Plant Cell Reports
Plant Cell Reports (1988) 7: 144-147
© Springer-Verlag 1988
Somatic embryogenesis and plant regeneration in two-year old cultures of Zea diploperennis B. Swedlund and R. D. Locy NPI, 417 Wakara Way, Salt Lake City, UT 84108, USA Received April 19, 1987 / Revised version received February 15, 1988 - Communicated by I. K. Vasil
ABSTRACT
r~TERIALS AND METHODS
Immature embryos and immature l e a f tissues were used to establish embryogenic cultures of Zea diploperennis. Callus was induced on media containing MS salts and vitamins, sucrose (2% for leaves, 6% for embryos), 5% coconut milk and 1-6 mg/l 2,4-D. Embryogenic callus was maintained by subculturing on media containing MS salts and vitamins, 2% sucrose, 500 mg/l casein hydrolysate and i mg/l 2,4-D. Regeneration occurred when the 2,4-D level was reduced to 0.25 mg/l. Kinetin added at 0°25 mg/l further stimulated regeneration. Root t i p squashes on 10 plants regenerated a f t e r 2 years in culture indicated a normal 2n=20 chromosome number.
Zea diploperennis plants were grown from seed to maturity in the greenhouse. These plants were s e l f pollinated and 10 to 12 days a f t e r p o l l i n a t i o n inflorescences with developing kernels were harvested. These were disinfested by immersion in 1.25% sodium hypochlorite (1:5 d i l u t i o n of commercial laundry bleach) for 20 minutes° After rinsing the i n f l o r e scences with s t e r i l e deionized water, immature embryos were excised and placed on Murashige and Skoog (1962) (MS) salts and vitamins supplemented with 6% sucrose, 5% coconut milk, 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.8% agar. Embryos were oriented so that the embryo axis was in contact with the media. Embryos ranged in size from I-2 mm in length o
INTRODUCTION Somatic embryogenesis and plant regeneration in v i t r o has been reported for most of the economicaTly important cereal grasses ( f o r review see Vasil 1985)o Various meristematic tissues; i . e o , immature leaves, immature inflorescences, immature and/or mature embryos have been used as explants to i n i t i a t e these cultures ( B r e t t e l l et a l . 1980, Hanning and Conger 1982, Nabors et a l . 1983, Ozias-Akins and gasil 1982, 1983a,b, Boyes and Vasil 1984, Sun and Chu 1986). Some species such as Pennisetum americanum produce embryogenic callus quite r e a d i l y from several tissue sources (Vasil and Vasil 1981, 1982, Heyser and Nabors 1982, Botti and Vasil 1983) while others such as Z e a ~ a r e more r e c a l c i t r a n t , y i e l d i n g e m b r y o g e n i c ~ l u s only from very specific stages of but a few tissues; i o e . , immature embryos (Green 1982, Lu et a l . 1982, 1983, Duncan et a l . 1985). Genotype also influences the ease with which embryogenic callus can be obtained. In maize for example, many genotypes have been shown to form embryogenic callus, however, the frequency and extent of callus formation can be highly variable (Lu et alo 1983, Duncan et a l . 1985, Tomes and Smith 1985). In an attempt to improve the e f f i c i e n c y of plant regeneration from tissue cultures of the genus Zea, we have investigated Zo diploperennis, a wild r-Te~ative of Z. mays ( l l t i s - e t a l . 1979, N t i s 1983)o We report here culture conditions for establishing embryogenic callus of Z. diploperennis and discuss i t s possible usefulnes~ as a model system for Zea ma~v__So Offprint requests to: B. Swedlund
Another group of plants was severely cropped in order to encourage t i l l e r i n g . New growth was removed from the plants below the uppermost node and disinfested as above. The outer leaves were removed and sections 2 mm in length were cut from the region approximately I cm below to 2 cm above the shoot apex. These sections were placed on the above media modified by lowering the 2,4-D and sucrose levels to I mg/1 and 2% respectively and by replacing the coconut milk with 500 mg/l casein hydrolysate. After approximately 4 weeks, white, compact callus, comparable to embryogenic callus of other grass species, was removed and placed on MS salts and vitamins with 2% sucrose, 1 mg/1 2,4-D and 500 mg/1 casein hydrolysateo After each subculture nonembryogenic f r i a b l e callus, roots and/or shoots were removed and discarded; the remaining compact callus was teased apart and small pieces were transferred to fresh media. For plant regeneration, compact white callus was transferred to media with 0 or 0.25 mg/1 2,4-D, with or without 0.25 mg/l kinetino When plantlets reached 2 cm or more in length they were transferred to half strength MS salts and vitamins with 2% sucrose and 0°25 mg/l a-naphthaleneacetic acid (NAA)o After approximately 3 weeks plants were transferred to soil in the greenhouse and kept in high humidity for 3-4 days° Embryogenic callus was transferred to fresh media for growth studies° Small portions of callus were weighed and transferred to fresh media° There-
