Plant Cell Reports (1984) 3:23-26
Plant Cell Reports © Springer-Verlag 1984
Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro Takashi Sasaki and Ke-iji Kainuma National Food Research Institute, P~O. Box 11, Tsukuba Science City, [baraki 305, Japan Received May 24, 1983 / Revised version received December 28, 1983 - Communicated by F. Constabel
ABSTRACT
MATERIALS
The r e g u l a t i o n of starch synthesis and e x o c e l l u l a r p o l y s a c c h a r i d e synthesis by GA 3 was studied with cells of sweet potato grown as s u s p e n s i o n in g l y c e r o l medium. In the p r e s e n c e of GA3, and under normal cell growth, starch formation was inhibited. The inc o r p o ria t i o n activity (starch synthesis) from ADP-[~4C] glucose or UDP-[14C] glucose with GA 3 treated cells was reduced. On the other hand, the synthesis of e x o c e l l u l a r polysaccharides c o m p o s e d of glucose, galactose, m a n n o s e and a r a b i n o s e etc., was s t i m u l a t e d and a clear i n c r e a s e of the M a n / A r a ratio was o b s e r v e d in the p r e s e n c e of GA 3. These results may indicate that GA 3 affects the r e g u l a t i o n of starch synthesis and exocellular p o l y s a c c h a r i d e synthesis.
Cell
INTRODUCTION Cells g r o w i n g as s u s p e n s i o n s in chemically defined m e d i a have been used in a n u m b e r of l a b o r a t o r i e s as a system for s tudy i n g aspects of the r e g u l a t i o n of metabolic activities in higher plants. Carbohydrate m e t a b o l i s m has been a focal point ever since the i n c e p t i o n of p l a n t cell culture. Increasingly, cell cultures are being used as a tool to e l u c i d a t e p l a n t c a r b o h y d r a t e synthesis. The e n d o g e n o u s formation of starch has been f o l l o w e d in cultu r e d cells (Thorpe and M e i e r 1972), and c y t o k i n i n s have been found to s t i m u l a t e accumulation. C o n d i t i o n s favoring the diff e r e n t i a t i o n process g r e a t l y e n h a n c e d both build up and b r e a k d o w n of starch. Also, starch d e p o s i t i o n in p o t a t o callus cultures was a f f e c t e d w h e n cells were t r a n s f e r r e d to a u x i n - f r e e m e d i u m s u p p l e m e n t e d with kinetin and sucrose at a high c o n c e n t r a t i o n (ObataSasamoto and Suzuki 1978}. R e l e a s e of polys a c c h a r i d e s into the m e d i u m d u r i n g the growth of cells in s u s p e n s i o n culture was o bser v e d (Liau and Boll 1972). Gibberellic acid was found to control the release of pectic acid and a p e r o x i d a s e (Fry 1980). However, the r e l a t i o n b e t w e e n starch biosynthesis and p o l y s a c c h a r i d e b i o s y n t h e s i s are still obscure. This p a p e r shows that the b i o s y n t h e s i s of starch and e x o c e l l u l a r p o l y s a c c h a r i d e s m i g h t be c o n t r o l l e d by GA3.
AND METHODS
culture Cell s u s p e n s i o n (Ipomoea batatas var a ~ e ~ Gamborg's at 25°C as d e s c r i b e d 1972).
