Plant Cell Reports
Plant Cell Reports (1986) 3:169-173
© Springer-Verlag 1986
Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides M a t t h i a s F l a d u n g and J o s e f H e s s e l b a c h Ma×-Planck-Institut For Zt~chtungsforschung, Abteilung PflanzenzOchtung, D-5000 K~ln 30, FRG Received December 13, 1985 / Revised version received March 10, 1986 - Communicated by H. L0rz
ABSTRACT Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcat u m T h u m b . , as w e l l as b a s a l p a r t s of l e a v e s and m e s o c o t y l s f r o m s t e r i l e s e e d l i n g s , a n d s e e d s f r o m P a n i c u m m i l i o i d e s N e e s ex. T r i n w e r e u s e d as e x p l a n t s to i n d u c e c a l l u s on a Murashige and Skoog medium supplemented w i t h 2.5 to !0 m g / l of 2 , 4 - D . S u b c u l t u r i n g of t h e w h i t e c a l l u s f r o m P. m i l i o i d e s a n d of the b r o w n c a l l u s f r o m P. b i s u l c a t u m on a m e d i u m c o n t a i n i n g 0.i m g / l 2 , 4 - D a n d i0 g/l s u c r o s e led in b o t h s p e c i e s to the a p p e a r a n c e of g r e e n s t r u c t u r e s f r o m w h i c h p l a n t s c o u l d be r e g e n e r a t e d . P l a n t s w e r e r e g e n e r a ted b y an o r g a n o g e n e t i c p r o c e s s in P. m i l i o i d e s , w h i l e P. b i s u l c a t u m p l a n t s w e r e r e generated both via organogenesis and somatic embryogenesis. 1032 a n d 94 p l a n t s , f r o m P. b i s u l c a t u m a n d P. m i l i o i d e s , r e s p e c t i v e l y , w e r e t r a n s f e r r e d i n t o soil, a n d a b o u t 90% of t h e m w e r e g r o w n to m a t u r i t y a n d set seeds.
Abbreviatations: MS, M u r a s h i g e a n d S k o o g m e d i u m (15)} 2,4-D, 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid} IAA, i n d o l e a c e t i c acid.
s i o n s ( 7 , 1 0 , 1 2 ) . In the e a r l y in v i t r o w o r k d o n e in t h i s genus, r e g e n e r a t i o n of p l a n t s v i a o r g a n o g e n e s i s w a s r e p o r t e d in P. m i l i a c e u m a n d P. a n t i d o l e (2,17). H e r e we d e s c r i b e the i n d u c t i o n a n d m a i n t e n a n c e of c a l l u s t i s s u e s in P. b i s u l c a t u m , a C-3 s p e c i e s , a n d in P. m i l i o i d e s , a C-3-C-4 intermediate species, and regenera t i o n of p l a n t s v i a s o m a t i c e m b r y o g e n e s i s and o r g a n o g e n e s i s in P. b i s u l c a t u m , a n d v i a o r g a n o g e n e s i s in P. m i l i o i d e s f o r the f i r s t time. T h e p o s s i b i l i t y of i s o l a t i n g s o m a c l o n a l v a r i a n t s a f t e r in v i t r o c u l t u r e a n d plant regeneration (8) w a s one of the app r o a c h e s a d o p t e d in s e a r c h i n g for p h o t o s y n thetic and photorespiratory mutants. Members of the R-2 g e n e r a t i o n w i l l be s c r e e n e d for s u c h m u t a t i o n s . The p h o t o s y n t h e t i c ones will be s c r e e n e d w i t h the h i g h - c h l o r o p h y l l - f l u o r e s c e n c e - y i e l d m e t h o d d u r i n g e x p o s u r e to U V l i g h t (13), w h i l e the p h o t o r e s p i r a t o r y ones w i l l be i s o l a t e d b y t h e i r i n a b i l i t y to g r o w in n o r m a l a i r c o n d i t i o n s (20). N o r m a l l y , such mutants are isolated after mutagen t r e a t m e n t of seeds, but n o w w e i n t e n d to s c r e e n for s u c h v a r i a n t s w i t h o u t p h y s i c a l or chemical mutagen treatment.