145 a f t e r , c a l l u s was weighed weekly f o r 3 weeks and the weight was recorded on a per plate basis. Percent dry weight was determined by subc u l t u r i n g s i m i l a r pieces of c a l l u s as f o r fresh weight studies° Callus from randomly selected plates was weighed to determine fresh weight, dried overnight in a 60 C oven, and then reweighed. Percent dry weight was calculated by d i v i d i n g the dry weight by the o r i g i n a l fresh weight. Chromosomes were counted in the parent plant and in 10 plants regenerated a f t e r 2 years in culture° The procedure was as described previously (Swedlund and Vasil 1985). For each p l a n t , at least 3 root t i p s , 3 c e l l s per root t i p , and a t o t a l of 10 c e l l s were examined° Zea diploperennis l i k e Zea mays has 2n=20 chromosomes. Callus pieces were f i x e d in 2.5% gluta{aldehyde and p o s t - f i x e d in 1% osmium t e t r o x i d e . These were then treated by immersion in a saturated solution of thiocarbohydrazide for 30 minutes followed by 30 minutes in i% osmium t e t r o x i d e . This l a t t e r step reduced charging of specimens in the electron microscope and therefore improved the c l a r i t y of micrographs. Specimens were dehydrated in ethanol, c r i t i c a l point dried, sputter coated with gold, and viewed and photographed in a Hitachi S-450 scanning electron microscope using normal procedures. RESULTS AND DISCUSSION Callus i n i t i a t i o n Within one week, the s c u t e l l a of immature embryos began to swell. Callus began to form on s c u t e l l a in the region most proximal to the coleorhizao I n i t i a l l y , almost a l l of the c a l l u s formed
Figure i . Embryoids (em), embryogenic c a l l u s (ec), nonembryogenic c a l l u s (nec), and a leaf ( I f ) from a precociously germinating embryoido Note shiny, watery appearance of the nonembryogenic c a l l u s , and white, h i g h l y organized appearance of the embryogenic callus°
was composed of small, densely cytoplasmic, starchy c e l l s subsequently shown to be embryogenic c e l l s . Approximately 75% of the embryos cultured gave rise to t h i s type of c a l l u s . Large, elongated, h i g h l y vacuolated c e l l s began to appear s h o r t l y afterward. These c e l l s subsequently did not form embryos and are referred to as nonembryogenic c e l l s . Nonembryogenic callus arose from nearly a l l of the stem and immature l e a f tissues cultured regardless of t h e i r p o s i t i o n in r e l a t i o n to the shoot meristem. However, embryogenic c a l l u s only arose from approximately 5% of the immature l e a f explants taken from the region 0.5 to 1.0 cm above the shoot apex. Such callus was always associated with developing vascular bundles as has been reported f o r other genera of the Gramineae (Vasil 1985)o Nonembryogenic c a l l u s began to appear a f t e r 7 days whereas embryogenic callus was not v i s i b l e u n t i l day 10. Culture maintenance and growth A f t e r 4-6 weeks on i n i t i a t i o n media, embryoids could be discerned along with less organized compact c a l l u s ( f i g . 1,2) and very unorganized nonembryogenic callus ( f i g . I ) . Compact c a l l u s , including well formed embryoids, was transferred to maintenance media. I n i t i a l l y , maintenance media contained 5% coconut milk. Since the coconut milk that we could r e a d i l y obtain varied considerably in q u a l i t y , we sought other more consistent organic addenda. Casein hydrolysate has often been used in cultures of cereals (Gray and Conger 1985) and has proved to be very acceptable f o r cultures of Z. dj]oloperennis. In subsequent studies casein hydroly'sate has replaced coconut milk f o r both i n i t i a t i o n and maintenance media.