cultures of sweet potato Kokei 14) were grown in PRL-4 m e d i u m in the dark p r e v i o u s l y (Sasaki et al
E x t r a c t i o n and d e t e r m i n a t i o n of starch The starch content of the tissues was d e t e r m i n e d by the g l u c o a m y l a s e - g l u c o s e oxidase m e t h o d after d i m e t h y l sulfoxide extraction (Sasaki 197~). Isolation and analysis of e x o c e l i u l a r polysaccharides The p o l y s a c c h a r i d e s in the culture filtrate, w h i c h p a s s e d through filter paper, were p r e c i p i t a t e d w i t h alcohol, and w a s h e d with alcohol-ether. The neutral sugars and uronic acid were s e p a r a t e d by paper chromatography with ethyl acetate - p y r i d i n e - H20 (12:5:4 V/V) and n - p r o p a n o l - p y r i d i n e acetic acid - H2 O (8:8:4:1), respectively. The q u a n t i t a t i v e analysis was carried out by gas c h r o m a t o g r a p h y after t r e a t m e n t with t r i f l u o r o a c e t i c acid, and the uronic acid was e s t i m a t e d by the c a r b a z o l m e t h o d (Bjorndal et al. 1967). A s s a y of the s t a r c ~ synthetase a c t i v i t y The f r a c t i o n of cell extract p r e c i p i tated by 30-65% (NH4)2SO 4 was used for the assay of enzyme activity. The A D P - g l u c o s e or U D P - q l u c o s e synthetase activity was assayed by [14C] glucose i n c o r p o r a t i o n from ADP-[14C] glucose or UDP-[14C] glucose into primer (Sasaki and Kainuma 1980). RESULTS Cell
AND D I S C U S S I O N
s u s p e n s i o n cultures of sweet potato Cells of sweet potato u t i l i z e d g l y c e r o l at the same e f f i c i e n c y as sucrose, glucose, fructose and starch, and grew well in the p r e s e n c e of r e s p e c t i v e carbon sucroses. But the cells did not grow in n u t r i e n t m e d i a c o n t a i n i n g lactate, D - g l u c o n a t e or citric acid as a source of carbon (Fig. i).
24 amylase action of the starch extracted from the cultured cells were similar to those of standard sweet potato starch.
30
---o-- Glc Inhibition of starch synthesis in G~3-treated cells Very little starch was present at zero time. Subsequently it increased rapidly to reach a maximum at 2 weeks and decreased. However, in the presence of GA 3 (3x10-5M), which was added in the m e d i u m at 1 week, starch synthesis was inhibited almost completely under the normal growth. On the other hand, NAA (3x10-SM), 6-benzyladenine (5x10-6M) and abscisic acid (3x10-5M) had no effect on growth and starch synthesis (Figs. 3 and 4).
O
~ 20 v n-
~w
i0
0
I
I
I
,. I
1
2
3
4
WEE'KS
Fig. i. sources culture
Growth curve in different carbon of sweet potato cell suspension
Starch synthesis in cell suspension culture Starch formation in cells increased steadily to attain a maximum value at 2 weeks, i.e. at the early stationaly phase, and decreased rapidly in both the glucose and glycerol medium (Fig. 2). However, starch formation in a glycerol medium was more (aPprox. a 2-fold increase) than in a glucose medium throughout the course of the experiment. This finding indicates the rapid conversion of glycerol (C3-compound) to starch. The biochemical characteristics, absorption maximum of the iodine complex and gluco
(a)
Fig. 3. Microscopic view of starch formation in GA treated cells. The starch in the cells (2 weeks} was stained by iodine. [a) control cells and (b) GA-treated cells.
~ ~
1.0 ~
Glc _
/
20
(b)
200
~
Control BA
N~
~
==
Gly
// 0,5
i0 ~
l
I
1
2
,.
I
3
WEEKS
Fig. 2. Starch synthesis from glycerol and glucose medium by sweet potato cultured cells
i00
0
I
I
1
1
2
3
WEEKS
Fig. 4. Effect of growth regulators on starch formation of sweet potato cells. growth regulator was added at one week.
The
25 In vitro starch synthetase a c t i v i t y in GA3treated cells The i n c o r p o r a t i o n a c t i v i t ~ 4 o f [14C]glucose into p r i m e r from ADP-[ C] or UDP[±4C] glucose was tested w i t h the cell-free extract of GA (3xl0-SM)-treated cells. The i n c o r p o r a t i o n a c t i v i t y had a t e n d e n c y to increase with the cell growth a t t a i n i n g maxim u m value at 2 weeks and then d e c l i n e d as in vitro starch synthesis in control cell. In G A 3 - t r e a t e d cell, starch s y n t h e t a s e activity from U D P - g l u c o s e d e c r e a s e d markedly, this period c o - i n c i d e d with the s u p r e s s i o n of starch f o r m a t i o n (Fig. 5) and the similar result was o b t a i n e d in the case of ADPglucose. These findings i n d i c a t e that the b i o s y n t h e s i s of starch s y n t h e t a s e m i g h t be r e p r e s s e d by GA 3. -
i00 STARCH
5O
-
~
~
EXOCELLULAR POLYSACCHARIDES
C~
~ 200 ..
---O--Control
8
~
_.~ ~-
,
. - - ~ - - + ~
~e~_ ~o
100
~.
1
.,,
2
~
WEEKS
~,5
}?
