e d in 2 , 4 - D - f r e e m e d i a . T h r e e t y p e s o f callus tissues w i t h v a r y i n g m o r p h o g e n e t i c p o t e n t i a l w e r e o b t a i n e d : (a) Compact callus- a hard,

compact

and

smooth

looking

callus,

which

c o n s i s t e d o f small, r o u n d a n d r i c h l y c y t o p l a s m i c cells, t h a t b e c a m e w h i t e in l a t e r stages a n d was s h o w n to be embryogenic

in n a t u r e

(Fig. 3); (b) Soft callus-a

friable, s e m i - t r a n s l u c e n t callus tissue t h a t c o n s i s t e d o f loose, l a r g e a n d e l o n g a t e d cells (Fig. 4) a n d

occa-

s i o n a l l y d e v e l o p e d r o o t s ; (c) Mucilaginous callus-a soft, g u m m y a n d s h i n y callus w h i c h was n o n - e m b r y o g e n i c in n a t u r e , c o u l d n o t be s u b c u l t u r e & a n d c o n s i s t e d o f e l o n g a t e d , h i g h l y d i s s o c i a t e d cells. T h e m a j o r i t y o f the callus o r i g i n a t e d f r o m the a b a x i a l (lower) s u r f a c e o f the e x p l a n t s (Figs. l-3) a l t h o u g h s o m e callus also f o r m e d f r o m the a d a x i a l surface. I n a b o u t 8 days, a n o d u l a r callus a r o s e w h i c h was c o v e r e d w i t h a t h i n layer o f l o o s e m u c i l a g i n o u s cells t h a t g a v e t h e callus a s h i n y a p p e a r a n c e .

In 2-3weeks,

white

c o m p a c t callus was f o r m e d w h i c h s u b s e q u e n t l y differentiated embryoids.

Fig. 1. Scanning electron micrograph of 10-day old cultured leaf blade with swollen veins (arrowhead) and callus (c) at the cut end ( x 35) Fig.2. Embryogenic callus formation in 2-week old cultured leaf sheath ( x 35) Fig.3. Embryogenic callus formed from the midrib after 20 days in culture ( x 6) Fig.4. Scanning electron micrograph of the soft callus showing elongated cells (x 110) Fig. 5. Globular proembryoid (g) and embryoids showing the formation of the scutellar notch (arrowheads) ( x 96) Fig.6. The beginning of the formation of a coleoptile (co and shoot meristem (s) in the notched area of the embryoids ( x 68)

WAI-JANE HO and INDRA K. VASIL: Somatic Embryogenesis in Sugarcane

Figs. I-6

(Saccharum officinarum L.). I.

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WAI-JANEHO and INDRAK. VASIL:Somatic Embryogenesisin Sugarcane (Saccharum officinarum L.). I.

Fig.7. Globular proembryoid (g) and embryoids with a tube-like coleoptile (cO and scutellum (sc) ( x 54) Fig.8. Proliferation of the scutellum and formation of secondary embryogeniccallus tissue (x 25) Fig.9. An embryoid showing the organization of two shoot meristems (arrowheads) ( x 145) Fig. 10. Leafyscutella (ls) with multiple shoot meristems at their bases ( x 60)

The leaf sheath (Fig. 2) and midrib (Fig. 3) were more suitable for callus induction and produced more compact and embryogenic callus than the leaf blade. The frequency and v i g o r of embryogenic callus formation depended on leaf age and the distance of the explants from the base of the leaf (Fig. 11). The youngest, innermost 1-2 leaves gave rise to a greater amount of soft callus than compact callus. The fourth and fifth leaves were found to be the most suitable for the induction of embryogenic callus and the formation of embryoids. Leaves older than the sixth leaf did not produce embryogenic callus, and formed only roots.

