PlantCell Reports
Plant Cell Reports (1987) 6:348-351
© Springer-Verlag 1987
Somatic embryogenesis and plant regeneration in Caricapapaya L. tissue culture derived from root explants M. H. Chen 1, p. j. Wang 1, and E. Maeda 2 Institute of Botany, Academia Sinica, Taipei, Taiwan 11529, R.O.C. 2 Faculty of Agriculture, Nagoya University, Nagoya 464, Japan Received April 7, 1987 / Revised version received July 6, 1987 - Communicated by M. Tabata
ABSTRACT The r e g e n e r a t i o n p o t e n t i a l of shoot tip, stem, leaf, c o t y l e d o n and root e x p l a n t s of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studied. Callus i n d u c t i o n of these two c u l t i v a r s of papaya showed that the shoot tips and stems are most s u i t a b l e for f o r m i n g callus, w h i l e leaves, cotyledons and roots are c o m p a r a tively difficult to induce callus. Callus induction also varied with the varities. Somatic e m b r y o g e n e s i s was o b t a i n e d from 3m o n t h - o l d root cultures. A m e d i u m c o n t a i n i n g half s t r e n g t h of MS i n o r g a n i c salts, 160 mg/l adenine sulfate, 1.0 mg/l NAA, 0.5 mg/l kinetin and 1.0 mg/l GA 3 was o p t i m a l for embryogenesis. The callus m a i n t a i n e d high regenerative capacity after two years of c u l t u r e on this medium. Plants d e r i v e d from somatic e m b r y o s were o b t a i n e d under greenhouse c o n d i t i o n s . ABBREVIATIONS MS: M u r a s h i g e and S k o o g ' s (1962) medium, NAA: N a p h t h a l e n e a c e t i c acid, IAA: I n d o l e - 3 - a c e t i c acid, GA: G i b b e r e l l i c acid, PRV: P a p a y a ring spot virus.
been u t i l i z e d for" callus induction and r e g e n e r a t i o n , (Litz and Conover, 1983; Arora and Singh, 1978a), a c o m p a r a t i v e study on the s u i t a b i l i t y of v a r i o u s e x p l a n t s has not been made. In this study, the callus i n d u c t i o n and r e g e n e r a t i o n p o t e n t i a l of v a r i o u s sources of explants from two papaya c u l t i v a r s were compared. We also d e s c r i b e in vitro i n d u c t i o n of s o m a t i c e m b r y o s in c u l t u r e d root of Carica papaya L., the l o n g - t e r m m a i n t e n a n c e of strong e m b r y o g e n e s i s capacity, and p l a n t l e t f o r m a t i o n from s o m a t i c embryos. MATERIAL
AND M E T H O D S
Plant M a t e r i a l s . Seeds of two papaya varities, C a r i c a papaya L. 'Solo' and 'Sunrise', were supplied by the F e n g - S h e n Tropical H o r t i c u l t u r a l E x p e r i m e n t S t a t i o n of T a i w a n A g r i c u l t u r a l R e s e a r c h Institute. The seeds, after being w a s h e d for 1 h in tap water, were i m m e r s e d in 75% ethanol for i min, and then in 0.1% sodium h y p o c h l o r i t e for i0 min. After several rinses with s t e r i l i z e d water, they were sown on MS agar m e d i u m in 2.5x13 cm test tubes. Shoot tip, stem, leaf, c o t y l e d o n and root (with r o o t - t i p removed) were e x c i s e d from 4 to 6 - w e e k - o l d s e e d l i n g s with three leaves (5 to 8 cm in height). They were cut into 3-5 mm s e g m e n t s and were cultured.