L0 ~
5
--~'
v
I 2
o
Fig. 6. Inhibition of starch synthesis and stimulation of e x o c e l l u l a r p o l y s a c c h a r i d e s synthesis by GA 3. Control cells ( + ] and G A 3 - t r e a t e d cells ( ~ ).
3
WEEKS
Fig.
~
5.
In vitro U D P - g l u c o s e specific s t a r c h synthesis a c t i v i t y in G A 3 - t r e a t e d cells. Growth of control cells ( ~ ) and G A 3 - t r e a t e d cells ( + ) . UDP-glucose specific a c t i v i t y in control cells ( [] ) and in G A 3 - t r e a t e d cells ( • ). -
Control
-
S t i m u l a t i o n of e x o c e l l u l a r p o l y s a c c h a r i d e synthesis in G A 3 - t r e a t e d cells E x o c e l l u l a r p o l y s a c c h a r i d e s were comm o n l y p r o d u c e d by the s u s p e n s i o n - c u l t u r e d sweet potato cells and these substances are thought to be a portion of the cell wall that has escaped into the medium. (Sasaki et al, 1978). In the p r e s e n c e of GA 3 (3x10-5M), the synthesis of e x o c e l l u l a r m a t e r i a l was s t i m u l a t e d steadily after GA 3 a d d i t i o n to reach a m a x i m u m at 2 weeks. It is interesting to note that there was a d e c r e a s e in starch c o n t e n t (av. 35 mg/20 ml medium) while at the same time there was a corresponding i n c r e a s e in e x o c e l l u l a r polysaccharide content (av. 32 mg/20 ml medium). This suggests that the substrates that act as p r e c u r s o r of starch m i g h t be utilized to p r o d u c e p o l y s a c c h a r i d e chains. The exocellular p o l y s a c c h a r i d e s were c o m p o s e d of galactose, glucose, arabinose, xylose, mannose, rhamnose, fucose and galacturonic acid. The ratio of m a n n o s e / a r a b i n o s e increased in the p o l y s a c c h a r i d e s from GA 3 treated cells (Figs. 6 and 7).
30-
z
'" 0 z O
+ GA
30-
0 Rham
Ara
Fuc
GIc
Man
Xyl
Gal
Fig. 7. Effect of GA 3 on ratio of sugar c o m p o n e h t of e x o c e l ± u l a r polysaccharides.
26 Regulation of starch synthesis and exocellular polysaccharides by GA3 The above observation led to the following concept of a possible regulation by GA 3 biosynthesis of starch and exocellular polysaccharides as shown in Fig. 8.
On the starch biosynthesis system from glycerol, GA 3 may have inhibited a process of enzyme protein biosynthesis relating with starch synthesis. On the other hand, the findings that biosynthesis of exocellular polysaccharides was stimulated and Man/Ara ratio increased by GA 3 suggest it may regulate polysaccharides synthesis. ACKNOWLEDGEMENT
G
l I I /
y /
c
e
r
o
l
~
~ ......... ~Inhibition Starch S y ~ t h e ~ ~
1
~ P o ~ l y s ~ ~ e s
Fig. 8 Schematic representation on ~-d~ion of starch and exocellular polysaccharides synthesis by GA 3.
The author thanks Mr. N. Shibuya for his advice on the polysaccharides analysis and Miss F. Ohmura for technical assistance. REFERENCES Bjorndal, H., Lindberg, B. and Svensson, S. (1967) Acta. Chem. Scand. 21: 1801-1805. Fry, S.C. (1980) Phytochemistry 19:735-740. Liau, D.F. and Boll, W.G. (1972) Can. J. Bot. 55:2031-2038. Obata-Sasamoto, H. and Suzuki, H. (1978) Z. Pflanzenphysiol. Bd 88:33-37. Sasaki, T., Tadokoro, K. and Suzuki, S. (1972) Biochem. J. 129:789-791. Sasaki, T., Kugo, A. and Kainuma, K. (1978) VI Symposium for Plant Tissue Culture Abstract 19 (Japan) Sasaki, T. (1979) In: Methods in Starch Science (M. Nakamura and S. Suzuki ed.), Asakura Shoten, Tokyo pp. 1-7. Sasaki, T. and Kainuma, K. (1980) Biochem. J. 189:381-383. Thorpe, A.T. and Meier, D.D. (1972) Physiol. Plant. 27:365-369.