Figure 20 SEM of a t y p i c a l somatic embryo on the surface of Z. diploperennis c a l l u s . F u l l y developed embryoids such as t h i s appeared more f r e q u e n t l y near the end of a subculture i n t e r v a l . Coleoptile ( c l ) , scutellum (sc).
146 At each subculture, nonembryogenic callus and any roots or shoots which developed were removed and discarded. Nonembryogenic callus not removed was found to overgrow embryogenic callus in 2-3 subcultures° Thus, the visual selection and culture of the compact embryogenic callus was necessary to maintain regenerable callus cultures. Subculture i n t e r v a l s of 3 weeks were most e f f i c i e n t in maintaining embryogenic callus cultures° During a 3 week subculture, callus growth, as measured by fresh weight gains, generally showed a s l i g h t i n i t i a l lag phase followed by exponential growth ( f i g ° 3). Percent dry weight peaked between one and two weeks and f e l l somewhat thereafter (fig° 4)° This correlates well with the formation of nonembryogenic callus which accumulates more r a p i d l y towards the end of the subculture i n t e r v a l . Subculture i n t e r v a l s of longer than 3 weeks resulted in a greater accumulation of nonembryogenic c e l l s and a general decrease in growth rate. Regeneration Regeneration occurred via somatic embryogenesis. Well developed embryoids were c h a r a c t e r i s t i c a l l y part of the embryogenic callus ( f i g . 1,2)o Near the end of a subculture i n t e r v a l , embryoids would occasionally germinate spontaneously ( f i g . 1). However, consistent regeneration occurred when the 2,4-D level was lowered to 0 or 0°25 mg/l. I~re embryoids developed and subsequently germinated on media with 0.25 mg/l 2,4-D than without 2,4-D. Plantlets developed more rapidly when kinetin was added to the media, but the number of embryoids germinating did not appear to increase. Each gram of callus yielded approximately 35 plants when the 2,4-D and kinetin levels were at 0.25 mn/l {data not shown).
Chromosome numbers Regenerated plants did not d i f f e r in chromosome number or general morphology from the original plants placed in culture° All had 2n=20 chromosomes. Control root t i p s , as well as root tips from regenerants, had a few c e l l s which contained 19 chromosomeso Although the number of plants examined c y t o l o g i c a l l y was small, t h e i r apparent chromosomal s t a b i l i t y is consistent with other reports in which plants were regenerated via somatic embryogenesis (Vasil 1983, Hanna et a l . 1984, Swedlund and Vasil 1985). Also, no gross morphological changes have been observed in any of nearly 300 soil grown regenerants. Many cereal species form some albinos upon regeneration (Gamborget a l . 1970, Dale and Dalton 1983, Swedlund and Vasil 1985) and albinism often increases with culture age (Chandler and Vasil 1984)o However no albinos have been observed in any of our cultures over the 3 years of callus growth. P r i o l i et a l . (1984) cultured apical and l a t e r a l meristems of Z. diploperennis and reported the formation of ~ semifriable Callus from which adventitious shoots arose. Regeneration could s t i l l be achieved a f t e r 3-4 subculture i n t e r v a l s . In contrast we report plant regeneration via somatic embryogenesis in a compact callus which has retained i t s regeneration capacity for more than 3 years (approximately 45 subculture i n t e r v a l s ) . This embryogenic callus can be obtained from both immature leaves and immature somatic embryos, grows reasonably f a s t , and in our hands, tends to be somewhat more p l i a b l e in culture than Z. mays. I t s apparent s t a b i l i t y and long-term r e g e n ~ r a ~ t y make i t an additional a t t r a c t i v e system for the study of somatic embryogenesis in cerealso
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Figure 3. Callus fresh weight gain during a typical subculture i n t e r v a l . Generally, a lag in fresh weight gain was followed by exponential growth. Data points are an average of 5 plates, 5 callus pieces per plate, replicated 3 times°
0.0
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Figure 4. Percent dry weight of callus over a subculture i n t e r v a l . Greatest percent dry weight was consistently one week a f t e r subculture. Data collected e s s e n t i a l l y as for Figure 3.