INTRODUCTION Papaya (Carica papaya L°) plants in T a i w a n have been almost c o m p l e t e l y i n f e c t e d by ring spot virus. This d i s e a s e has become the single most serious p r o b l e m of the T a i w a n papaya industry. T h e r e f o r e , it is of g r e a t importance to a c h i e v e a method of papaya regeneration in order to breed ring spot virus r e s i s t a n t strains by m e a n s of tissue culture. R e c e n t a c h i e v e m e n t s in papaya tissue c u l t u r e have been r e v i e w e d by Litz (1984). E m b r y o g e n e s i s in papaya callus has been o b t a i n e d from s e e d l i n g p e t i o l e s e g m e n t s (DeB r u i j n e et al., 1974) and stem s e g m e n t s (Yie and Liaw, 1977). These two r e p o r t s d e s c r i b e a three-step and a t w o - s t e p culture procedure, respectively, to obtain somatic embryos. In these methods, it is n e c e s s a r y to t r a n s f e r the callus to a n o t h e r m e d i u m for e m b r y o g e n e s i s . A l t h o u g h v a r i o u s e x p l a n t s have
Offprint requests to: E. Maeda
C u l t u r e M e d i a and E n v i r o n m e n t . A modified M u r a s h i g e and Skoog (1962) m e d i u m was used as basal medium. This m e d i u m c o n t a i n e d half s t r e n g t h i n o r g a n i c salts, 0.5 mg/l t h i a m i n e HCI, 1.0 mg/l pyridoxin-HCl, 5.0 mg/l n i c o t i n i c acid, 2.0 mg/l glycine, 1.0 mg/l casein h y d r o l y s a t e , I00 mg/l m y o - i n o s i t o l , 160 mg/l a d e n i n e sulfate, 30 g/l sucrose, and 8 g/l agar. D i f f e r e n t c o m b i n a t i o n s of 1.0 mg/l NAA with k i n e t i n (0, 0.5 mg/l) and gibberellic acid (GA 3) (0, 1.0 mg/l) were used as s u p p l e m e n t s . The pH was a d j u s t e d ,to pH 5.8 with IN KOH and IN HCI before a d d i n g agar. Sterilization was by a u t o c l a v i n g at i.i k g / c m 2 (I12°C) for i0 min. GA 3 was f i l t e r sterilized. E m b r y o g e n i c callus was s u b c u l t u r e d on medium containing NAA, kinetin, and GA 3. G e r m i n a t e d s o m a t i c e m b r y o s were i s o l a t e d and c u l t u r e d on m e d i u m c o n t a i n i n g NAA only.
349 C u l t u r e s were m a i n t a i n e d at 25 ± 2°C in a growth room with 16 h light (2000 lux) and 8 h darkness. R e - e s t a b l i s h m e n t of P l a n t s in Soil. The in vitro r e g e n e r a t e d p l a n t l e t s were r e m o v e d from c u l t u r e tubes, w a s h e d free of agar m e d i u m and potted in moist m i x t u r e of sand : soil (I/i : v/v), and c o v e r e d with a beaker to m a i n t a i n high humidity. One p e r c e n t IAA was sprayed on the p l a n t l e t s for two weeks. The p l a n t l e t s were then t r a n s p l a n t e d to soil and c u l t u r e d under g r e e n h o u s e c o n d i t i o n s . RESULTS C a l l u s was i n d u c e d from p a p a y a shoot tip, stem, leaf, c o t y l e d o n and root c u l t u r e s (Table i). Most of the e x p l a n t s formed callus at the cut ends after i0 days. C o n s i d e r a b l e v a r i a t i o n in the d e v e l o p m e n t of callus among single e x p l a n t s was observed. The result of callus formation after 3 weeks, and the regeneration of s o m a t i c embryos after 3 m o n t h s are shown in T a b l e I. O p t i m a l callus formation took place on 1/2 MS m e d i u m c o n t a i n i n g 1.0 mg/l NAA and 0.5 mg/l kinetin. Shoot tips and stems are most suitable e x p l a n t s for f o r m i n g callus, while leaves, c o t y l e d o n s and roots are c o m p a r a t i v e l y recalcitrant to callus induction. C a l l u s was i n i t i a t e d m a i n l y from the base of the shoot tip, and at the cut end of stem segments. Those e x p l a n t s w i t h o u t callus p r o l i f e r a t i o n were u s u a l l y s w o l l e n p r e s u m a b l y due to e x p a n sion growth of tissue. Roots were o c c a s i o n a l l y from the vein region of s w o l l e n leaf explants. C a l l u s i n d u c t i o n also varied with d i f f e r e n t cultivars; the e x p l a n t s from 'solo' p r o d u c e d more callus than the e x p l a n t s from 'Sunrise' on m e d i u m c o n t a i n i n g 1.0 mg/l NAA.