MATERIAL
AND METHODS
INTRODUCTION In m a n y s p e c i e s w i t h i n the m i l l e t s c a l lus i n d u c t i o n w a s r e p o r t e d a n d r e g e n e r a n t s were obtained via organogenesis and somatic embryogenesis (19). A l l t h e s e s p e c i e s , s u c c e s s f u l l y c u l t i v a t e d in v i t r o , a r e c h a r a c t e r i z e d b y h a v i n g the C-4 t y p e of p h o t o s y n t h e s i s (5,16). In the g e n u s P a n i c u m , s p e c i e s w i t h C - 3 a n d C-4 t y p e of p h o t o s y n t h e s i s have been characterized (14). A d d i t i o n a l l y socalled C-3-C-4 intermediate species have b e e n f o u n d (4). C a l l u s i n d u c t i o n w i t h i n the P a n i c u m s p e c i e s w a s r e p o r t e d o n l y for C-4 s p e c i e s (5,16): in P. m a x i m u m ( 2 , 9 , 1 1 ) , P. m i l i a c e u m ( 2 , 7 , 1 7 , 1 8 ) , P. m i l i a r e (18) a n d P. a n t i d o l e (2). R e g e n e r a t i o n of p l a n t s is p o s s i b l e v i a somatic embryogenesis in P. m a x i m u m a n d P. miliare starting from calli obtained from y o u n g l e a v e s , m a t u r e and i m m a t u r e e m b r y o s , a n d young inflorescences ( 9 , 1 1 , 1 8 ) ; in P. m a x i m u m a n d in P. m i l i a c e u m f r o m c e l l s u s p e n -
Offprint requests to: J. Hesselbach
S e e d s of P. b i s u l c a t u m T h u m b (PI 1 9 4 8 6 1 ) a n d P. m i l i o i d e s N e e s ex. T r i n (PI 2 8 4 2 2 0 ) w e r e o b t a i n e d f r o m the S o u t h e r n R e g i o n a l P l a n t I n t r o d u c t i o n S t a t i o n , G e o r g i a , USA. S e e d s w e r e s u r f a c e s t e r i l i z e d w i t h 70% a l c o h o l for 1 min, f o l l o w e d b y 0 . 1 % H g C I - 2 / T w e e n 20 for 3 m i n and g e r m i n a t e d e i t h e r on MS a g a r w i t h o u t h o r m o n e s or d i r e c t l y on c a l lus i n d u c t i o n m e d i u m (MS w i t h 5 m g / l 2,4-D, 30g/l sucrose). Twenty day old seedlings grown without hormones were dissected into l e a v e s w i t h (P. m i l i o i d e s ) or w i t h o u t b a s i s , w h i c h w e r e cut i n t o t h r e e to f i v e p i e c e s , a n d m e s o c o t y l . T h e s e e x p l a n t s w e r e u s e d to i n i t i a t e c a l l u s f o r m a t i o n on c a l l u s i n d u c t i o n m e d i u m (MS w i t h 2 . 5 - 1 0 m g / l 2,4-D, 30 g/l s u c r o s e ) C a l l i w e r e o b t a i n e d w i t h i n two to t h r e e w e e k s ; t h e y w e r e s u b c u l t u r e d on MS, c o n t a i n ing i0 or 1 g / l s u c r o s e a n d 1 or 0.i m g / l 2 , 4 - D . A f t e r 1 to 3 s u b c u l t u r e s g r e e n s h o o t b u d s or s m a l l p l a n t l e t s d e r i v e d f r o m s o m a t i c
170 e m b r y o s w e r e t r a n s f e r r e d on 2 , 4 - D f r e e m e d i u m (MS, w i t h 20 g / l sucrose, w i t h o u t h o r m o n e s or w i t h 2 m g / l Z e a t i n ) . In b o t h s p e c i e s r e g e n e r a n t s w e r e o b t a i n e d d u r i n g i0 to 12 s u b c u l t u r e s of the e m b r y o g e n i c c a l l u s . S u b c u l t u r e s w e r e d o n e e v e r y 3 to 4 w e e k s . All media and glass culture vessels were sterilized by autoclaving. The cultures were i n c u b a t e d in a g r o w t h r o o m at 2 4 ~ 0 . 2 ° C , exp o s e d to a r t i f i c i a l l i g h t ( O S R A M N a t u r a L 5 8 W / 25) of 11.07 W / m 2 w i t h l i g h t / d a r k c y c l e s of 13.5/10.5 hours.
w a s f o r m e d at a l l 2 , 4 - D l e v e l s t e s t e d s t a r t ing f r o m b a s a l p a r t s of the l e a v e s and f r o m m e s o c o t y l s . No c a l l u s i n d u c t i o n w a s a c h i e v e d f r o m the u p p e r p a r t s of the l e a v e s . T h e o n l y 2 , 4 - D l e v e l t e s t e d for s e e d s as e x p l a n t s w a s 5 m g / l . O n l y a b o u t 5% of t h e s e c u l t e r e d explants formed callus. In P. b i s u l c a t u m , a b r o w n , g u m m y c a l l u s was visible only when explants were used from m e s o c o t y l s or seeds. N o c a l l u s i n d u c t i o n w a s a c h i e v e d f r o m l e a v e s w i t h o u t b a s i s . At 2.5 m g / l 2 , 4 - D m e s o c o t y l e x p l a n t s g a v e the h i g h est p e r c e n t a g e of c a l l u s f o r m a t i o n . T h e p e r c e n t a g e r a n g e d f r o m 76% at 2.5 m g / l 2 , 4 - D to 7% at i0 m g / l . T h e o n l y 2 , 4 - D l e v e l t e s t e d for s e e d s a g a i n w a s 5 m g / 1 . B u t h e r e w e got a m u c h h i g h e r p e r c e n t a g e of c a l l u s i n d u c t i o n t h a n in P. m i l i o i d e s . A r e p e t i t i o n of c a l l u s i n d u c t i o n f r o m m e s o c o t y l in b o t h s p e c i e s gave
RESULTS Callus induction was achieved from s e v e r a l e x p l a n t s of the two P a n i c u m s p e c i e s ( T a b l e i). In P. m i l i o i d e s , a w h i t e c a l l u s
Table
i. C a l l u s i n d u c t i o n (% of c u l t u r e d e x p l a n t s , absolute numbers) from different explants s p e c i e s on m e d i a c o n t a i n i n g v a r i o u s 2 , 4 - D
2,4-D (mg/l)
P. milioides meso-
seed
cotyl
basis
Table
P. bJsulcatum
leaf with
in b r a c k e t s the of t w o P a n i c u m concentrations.