147 ACKNOWLEDGEMENTS We wish to thank Dr. Edward King for advice and assistance with SEM. The technical assistance of Melanie Sesin and Tracey Hill is also gratefully acknowledged° REFERENCES Botti C, Vasil IK (1983) Z. Pflanzenphysiol. 111:319-325 8oyes CJ, Vasil IK (1984) Plant Sci. Letto 35: 153-157 Brettell RIS, Wernicke, W, Thomas E (1980) Protoplasma 104:141-145 Chandler SF, Vasil IK (1984) Jo Plant Physiol. 117:147-156 Dale PJ, Dalton SJ (1983) Z. Pflanzenphysiol. 111:39-45 Duncan DR, Williams ME, Zehr BE, Widholm JM (1985) Planta 165:322-332 Gamborg OL, Constabel F, Miller RA (1970) Planta 95:355-358 Gray DJ, Conger BV (1985) Plant Cell Tisso Org. Cult. 4:123-133 Green CE (1982) In: Fujiwara A (ed) Proco 5th Into Congro Plant Tissue Culture, Maruzen Co., Ltdo, Tokyo, pp 107-108 Hanna WW, Lu C, Vasil IK (1984) Theor. Appl. Genet. 67:155-159 Hanning GE, Conger BV (1982) Theor. Appl. Genet. 63:155-159 Heyser JW, Nabors MW (1982) Crop Sci. 22:1070-1074 l l t i s HH (1983) Science 222:886-894
l l t i s HH, Doebley JF, Guzman MR, Pazy B (1979) Science 203:186-188 Lu C, Vasil IK, Ozias-Akins P (1982) Theor. Appl. Geneto 62:109-112 Lu C, Vasil V, Vasil IK (1983) Theor. Applo Geneto 66:285-289 Murashige T, Skoog F (1962) Physiolo Plant° 15: 473-497 Nabors MW, Heyser JW, Dykes TA, DeMott KJ (1983) Planta 157:385-391 Ozias-Akins P, Vasil IK (1982) Protoplasma 110:95-105 Ozias-Akins P, Vasil IK (19~3a) Protoplasma 115:104-113 Ozias-Akins P, Vasil IK (1983b) Protoplasma 117:40-44 Prioli LM, Silva WJ, Sondahl MR (1984) Jo Plant Physiolo 117:185-190 Sun CS, Chu CC (1986) Plant Cell Tisso Org. Cult. 5:175-178 Swedlund B, Vasil IK (1985) Theor. Applo Geneto 69:575-581 Tomes DT, Smith OS (1985) Theor. Appl. Geneto 70:505-509 Vasil IK (1983) In: Lurquin, P, Kleinhofs, A (eds) Genetic Engineering in Eukaryotes, Plenum, New York pp 233-252 Vasil IK (1985) In: Henke, RR, Hughes, KW, Constantin, MP, Hollaender, A (eds) Tissue Culture in Forestry and Agriculture, Plenum, New York pp 31-47 Vasil V, Vasil IK (1981) Amero Jo Boto 68:864-872 Vasil V, Vasil IK (1982) Boto Gazo 143:454-465
Plant Cell Reports (1988) 7: 144-147
© Springer-Verlag 1988
Somatic embryogenesis and plant regeneration in two-year old cultures of Zea diploperennis B. Swedlund and R. D. Locy NPI, 417 Wakara Way, Salt Lake City, UT 84108, USA Received April 19, 1987 / Revised version received February 15, 1988 - Communicated by I. K. Vasil
ABSTRACT
r~TERIALS AND METHODS
Immature embryos and immature l e a f tissues were used to establish embryogenic cultures of Zea diploperennis. Callus was induced on media containing MS salts and vitamins, sucrose (2% for leaves, 6% for embryos), 5% coconut milk and 1-6 mg/l 2,4-D. Embryogenic callus was maintained by subculturing on media containing MS salts and vitamins, 2% sucrose, 500 mg/l casein hydrolysate and i mg/l 2,4-D. Regeneration occurred when the 2,4-D level was reduced to 0.25 mg/l. Kinetin added at 0°25 mg/l further stimulated regeneration. Root t i p squashes on 10 plants regenerated a f t e r 2 years in culture indicated a normal 2n=20 chromosome number.