E m b r y o g e n e s i s o c c u r r e d only on call± from root culture. Stem and shoot tip e x p l a n t s showed a b u n d a n t callus p r o l i f e r a t i o n , however, no e m b r y o g e n e s i s was o b s e r v e d (Table i). A l t h o u g h a small q u a n t i t y of callus was i n d u c e d from root e x p l a n t s after one month; some of the b r o w n i n g e x p l a n t s p r o d u c e d callus later on (Fig. i). S o m a t i c embryo f o r m a t i o n from root callus occurred (Fig. 2) after three m o n t h s when c o m p a c t and hard s t r u c t u r e s were seen on the s u r f a c e of the callus. D i f f e r e n t stages of d e v e l o p i n g e m b r y o s were observed on the s u r f a c e of y e l l o w callus (Fig. 3-6). The e m b r y o g e n i c callus r e g e n e r a t e d more than one h u n d r e d s o m a t i c e m b r y o s on each e x p l a n t when s u b c u l t u r e d on the medium. For both c u l t i v a r s of papaya the o p t i m a l m e d i u m for e m b r y o g e n e s i s was 1/2 MS m o d i f i e d m e d i u m with 1.0 mg/l NAA, 0.5 mg/l kinetin, and 1.0 mg/l GA3, in w h i c h 30% of the root c u l t u r e s of 'Sunrise' and 20% of the root c u l t u r e s of 'Solo' r e g e n e r a t e d embryos. C a l l u s and somatic embryo f o r m a t i o n was s u s t a i n e d for more than two years by s u b c u l t u r i n g p o r t i o n s of callus with s e v e r a l somatic embryos. During the s u b c u l t u r e of e m b r y o g e n i c callus and somatic embryos, new callus and e m b r y o i d s were formed, a few s o m a t i c e m b r y o s d e v e l o p e d h y p o c o t y l , t r i - l o b e d leaves (Fig. 7) and roots; roots were generally formed d i r e c t l y from the callus and not from s o m a t i c embryos. The shoots p r o d u c e d roots after they were i s o l a t e d and t r a n s f e r e d into NAA c o n t a i n ing m e d i u m for one m o n t h (Fig. 8). More than forty rooted plantlets were successfully t r a n s f e r r e d to pots and c u l t u r e d under greenh o u s e c o n d i t i o n s (Fig. 9).
Table I. E f f e c t s of growth r e g u l a t o r s on c a l l u s i n g ( a ) and somatic e m b r y o g e n e s i s (E) in tissue c u l t u r e of two p a p a y a c u l t i v a r s (Solo and Sunrise). The 1/2 MS m e d i u m (1962) was used as the basal medium. Data were c o l l e c t e d after 1 m o n t h c u l t u r i n g in 16 h light (2000 lux) and 8 h d a r k n e s s at 25 ± 2°C.
NAA
Explants Shoot tip Stem Leaf Cotyledon Root
(a)
1.0 mg/l
Solo/Sunrise ++/+ ++/+ ++/0 0/+ ++(E)/+
degree of c a l l u s i n g : +++, abundant.
O, not visible;
NAA Kinetin
1.0 0.5
Solo/Sunrise +++/+++ +++/+++ +/++ 0/++ +/+
+, limited,
NAA 1.0 GA 3 1.0
NAA Kinetin GA 3
Solo/Sunrise
Solo/Sunrise
++/++ 0/+ 0/0 0/0 O(E)/O(E)
/++ +/++ 0/0 0/0 O(E)/+(E)
u s u a l l y at the cut surface;
1.0 0.5 1.0
++, l o c a l i z e d ;
350
Figs. i-9. C a l l u s induction, s o m a t i c e m b r y o g e n e s i s and plant r e g e n e r a t i o n from root e x p l a n t s of papaya. i. C a l l u s (C) induced from the browning root e x p l a n t after l m o n t h of culture. 2. E m b r y o g e n i c callus (EC) a p p e a r e d on root callus after 3 m o n t h of culture. 3. S o m a t i c e m b r y o s (SE) on the surface of root callus. 4. B i p o l a r t o r p e d o - s h a p e d embryo (SE).
