Plant Cell Reports
Plant Cell Reports (1984) 3:112-115
© Springer-Verlag 1984
Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.) Jang R. Liu and Daniel J. Cantliffe Vegetable Crops Department, IFAS, University of Florida, GainesviUe, FL 32611, USA Received March 13, 1984/Revised version received May 7, 1984 - Communicated by L. K. Vasil
Abstract Leaf, s h o o t - t i p , stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, b r i g h t - to pale-yellow, was compact and organized, while the other was d u l l - y e l l o w and f r i a b l e . The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids r e a d i l y developed into a torpedo-shape before germination. The p l a n t l e t s were transplanted to soil where they flowered and formed storage roots at maturity. Abbreviation: 2,4-D = 2,4-dichlorophenoxyacetic acid Introduction Achievement of crop improvement through plant cell and tissue culture techniques is dependent upon success in plant regeneration. The sweet potato is an important food crop which provides carbohydrate and protein to a large sector of the world population. Annual world food production of sweet potato in tons ranks s i x t h , d i r e c t l y behind barley (Harlan 1976). Sweet potato also has potential as a biomass species for methane and ethanol production. Despite i t s enormous significance in world a g r i c u l t u r e , studies related to cell and tissue culture of this species have been few. Adventitious shoots were induced from callus derived from root discs (Yamaguchi and Nakajima 1973), or from l e a f explants (Sehgal 1975). I t was reported that roots (Kobayashi and Shikata 1975), shoots (Sehgal 1978), and embryoids (Tsay and Tseng 1979) were induced from anther-derived callus. Petiole protoplasts and l e a f mesophyll c e l l s isolated enzymatically formed callus from which roots were produced (Bidney and Shepard 1980). This paper describes the formation of callus, embryoids and plants from various explants of sweet potato. Materials and Methods Roots of sweet potato (Ipomea batatas Poir.) c u l t i v a r s White Star and GaTG 3 were potted in a soiless mix ( p e a t : v e r m i c u l i t e : p e r l i t e ) and maintained in growth chambers at 27°C day/22°C night under a 16 h photoperiod. The plants were
Offprint requests to: J. R. Liu
f e r t i l i z e d weekly and watered as needed. Stock plants were maintained in the growth chamber for periods of up to one year, however they were kept in a highly vegetative state by constantly cutting back the sterns. Plants developed from in v i t r o shoot-tip cultures were used as a source of l e a f and shoot-tip explants. Shoot-tips were excised from highly p r o l i f e r a t i n g plants in growth chambers and the f u l l y expanded leaves were removed. The remaining part of the shoot-tip was disinfected with I0% Clorox for lO min under p a r t i a l vacuum, Procedures a f t e r d i s i n f e c t i o n were conducted under s t e r i l e conditions. Shoot-tips containing a few young leaves were rinsed 3 times with s t e r i l e water and trimmed to 5-I0 mm-long s t r i p s . They were placed onto basal medium containing Murashige and Skoog (1962) inorganic s a l t s , lO0 mg/l m y o - i n o s i t o l , 0.4 mg/l thiamine-HCl, 30 g/l sucrose, and 8 g/l Phytagar (GIBCO) in lO0 x 15 mm p l a s t i c Petri dishes. In some experiments, 0.3 mg/l 6-benzylaminopurine was added to basal medium to accelerate the growth of shoot-tips. Each dish contained 7 shoot-tips and was wrapped with Parafilm. All media were adjusted to pH 5.8 befsre autoclaving in 200-ml aliquots for 15 Bin at 121 C. The cultures were incubated at 27 C under 16 h of cool-white fluorescent l i g h t at an i n t e n s i t y of l,O00 lux. One week a f t e r incubation, leaves started to expand from the shoot-tips and formed roots at the basal end. After 2 to 4 weeks of incubation, well-expanded leaves, lO-15 mm-long, were cut into 2 mm-wide strips with l e a f margins i n t a c t . An explant consisted of a l l of the strips taken from one l e a f . Leaf explants were placed with e i t h e r the abaxial or adaxial surface down on callus induction medium, i . e . , basal medium plus 60 g/l sucrose and 2,4-D at 0.5 to 4.0 mg/l. Shoot-tips, 3-5 mm-long, containing 4-5 pairs of l e a f primordia and young leaves, were separately excised for culture in v i t r o . Stem segments, 5 mm-long, were taken from a 15 cm-long internodal section d i r e c t l y behind the apex on plants maintained in growth chambers. They were disinfected as described above. Freshly harvested roots, lO-15 cm-long, were disinfected with 5% Clorox for 30 min, dried and cut transversely into 20 mm-long slices. A series of cores with the cambium layer i n t a c t were excised from the slices with a No.l cork borer. These cores were cut transversely into 5 mm-long segments and used as root explants.
