Effect of plant growth regulators on somatic embryogenesis in leaf ...

PlantCell Reports

Plant Cell Reports (1991) t0:179-182

9 Springer-Verlag 1991

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora T. Hatanaka t, O. Arakawa 2, T. Yasuda 2, N. Uchida 2, and T. Yamaguchi 2 Graduate School of Science and Technology 1, Department of Agriculture and Horticulture 2, Kobe University, Rokko, Kobe 657, Japan Received December 14, 1990/Revised version received M a r c h 13, 1991 - Communicated by J. K. Vasil

ABSTRACT The effects of plant growth regulators on somatic embryogenesis were studied in l e a f cultures of C o f f e a canephora. The maximum n u m b e r of s o m a t i c embryos were obtained on m e d i a that c o n t a i n e d only cytokinln as a plant growth regulator. A l l of the a u x i n s t e s t e d (NAA, IBA, IAA and 2,4-D) inhibited the formation of embryos. The optimal c o n c e n t r a t i o n of each cytokinin (2-iP, BA and kinetin) for somatic e m b r y o g e n e s i s was 5 ~M. Under optimal conditions, each explant formed more than i00 e m b r y o l d s w i t h little callus and few adventitious roots. E m b r y o i d s were formed only at the cut edges of the leaf discs. C y t o k i n i n s were a b s o r b e d only at the cut edges of leaf discs that were in contact with the medium, and were not t r a n s p o r t e d to other parts of the explant. ABBREVIATIONS: 2-iP : i s o - p e n t e n y l a d e n i n e , BA : benzyladenine, N A A : l - n a p h t h a ~ e n e a c e t i e acid, IBA : i n d o l e - 3 - b u t y r l c acid, IAA : i n d o l e - 3 - a c e t l c acid, 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 INTRODUCTION C o f f e a c a n e p h o r a is a commercial crop used for the m a n u f a c t u r e of instant coffee and is k n o w n for its resistance to coffee leaf rust. Since C__L e a n e p h o r a is self-incompatible, the p r o p a g a t i o n of p r o d u c t i v e mature trees has t o depend on v e g e t a t i v e propagation, for example. by c u t t i n g and grafting. A method for rapid propagation via tissue culture would be very u s e f u l for the p r o p a g a t i o n of this crop. T i s s u e culture of coffee was carried out by S t a r i t s k y (1970) at first, and. t h e n Herman and Haas (1975) reported the regeneration of plants of C. arablca. Later, Sondahl et al. (1981) studied somatic embryogenesis in C_u, arabica. T h e m e t h o d s u s e d by t h e m i n v o l v e d c o m b i n a t i o n s of auxins and cytokinins as plant g r o w t h regulators, and required long periods of time for regeneration. Furthermore, the rate of r e g e n e r a t i o n was very low. Earlier, we have

Offprint requests to. T. Yasuda

descrived the p r o d u c t i o n of embryogenic callus in a defined m e d i u m that contained only benzyladenine (BA) as the p l a n t g r o w t h r e g u l a t o r from leaf cultures of Coffea arabica (Yasuda et al. 1985). Coffea e a n e p h o r a is the first species in the g e n u s C o f f e a in w h i c h regeneration by tissue culture has been successful (Staritsky 1970). P i e r s o n et al. (1983) reported embryogenesis in C_~. canephora when a combination of 1 mg/l 2-iP and 5 mg/l IBA was used, but they did not discuss the effects of the kind and concentration of t h e p l a n t growth regulators on embryogenesis. We h a v e s t u d i e d effects of various plant growth regulators in o r d e r to establish a more efficient method, and discuss the importance of eytokinin on somatic embryogenesis in leaf cultures of C__u. c_anephora, MATERIALS