Zea diploperennis plants were grown from seed to maturity in the greenhouse. These plants were s e l f pollinated and 10 to 12 days a f t e r p o l l i n a t i o n inflorescences with developing kernels were harvested. These were disinfested by immersion in 1.25% sodium hypochlorite (1:5 d i l u t i o n of commercial laundry bleach) for 20 minutes° After rinsing the i n f l o r e scences with s t e r i l e deionized water, immature embryos were excised and placed on Murashige and Skoog (1962) (MS) salts and vitamins supplemented with 6% sucrose, 5% coconut milk, 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.8% agar. Embryos were oriented so that the embryo axis was in contact with the media. Embryos ranged in size from I-2 mm in length o
INTRODUCTION Somatic embryogenesis and plant regeneration in v i t r o has been reported for most of the economicaTly important cereal grasses ( f o r review see Vasil 1985)o Various meristematic tissues; i . e o , immature leaves, immature inflorescences, immature and/or mature embryos have been used as explants to i n i t i a t e these cultures ( B r e t t e l l et a l . 1980, Hanning and Conger 1982, Nabors et a l . 1983, Ozias-Akins and gasil 1982, 1983a,b, Boyes and Vasil 1984, Sun and Chu 1986). Some species such as Pennisetum americanum produce embryogenic callus quite r e a d i l y from several tissue sources (Vasil and Vasil 1981, 1982, Heyser and Nabors 1982, Botti and Vasil 1983) while others such as Z e a ~ a r e more r e c a l c i t r a n t , y i e l d i n g e m b r y o g e n i c ~ l u s only from very specific stages of but a few tissues; i o e . , immature embryos (Green 1982, Lu et a l . 1982, 1983, Duncan et a l . 1985). Genotype also influences the ease with which embryogenic callus can be obtained. In maize for example, many genotypes have been shown to form embryogenic callus, however, the frequency and extent of callus formation can be highly variable (Lu et alo 1983, Duncan et a l . 1985, Tomes and Smith 1985). In an attempt to improve the e f f i c i e n c y of plant regeneration from tissue cultures of the genus Zea, we have investigated Zo diploperennis, a wild r-Te~ative of Z. mays ( l l t i s - e t a l . 1979, N t i s 1983)o We report here culture conditions for establishing embryogenic callus of Z. diploperennis and discuss i t s possible usefulnes~ as a model system for Zea ma~v__So Offprint requests to: B. Swedlund
Another group of plants was severely cropped in order to encourage t i l l e r i n g . New growth was removed from the plants below the uppermost node and disinfested as above. The outer leaves were removed and sections 2 mm in length were cut from the region approximately I cm below to 2 cm above the shoot apex. These sections were placed on the above media modified by lowering the 2,4-D and sucrose levels to I mg/1 and 2% respectively and by replacing the coconut milk with 500 mg/l casein hydrolysate. After approximately 4 weeks, white, compact callus, comparable to embryogenic callus of other grass species, was removed and placed on MS salts and vitamins with 2% sucrose, 1 mg/1 2,4-D and 500 mg/1 casein hydrolysateo After each subculture nonembryogenic f r i a b l e callus, roots and/or shoots were removed and discarded; the remaining compact callus was teased apart and small pieces were transferred to fresh media. For plant regeneration, compact white callus was transferred to media with 0 or 0.25 mg/1 2,4-D, with or without 0.25 mg/l kinetino When plantlets reached 2 cm or more in length they were transferred to half strength MS salts and vitamins with 2% sucrose and 0°25 mg/l a-naphthaleneacetic acid (NAA)o After approximately 3 weeks plants were transferred to soil in the greenhouse and kept in high humidity for 3-4 days° Embryogenic callus was transferred to fresh media for growth studies° Small portions of callus were weighed and transferred to fresh media° There-