113 Five explants of leaves, shoot-tips, stems, and roots were placed separately on callus induction medium in lO0 x 15 mm Petri dishes. All cultures were incubated at 27°C in the dark and observed p e r i o d i c a l l y under a dissecting microscope for embryogenic callus formation.
Table I.
Embryogenic callus formation from sweet potato explants on medium containing l.O mg/l 2,4-D.
Percentage of explants forming embryogenic callus
Results
Cultivar
One to 2 weeks a f t e r incubation, l e a f , shoot-tips, and stem explants started to lose t h e i r bright-green or purple color and to form d u l l - y e l l o w , f r i a b l e callus on t h e i r surface. This callus did not give rise to embryoids. Three to 8 weeks a f t e r incubation, pale- to b r i g h t - y e l l o w , compact and organized callus emerged from the surface of explants (Fig. l a ) . When observed with a dissecting microscope, the callus was d i s t i n c t by color and texture from the f r i a b l e c a l l u s . When observed under a l i g h t microscope, the locus was composed of t i g h t l y packed isodiametric c e l l s which contained numerous starch grains. This callus was embryogenic: i t developed into globular embryoids, which developed further into heart-shaped embryoids, and which were usually attached to the callus through suspensor-like structures. The abaxial surface of the l e a f explants appeared to form more embryogenic callus than the adaxial surface. Young leaves and l e a f primordia of the shoot-tip explant produced embryogenic callus The cut edge of the strips of l e a f explant did not produce embryogenic callus while in stem and root explants i t did. Portions of the explant submerged in the medium did not produce embryogenic callus. As long as the cultures remained on callus induction medium, most embryoids did not develop beyond the torpedo-shaped stage (Fig. I b ) . Other embryoids developed continuously by budding from the surface of cotyledons and hypocotyls of embryoids, along with p r o l i f e r a t i o n of both of the embryogenic and non-embryogenic callus. When transferred to basal medium, heart-shaped embryoids r e a d i l y developed into torpedo-shaped embryoids (Fig. Ic) and then germinated (Fig. Id). Regenerated p l a n t l e t s often had poorly developed cotyledons and/or hypocotyls. Over 40% of cotyledon and hypocotyl segments from regenerated p l a n t l e t s also produced embryogenic callus on medium containing l.O mg/l 2,4-D. Each p l a n t l e t was placed on basal medium in 25 x 150 mm test tubes under l i g h t . At least 50 plants of each c u l t i v a r were transplanted to soil when they were 5-10 cm-high. The plants grew rapidly (Fig. le) and a f t e r 5 months in growth chambers, when they were over 3 m-long, they flowered, and produced storage roots (15 cm and larger in diameter). The plants derived via tissue culture were phenot y p i c a l l y the same as the donor plant. No embryogenic callus was formed from explants on basal medium. Embryogenic callus was generally not produced from l e a f and shoot-tip explants on media containing over 2.0 mg/l 2,4-D, but media containing 0.5-2.0 mg/l 2,4-D were ideal for induction of embryogenic callus. Embryogenic callus was observed from stem and root explants only on medium containing l.O mg/l 2,4-D. Leaf explants produced embryogenic callus at much higher rates than s h o o t - t i p , stem, and root callus (Table l ) . When embryogenic callus was subcultured on callus induction medium containing 2.0 mg/l 2,4-D supplemented with 2.0 mg/l k i n e t i n and 20% (v/v) deproteinized coconut water (GIBCO), s a t i s f a c t o r y m i t o t i c d i v i s i o n of the callus was
White Star GaTG 3
Leaf a
Shoot-tip b
23 22
3 5
Stem b
Root b
l l
-
a I00 (White Star) and 37 (GaTG 3) explants respectively. b I00-200 e x p l a n t s , c
Not c u l t u r e d .