5. M a t u r e somatic embryos fully formed c o t y l e d o n s .
with
6. N u m e r o u s callus.
on root
somatic
7. A shoot with hypocotyl. 8. A plant roots. 9. A plant
DISCUSSION Embryogenesis and o r g a n o g e n e s i s have been o b t a i n e d from calli d e r i v e d from p a p a y a seedling petiole (Arora, 1978a) and stem s e g m e n t s (Yie and Liaw, 1977). However, no embryogenesis was o b s e r v e d on s e e d l i n g c o t y l e d o n c u l t u r e (Litz and Conover, 1983). Our r e s u l t s show that it is easier to induce callus on shoot tips and stem s e g m e n t s in the same c u l t u r e m e d i u m and for the root segm e n t s it is easier to induce embryogenesis on GA3 c o n t a i n i n g medium. This shows that the s u c c e s s of callus i n d u c t i o n and e m b r y o g e n e s i s of p a p a y a is d e p e n d e n t on s o u r c e s of the e x p l a n t and the m e d i u m c o m p o n e n t s . O p t i m a l papaya callus f o r m a t i o n from the v a r i o u s e x p l a n t s was o b t a i n e d on a m e d i u m c o n t a i n i n g both NAA and kinetin. The r e s u l t s of Arora and Singh (1978a, b) and Litz and Conover (1983) indicate that a d d i t i o n of c y t o k i n i n to the NAA m e d i u m i n c r e a s e s the g r o w t h of callus. In the r e p o r t s of Yie a n d Liaw (1977) and Arora and S i n g h (1978b), callus d e r i v e d from papaya s e e d l i n g stem s e g m e n t s can give rise to a d v e n t i t i o u s shoots or embryos. But in our study, no r e g e n e r a t i o n was o b s e r v e d on any o f the stem cultures. T h i s may be due to the m e d i u m c o n t a i n i n g high
somatic
with
grown
embryos
tri-lobed
well
leaf and
developed
in soil.
auxin (1.O mg/1), and the lack of t r a n s f e r ring the c a l l u s to m e d i u m with higher concentrations of c y t o k i n i n (i.0 - 2.0 mg/l kinetin) and lower c o n c e n t r a t i o n s of a u x i n (0 - 0.05 mg/l IAA) (Yie and Liaw, 1977), w h i c h is c r i t i c a l for r e g e n e r a t i o n . D e B r u i j n e et al. (1974) s u g g e s t e d that embryogenesis seemed to be d e p e n d e n t on a relatively low s u c r o s e c o n c e n t r a t i o n . Litz and C o n o v e r (1982) have d e m o n s t r a t e d that a m e d i u m c o n t a i n i n g coconut water is n e c e s s a r y for e m b r y o g e n e s i s in ovule callus. The r e s u l t s of the p r e s e n t study show that e m b r y o g e n e s i s of p a p a y a root e x p l a n t can be o b t a i n e d on a d e f i n e d m e d i u m with the usual c o n c e n t r a t i o n (3%) of s u c r o s e and w i t h o u t the a d d i t i o n of c o c o n u t milk. The papaya root c u l t u r e s p r o d u c e d s o m a t i c e m b r y o s on the m e d i u m s u p p l e m e n t e d w i t h NAA only. We c o n s i d e r that auxin is e s s e n t i a l for i n d u c t i o n of e m b r y o g e n i c c a l l u s in p a p a y a as in wild c a r r o t ( H a l p e r i n and W e t h e r e l l , 1964). K i n e t i n is p r o b a b l y not c r i t i c a l for i n d u c t i o n of e m b r y o g e n i c callus, b u t the p r e s e n c e of GA 3 in the m e d i u m i n c r e a s e s e m b r y o g e n e s i s . The result is s i m i l a r to t~hat o b t a i n e d in a l f a l f a (Kao and M i c h a y l u k , 1981) w h i c h shows that g i b b e r e l l i c acid, t o g e t h e r w i t h auxin, plays an e s s e n t i a l role in the d e t e r m i n a t i o n of e m b r y o g e n y .
351 Embryogenesis provides a simple and quick method to obtain a large number of plants. Papaya root culture can be used for rapid p r o p a g a t i o n for rare and special plants. Because there is more than one root in a plant, the seedling can still live after some roots have been excised.
ACKNOWLEDGEMENTS
This work was supported by the of Agriculture, Taiwan, R.O.C., The thank Mr. D. N. Hwang of F e n g - S h e n H o r t i c u l t u r a l E x p e r i m e n t Station for materials.