145 a f t e r , c a l l u s was weighed weekly f o r 3 weeks and the weight was recorded on a per plate basis. Percent dry weight was determined by subc u l t u r i n g s i m i l a r pieces of c a l l u s as f o r fresh weight studies° Callus from randomly selected plates was weighed to determine fresh weight, dried overnight in a 60 C oven, and then reweighed. Percent dry weight was calculated by d i v i d i n g the dry weight by the o r i g i n a l fresh weight. Chromosomes were counted in the parent plant and in 10 plants regenerated a f t e r 2 years in culture° The procedure was as described previously (Swedlund and Vasil 1985). For each p l a n t , at least 3 root t i p s , 3 c e l l s per root t i p , and a t o t a l of 10 c e l l s were examined° Zea diploperennis l i k e Zea mays has 2n=20 chromosomes. Callus pieces were f i x e d in 2.5% gluta{aldehyde and p o s t - f i x e d in 1% osmium t e t r o x i d e . These were then treated by immersion in a saturated solution of thiocarbohydrazide for 30 minutes followed by 30 minutes in i% osmium t e t r o x i d e . This l a t t e r step reduced charging of specimens in the electron microscope and therefore improved the c l a r i t y of micrographs. Specimens were dehydrated in ethanol, c r i t i c a l point dried, sputter coated with gold, and viewed and photographed in a Hitachi S-450 scanning electron microscope using normal procedures. RESULTS AND DISCUSSION Callus i n i t i a t i o n Within one week, the s c u t e l l a of immature embryos began to swell. Callus began to form on s c u t e l l a in the region most proximal to the coleorhizao I n i t i a l l y , almost a l l of the c a l l u s formed
Figure i . Embryoids (em), embryogenic c a l l u s (ec), nonembryogenic c a l l u s (nec), and a leaf ( I f ) from a precociously germinating embryoido Note shiny, watery appearance of the nonembryogenic c a l l u s , and white, h i g h l y organized appearance of the embryogenic callus°
was composed of small, densely cytoplasmic, starchy c e l l s subsequently shown to be embryogenic c e l l s . Approximately 75% of the embryos cultured gave rise to t h i s type of c a l l u s . Large, elongated, h i g h l y vacuolated c e l l s began to appear s h o r t l y afterward. These c e l l s subsequently did not form embryos and are referred to as nonembryogenic c e l l s . Nonembryogenic callus arose from nearly a l l of the stem and immature l e a f tissues cultured regardless of t h e i r p o s i t i o n in r e l a t i o n to the shoot meristem. However, embryogenic c a l l u s only arose from approximately 5% of the immature l e a f explants taken from the region 0.5 to 1.0 cm above the shoot apex. Such callus was always associated with developing vascular bundles as has been reported f o r other genera of the Gramineae (Vasil 1985)o Nonembryogenic c a l l u s began to appear a f t e r 7 days whereas embryogenic callus was not v i s i b l e u n t i l day 10. Culture maintenance and growth A f t e r 4-6 weeks on i n i t i a t i o n media, embryoids could be discerned along with less organized compact c a l l u s ( f i g . 1,2) and very unorganized nonembryogenic callus ( f i g . I ) . Compact c a l l u s , including well formed embryoids, was transferred to maintenance media. I n i t i a l l y , maintenance media contained 5% coconut milk. Since the coconut milk that we could r e a d i l y obtain varied considerably in q u a l i t y , we sought other more consistent organic addenda. Casein hydrolysate has often been used in cultures of cereals (Gray and Conger 1985) and has proved to be very acceptable f o r cultures of Z. dj]oloperennis. In subsequent studies casein hydroly'sate has replaced coconut milk f o r both i n i t i a t i o n and maintenance media.