2.5
4.7 (150)
42 (220)
5.0
2.0 (150)
53 (223)
/
/
5.2 (135)
leaf without
meso-
basis
cotyl
0 (151)
76 (268)
0 (150)
45 (266)
seed
/
/
75 (128)
7.5
6.0 (149)
60 (219)
/
/
0 (150)
12 (256)
/
/
I0.0
4.7 (150)
43 (230)
/
/
0 (150)
7 (260)
/
/
2. G r o w t h of c a l l i on s u b c u l t u r e m e d i a (++ v e r y g o o d g r o w t h ; + g o o d g r o w t h ; - no g r o w t h ) , a n d the c a p a c i t y of p l a n t r e g e n e r a t i o n d u r i n g the f i r s t or the f o l l o w i n g s u b c u l t u r e s (o o r g a n o g e n e s i s ; e e m b r y o g e n e s i s )
Species £xplant 2,4-D (mg/l) Of induction
medium
Subculture medium for callus growth and plant regeneration Sucrose 10g/l 2,4-D 0.1mg/l Sucrose I g l l Sucrose 10g/1 2,4-D Img/] Callus Regeneration n ° of plants regenerated 2,4-D Img/l Callus growth(1) Callus growth(1) growth p r o c e s s per s i n g l e e x p ] a n t t o t a l C a l l u s growth(I) Sucrose
lg/l
2,4-D 0.1 mg/l
P. milloJdes Leaf with basis
2.5 5.0 7.5 10.0
+/-
+
o
1
2
4/-
4
o
1
2
~/-
+÷
+/-
+
o
1-4
7
o
1
1
o
1-26
e,o e,o
42-82
Mesocotyl 4
2.5
5.0 7.5
44
10.0
÷
Seed
+/-
5.0
82
P. bisu] Catum Mesocotyl
2.5 5.0 7.5 10.0
+/-
÷
~+
.+]-
÷
+
+/+[-
4/--
4/4]-
27-291
835 124 ./-
Seed
./-
5.0
(1)
no p l a n t s
regenerated
o under
these conditions
73
73
171
Figure
~. Plant regeneration from c u l t u r e d calli of P. milioides
and P. b i s u l c a t u m .
A-B. O r g a n o g e n e s i s callusl
C-F. O r g a n o g e n e s i s callus
in P. milioides~
A_t Green spotter
B__~.R e g e n e r a t e d plantlets. in P. bisulcatum~
(g) developed
green shoot buds E. Regenerants
G-H. Different plantlet
(b)~ D_t. R e g e n e r a t e d
~n agarl
two m o r p h o l o g i c a l
C. The gummy
a w h i t e callus
(w) with plantlets~
F. R e g e n e r a n t s
in soil:
phenotypes.
stages during the d e v e l o p m e n t of a from a somatic embryo
in P. bisulcatum.
172 similar results. For both species the first s u b c u l t u r e m e d i u m c o n t a i n e d reduced levels both of sucrose and 2,4-D to s t i m u l a t e p o s s i b l e r e g e n e r a t i o n processes (Table 2). A s u p e r i o r g r o w t h was observed on the m e d i u m w i t h i0 g/l sucrose in the p r e s e n c e of 1 or 0.I mg/l 2,4-D. For both species r e g e n e r a t i o n of plants was p o s s i b l e only from calli s u b c u l t u r e d on a m e d i u m cont a i n i n g I0 g/l sucrose and 0.i mg/l 2 , 4 - D (see the 4th and f o l l o w i n g c o l u m n s of T a b l e 2). On this m e d i u m the first subculture, as well as the others w h i c h followed, p e r m i t t e d the r e c o v e r y of small but a l r e a d y well organized plantlets. In P. m i l i o i d e s w h i t e calli grown on 10g/l sucrose and in the p r e s e n c e of 0.i mg/l 2,4-D, d e v e l o p e d green spots. Plant p r i m o r d i a originated from such green s t r u c t u r e s and this was followed by an o r g a n o g e n e t i c type of d e v e l o p ment (Fig. 1 A-B). O r g a n o g e n e s i s was completed on the medium; from there, for further root d e v e l o p m e n t , 4 to 6 cm p l a n t l e t s w e r e t r a n s f e r r e d to a h o r m o n free medium. C a l l i from leaves, m e s o c o t y l s and seeds were all able to r e g e n e r a t e p l a n t l e t s only when they were induced at c o n c e n t r a t i o n s of 2.5 or 5 m g / l of 2,4-D. Based on a c t u a l n u m b e r s of plants regenerated, calli from seeds induced at 5 m g / l 2,4-D were, under the c o n d i t i o n s tested, the best source of tissue for plant r e g e n e r a t i o n in this species. About 10% of the P. b i s u l c a t u m calli c u l t u r e d on I0 g/l sucrose and 0.i mg/l 2,4-D o r i g i n a t e d white calli w i t h green s h o o t b u d s or formed somatic embryos. O r g a n o g e n e s i s was obtained from calli induced from m e s o c o t y l s at 2.5 or 5 mg/l 2,4-D and from seeds (Fig. 1 C-F). In this species true cases of somatic e m b r y o g e n e s i s were observed; isolated somatic embryos g e r m i n a t e d and d e v e l o p e d single p l a n t lets (Fig. 1 G-H). The most useful source of tissue for plant r e g e n e r a t i o n in P. bisulcarum r e v e a l e d calli from m e s o c o t y l induced at 2.5 m g / l of 2,4~D. After 1 to 3 s u b c u l t u r e s on MS w i t h o u t hormones, the well r o o t e d p l a n t l e t s of b o t h species r e a c h e d a height of about 15 cm when they were t r a n s p l a n t e d from agar to soil; t h o r o u g h w a s h i n g of the r o o t s b e f o r e transp l a n t i n g was c r u c i a l to give a success rate of about 95%. The d e v e l o p m e n t of p l a n t s from e m b r y o g e n i c calli and the g r o w t h of the regenerants under sterile c o n d i t i o n s w e r e m u c h slower in P. m i l i o i d e s than in P. bisulcatum. Therefore, up to now, we have been able to r e g e n e r a t e to m a t u r i t y and seed set 1032 plants in 8 m o n t h s and 94 p l a n t s in 6 m o n t h s from P. b i s u l c a t u m and P. m i ! i o i d e s , r e s p e c tively, w h i c h is a p p r o x i m a t e l y 90% of success in r e g e n e r a t i n g plants. The c a p a c i t y of the e m b r y o g e n i c c a l l u s to d e v e l o p p l a n t l e t s decreased w i t h time of c u l t i v a t i o n in b o t h species. In the first g e n e r a t i o n of r e g e n e r a n t s (R-l) in both species we noted a s t r i k i n g p h e n o t y p i c uniformity. Only in P. b i s u l c a t u m two d i f f e r e n t p h e n o t y p e s were o b s e r v e d w i t h i n the regenerants: a tall, fast g r o w i n g type w i t h o u t panicles, r e s e m b l i n g the w i l d type, and a m o r e b u s h y type w i t h m a n y p a n i c l e s two m o n t h s after t r a n s p l a n t i n g into soil (Fig. l F).