AND M E T H O D S

M a t e r i a l s and p r e p a r a t i o n of explants. The plant material was collected from 3year-old trees of Coffea canephora Pierre ex Frohner, which were growing in a greenhouse. Y o u n g l e a v e s (7.5-10 cm long), the second from the top, were washed twice with a 1% solution of T w e c n 20. They were disinfected in a 1% solution of sodium h y p o c h l o r i t e that c o n t a i n e d 0.5% T w e e n 20 for 20 minutes and then rinsed 5 times with sterilized d i s t i l l e d water. Leaf discs (5 mm in diameter) were punched out in a 1% solution of sodium ascorbate, and placed on agar solidified media. N u t r i e n t m e d i u m and culture conditions Tile d e f i n e d b a s a l m e d i u m c o n t a i n e d 1/4 strength m a c r o salts and half strength m i c r o salts of MS m e d i u m (Murashlge and S k o o g 1962), organic c o n s t i t u e n t s of B5 m e d i u m (Gamborg et al. 1968), and 30 g/l sucrose (Table i). This basal medium was supplemented with growth regulators and ~ts DH was adjusted to 5.7 by a d d i t i o n of Na0H or HCI. The agar solidified m e d i u m (15 ml) was dispensed in culture vials (35 m m in d i a m e t e r x 75 m m in h e i g h t ) . The vials were closed with a l u m i n u m caps and auto-

t80 T a b l e i. Basal m e d i u m used for tissue culture of coffee leaves, m o d i f i e d version of M u r a s h i g e & S k o o g ' s medium. Basal m e d i u m M a c r o salts NH4NO 3 KNO 3 MgSO4" 7H20

KH2PO4 CaCI 2 9 2H20 M i c r o salts H3BO 3 MnSO 4 - 4H20 ZnSO 4 " 7 H 2 0 N a 2 M o O 4 92H20 CuSO4"5H20 Fe-Na-EDTA Organic constituents Inositol N i c o t i n i c acid P y r i d o x i n e HCI T h i a m i n e HC1 Sucrose Agar pH = 5.7

(mg/l)

412. S 475 ~0 92.5

85.0 ii0.0

3. i Ii. 15 4.3 0. 125 0.05 21.0 i00.0 1.0 1.0 I0.0 30,000,0 9,000,0

claved for 12 m i n u t e s at 120"C, C u l t u r e s were m a i n t a i n e d with 14 hours of l i g h t a n d i0 h o u r s of d a r k n e s s daily, u n d e r fluorescent light with a intensity o f 30 ~ m o l m - 2 s -I a t 25" C. Visible embryoids (> a b o u t 0.5 m m long) were c o u n t e d after a b o u t 2 m o n t h s of culture.. E f f e c t s of v a r i o u s plant growth regulators on embryogenesls. The leaf discs were c u l t u r e d on m e d i a that contained various plant growth r e g u l a t o r s at different concentrations, namely, the cytokinins 2~iP, BA and kinetin, and the auxins NAA, IBA, IAA and 2,4-D. The c o n c e n t r a t i o n s of each are given in the legends to F i g u r e s i, 2 and 3, M e t h o d s of i n c u b a t i o n T h e leaf discs were i n c u b a t e d in d i f f e r e n t w a y s as shown d i a g r a m m a t i c a l l y in Figure 5. The culture medium contained 5 ~M 2-iP as plant growth regulator. C o n d i t i o n s w e r e as follows: A. T h e l e a f d i s c w a s p l a c e d w i t h the u p p e r e p i d e r m i s in contact with the medium. B. Half of the disc was immersed v e r t i c a l l y in the medium. C. The entire disc was immersed v e r t i c a l l y in the medium. D. The entire disc was i m m e r s e d h o r i z o n t a l l y in the medium. Autoradiography Halves of leaf discs were immersed vertic a l l y in m e d i u m that c o n t a i n e d 5 # M benzyl[814C] adenine (HA, specific activity 37 KBq/~mol; Amersham Japan, Tokyo). Explants were a n a l y z e d at intervals of I0 days. They were dried between filter papers and exposes to K o d a k D i a g n o s t i c Film (X-OMAT, AR) for one

day.