Figure 20 SEM of a t y p i c a l somatic embryo on the surface of Z. diploperennis c a l l u s . F u l l y developed embryoids such as t h i s appeared more f r e q u e n t l y near the end of a subculture i n t e r v a l . Coleoptile ( c l ) , scutellum (sc).
146 At each subculture, nonembryogenic callus and any roots or shoots which developed were removed and discarded. Nonembryogenic callus not removed was found to overgrow embryogenic callus in 2-3 subcultures° Thus, the visual selection and culture of the compact embryogenic callus was necessary to maintain regenerable callus cultures. Subculture i n t e r v a l s of 3 weeks were most e f f i c i e n t in maintaining embryogenic callus cultures° During a 3 week subculture, callus growth, as measured by fresh weight gains, generally showed a s l i g h t i n i t i a l lag phase followed by exponential growth ( f i g ° 3). Percent dry weight peaked between one and two weeks and f e l l somewhat thereafter (fig° 4)° This correlates well with the formation of nonembryogenic callus which accumulates more r a p i d l y towards the end of the subculture i n t e r v a l . Subculture i n t e r v a l s of longer than 3 weeks resulted in a greater accumulation of nonembryogenic c e l l s and a general decrease in growth rate. Regeneration Regeneration occurred via somatic embryogenesis. Well developed embryoids were c h a r a c t e r i s t i c a l l y part of the embryogenic callus ( f i g . 1,2)o Near the end of a subculture i n t e r v a l , embryoids would occasionally germinate spontaneously ( f i g . 1). However, consistent regeneration occurred when the 2,4-D level was lowered to 0 or 0°25 mg/l. I~re embryoids developed and subsequently germinated on media with 0.25 mg/l 2,4-D than without 2,4-D. Plantlets developed more rapidly when kinetin was added to the media, but the number of embryoids germinating did not appear to increase. Each gram of callus yielded approximately 35 plants when the 2,4-D and kinetin levels were at 0.25 mn/l {data not shown).
Chromosome numbers Regenerated plants did not d i f f e r in chromosome number or general morphology from the original plants placed in culture° All had 2n=20 chromosomes. Control root t i p s , as well as root tips from regenerants, had a few c e l l s which contained 19 chromosomeso Although the number of plants examined c y t o l o g i c a l l y was small, t h e i r apparent chromosomal s t a b i l i t y is consistent with other reports in which plants were regenerated via somatic embryogenesis (Vasil 1983, Hanna et a l . 1984, Swedlund and Vasil 1985). Also, no gross morphological changes have been observed in any of nearly 300 soil grown regenerants. Many cereal species form some albinos upon regeneration (Gamborget a l . 1970, Dale and Dalton 1983, Swedlund and Vasil 1985) and albinism often increases with culture age (Chandler and Vasil 1984)o However no albinos have been observed in any of our cultures over the 3 years of callus growth. P r i o l i et a l . (1984) cultured apical and l a t e r a l meristems of Z. diploperennis and reported the formation of ~ semifriable Callus from which adventitious shoots arose. Regeneration could s t i l l be achieved a f t e r 3-4 subculture i n t e r v a l s . In contrast we report plant regeneration via somatic embryogenesis in a compact callus which has retained i t s regeneration capacity for more than 3 years (approximately 45 subculture i n t e r v a l s ) . This embryogenic callus can be obtained from both immature leaves and immature somatic embryos, grows reasonably f a s t , and in our hands, tends to be somewhat more p l i a b l e in culture than Z. mays. I t s apparent s t a b i l i t y and long-term r e g e n ~ r a ~ t y make i t an additional a t t r a c t i v e system for the study of somatic embryogenesis in cerealso
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Figure 3. Callus fresh weight gain during a typical subculture i n t e r v a l . Generally, a lag in fresh weight gain was followed by exponential growth. Data points are an average of 5 plates, 5 callus pieces per plate, replicated 3 times°
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3.0
WEEK
Figure 4. Percent dry weight of callus over a subculture i n t e r v a l . Greatest percent dry weight was consistently one week a f t e r subculture. Data collected e s s e n t i a l l y as for Figure 3.
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