Leaf explants nearest the shoot apex showed some darkening in culture and formed only a small amount of white compact callus. Explants taken within 5 cm from the leaf base showed little difference in their ability to form embryogenic callus. The young stem near the shoot tip became dark purplish, and gave rise to only soft and mucilaginous calli. Well organized embryoids were obtained within 30 55 days in media containing 0.5 3.0rag/1 2,4-D. The embryoids appeared earlier at low 2,4-D concentrations than at high levels of 2,4-D. Many globular structures were seen on the periphery of white embryo-

WAI-JANEHO and INDRAK. VAS~L:Somatic Embryogenesisin Sugarcane (Saccharum ofji'cinarum L.). I. genic callus. Later, a lateral notch became apparent as the scutellum was formed (Fig. 5). This was followed by the organization of a shoot meristem and coleoptile in the notched area (Fig. 6). Further development of the notched structures resulted in the formation of many somatic embryos. These had a scutellum, a tube-like coleoptile surrounding the shoot meristem (Fig. 7) and a coleorhiza enclosing a root meristem.

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3.2. The Establishment of Plantlets Well-developed embryoids were transferred to the basal medium with or without 0.002 0.02 mg/1 zeatin or lmg/1 G A to promote germination. A strong root system was formed by transfer of the germinating embryoids to a medium with half-strength MS salts and 6% sucrose. High sucrose concentrations also stimulated root differentiation as well as root growth. Once established, the plantlets were transplanted to potting soil, acclimatized in a 27 ~ growth chamber with a 16-hour photoperiod for 2-3 weeks and then grown to maturity.

60-

3.3. Nutritional Requirements for Callus Initiation and Embryoid Differentiation

5O

(a) Growth Regulators

4O

50

z .J

scutellum of embryoids with a broad meristematic zone. The trichomes were characteristic of the scutellum and different from those formed on leaves.

70

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173

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0

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0

DISTANCE

L

1

i

i

i

i

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FROM

APICAL

MERISTEM

(cm)

F i g . l l . The ability of segments of the fourth leaf to form an embryogenic callus as a function of distance form the apical meristem

A number of embryoids showed atypical development, such as secondary proliferation of the embryoid scutellum (Fig. 8) and leafy scutella. Embryoids with a broad meristematic zone formed multiple shoot meristems (Fig. 9) and gave rise to two or more shoots/plants. Embryoids with fasciated scutella and multiple shoot meristems frequently were observed when cultures were initiated in low 2,4-D medium (Fig. 10). Cultures kept at 0.5 mg/1 2,4-D in light often showed the development of some green leafy scutella before the shoot meristem had organized. In such cases of precocious germination embryoids formed only a leafy scutellum, but no shoot or root. Many trichomes were seen on the proliferating scutellum of embryoids (Fig. 8), on the leafy scutellum (Fig. 10) and on the

The formation of embryogenic callus on cultured leaf segments was inversely proportional to the concentration of 2,4-D in the medium. The optimum concentration for embryogenic callus formation was found to be 0.5 1.5 mg/1. However, root development and the formation of leafy scutella were more common in low 2,4-D media (0.5 mg/1). The maximum fresh weight of the callus was achieved in one month on media with 1.5 rag/12,4-D. However, 2.5 3.0 mg/12,4-D was best for the maintenance of embryogenic callus in an undifferentiated state. Embryogenic callus could be subcultured at 4 week intervals for more than a year in a medium containing 2.5 3.0 rag/1 2,4-D together with 5 ~ CM and 50 mg/1 arginine. Addition of 0.002 mg/1 zeatin decreased the growth of the embryogenic callus and caused it to become less compact and less organized. Optimum embryoid formation occurred at 2,4-D concentrations lower than those required for optimum growth of embryogenic callus. Well developed embryoids were obtained from embryogenic callus when 2,4-D in the medium was gradually lowered by either prolonged growth of the callus on the same medium without subculture or transfer of the callus to a medium containing lower concentrations of 2,4-D (0.25-0.5 rag/l). Other growth regulators were also tested. Picloram (0.1-6.0rag/i) was not effective for the induction of embryogenic callus. A combination of N A A (1 rag/l), 2,4-D (0.5rag/i), and BAP (0.5 rag/l) gave very poor