RESULTS AND D I S C U S S I O N The e f f e c t s of p l a n t growth r e g u l a t o r s on embryogenesis were examined in Coffea canehp~Lg_L r a. Whenmedia t h a t c o n t a i n e d 5 ~M 2 - i P or BA and v a r i o u s c o n c e n t r a t i o n s of NAA were u s e d , e f f e c t i v e e m b r y o g e n e s i s was o b s e r v e d on t h e media t h a t c o n t a i n e d c y t o k i n i n s a l o n e . Init i a l l y , small globular structures a p p e a r e d , and then embryoids were observed after 5 w e e k s of culture. NAA markedly inhibited somatic embryogenesis.in direct proportion to its concentration, but it promoted formation of adventitious roots and non-embryogenic callus (Figure I). 2-iP was more effective than BA for embryogenesis, When the explants were cultured on media that contained 5 ~ M 2-iP with various concentrations of IBA, IAA or 2,4-D, e m b r y o g e n e s i s was i n h i b i t e d as w h e n N A A w a s p r e s e n t , w i t h some d i f f e r e n c e in the e x t e n t of i n h i b i t i o n (Figure 2). Both NAA and 2,4-D were inhibitory even at c o n c e n t r a t i o n s as low as 5 nM. Auxin was not n e e d e d at all a n d c y t o k i n i n was the essential factor for somatic e m b r y o g e n e s i s in leaf c u l t u r e s of C. ean eRhora. Next, d i s c s were c u l t u r e d on m e d l a that contained different c o n c e n t r a t i o n s of cytokinins w i t h o u t auxin, and 5 ~ M was found to be the optimal c o n c e n t r a t i o n for e m b r y o g e n e s l s of all t e s t e d c y t o k i n i n s (Figure 3). E m b r y o g e n e s i s from leaves of C. canephora is i n d u c e d e a s i l y a n d r a p i d l y by o u r s i m p l e p r o c e d u r e , w l t h c y t o k i n i n as the sole p l a n t growth regulator (as compared w i t h embryogenesis from C. arabica). When we did not include a u x i n s i n the m e d i u m , l i t t l e c a l l u s a n d f e w a d v e n t i t i o u s roots were formed. The number of explants With embryoids was 34% at 67 days of

210 " 200 -

o

E

d

z

X

2-iP [~IBA

120"

80 4o

0

5

50

500nM

Concentration of NAA Figure i. Effects of two cytokinins with various concentrations of NAA on the production of embryoids. Leaf discs from a single leaf were cultured on media that contained 5 ~M 2-iP or BA and various concentrations of NAA. The incubation condition corresponded to A in Figure 5. The numbers of embryoids were counted after 6 weeks of culture, vertical bars represent the standard errors of the means for 9 to 12 replicates.

181 90

I BA

I

80

160,

T

60

4G

if;

[~ T

E

20

2,4-D

/_

12(]

L

6o

~

I AA

70

-I-

8O

20

d Z 0

0.5

"

"

5

0

50 pM

0

0.5

5

0

5%M

5

50

50%M

Concentration of auxin Figure 2. E f f e c t s of auxins on the p r o d u c t i o n of embryoids. The culture m e d i a contained 5 ~ M 2-iP and v a r i o u s c o n c e n t r a t i o n s of IBA, IAA or 2,4-D. The incubation conditions c o r r e s p o n d e d to B in Figure 5. The numbers of embryoids were counted after 2 months of culture. Vertical bars represent the standard errors of the means for 9 to 12 replicates. Each graph shows the results o b t a i n e d with discs from a single leaf.

2-iP

110 100

"E

BA

80

30 F

~- Kinetin

60

~- 8o

20, .~-

60

E (IJ

40

40

1 10-

20

d

Z

20 1

0 5

25

pM

1

5

25 pM

1

5

25pM

Concentration of cytokinin Figure 3. Effects of various c o n c e n t r a t i o n s of various cytokinins on the p r o d u c t i o n of embryoids. The culture media c o n t a i n e d different concentrations of 2-iP, BA or k i n e t i n but no auxins. The i n c u b a t i o n conditions c o r r e s p o n d e d to B in Figure 5. The numbers of embryoids were counted after 2 months of culture. V e r t i c a l bars represent the standard errors of the means for 9 to 20 replicates. E a c h graph shows results obtained with discs from a single leaf.