Council authors Tropical supplying
REFERENCES
Arora
IK, Singh RN (1978a) Scientia Hort. 8: 357-361 Arora IK, Singh RN (1978b) Curt. Sci. 47: 867-868 D e B r u i j n e E, DeLanghe E, Van RR (1974) Int. Symp. Fytofarm. Fytiat. 26: 637-645 Halperin W, W e t h e r e l l DR (1964) Am. J. Bot. 51: 274-283 Kao KN, M i c h a y l u k MR (1981) In Vitro 17: 645-648 Litz RE, Conover RA (1982) Plant 8ci. Lett. 26: 153-158 Litz RE, Conover RA (1983) Scientia Hort. 19: 287-293 Litz RE (1984) In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of Plant Cell Culture, Vol 2, Macmillan, New York, pp 349-368 M u r a s h i g e T, Skoog F (1962) Physiol. Plant. 15: 482-497 Yie ST, Liaw SI (1977) In Vitro 13: 564-567
Plant Cell Reports (1987) 6:348-351
© Springer-Verlag 1987
Somatic embryogenesis and plant regeneration in Caricapapaya L. tissue culture derived from root explants M. H. Chen 1, p. j. Wang 1, and E. Maeda 2 Institute of Botany, Academia Sinica, Taipei, Taiwan 11529, R.O.C. 2 Faculty of Agriculture, Nagoya University, Nagoya 464, Japan Received April 7, 1987 / Revised version received July 6, 1987 - Communicated by M. Tabata
ABSTRACT The r e g e n e r a t i o n p o t e n t i a l of shoot tip, stem, leaf, c o t y l e d o n and root e x p l a n t s of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studied. Callus i n d u c t i o n of these two c u l t i v a r s of papaya showed that the shoot tips and stems are most s u i t a b l e for f o r m i n g callus, w h i l e leaves, cotyledons and roots are c o m p a r a tively difficult to induce callus. Callus induction also varied with the varities. Somatic e m b r y o g e n e s i s was o b t a i n e d from 3m o n t h - o l d root cultures. A m e d i u m c o n t a i n i n g half s t r e n g t h of MS i n o r g a n i c salts, 160 mg/l adenine sulfate, 1.0 mg/l NAA, 0.5 mg/l kinetin and 1.0 mg/l GA 3 was o p t i m a l for embryogenesis. The callus m a i n t a i n e d high regenerative capacity after two years of c u l t u r e on this medium. Plants d e r i v e d from somatic e m b r y o s were o b t a i n e d under greenhouse c o n d i t i o n s . ABBREVIATIONS MS: M u r a s h i g e and S k o o g ' s (1962) medium, NAA: N a p h t h a l e n e a c e t i c acid, IAA: I n d o l e - 3 - a c e t i c acid, GA: G i b b e r e l l i c acid, PRV: P a p a y a ring spot virus.
been u t i l i z e d for" callus induction and r e g e n e r a t i o n , (Litz and Conover, 1983; Arora and Singh, 1978a), a c o m p a r a t i v e study on the s u i t a b i l i t y of v a r i o u s e x p l a n t s has not been made. In this study, the callus i n d u c t i o n and r e g e n e r a t i o n p o t e n t i a l of v a r i o u s sources of explants from two papaya c u l t i v a r s were compared. We also d e s c r i b e in vitro i n d u c t i o n of s o m a t i c e m b r y o s in c u l t u r e d root of Carica papaya L., the l o n g - t e r m m a i n t e n a n c e of strong e m b r y o g e n e s i s capacity, and p l a n t l e t f o r m a t i o n from s o m a t i c embryos. MATERIAL
AND M E T H O D S
Plant M a t e r i a l s . Seeds of two papaya varities, C a r i c a papaya L. 'Solo' and 'Sunrise', were supplied by the F e n g - S h e n Tropical H o r t i c u l t u r a l E x p e r i m e n t S t a t i o n of T a i w a n A g r i c u l t u r a l R e s e a r c h Institute. The seeds, after being w a s h e d for 1 h in tap water, were i m m e r s e d in 75% ethanol for i min, and then in 0.1% sodium h y p o c h l o r i t e for i0 min. After several rinses with s t e r i l i z e d water, they were sown on MS agar m e d i u m in 2.5x13 cm test tubes. Shoot tip, stem, leaf, c o t y l e d o n and root (with r o o t - t i p removed) were e x c i s e d from 4 to 6 - w e e k - o l d s e e d l i n g s with three leaves (5 to 8 cm in height). They were cut into 3-5 mm s e g m e n t s and were cultured.