174

WAI-JANEHO and INDRAK. VASIL:Somatic Embryogenesisin Sugarcane (SaccharumofficinarumL.). I.

results, and the explants produced more mucilaginous and soft callus than white compact callus. The differentiation of the embryoids was also inhibited. Growth retardants such as ABA (0.01 0.2 mg/1), A Z G (0.01-0.2 rag/l) and CCC (0.1-0.5 mg/1) prevented precocious germination of the embryoids and allowed them to mature. (b) Inorganic Components In MS medium, 81~ of the explants formed embryogenic callus. The N 6 medium was approximately 40~ less effective than the MS medium for the induction of embryogenic callus and the formation of embryoids. The MS medium contains 60mM of total nitrogen (20.6 mM NH4 + and 39.4 m M NO 3-). When NH4+ was completely removed from MS medium, neither 40 nor 60 mM N O 3- supported initiation and/or growth of embryogenic callus. Only soft and mucilaginous calli were formed. When NH4 + was the only nitrogen source available, 20 mM NH4+ enabled 6.1~ of the explants to produce embryogenic callus but the differentiation of embryoids on this medium was very poor. However, 60 mM NH4 + as the only nitrogen source in the MS medium allowed about 65~ of the leaf explants to form embryogenic callus and subsequently embryoids. In the MS medium ammonium nitrogen evidently was more important than the nitrate nitrogen for embryogenic callus and embryoid formation, but NH4+ alone was not as effective as the combination of both NH4+ and NO 3 -. Darkening of the embryogenic callus was prevented by incorporating 1~o charcoal into the medium. Charcoal also stimulated growth of compact callus and the subsequent formation of embryoids. (c) Organic Supplements The addition of 5% CM to the culture medium did not signilicantly enhance the formation of embryogenic callus. Also, it inhibited subsequent development of embryoids but promoted precocious germination. Yeast extract (1 mg/1) slightly increased production of

embryogenic callus. Differentiation of embryoids from callus which had been subcultured several times was promoted by the addition of 500-1,000 mg/1 YE. The promotive effect of YE was further enhanced with the addition of 50mg/1 arginine and 1~ charcoal to the medium. Incorporation of 1 g/1 glutamic acid, or 50 mg/1 arginine, or 500-1,000 mg/1 CH to MS medium, however, did not increase the formation of embryogenic callus and embryoids. High sucrose (4-8~) concentrations promoted root development and decreased the amount of white compact callus formed. Higher levels of sucrose (6-8~) also prevented precocious germination of embryoids.

3.4. Ontogeny of Somatic Embryos Anatomical studies of the cultured leaf segments (Fig. 12) revealed that the innermost two leaves usually did not have distinct differentiation of xylem and phloem. In the third leaf well differentiated vascular tissues were present only in the midrib. In the fourth and fifth leaves, fully differentiated vascular tissues were present in the leaf sheath, the midrib and many large veins in the leaf blade. In culture, cell division activity was highest in segments from the fourth and fifth leaves which showed advanced differentiation of vascular bundles. Isolated dividing cells were seen in xylem and phloem parenchyma, bundle sheath, mesophyll and epidermis in explants cultured on 2,4-D media for one day. In two days, the large vein (Fig. 13) and edges of the leaf showed active cell divisions which resulted in tissue swelling. The centers of cell division were primarily in the abaxial mesophyll cells surrounding the vascular bundles and secondarily in the phloem parenchyma. These divisions formed nodular callus on the abaxial surface of the leaf after 8 days (Figs. 14 and 15). Occasionally, divisions in the xylem parenchyma and the surrounding mesophyll cells formed callus also on the adaxial surface of the leaf (Fig. 16). By 8 days, a cambium-like zone was formed within the