Figure 4. The development of somatic embryos from coffee leaves, a: A globular embryo, b: A torpedo-stage embryo, c and d: Cotyledonary embryos. The bar represents 1 mm.

culture in the report of Pierson et al. (1983), while it was almost 100% at 40 days by our procedure. Each of the explants formed more than i00 embryoids under the optimal conditions, namely, when young tender leaves were cultured on medium that contained 5 #M 2-iP without auxin. Embryoids developed from globular e m b r y o s t o t o r p e d o - s t a g e embryos and finally into cotyledonary embryos (Figure 4). These embryos were similar to zygotic embryos formed in mature seeds of coffee. They were transferred t o f r e s h m e d i a a n d g r e w up t o p l a n t l e t s . Almost all of the plantlets could be transplanted to soil after they reached a height of 3 t o 4 cm, t h e n we c o u l d o b t a i n c o f f e e p l a n t s , normal in appearance. They are growing vigor-

182

A

B

C

D

Figure 5. Effects of conditions of incubation on embryogenesis. A: Embryoids were not formed at some points on the cut edges where they were not in contact with the medium as a result of curling. B: Embryolds were formed only at edges in the lower half of the disc that was half-immersed in the medium. C, D: Embryoids were formed along almost the entire cut edge. Table 2. Effects of conditions the production of embryolds. Incubation conditions

A B C D

of incubation on

Number of embryoids (N/explant) 303+

3.5

31.7+ 6.9 55.3+ 6.6 58.7 + 11.6

The leaf discs were cultured on m e d i u m that c o n t a i n e d 5 ~ M 2-1P. The n u m b e r s of embryoids were counted after 2 months of culture. Values represent the means + S,E. of 9 replicates.

Figure 7. Autoradlographs of leaf discs cultured on the medium supplemented with [14C]-BA. The e x p l a n t s w e r e c u l t u r e d on m e d i u m that contained 5 ~ M [14C]-BA for 20 days (a) and 40 days (b). The conditions of incubation corresponded to B in Figure 5. Radioactivity absorbed was 63.98 Bq (20 days) and 59.76 Bq (40 days) per disc. Bars represent 5 mm. medium in places where the edges of the discs had curled up and away from the medium. Conditions C and D resulted in many embryoids (Table 2). Under condition B, embryolds were formed in the lower half of the cut edges immersed in the medium (Figure 6). Figure 7 shows the autoradiographs of leaf discs cultured on medium that contained 14Clabeled BA for 20 and 40 days. Radioactivity was detected only at the edges of the discs that were submerged in the medium, Same results were obtained after i0 and 30 days of treatments (data not shown). Emhryogenesis occurred only in the regions where cytokinin was in contact with cut edges of coffee leaves. This indicates that cytoklnlns are not absorbed through the leaf epidermis, and tha~ cytokinins absorbed remain at the cut edges are not t r a n s p o r t e d into the leaf tissue. The areas directly exposed to eytokinins are the only embryo forming areas. Thus, cytokinins Clearly play an important role in somatic embryogenesis in coffee. ACKNOWLEDGEMENT

We are grateful to Professor H. imasekl Nagoya University for useful suggestions.

in

REFERENCES

Figure 6. E x a m p l e of f o r m a t i o n of embryoid along the edge of the lower half of discs after 2 months of culture. The conditions of incubation corresponded to B in Figure 5. The bar represents 5 mm. ously in a greenhouse. Our results indicate that C__~.~ h o r a leaves should be very useful for the establishmeng of a clonal propagation system and for studies of the m e c h a n i s m of somatic embryogenesls. When the leaf discs were cultured under different conditions, embryoids were formed only at the cut edges of the discs that were in contact with the m e d i u m (Figure 5). Under condition A, embryoids were not formed at the cut edges that were not in contact with the

i) Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 5 0 : 1 5 1 - 1 5 8 2) H e r m a n FRP, Haas GJ (1975) HortScl. I0: 588-589 3) Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 4) Pierson ES, v a n L a m m e r s o n AAN, Schel JHN, Staritsky G (1983) Protoplasma 1 1 5 : 2 1 7 - 2 2 1 5) Sondahl MR, Monaco LC, Sharp WR (1981) In: Thorpe TA (ed) Plant tissue culture, Academic Press New York, pp 325-347 6) S t a r i t s k y G (1970) Aeta. Bot. Neerl. 19: 509-514 7) Yasuda T, Fujll Y, Yamaguchl T ( 1 9 8 5 ) Plant Cell Physiol. 2 6 : 5 9 5 - 5 9 7