sustained and a low rate of heart or torpedo-shaped embryoid formation was observed (Fig. I f ) . At each subculture, non-embryogenic f r i a b l e callus which was attached to the embryogenic callus was r o u t i n e l y removed. When subcultured monthly, i t retained i t s embryogenic capacity for over 9 months. The addition of e i t h e r k i n e t i n (O.Ol-O.l mg/l), glutamine (200-800 mg/l), proline (lO.O-l,O00 mg/l), or coconut water (5-20%), to callus induction medium containing l.O mg/l 2,4-D did not increase the frequency of embryogenic callus formation from stem explants. Flower buds at various developmental stages were excised and disinfected as previously described for shoot-tips. After c u l t u r i n g in the same manner as shoot-tip explants, no embryogenic callus was obtained. Discussion The formation of the embryogenic callus in this study was similar to that reported in Gramineae (e.g., Pennisetum; Haydu and Vasil 1981) and Rubiaceae (e.g., coffee; Staritsky 1970). I t was compact and composed of isodiametric, starch-rich c e l l s . Conversion of the embryogenic to non-embryogenic callus Was i r r e v e r s i b l e , since prolonged culture did not induce embryogenic from non-embryogenic c a l l u s . Likewise, non-embryogenic callus derived d i r e c t l y from the explant did not become embryogenic. Under conditions s i m i l a r to the present work, Sehgal (1975) observed only f r i a b l e callus from l e a f explants. When transferred to medium containing e i t h e r adenine or k i n e t i n , the callus gave rise to shoot buds and roots. Atypical p l a n t l e t s appear to be due to a lack of proper growth conditions rather than genetic change since mature plants showed no phenotypic abnormalities. Ozias-Akins and Vasil (1982) proposed that atypical p l a n t l e t s of wheat resulted from precocious germination of somatic embryos. Genetic variants are often observed in plants regenerated from callus cultures. However no prominent evidence has been reported that somatic embryogenesis induced through an intermediary callus leads to genetic v a r i a b i l i t y in the regenerated plant. I t is possible to recover n a t u r a l l y occurring mutant c e l l s from vegetatively propagated plants, such as sweet potato, in which c e l l u l a r v a r i a t i o n presumably accumulates, and produce whole plants via somatic embryogenesis. Regenerated plants from n a t u r a l l y occurring mutant c e l l s are usually much more valuable in crop improvement ( e . g . , cold-, heat-, s a l t - , and disease-resistant selections) than those from mutagen-induced mutant c e l l s . Furthermore, somatic embryos are of single cell o r i g i n (Haccius 1978), so regenerated plants are not l i k e l y to be chimeral. Large numbers of
114
Fig. I. Developmental sequence of somatic embryo and plantlet formation in sweet potato. (a) Embryogenic callus (arrows) arising from leaf tissue (xl8), (b) Proliferating embryogenic callus from leaf tissue showing globular, heart- and torpedo-shaped embryoids (xl4), (c) Numerous heart- and torpedo-shaped embryoids from embryogenic callus (x7.5), (d) Germination of embryoids into plantlets (x7), (e) Maturing plants regernerated from embryogenic callus, (f) Subcultured embryogenic callus with a low rate of embryoid production (xl5)
embryoids can be developed in suspension culture and might enable an otherwise vegetatively propagated crop to be seeded mechanically with the aid of techniques such as f l u i d d r i l l i n g . Propagation of sweet potato in this way is under investigation.
Acknowledgements We thank the Gas Research I n s t i t u t e , Chicago, IL. for partial support of this research. Florida Agricultural Experiment Station Journal Series 5453.