Fig. 12. Cross section of furled leaves at the time of culture (x 19) Fig. 13. Cell divisionin epidermal and sub-epidermalcells causing swelling of a larger vein in 2-day old cultured leaf segment ( x 225) Fig. 14. Radial files of cells produced by the cambium-likezone and slimy, loose cells on the surface ( x 75) Fig. 15. Cross section of 8-day old cultured leaf segments showing callus formation from leaf margin (arrowhead) and veins ( x 66) Fig.16. Formation of embryogeniccallus on both surfaces of the leaf blade in 20-day old culture (x 45) Fig. 17. Cell divisions (arrowhead) near the surface of the embryogeniccallus (x 440)


Figs. 12-17


175

176


(Saceharum officinarum L.). I.

Fig. 18. An embryogenic cell (arrowhead) with dense cytoplasm, conspicuous nucleus and thickened cell wall at the periphery of the embryogenic callus ( x 570) Figs. 19-22. Discrete groups ( = proembryoids) of two, three or more cells within a common thick wall at the periphery of embryogenic callus. Fig. 19 ( x 525) ; Fig.20 ( x 510); Fig.21 ( x 725) ; Fig.22 ( x 510)

Fig. 23. A globular proembryoid with uniseriate suspensor and periclinal division (arrowhead) in the peripheral cells of the embryoid proper indicating the beginning of the differentiation of an epidermis ( x 570) Fig.24. A globular proembryoid with a biseriate suspensor attached to the surface of mucilaginous callus ( x 340) Fig.25 and 26. Embryoids showing initiation of scutellar notch ( x 275, x 280) Fig.27. The differentiation of the procambial strand (arrowheads) in the scutellum ( x 165) Fig. 28. An embryoid with scutellum (sc), a shoot meristem with several embryonic leaves enclosed within the coleoptile (c/), and a root meristem with root cap surrounded by the coleorhiza (cr) ( x 60)

WAI-JANE H o and [NDRA K. VASIL: Somatic Embryogenesis in Sugarcane

Figs. 23-28


177

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WAI-JANEHO and ][NDRAK. VASIL"SomaticEmbryogenesisin Sugarcane(Saccharum officinarum L.). I.

nodular callus (Fig. 14), and the outer layers of the callus became loose and mucilaginous. Embryogenic callus was derived from continued proliferation of the cambium-like zone. Extensive cell division activity at the periphery of the embryogenic callus resulted in several layers of small densely cytoplasmic cells with conspicuous nuclei (Fig. 17). The surface of the callus became irregular as a result of extensive localized cell divisions. Embryogenic cells with conspicuous nuclei, dense cytoplasm and thickened cell walls (Fig. 18) situated at the periphery of the embryogenic callus underwent internal segmenting divisions. Proembryoids with two, three, four or more cells were seen enclosed in a common thick wall (Figs. 19-22). Continued divisions formed globular proembryoids (Figs. 23 and 24). The differentiation of dermatogen was indicated by periclinal divisions in the peripheral cells of early globular proembryoids (Fig. 23). Proembryoids and embryoids were attached to the callus surface by a small suspensor (Figs. 23 26). However, in many cases the suspensor was either not clearly distinguishable or present as a broad multicellular structure. Further development of the proembryoids was characterized by the formation of a lateral notch in the terminal end (Figs.25 and 26) which indicated the initiation of the scutellum. Following the deepening of the notch, a procambium differentiated in the scutellum (Fig. 27). The shoot apical meristem was organized in the region of the notch and later a root meristem was organized in the basal part of the embryoid. The lateral scutellum consisted of large cells with conspicuous starch grains (Fig. 28). A well-developed embryoid showed a coleoptile enclosing a shoot meristem with several embryonic leaves at one end and a root meristem with root cap enclosed in a coleorhiza at the opposite end (Fig. 28). 4. Discussion