DISCUSSION Callus induction for both P. m i l i o i d e s and P. b i s u l c a t u m must a p p a r e n t l y be done on m e d i a w i t h c o n c e n t r a t i o n s of 2,4-D not higher than 5 m g / l (21). Under such c o n d i t i o n s and among the explants tested for P. milioides, m e s o c o t y l s and basal parts of the l e a v e s are capable of p r o d u c i n g calli w h i c h r e g e n e r a t e plants. In P. bisulcatum, m e s o c o t y l s seem to be the m o r e p r o m i s i n g source of explants. Compared to P. m i l i o i d e s and P. bisulcatum, only P. maximum, a species of this genus whose b e h a v i o r in vitro has been c a r e f u l l y studied (9,10,Ii,~'), can p r o d u c e r e g e n e r a b l e calli even at c o n c e n t r a t i o n s of 2,4-D as high as 10 mg/l. P. m i l i o i d e s and P, b i s u l c a t u m seem to be m o r e similar to P, miliaceum, P. m i l i a r e and P. antidole w h e r e the levels of 2,4-D for callus induction is f r e q u e n t l y r e p o r t e d to be not higher than 2.5 mg/l (2,7,18). O p t i m a l conditions, among those tested, for s u b c u l t ~ r i n g calli and r e g e n e r a t i n g p l a n t s of P. m i l i o i d e s and P. b i s u l c a t u m were i0 g/1 sucrose and 0.i mg/l 2,4-D. On such a medium, the calli increase in size while p r o d u c i n g green areas and d e v e l o p p l a n t l e t s via o r g a n o g e n e s i s and e m b r y o g e n e s i s . Such cultures can s u c c e s s f u l l y be s u b c u l t u r e d further on the same medium. In the first series of callus induction, e m b r y o g e n i c callus of P. bisulcatum p r o d u c e d 1032 p l a n t l e t s d u r i n g i0 to 12 s u b c u l t u r e s of the e m b r y o g e n i c callus w i t h i n 6 to 8 months. The r e g e n e r a t i o n p r o c e s s of P. m i l i o i d e s is m u c h slower, so far we have been able to r e g e n e r a t e to m a t u r i t y only 94 p l a n t s d u r i n g i0 to 12 subcultures of the e m b r y o g e n i c callus w i t h i n 6 months. Up to naw, in a second series of callus induction, e m b r y o g e n i c callus of b o t h species p r o d u c e d p l a n t l e t s d u r i n g 6 months, but they are not included in the number of r e g e n e r a n t s m e n t i o n e d above. These calli so far possess a high plant r e g e n e r a t i o n capacity. The c o n t i n u o u s a v a i l a b i l i t y of plants for i s o l a t i n g s o m a c l o n a l v a r i a n t s after in v i t r o c u l t u r e and plant r e g e n e r a t i o n (8) is the p r e r e q u i s i t e for s e l e c t i n g p h o t o s y n t h e tic and p h o t o r e s p i r a t o r y v a r i a n t s in the R-2 generation. R-I plants o b t a i n e d in both species a p p e a r e d rather stable in their phenotype, a s i t u a t i o n a l r e a d y found in other r e g e n e r a t e d P a n i c u m species (9,11,18), a l t h o u g h two d i f f e r e n t p h e n o t y p e s for g r o w t h habit could be o b s e r v e d in P. bisulcatum.
ACKNOWLEDGEMENT We w o u l d like to g r a t e f u l l y t h a n k Mrs B. P i e g e l e r and S. Franken for e x c e l l e n t t e c h n i c a l assistance.