115 References Bidney DL, Shepard JF (1980) Plant Sci Let 18:335342 Haccius B (1978) Phytomorphology 28:74-81 Harlan JR (1976) Sci Am 235:89-97 Haydu Z, Vasil IK (1981) Theor Appl Genet 59:269-273 Kobayashi M, Shikata SI (1975) Bull Chugoko Natl Agric Exp Stn Set A24:109-124 Murashige T, Skoog F (1962) Physiol Plant 15:473-497
Ozias-Akins P, Vasil IK (1982) Protoplasma II0:95-I05 Sehgal CB (1975) Beitr Biol Pflanzen 51:47-52 Sehgal CB (1978) Z Pflanzenphysiol Bd 88:349-352 Staritsky G (1970) Acta Bot Neerl 19:509-514 Tsay HS, Tseng MT (1979) Bot Bull Academia Sinica 20:I17-122 Yamaguchi J, Nakajima T (1973) In: Proceedings of the 8th international conference on plant growth substances, Tokyo, pp l121-1127
Plant Cell Reports (1984) 3:112-115
© Springer-Verlag 1984
Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.) Jang R. Liu and Daniel J. Cantliffe Vegetable Crops Department, IFAS, University of Florida, GainesviUe, FL 32611, USA Received March 13, 1984/Revised version received May 7, 1984 - Communicated by L. K. Vasil
Abstract Leaf, s h o o t - t i p , stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, b r i g h t - to pale-yellow, was compact and organized, while the other was d u l l - y e l l o w and f r i a b l e . The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids r e a d i l y developed into a torpedo-shape before germination. The p l a n t l e t s were transplanted to soil where they flowered and formed storage roots at maturity. Abbreviation: 2,4-D = 2,4-dichlorophenoxyacetic acid Introduction Achievement of crop improvement through plant cell and tissue culture techniques is dependent upon success in plant regeneration. The sweet potato is an important food crop which provides carbohydrate and protein to a large sector of the world population. Annual world food production of sweet potato in tons ranks s i x t h , d i r e c t l y behind barley (Harlan 1976). Sweet potato also has potential as a biomass species for methane and ethanol production. Despite i t s enormous significance in world a g r i c u l t u r e , studies related to cell and tissue culture of this species have been few. Adventitious shoots were induced from callus derived from root discs (Yamaguchi and Nakajima 1973), or from l e a f explants (Sehgal 1975). I t was reported that roots (Kobayashi and Shikata 1975), shoots (Sehgal 1978), and embryoids (Tsay and Tseng 1979) were induced from anther-derived callus. Petiole protoplasts and l e a f mesophyll c e l l s isolated enzymatically formed callus from which roots were produced (Bidney and Shepard 1980). This paper describes the formation of callus, embryoids and plants from various explants of sweet potato. Materials and Methods Roots of sweet potato (Ipomea batatas Poir.) c u l t i v a r s White Star and GaTG 3 were potted in a soiless mix ( p e a t : v e r m i c u l i t e : p e r l i t e ) and maintained in growth chambers at 27°C day/22°C night under a 16 h photoperiod. The plants were
Offprint requests to: J. R. Liu
f e r t i l i z e d weekly and watered as needed. Stock plants were maintained in the growth chamber for periods of up to one year, however they were kept in a highly vegetative state by constantly cutting back the sterns. Plants developed from in v i t r o shoot-tip cultures were used as a source of l e a f and shoot-tip explants. Shoot-tips were excised from highly p r o l i f e r a t i n g plants in growth chambers and the f u l l y expanded leaves were removed. The remaining part of the shoot-tip was disinfected with I0% Clorox for lO min under p a r t i a l vacuum, Procedures a f t e r d i s i n f e c t i o n were conducted under s t e r i l e conditions. Shoot-tips containing a few young leaves were rinsed 3 times with s t e r i l e water and trimmed to 5-I0 mm-long s t r i p s . They were placed onto basal medium containing Murashige and Skoog (1962) inorganic s a l t s , lO0 mg/l m y o - i n o s i t o l , 0.4 mg/l thiamine-HCl, 30 g/l sucrose, and 8 g/l Phytagar (GIBCO) in lO0 x 15 mm p l a s t i c Petri dishes. In some experiments, 0.3 mg/l 6-benzylaminopurine was added to basal medium to accelerate the growth of shoot-tips. Each dish contained 7 shoot-tips and was wrapped with Parafilm. All media were adjusted to pH 5.8 befsre autoclaving in 200-ml aliquots for 15 Bin at 121 C. The cultures were incubated at 27 C under 16 h of cool-white fluorescent l i g h t at an i n t e n s i t y of l,O00 lux. One week a f t e r incubation, leaves started to expand from the shoot-tips and formed roots at the basal end. After 2 to 4 weeks of incubation, well-expanded leaves, lO-15 mm-long, were cut into 2 mm-wide strips with l e a f margins i n t a c t . An explant consisted of a l l of the strips taken from one l e a f . Leaf explants were placed with e i t h e r the abaxial or adaxial surface down on callus induction medium, i . e . , basal medium plus 60 g/l sucrose and 2,4-D at 0.5 to 4.0 mg/l. Shoot-tips, 3-5 mm-long, containing 4-5 pairs of l e a f primordia and young leaves, were separately excised for culture in v i t r o . Stem segments, 5 mm-long, were taken from a 15 cm-long internodal section d i r e c t l y behind the apex on plants maintained in growth chambers. They were disinfected as described above. Freshly harvested roots, lO-15 cm-long, were disinfected with 5% Clorox for 30 min, dried and cut transversely into 20 mm-long slices. A series of cores with the cambium layer i n t a c t were excised from the slices with a No.l cork borer. These cores were cut transversely into 5 mm-long segments and used as root explants.