4.1. Somatic Embryogenesis in Sugarcane

In most of the previous studies also tissue cultures of sugarcane were initiated from young leaves, although often the terminal shoot meristem was included. Plant regeneration in the resulting callus cultures reportedly occurred by de novo organization of shoot meristems. Some authors have mentioned somatic embryogenesis in their reports but without substantiation (L~v and CHEN 1974, NADAR et al. 1978, ZENC 1979). No developmental stages or characteristic parts of the grass embryo were described, except the presence of a "coleoptile-like" structure (LIU and C HEN1974). Terms

such as "meristemoid", "shoot meristem", and "somatic embryo" often were used interchangeably to describe the same structure (LIu and CI4EN 1974), and claims were made of the origin of the somatic embryos from single cells without experimental evidence or justification (NADARet al. 1978). The present study provides definitive evidence of the regeneration of plants from somatic embryos formed in tissue cultures of sugarcane (see also Ho and VastI. 1983). In all of the previous studies on sugarcane, plant regeneration probably was also by somatic embryogenesis, although this might not have been recognized for the following reasons: (1) No detailed morphological or developmental studies of plant regeneration were carried out. (2) The somatic embryos formed in vitro were often atypical and unlike zygotic embryos, and either were not recognized or were misinterpreted. (3) Precocious germination of embryoids may have taken place before they could be morphologically identified as embryoids. (4) In many of the embryoids the root meristem was either poorly developed or absent. Such germinating embryoids could easily have been confused with growing shoot buds. (5) Cytokinins were often added to the medium to induce shoot regeneration. This resulted in the formation of multiple shoot meristems in the embryoids as well as their precocious germination. (6) Many axillary buds developed following germination of the embryoids and added to the illusion that plant regeneration was taking place by the organization of shoot meristems.

4.2. Factors Controlling the Formation of Embryogenic Callus from Leaf

The developmental stage of each explant, both in relation to the age of the donor leaf and position on the leaf itself, strongly influenced the induction of embryogenic callus. In Panicum (Lu and VAS~I~ 1981) and Pennisetum (HAYDU and VASIL 1981), also callus and embryoid formation were shown to be related to the developmental stage of leaves at the time of culture. The leaf sheath portion of sugarcane had a better capacity for callus and embryoid formation than the leaf blade. The potential of the leaf sheath to form embryoids also has been demonstrated in Panicum (L~5 and VASIL 198l). CHAGVARDIEFFet al. (1981) too have described the variations in callus formation when leaf explants were taken from different levels of developing sugarcane leaves. Auxin plays a very important role in the induction of

WAI-JANEHO and INDRAK. VASIL:Somatic Embryogenesisin Sugarcane (Saccharum officinarum L.). I. embryogenic callus in sugarcane. Of all the growth regulators, 2,4-D has been shown to be the most potent for the induction of callus and formation of somatic embryos in cell and tissue cultures of grasses (VASIL e t al. 1982), including sugarcane. A wide range of 2,4-D concentrations (0.5 3.0 mg/1) was found to be suitable for induction of embryogenic callus. However, the appearance of embryogenic callus was delayed and more mucilaginous callus was produced in media containing high 2,4-D concentrations. In sorghum also increasing the concentration of 2,4-D encouraged the development of a gelatinous callus from the embryogenic callus (BRETTELLet al. 1980). Cytokinins had an inhibitory effect on sugarcane callus growth and such an effect also has been reported in other gramineous species (DumTset aI. 1975, DALEand DEAMBROGIO 1979). However, after the formation of proembryoids, cytokinins caused the initiation of multiple shoot meristems and precocious germination.

179

Although somatic embryos develop from single cells, such cells are neither physically isolated initially, nor surrounded by a specialized nutrient millieu as is the zygote. The physiological gradients and directed nutrient supplies available to the zygote are also lacking in a tissue culture system. It is not surprising, therefore, that embryoids formed in vitro often do not precisely follow the pattern of development of a zygotic embryo, particularly during the early stages of cell division and differentiation.