REFERENCES 1 A m m i r a t o PV (1984 In: Evans DA, Sharp WA, A m m i r a t o PV, Y a m a d a Y (eds) H a n d b o c k of plant cell culture, Vol I, M a c m i l l a n Publ i s h i n g Co, pp 82-123 2 Bajaj YPS, Sidhu BS, Dubey VK (1981) E u p h y t i c a 30: 135-140 3 B h o j w a n i SS, Razdan MK (1983) D e v e l o p m e n t s in Crop Science (5): Plant Tissue Culture. Elsevier, Amsterdam, Oxford, New York, Tokyo, pp 91-112
173 4 Brown RH, Brown WV (1975) Crop Science 15: 681-685 5 Downton WJS (1975) P h o t o s y n t h e t i c a 9: 96105 6 Haccius B (1978) P h y t o m o r p h o l o g y 28: 74-81 7 H e y s e r JW (1983) Z P f l a n z e n p h y s i o l 113: 293-299 8 Larkin PJ, Scowcroft WR (1981) Theor Appl Gen 60: 197-214 9 Lu C, Vasil IK 1981a) Theor Appl Gen 59: 275-280 i0 Lu C, V a s i l IK 1981b) Ann Bot 48: 543-548 ii Lu C, Vasil IK 1982) Amer J Bot 69: 77-81 12 Lu C, Vasil V, Vasil IK (1981) Z P f l a n z e n p h y s i o l 104: 311-318 13 Miles D (1982) In: E d e l m a n M, H a l l i c k RB, Chua NH (eds) M e t h o d s in C h l o r o p l a s t Molecular Biology, E l s e v i e r B i o m e d i c a l Press, Amsterdam, pp 75-107 14 Moss DN, K r e n z e r Jr EG, Brun WA (1969) Science 164: 187-188
15 M u r a s h i g e T, Skoog F (1962) Physiol Plant 15: 473-479 16 R a g h a v e n d r a AS, Das VSR (1978) P h o t o s y n t h e tica 12: 200-208 17 Rangan TS (1974) Z P f l a n z e n p h y s i o l 72: 456-459 18 Rangan TS Vasil IK (1983) Z P f l a n z e n p h y siol 109: 49-53 19 Rangan TS Vasil IK (1984) In: A m m i r a t o PV, Evans DA,. Sharp WR, Y a m a d a Y (eds) Handbook of plant cell culture, Vol III, M a c m i l l a n P u b l i s h i n g Company, New York, pp 126-150 20 S o m e r v i l l e CR, Ogren WL (1982) In: E d e l m a n M, H a l l i c k RB, Chua NH (eds) M e t h o d s in C h l o r o p l a s t M o l e c u l a r Biology, E l s e v i e r B i o m e d i c a l Press, Amsterdam, pp 129-138 21 Vasil V, Vasil IK (1984) In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, Vol I, A c a d e m i c Press, pp 36-42
Plant Cell Reports (1986) 3:169-173
© Springer-Verlag 1986
Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides M a t t h i a s F l a d u n g and J o s e f H e s s e l b a c h Ma×-Planck-Institut For Zt~chtungsforschung, Abteilung PflanzenzOchtung, D-5000 K~ln 30, FRG Received December 13, 1985 / Revised version received March 10, 1986 - Communicated by H. L0rz
ABSTRACT Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcat u m T h u m b . , as w e l l as b a s a l p a r t s of l e a v e s and m e s o c o t y l s f r o m s t e r i l e s e e d l i n g s , a n d s e e d s f r o m P a n i c u m m i l i o i d e s N e e s ex. T r i n w e r e u s e d as e x p l a n t s to i n d u c e c a l l u s on a Murashige and Skoog medium supplemented w i t h 2.5 to !0 m g / l of 2 , 4 - D . S u b c u l t u r i n g of t h e w h i t e c a l l u s f r o m P. m i l i o i d e s a n d of the b r o w n c a l l u s f r o m P. b i s u l c a t u m on a m e d i u m c o n t a i n i n g 0.i m g / l 2 , 4 - D a n d i0 g/l s u c r o s e led in b o t h s p e c i e s to the a p p e a r a n c e of g r e e n s t r u c t u r e s f r o m w h i c h p l a n t s c o u l d be r e g e n e r a t e d . P l a n t s w e r e r e g e n e r a ted b y an o r g a n o g e n e t i c p r o c e s s in P. m i l i o i d e s , w h i l e P. b i s u l c a t u m p l a n t s w e r e r e generated both via organogenesis and somatic embryogenesis. 1032 a n d 94 p l a n t s , f r o m P. b i s u l c a t u m a n d P. m i l i o i d e s , r e s p e c t i v e l y , w e r e t r a n s f e r r e d i n t o soil, a n d a b o u t 90% of t h e m w e r e g r o w n to m a t u r i t y a n d set seeds.
Abbreviatations: MS, M u r a s h i g e a n d S k o o g m e d i u m (15)} 2,4-D, 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid} IAA, i n d o l e a c e t i c acid.
s i o n s ( 7 , 1 0 , 1 2 ) . In the e a r l y in v i t r o w o r k d o n e in t h i s genus, r e g e n e r a t i o n of p l a n t s v i a o r g a n o g e n e s i s w a s r e p o r t e d in P. m i l i a c e u m a n d P. a n t i d o l e (2,17). H e r e we d e s c r i b e the i n d u c t i o n a n d m a i n t e n a n c e of c a l l u s t i s s u e s in P. b i s u l c a t u m , a C-3 s p e c i e s , a n d in P. m i l i o i d e s , a C-3-C-4 intermediate species, and regenera t i o n of p l a n t s v i a s o m a t i c e m b r y o g e n e s i s and o r g a n o g e n e s i s in P. b i s u l c a t u m , a n d v i a o r g a n o g e n e s i s in P. m i l i o i d e s f o r the f i r s t time. T h e p o s s i b i l i t y of i s o l a t i n g s o m a c l o n a l v a r i a n t s a f t e r in v i t r o c u l t u r e a n d plant regeneration (8) w a s one of the app r o a c h e s a d o p t e d in s e a r c h i n g for p h o t o s y n thetic and photorespiratory mutants. Members of the R-2 g e n e r a t i o n w i l l be s c r e e n e d for s u c h m u t a t i o n s . The p h o t o s y n t h e t i c ones will be s c r e e n e d w i t h the h i g h - c h l o r o p h y l l - f l u o r e s c e n c e - y i e l d m e t h o d d u r i n g e x p o s u r e to U V l i g h t (13), w h i l e the p h o t o r e s p i r a t o r y ones w i l l be i s o l a t e d b y t h e i r i n a b i l i t y to g r o w in n o r m a l a i r c o n d i t i o n s (20). N o r m a l l y , such mutants are isolated after mutagen t r e a t m e n t of seeds, but n o w w e i n t e n d to s c r e e n for s u c h v a r i a n t s w i t h o u t p h y s i c a l or chemical mutagen treatment.