113 Five explants of leaves, shoot-tips, stems, and roots were placed separately on callus induction medium in lO0 x 15 mm Petri dishes. All cultures were incubated at 27°C in the dark and observed p e r i o d i c a l l y under a dissecting microscope for embryogenic callus formation.
Table I.
Embryogenic callus formation from sweet potato explants on medium containing l.O mg/l 2,4-D.
Percentage of explants forming embryogenic callus
Results
Cultivar
One to 2 weeks a f t e r incubation, l e a f , shoot-tips, and stem explants started to lose t h e i r bright-green or purple color and to form d u l l - y e l l o w , f r i a b l e callus on t h e i r surface. This callus did not give rise to embryoids. Three to 8 weeks a f t e r incubation, pale- to b r i g h t - y e l l o w , compact and organized callus emerged from the surface of explants (Fig. l a ) . When observed with a dissecting microscope, the callus was d i s t i n c t by color and texture from the f r i a b l e c a l l u s . When observed under a l i g h t microscope, the locus was composed of t i g h t l y packed isodiametric c e l l s which contained numerous starch grains. This callus was embryogenic: i t developed into globular embryoids, which developed further into heart-shaped embryoids, and which were usually attached to the callus through suspensor-like structures. The abaxial surface of the l e a f explants appeared to form more embryogenic callus than the adaxial surface. Young leaves and l e a f primordia of the shoot-tip explant produced embryogenic callus The cut edge of the strips of l e a f explant did not produce embryogenic callus while in stem and root explants i t did. Portions of the explant submerged in the medium did not produce embryogenic callus. As long as the cultures remained on callus induction medium, most embryoids did not develop beyond the torpedo-shaped stage (Fig. I b ) . Other embryoids developed continuously by budding from the surface of cotyledons and hypocotyls of embryoids, along with p r o l i f e r a t i o n of both of the embryogenic and non-embryogenic callus. When transferred to basal medium, heart-shaped embryoids r e a d i l y developed into torpedo-shaped embryoids (Fig. Ic) and then germinated (Fig. Id). Regenerated p l a n t l e t s often had poorly developed cotyledons and/or hypocotyls. Over 40% of cotyledon and hypocotyl segments from regenerated p l a n t l e t s also produced embryogenic callus on medium containing l.O mg/l 2,4-D. Each p l a n t l e t was placed on basal medium in 25 x 150 mm test tubes under l i g h t . At least 50 plants of each c u l t i v a r were transplanted to soil when they were 5-10 cm-high. The plants grew rapidly (Fig. le) and a f t e r 5 months in growth chambers, when they were over 3 m-long, they flowered, and produced storage roots (15 cm and larger in diameter). The plants derived via tissue culture were phenot y p i c a l l y the same as the donor plant. No embryogenic callus was formed from explants on basal medium. Embryogenic callus was generally not produced from l e a f and shoot-tip explants on media containing over 2.0 mg/l 2,4-D, but media containing 0.5-2.0 mg/l 2,4-D were ideal for induction of embryogenic callus. Embryogenic callus was observed from stem and root explants only on medium containing l.O mg/l 2,4-D. Leaf explants produced embryogenic callus at much higher rates than s h o o t - t i p , stem, and root callus (Table l ) . When embryogenic callus was subcultured on callus induction medium containing 2.0 mg/l 2,4-D supplemented with 2.0 mg/l k i n e t i n and 20% (v/v) deproteinized coconut water (GIBCO), s a t i s f a c t o r y m i t o t i c d i v i s i o n of the callus was
White Star GaTG 3
Leaf a
Shoot-tip b
23 22
3 5
Stem b
Root b
l l
-
a I00 (White Star) and 37 (GaTG 3) explants respectively. b I00-200 e x p l a n t s , c
Not c u l t u r e d .