References BRETTELL, R. I. S., WERNICKE,W., THOMAS,E., 1980: Embryo-

genesisfromcultured immatureinflorescenceof Sorghum bicolor. Protoplasma 104, 141- 148. CHAGVARDIEFF,P., BONNEL, E., DEMARLY,Y., 1981 : La culture in

vitro de tissus somatiques de canne a sucre (Saccharum sp.). Agron. Trop. 36, 266-278. CHU, C. C., WANG,C. C., SUN,C. S., HSU, C.,YIN, K. C., CHU, C.Y.,

4.3. Ontogeny o f Somatic Embryos Evidence presented in this report shows that somatic embryos develop from single embryogenic cells, like those of Pennisetum americanum (VASlL and VASIL 1982). Tissue culture techniques have been applied in the improvement of the vegetatively propagated sugarcane primarily by selecting mutants and/or variants followed by clonal propagation (HEINZ et al. 1977). Plant regeneration through somatic embryos, which develop from single cells, should produce non-chimeric mutants which may be more useful in plant breeding programs. The fact that HEINZ (1973) observed only rare sectorial chimeras in induced mutants recovered from tissue and cell cultures of sugarcane supports our belief that in most of the previous investigations on sugarcane also plants were obtained only by somatic embryogenesis (see section 4.1.). The establishment of polarity in somatic embryos was delayed until after a few internal segmenting divisions had taken place in the embryogenic cell during the initial stages of embryoid development. The root meristem was often organized late and was sometimes absent. Similarly, the suspensor was either absent, or when present varied from a uni- or biseriate suspensor to a broad multicellular structure. These phenomena are common during the formation of nonzygotic embryos in angiosperms (HACCiUS and BHANDARI 1975). The variations observed in the development of somatic embryos of sugarcane are, therefore, neither unusual nor unexpected.

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regeneration from leaf tissues and anthers of Pennisetum purpureum Schum. Theor. appl. Genet. 59, 269--273. HEINZ, D. J., t973: Sugarcane improvement through induced mutations using vegetative propagules and cell culture techniques. In: Proc. Induced Mutations in VegetativelyPropagated Plants PL 501/5, 53 - 59. - KRISHNAMURTHI,M., NICKELL,L. G., MARETZK1,A., 1977:Cell, tissue and organ culture in sugarcane improvement. In: Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture (REINERT,J., BAJAJ,Y. P. S., eds.), pp. 3-17. BerlinHeidelberg-New York: Springer. HENDRE, R. R., MASCARENHAS,A. F., NADGIR,A. L., PATHAK,M., JAGANNATHAN,g., 1975:Growth of mosaicvirus-free sugarcane plants from apical meristem. Indian Phytopathol. 28, 175-178. Ho, W. J., VASlL,I. K., I983: Somatic embryogenesisin sugarcane (Saecharurn officinarum L.). II. The growth of and plant regeneration from embryogeniccell suspension cultures. Ann. Bot. 51, 719 - 726. KALAW,E. P., TOLENTINO,E. G., ROSARIO,E. L., 1977:Evaluation of sugarcaneclonesderivedformcallus tissues. PhilippineJ. Crop Sci. 2, 195-196. KOGA,Y., KUDO,M., 1977:An efficientmethod of producing callus and plantlets by tassel culture for sugarcane breeding. JARQ 11, 193-197.

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VASIL, I. K., 1982a: Plant cell culture and somatic cell genetics of cereals and grasses. In: Plant Improvement and Somatic Cell Genetics (VASIL, I. K., SCOWCROFT, W. R., FREY, K. J., eds.), pp. 179--203. New York: Academic Press. 1982 b: Somatic embryogenesis and plant regeneration in cereals and grasses. In: Plant Tissue Culture 1982 (FuJIWARA, A., ed.), pp. 101 - 104. Tokyo: Maruzen.

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