MATERIAL
AND METHODS
INTRODUCTION In m a n y s p e c i e s w i t h i n the m i l l e t s c a l lus i n d u c t i o n w a s r e p o r t e d a n d r e g e n e r a n t s were obtained via organogenesis and somatic embryogenesis (19). A l l t h e s e s p e c i e s , s u c c e s s f u l l y c u l t i v a t e d in v i t r o , a r e c h a r a c t e r i z e d b y h a v i n g the C-4 t y p e of p h o t o s y n t h e s i s (5,16). In the g e n u s P a n i c u m , s p e c i e s w i t h C - 3 a n d C-4 t y p e of p h o t o s y n t h e s i s have been characterized (14). A d d i t i o n a l l y socalled C-3-C-4 intermediate species have b e e n f o u n d (4). C a l l u s i n d u c t i o n w i t h i n the P a n i c u m s p e c i e s w a s r e p o r t e d o n l y for C-4 s p e c i e s (5,16): in P. m a x i m u m ( 2 , 9 , 1 1 ) , P. m i l i a c e u m ( 2 , 7 , 1 7 , 1 8 ) , P. m i l i a r e (18) a n d P. a n t i d o l e (2). R e g e n e r a t i o n of p l a n t s is p o s s i b l e v i a somatic embryogenesis in P. m a x i m u m a n d P. miliare starting from calli obtained from y o u n g l e a v e s , m a t u r e and i m m a t u r e e m b r y o s , a n d young inflorescences ( 9 , 1 1 , 1 8 ) ; in P. m a x i m u m a n d in P. m i l i a c e u m f r o m c e l l s u s p e n -
Offprint requests to: J. Hesselbach
S e e d s of P. b i s u l c a t u m T h u m b (PI 1 9 4 8 6 1 ) a n d P. m i l i o i d e s N e e s ex. T r i n (PI 2 8 4 2 2 0 ) w e r e o b t a i n e d f r o m the S o u t h e r n R e g i o n a l P l a n t I n t r o d u c t i o n S t a t i o n , G e o r g i a , USA. S e e d s w e r e s u r f a c e s t e r i l i z e d w i t h 70% a l c o h o l for 1 min, f o l l o w e d b y 0 . 1 % H g C I - 2 / T w e e n 20 for 3 m i n and g e r m i n a t e d e i t h e r on MS a g a r w i t h o u t h o r m o n e s or d i r e c t l y on c a l lus i n d u c t i o n m e d i u m (MS w i t h 5 m g / l 2,4-D, 30g/l sucrose). Twenty day old seedlings grown without hormones were dissected into l e a v e s w i t h (P. m i l i o i d e s ) or w i t h o u t b a s i s , w h i c h w e r e cut i n t o t h r e e to f i v e p i e c e s , a n d m e s o c o t y l . T h e s e e x p l a n t s w e r e u s e d to i n i t i a t e c a l l u s f o r m a t i o n on c a l l u s i n d u c t i o n m e d i u m (MS w i t h 2 . 5 - 1 0 m g / l 2,4-D, 30 g/l s u c r o s e ) C a l l i w e r e o b t a i n e d w i t h i n two to t h r e e w e e k s ; t h e y w e r e s u b c u l t u r e d on MS, c o n t a i n ing i0 or 1 g / l s u c r o s e a n d 1 or 0.i m g / l 2 , 4 - D . A f t e r 1 to 3 s u b c u l t u r e s g r e e n s h o o t b u d s or s m a l l p l a n t l e t s d e r i v e d f r o m s o m a t i c
170 e m b r y o s w e r e t r a n s f e r r e d on 2 , 4 - D f r e e m e d i u m (MS, w i t h 20 g / l sucrose, w i t h o u t h o r m o n e s or w i t h 2 m g / l Z e a t i n ) . In b o t h s p e c i e s r e g e n e r a n t s w e r e o b t a i n e d d u r i n g i0 to 12 s u b c u l t u r e s of the e m b r y o g e n i c c a l l u s . S u b c u l t u r e s w e r e d o n e e v e r y 3 to 4 w e e k s . All media and glass culture vessels were sterilized by autoclaving. The cultures were i n c u b a t e d in a g r o w t h r o o m at 2 4 ~ 0 . 2 ° C , exp o s e d to a r t i f i c i a l l i g h t ( O S R A M N a t u r a L 5 8 W / 25) of 11.07 W / m 2 w i t h l i g h t / d a r k c y c l e s of 13.5/10.5 hours.
w a s f o r m e d at a l l 2 , 4 - D l e v e l s t e s t e d s t a r t ing f r o m b a s a l p a r t s of the l e a v e s and f r o m m e s o c o t y l s . No c a l l u s i n d u c t i o n w a s a c h i e v e d f r o m the u p p e r p a r t s of the l e a v e s . T h e o n l y 2 , 4 - D l e v e l t e s t e d for s e e d s as e x p l a n t s w a s 5 m g / l . O n l y a b o u t 5% of t h e s e c u l t e r e d explants formed callus. In P. b i s u l c a t u m , a b r o w n , g u m m y c a l l u s was visible only when explants were used from m e s o c o t y l s or seeds. N o c a l l u s i n d u c t i o n w a s a c h i e v e d f r o m l e a v e s w i t h o u t b a s i s . At 2.5 m g / l 2 , 4 - D m e s o c o t y l e x p l a n t s g a v e the h i g h est p e r c e n t a g e of c a l l u s f o r m a t i o n . T h e p e r c e n t a g e r a n g e d f r o m 76% at 2.5 m g / l 2 , 4 - D to 7% at i0 m g / l . T h e o n l y 2 , 4 - D l e v e l t e s t e d for s e e d s a g a i n w a s 5 m g / 1 . B u t h e r e w e got a m u c h h i g h e r p e r c e n t a g e of c a l l u s i n d u c t i o n t h a n in P. m i l i o i d e s . A r e p e t i t i o n of c a l l u s i n d u c t i o n f r o m m e s o c o t y l in b o t h s p e c i e s gave
RESULTS Callus induction was achieved from s e v e r a l e x p l a n t s of the two P a n i c u m s p e c i e s ( T a b l e i). In P. m i l i o i d e s , a w h i t e c a l l u s
Table
i. C a l l u s i n d u c t i o n (% of c u l t u r e d e x p l a n t s , absolute numbers) from different explants s p e c i e s on m e d i a c o n t a i n i n g v a r i o u s 2 , 4 - D
2,4-D (mg/l)
P. milioides meso-
seed
cotyl
basis
Table
P. bJsulcatum
leaf with
in b r a c k e t s the of t w o P a n i c u m concentrations.