Plant Cell Reports © Springer-Verlag 1984
Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro Takashi Sasaki and Ke-iji Kainuma National Food Research Institute, P~O. Box 11, Tsukuba Science City, [baraki 305, Japan Received May 24, 1983 / Revised version received December 28, 1983 - Communicated by F. Constabel
ABSTRACT
MATERIALS
The r e g u l a t i o n of starch synthesis and e x o c e l l u l a r p o l y s a c c h a r i d e synthesis by GA 3 was studied with cells of sweet potato grown as s u s p e n s i o n in g l y c e r o l medium. In the p r e s e n c e of GA3, and under normal cell growth, starch formation was inhibited. The inc o r p o ria t i o n activity (starch synthesis) from ADP-[~4C] glucose or UDP-[14C] glucose with GA 3 treated cells was reduced. On the other hand, the synthesis of e x o c e l l u l a r polysaccharides c o m p o s e d of glucose, galactose, m a n n o s e and a r a b i n o s e etc., was s t i m u l a t e d and a clear i n c r e a s e of the M a n / A r a ratio was o b s e r v e d in the p r e s e n c e of GA 3. These results may indicate that GA 3 affects the r e g u l a t i o n of starch synthesis and exocellular p o l y s a c c h a r i d e synthesis.
Cell
INTRODUCTION Cells g r o w i n g as s u s p e n s i o n s in chemically defined m e d i a have been used in a n u m b e r of l a b o r a t o r i e s as a system for s tudy i n g aspects of the r e g u l a t i o n of metabolic activities in higher plants. Carbohydrate m e t a b o l i s m has been a focal point ever since the i n c e p t i o n of p l a n t cell culture. Increasingly, cell cultures are being used as a tool to e l u c i d a t e p l a n t c a r b o h y d r a t e synthesis. The e n d o g e n o u s formation of starch has been f o l l o w e d in cultu r e d cells (Thorpe and M e i e r 1972), and c y t o k i n i n s have been found to s t i m u l a t e accumulation. C o n d i t i o n s favoring the diff e r e n t i a t i o n process g r e a t l y e n h a n c e d both build up and b r e a k d o w n of starch. Also, starch d e p o s i t i o n in p o t a t o callus cultures was a f f e c t e d w h e n cells were t r a n s f e r r e d to a u x i n - f r e e m e d i u m s u p p l e m e n t e d with kinetin and sucrose at a high c o n c e n t r a t i o n (ObataSasamoto and Suzuki 1978}. R e l e a s e of polys a c c h a r i d e s into the m e d i u m d u r i n g the growth of cells in s u s p e n s i o n culture was o bser v e d (Liau and Boll 1972). Gibberellic acid was found to control the release of pectic acid and a p e r o x i d a s e (Fry 1980). However, the r e l a t i o n b e t w e e n starch biosynthesis and p o l y s a c c h a r i d e b i o s y n t h e s i s are still obscure. This p a p e r shows that the b i o s y n t h e s i s of starch and e x o c e l l u l a r p o l y s a c c h a r i d e s m i g h t be c o n t r o l l e d by GA3.
AND METHODS
culture Cell s u s p e n s i o n (Ipomoea batatas var a ~ e ~ Gamborg's at 25°C as d e s c r i b e d 1972).