sustained and a low rate of heart or torpedo-shaped embryoid formation was observed (Fig. I f ) . At each subculture, non-embryogenic f r i a b l e callus which was attached to the embryogenic callus was r o u t i n e l y removed. When subcultured monthly, i t retained i t s embryogenic capacity for over 9 months. The addition of e i t h e r k i n e t i n (O.Ol-O.l mg/l), glutamine (200-800 mg/l), proline (lO.O-l,O00 mg/l), or coconut water (5-20%), to callus induction medium containing l.O mg/l 2,4-D did not increase the frequency of embryogenic callus formation from stem explants. Flower buds at various developmental stages were excised and disinfected as previously described for shoot-tips. After c u l t u r i n g in the same manner as shoot-tip explants, no embryogenic callus was obtained. Discussion The formation of the embryogenic callus in this study was similar to that reported in Gramineae (e.g., Pennisetum; Haydu and Vasil 1981) and Rubiaceae (e.g., coffee; Staritsky 1970). I t was compact and composed of isodiametric, starch-rich c e l l s . Conversion of the embryogenic to non-embryogenic callus Was i r r e v e r s i b l e , since prolonged culture did not induce embryogenic from non-embryogenic c a l l u s . Likewise, non-embryogenic callus derived d i r e c t l y from the explant did not become embryogenic. Under conditions s i m i l a r to the present work, Sehgal (1975) observed only f r i a b l e callus from l e a f explants. When transferred to medium containing e i t h e r adenine or k i n e t i n , the callus gave rise to shoot buds and roots. Atypical p l a n t l e t s appear to be due to a lack of proper growth conditions rather than genetic change since mature plants showed no phenotypic abnormalities. Ozias-Akins and Vasil (1982) proposed that atypical p l a n t l e t s of wheat resulted from precocious germination of somatic embryos. Genetic variants are often observed in plants regenerated from callus cultures. However no prominent evidence has been reported that somatic embryogenesis induced through an intermediary callus leads to genetic v a r i a b i l i t y in the regenerated plant. I t is possible to recover n a t u r a l l y occurring mutant c e l l s from vegetatively propagated plants, such as sweet potato, in which c e l l u l a r v a r i a t i o n presumably accumulates, and produce whole plants via somatic embryogenesis. Regenerated plants from n a t u r a l l y occurring mutant c e l l s are usually much more valuable in crop improvement ( e . g . , cold-, heat-, s a l t - , and disease-resistant selections) than those from mutagen-induced mutant c e l l s . Furthermore, somatic embryos are of single cell o r i g i n (Haccius 1978), so regenerated plants are not l i k e l y to be chimeral. Large numbers of
114
Fig. I. Developmental sequence of somatic embryo and plantlet formation in sweet potato. (a) Embryogenic callus (arrows) arising from leaf tissue (xl8), (b) Proliferating embryogenic callus from leaf tissue showing globular, heart- and torpedo-shaped embryoids (xl4), (c) Numerous heart- and torpedo-shaped embryoids from embryogenic callus (x7.5), (d) Germination of embryoids into plantlets (x7), (e) Maturing plants regernerated from embryogenic callus, (f) Subcultured embryogenic callus with a low rate of embryoid production (xl5)
embryoids can be developed in suspension culture and might enable an otherwise vegetatively propagated crop to be seeded mechanically with the aid of techniques such as f l u i d d r i l l i n g . Propagation of sweet potato in this way is under investigation.
Acknowledgements We thank the Gas Research I n s t i t u t e , Chicago, IL. for partial support of this research. Florida Agricultural Experiment Station Journal Series 5453.
115 References Bidney DL, Shepard JF (1980) Plant Sci Let 18:335342 Haccius B (1978) Phytomorphology 28:74-81 Harlan JR (1976) Sci Am 235:89-97 Haydu Z, Vasil IK (1981) Theor Appl Genet 59:269-273 Kobayashi M, Shikata SI (1975) Bull Chugoko Natl Agric Exp Stn Set A24:109-124 Murashige T, Skoog F (1962) Physiol Plant 15:473-497
Ozias-Akins P, Vasil IK (1982) Protoplasma II0:95-I05 Sehgal CB (1975) Beitr Biol Pflanzen 51:47-52 Sehgal CB (1978) Z Pflanzenphysiol Bd 88:349-352 Staritsky G (1970) Acta Bot Neerl 19:509-514 Tsay HS, Tseng MT (1979) Bot Bull Academia Sinica 20:I17-122 Yamaguchi J, Nakajima T (1973) In: Proceedings of the 8th international conference on plant growth substances, Tokyo, pp l121-1127