2.5
4.7 (150)
42 (220)
5.0
2.0 (150)
53 (223)
/
/
5.2 (135)
leaf without
meso-
basis
cotyl
0 (151)
76 (268)
0 (150)
45 (266)
seed
/
/
75 (128)
7.5
6.0 (149)
60 (219)
/
/
0 (150)
12 (256)
/
/
I0.0
4.7 (150)
43 (230)
/
/
0 (150)
7 (260)
/
/
2. G r o w t h of c a l l i on s u b c u l t u r e m e d i a (++ v e r y g o o d g r o w t h ; + g o o d g r o w t h ; - no g r o w t h ) , a n d the c a p a c i t y of p l a n t r e g e n e r a t i o n d u r i n g the f i r s t or the f o l l o w i n g s u b c u l t u r e s (o o r g a n o g e n e s i s ; e e m b r y o g e n e s i s )
Species £xplant 2,4-D (mg/l) Of induction
medium
Subculture medium for callus growth and plant regeneration Sucrose 10g/l 2,4-D 0.1mg/l Sucrose I g l l Sucrose 10g/1 2,4-D Img/] Callus Regeneration n ° of plants regenerated 2,4-D Img/l Callus growth(1) Callus growth(1) growth p r o c e s s per s i n g l e e x p ] a n t t o t a l C a l l u s growth(I) Sucrose
lg/l
2,4-D 0.1 mg/l
P. milloJdes Leaf with basis
2.5 5.0 7.5 10.0
+/-
+
o
1
2
4/-
4
o
1
2
~/-
+÷
+/-
+
o
1-4
7
o
1
1
o
1-26
e,o e,o
42-82
Mesocotyl 4
2.5
5.0 7.5
44
10.0
÷
Seed
+/-
5.0
82
P. bisu] Catum Mesocotyl
2.5 5.0 7.5 10.0
+/-
÷
~+
.+]-
÷
+
+/+[-
4/--
4/4]-
27-291
835 124 ./-
Seed
./-
5.0
(1)
no p l a n t s
regenerated
o under
these conditions
73
73
171
Figure
~. Plant regeneration from c u l t u r e d calli of P. milioides
and P. b i s u l c a t u m .
A-B. O r g a n o g e n e s i s callusl
C-F. O r g a n o g e n e s i s callus
in P. milioides~
A_t Green spotter
B__~.R e g e n e r a t e d plantlets. in P. bisulcatum~
(g) developed
green shoot buds E. Regenerants
G-H. Different plantlet
(b)~ D_t. R e g e n e r a t e d
~n agarl
two m o r p h o l o g i c a l
C. The gummy
a w h i t e callus
(w) with plantlets~
F. R e g e n e r a n t s
in soil:
phenotypes.
stages during the d e v e l o p m e n t of a from a somatic embryo
in P. bisulcatum.
172 similar results. For both species the first s u b c u l t u r e m e d i u m c o n t a i n e d reduced levels both of sucrose and 2,4-D to s t i m u l a t e p o s s i b l e r e g e n e r a t i o n processes (Table 2). A s u p e r i o r g r o w t h was observed on the m e d i u m w i t h i0 g/l sucrose in the p r e s e n c e of 1 or 0.I mg/l 2,4-D. For both species r e g e n e r a t i o n of plants was p o s s i b l e only from calli s u b c u l t u r e d on a m e d i u m cont a i n i n g I0 g/l sucrose and 0.i mg/l 2 , 4 - D (see the 4th and f o l l o w i n g c o l u m n s of T a b l e 2). On this m e d i u m the first subculture, as well as the others w h i c h followed, p e r m i t t e d the r e c o v e r y of small but a l r e a d y well organized plantlets. In P. m i l i o i d e s w h i t e calli grown on 10g/l sucrose and in the p r e s e n c e of 0.i mg/l 2,4-D, d e v e l o p e d green spots. Plant p r i m o r d i a originated from such green s t r u c t u r e s and this was followed by an o r g a n o g e n e t i c type of d e v e l o p ment (Fig. 1 A-B). O r g a n o g e n e s i s was completed on the medium; from there, for further root d e v e l o p m e n t , 4 to 6 cm p l a n t l e t s w e r e t r a n s f e r r e d to a h o r m o n free medium. C a l l i from leaves, m e s o c o t y l s and seeds were all able to r e g e n e r a t e p l a n t l e t s only when they were induced at c o n c e n t r a t i o n s of 2.5 or 5 m g / l of 2,4-D. Based on a c t u a l n u m b e r s of plants regenerated, calli from seeds induced at 5 m g / l 2,4-D were, under the c o n d i t i o n s tested, the best source of tissue for plant r e g e n e r a t i o n in this species. About 10% of the P. b i s u l c a t u m calli c u l t u r e d on I0 g/l sucrose and 0.i mg/l 2,4-D o r i g i n a t e d white calli w i t h green s h o o t b u d s or formed somatic embryos. O r g a n o g e n e s i s was obtained from calli induced from m e s o c o t y l s at 2.5 or 5 mg/l 2,4-D and from seeds (Fig. 1 C-F). In this species true cases of somatic e m b r y o g e n e s i s were observed; isolated somatic embryos g e r m i n a t e d and d e v e l o p e d single p l a n t lets (Fig. 1 G-H). The most useful source of tissue for plant r e g e n e r a t i o n in P. bisulcarum r e v e a l e d calli from m e s o c o t y l induced at 2.5 m g / l of 2,4~D. After 1 to 3 s u b c u l t u r e s on MS w i t h o u t hormones, the well r o o t e d p l a n t l e t s of b o t h species r e a c h e d a height of about 15 cm when they were t r a n s p l a n t e d from agar to soil; t h o r o u g h w a s h i n g of the r