cultures of sweet potato Kokei 14) were grown in PRL-4 m e d i u m in the dark p r e v i o u s l y (Sasaki et al
E x t r a c t i o n and d e t e r m i n a t i o n of starch The starch content of the tissues was d e t e r m i n e d by the g l u c o a m y l a s e - g l u c o s e oxidase m e t h o d after d i m e t h y l sulfoxide extraction (Sasaki 197~). Isolation and analysis of e x o c e l i u l a r polysaccharides The p o l y s a c c h a r i d e s in the culture filtrate, w h i c h p a s s e d through filter paper, were p r e c i p i t a t e d w i t h alcohol, and w a s h e d with alcohol-ether. The neutral sugars and uronic acid were s e p a r a t e d by paper chromatography with ethyl acetate - p y r i d i n e - H20 (12:5:4 V/V) and n - p r o p a n o l - p y r i d i n e acetic acid - H2 O (8:8:4:1), respectively. The q u a n t i t a t i v e analysis was carried out by gas c h r o m a t o g r a p h y after t r e a t m e n t with t r i f l u o r o a c e t i c acid, and the uronic acid was e s t i m a t e d by the c a r b a z o l m e t h o d (Bjorndal et al. 1967). A s s a y of the s t a r c ~ synthetase a c t i v i t y The f r a c t i o n of cell extract p r e c i p i tated by 30-65% (NH4)2SO 4 was used for the assay of enzyme activity. The A D P - g l u c o s e or U D P - q l u c o s e synthetase activity was assayed by [14C] glucose i n c o r p o r a t i o n from ADP-[14C] glucose or UDP-[14C] glucose into primer (Sasaki and Kainuma 1980). RESULTS Cell
AND D I S C U S S I O N
s u s p e n s i o n cultures of sweet potato Cells of sweet potato u t i l i z e d g l y c e r o l at the same e f f i c i e n c y as sucrose, glucose, fructose and starch, and grew well in the p r e s e n c e of r e s p e c t i v e carbon sucroses. But the cells did not grow in n u t r i e n t m e d i a c o n t a i n i n g lactate, D - g l u c o n a t e or citric acid as a source of carbon (Fig. i).
24 amylase action of the starch extracted from the cultured cells were similar to those of standard sweet potato starch.
30
---o-- Glc Inhibition of starch synthesis in G~3-treated cells Very little starch was present at zero time. Subsequently it increased rapidly to reach a maximum at 2 weeks and decreased. However, in the presence of GA 3 (3x10-5M), which was added in the m e d i u m at 1 week, starch synthesis was inhibited almost completely under the normal growth. On the other hand, NAA (3x10-SM), 6-benzyladenine (5x10-6M) and abscisic acid (3x10-5M) had no effect on growth and starch synthesis (Figs. 3 and 4).
O
~ 20 v n-
~w
i0
0
I
I
I
,. I
1
2
3
4
WEE'KS
Fig. i. sources culture
Growth curve in different carbon of sweet potato cell suspension
Starch synthesis in cell suspension culture Starch formation in cells increased steadily to attain a maximum value at 2 weeks, i.e. at the early stationaly phase, and decreased rapidly in both the glucose and glycerol medium (Fig. 2). However, starch formation in a glycerol medium was more (aPprox. a 2-fold increase) than in a glucose medium throughout the course of the experiment. This finding indicates the rapid conversion of glycerol (C3-compound) to starch. The biochemical characteristics, absorption maximum of the iodine complex and gluco
(a)
Fig. 3. Microscopic view of starch formation in GA treated cells. The starch in the cells (2 weeks} was stained by iodine. [a) control cells and (b) GA-treated cells.
~ ~
1.0 ~
Glc _
/
20
(b)
200
~
Control BA
N~
~
==
Gly
// 0,5
i0 ~
l
I
1
2
,.
I
3
WEEKS
Fig. 2. Starch synthesis from glycerol and glucose medium by sweet potato cultured cells
i00
0
I
I
1
1
2
3
WEEKS
Fig. 4. Effect of growth regulators on starch formation of sweet potato cells. growth regulator was added at one week.
The
25 In vitro starch synthetase a c t i v i t y in GA3treated cells The i n c o r p o r a t i o n a c t i v i t ~ 4 o f [14C]glucose into p r i m e r from ADP-[ C] or UDP[±4C] glucose was tested w i t h the cell-free extract of GA (3xl0-SM)-treated cells. The i n c o r p o r a t i o n a c t i v i t y had a t e n d e n c y to increase with the cell growth a t t a i n i n g maxim u m value at 2 weeks and then d e c l i n e d as in vitro starch synthesis in control cell. In G A 3 - t r e a t e d cell, starch s y n t h e t a s e activity from U D P - g l u c o s e d e c r e a s e d markedly, this period c o - i n c i d e d with the s u p r e s s i o n of starch f o r m a t i o n (Fig. 5) and the similar result was o b t a i n e d in the case of ADPglucose. These findings i n d i c a t e that the b i o s y n t h e s i s of starch s y n t h e t a s e m i g h t be r e p r e s s e d by GA 3. -
i00 STARCH
5O
-
~
~
EXOCELLULAR POLYSACCHARIDES
C~
~ 200 ..
---O--Control
8
~
_.~ ~-
,
. - - ~ - - + ~
~e~_ ~o
100
~.
1
.,,
2
~
WEEKS
~,5
}?
L0 ~
5
--~'
v
I 2
o
Fig. 6. Inhibition of starch synthesis and stimulation of e x o c e l l u l a r p o l y s a c c h a r i d e s synthesis by GA 3. Control cells ( + ] and G A 3 - t r e a t e d cells ( ~ ).
3
WEEKS
Fig.
~
5.
In vitro U D P - g l u c o s e specific s t a r c h synthesis a c t i v i t y in G A 3 - t r e a t e d cells. Growth of control cells ( ~ ) and G A 3 - t r e a t e d cells ( + ) . UDP-glucose specific a c t i v i t y in control cells ( [] ) and in G A 3 - t r e a t e d cells ( • ). -
Control
-
S t i m u l a t i o n of e x o c e l l u l a r p o l y s a c c h a r i d e synthesis in G A 3 - t r e a t e d cells E x o c e l l u l a r p o l y s a c c h a r i d e s were comm o n l y p r o d u c e d by the s u s p e n s i o n - c u l t u r e d sweet potato cells and these substances are thought to be a portion of the cell wall that has escaped into the medium. (Sasaki et al, 1978). In the p r e s e n c e of GA 3 (3x10-5M), the synthesis of e x o c e l l u l a r m a t e r i a l was s t i m u l a t e d steadily after GA 3 a d d i t i o n to reach a m a x i m u m at 2 weeks. It is interesting to note that there was a d e c r e a s e in starch c o n t e n t (av. 35 mg/20 ml medium) while at the same time there was a corresponding i n c r e a s e in e x o c e l l u l a r polysaccharide content (av. 32 mg/20 ml medium). This suggests that the substrates that act as p r e c u r s o r of starch m i g h t be utilized to p r o d u c e p o l y s a c c h a r i d e chains. The exocellular p o l y s a c c h a r i d e s were c o m p o s e d of galactose, glucose, arabinose, xylose, mannose, rhamnose, fucose and galacturonic acid. The ratio of m a n n o s e / a r a b i n o s e increased in the p o l y s a c c h a r i d e s from GA 3 treated cells (Figs. 6 and 7).
30-
z
'" 0 z O
+ GA
30-
0 Rham
Ara
Fuc
GIc
Man
Xyl
Gal
Fig. 7. Effect of GA 3 on ratio of sugar c o m p o n e h t of e x o c e l ± u l a r polysaccharides.
26 Regulation of starch synthesis and exocellular polysaccharides by GA3 The above observation led to the following concept of a possible regulation by GA 3 biosynthesis of starch and exocellular polysaccharides as shown in Fig. 8.
On the starch biosynthesis system from glycerol, GA 3 may have inhibited a process of enzyme protein biosynthesis relating with starch synthesis. On the other hand, the findings that biosynthesis of exocellular polysaccharides was stimulated and Man/Ara ratio increased by GA 3 suggest it may regulate polysaccharides synthesis. ACKNOWLEDGEMENT
G
l I I /
y /
c
e
r
o
l
~
~ ......... ~Inhibition Starch S y ~ t h e ~ ~
1
~ P o ~ l y s ~ ~ e s
Fig. 8 Schematic representation on ~-d~ion of starch and exocellular polysaccharides synthesis by GA 3.
The author thanks Mr. N. Shibuya for his advice on the polysaccharides analysis and Miss F. Ohmura for technical assistance. REFERENCES Bjorndal, H., Lindberg, B. and Svensson, S. (1967) Acta. Chem. Scand. 21: 1801-1805. Fry, S.C. (1980) Phytochemistry 19:735-740. Liau, D.F. and Boll, W.G. (1972) Can. J. Bot. 55:2031-2038. Obata-Sasamoto, H. and Suzuki, H. (1978) Z. Pflanzenphysiol. Bd 88:33-37. Sasaki, T., Tadokoro, K. and Suzuki, S. (1972) Biochem. J. 129:789-791. Sasaki, T., Kugo, A. and Kainuma, K. (1978) VI Symposium for Plant Tissue Culture Abstract 19 (Japan) Sasaki, T. (1979) In: Methods in Starch Science (M. Nakamura and S. Suzuki ed.), Asakura Shoten, Tokyo pp. 1-7. Sasaki, T. and Kainuma, K. (1980) Biochem. J. 189:381-383. Thorpe, A.T. and Meier, D.D. (1972) Physiol. Plant. 27:365-369.