o o t s b e f o r e transp l a n t i n g was c r u c i a l to give a success rate of about 95%. The d e v e l o p m e n t of p l a n t s from e m b r y o g e n i c calli and the g r o w t h of the regenerants under sterile c o n d i t i o n s w e r e m u c h slower in P. m i l i o i d e s than in P. bisulcatum. Therefore, up to now, we have been able to r e g e n e r a t e to m a t u r i t y and seed set 1032 plants in 8 m o n t h s and 94 p l a n t s in 6 m o n t h s from P. b i s u l c a t u m and P. m i ! i o i d e s , r e s p e c tively, w h i c h is a p p r o x i m a t e l y 90% of success in r e g e n e r a t i n g plants. The c a p a c i t y of the e m b r y o g e n i c c a l l u s to d e v e l o p p l a n t l e t s decreased w i t h time of c u l t i v a t i o n in b o t h species. In the first g e n e r a t i o n of r e g e n e r a n t s (R-l) in both species we noted a s t r i k i n g p h e n o t y p i c uniformity. Only in P. b i s u l c a t u m two d i f f e r e n t p h e n o t y p e s were o b s e r v e d w i t h i n the regenerants: a tall, fast g r o w i n g type w i t h o u t panicles, r e s e m b l i n g the w i l d type, and a m o r e b u s h y type w i t h m a n y p a n i c l e s two m o n t h s after t r a n s p l a n t i n g into soil (Fig. l F).
DISCUSSION Callus induction for both P. m i l i o i d e s and P. b i s u l c a t u m must a p p a r e n t l y be done on m e d i a w i t h c o n c e n t r a t i o n s of 2,4-D not higher than 5 m g / l (21). Under such c o n d i t i o n s and among the explants tested for P. milioides, m e s o c o t y l s and basal parts of the l e a v e s are capable of p r o d u c i n g calli w h i c h r e g e n e r a t e plants. In P. bisulcatum, m e s o c o t y l s seem to be the m o r e p r o m i s i n g source of explants. Compared to P. m i l i o i d e s and P. bisulcatum, only P. maximum, a species of this genus whose b e h a v i o r in vitro has been c a r e f u l l y studied (9,10,Ii,~'), can p r o d u c e r e g e n e r a b l e calli even at c o n c e n t r a t i o n s of 2,4-D as high as 10 mg/l. P. m i l i o i d e s and P, b i s u l c a t u m seem to be m o r e similar to P, miliaceum, P. m i l i a r e and P. antidole w h e r e the levels of 2,4-D for callus induction is f r e q u e n t l y r e p o r t e d to be not higher than 2.5 mg/l (2,7,18). O p t i m a l conditions, among those tested, for s u b c u l t ~ r i n g calli and r e g e n e r a t i n g p l a n t s of P. m i l i o i d e s and P. b i s u l c a t u m were i0 g/1 sucrose and 0.i mg/l 2,4-D. On such a medium, the calli increase in size while p r o d u c i n g green areas and d e v e l o p p l a n t l e t s via o r g a n o g e n e s i s and e m b r y o g e n e s i s . Such cultures can s u c c e s s f u l l y be s u b c u l t u r e d further on the same medium. In the first series of callus induction, e m b r y o g e n i c callus of P. bisulcatum p r o d u c e d 1032 p l a n t l e t s d u r i n g i0 to 12 s u b c u l t u r e s of the e m b r y o g e n i c callus w i t h i n 6 to 8 months. The r e g e n e r a t i o n p r o c e s s of P. m i l i o i d e s is m u c h slower, so far we have been able to r e g e n e r a t e to m a t u r i t y only 94 p l a n t s d u r i n g i0 to 12 subcultures of the e m b r y o g e n i c callus w i t h i n 6 months. Up to naw, in a second series of callus induction, e m b r y o g e n i c callus of b o t h species p r o d u c e d p l a n t l e t s d u r i n g 6 months, but they are not included in the number of r e g e n e r a n t s m e n t i o n e d above. These calli so far possess a high plant r e g e n e r a t i o n capacity. The c o n t i n u o u s a v a i l a b i l i t y of plants for i s o l a t i n g s o m a c l o n a l v a r i a n t s after in v i t r o c u l t u r e and plant r e g e n e r a t i o n (8) is the p r e r e q u i s i t e for s e l e c t i n g p h o t o s y n t h e tic and p h o t o r e s p i r a t o r y v a r i a n t s in the R-2 generation. R-I plants o b t a i n e d in both species a p p e a r e d rather stable in their phenotype, a s i t u a t i o n a l r e a d y found in other r e g e n e r a t e d P a n i c u m species (9,11,18), a l t h o u g h two d i f f e r e n t p h e n o t y p e s for g r o w t h habit could be o b s e r v e d in P. bisulcatum.
ACKNOWLEDGEMENT We w o u l d like to g r a t e f u l l y t h a n k Mrs B. P i e g e l e r and S. Franken for e x c e l l e n t t e c h n i c a l assistance.
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