INTRODUCTION Papaya (Carica papaya L°) plants in T a i w a n have been almost c o m p l e t e l y i n f e c t e d by ring spot virus. This d i s e a s e has become the single most serious p r o b l e m of the T a i w a n papaya industry. T h e r e f o r e , it is of g r e a t importance to a c h i e v e a method of papaya regeneration in order to breed ring spot virus r e s i s t a n t strains by m e a n s of tissue culture. R e c e n t a c h i e v e m e n t s in papaya tissue c u l t u r e have been r e v i e w e d by Litz (1984). E m b r y o g e n e s i s in papaya callus has been o b t a i n e d from s e e d l i n g p e t i o l e s e g m e n t s (DeB r u i j n e et al., 1974) and stem s e g m e n t s (Yie and Liaw, 1977). These two r e p o r t s d e s c r i b e a three-step and a t w o - s t e p culture procedure, respectively, to obtain somatic embryos. In these methods, it is n e c e s s a r y to t r a n s f e r the callus to a n o t h e r m e d i u m for e m b r y o g e n e s i s . A l t h o u g h v a r i o u s e x p l a n t s have
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C u l t u r e M e d i a and E n v i r o n m e n t . A modified M u r a s h i g e and Skoog (1962) m e d i u m was used as basal medium. This m e d i u m c o n t a i n e d half s t r e n g t h i n o r g a n i c salts, 0.5 mg/l t h i a m i n e HCI, 1.0 mg/l pyridoxin-HCl, 5.0 mg/l n i c o t i n i c acid, 2.0 mg/l glycine, 1.0 mg/l casein h y d r o l y s a t e , I00 mg/l m y o - i n o s i t o l , 160 mg/l a d e n i n e sulfate, 30 g/l sucrose, and 8 g/l agar. D i f f e r e n t c o m b i n a t i o n s of 1.0 mg/l NAA with k i n e t i n (0, 0.5 mg/l) and gibberellic acid (GA 3) (0, 1.0 mg/l) were used as s u p p l e m e n t s . The pH was a d j u s t e d ,to pH 5.8 with IN KOH and IN HCI before a d d i n g agar. Sterilization was by a u t o c l a v i n g at i.i k g / c m 2 (I12°C) for i0 min. GA 3 was f i l t e r sterilized. E m b r y o g e n i c callus was s u b c u l t u r e d on medium containing NAA, kinetin, and GA 3. G e r m i n a t e d s o m a t i c e m b r y o s were i s o l a t e d and c u l t u r e d on m e d i u m c o n t a i n i n g NAA only.
349 C u l t u r e s were m a i n t a i n e d at 25 ± 2°C in a growth room with 16 h light (2000 lux) and 8 h darkness. R e - e s t a b l i s h m e n t of P l a n t s in Soil. The in vitro r e g e n e r a t e d p l a n t l e t s were r e m o v e d from c u l t u r e tubes, w a s h e d free of agar m e d i u m and potted in moist m i x t u r e of sand : soil (I/i : v/v), and c o v e r e d with a beaker to m a i n t a i n high humidity. One p e r c e n t IAA was sprayed on the p l a n t l e t s for two weeks. The p l a n t l e t s were then t r a n s p l a n t e d to soil and c u l t u r e d under g r e e n h o u s e c o n d i t i o n s . RESULTS C a l l u s was i n d u c e d from p a p a y a shoot tip, stem, leaf, c o t y l e d o n and root c u l t u r e s (Table i). Most of the e x p l a n t s formed callus at the cut ends after i0 days. C o n s i d e r a b l e v a r i a t i o n in the d e v e l o p m e n t of callus among single e x p l a n t s was observed. The result of callus formation after 3 weeks, and the regeneration of s o m a t i c embryos after 3 m o n t h s are shown in T a b l e I. O p t i m a l callus formation took place on 1/2 MS m e d i u m c o n t a i n i n g 1.0 mg/l NAA and 0.5 mg/l kinetin. Shoot tips and stems are most suitable e x p l a n t s for f o r m i n g callus, while leaves, c o t y l e d o n s and roots are c o m p a r a t i v e l y recalcitrant to callus induction. C a l l u s was i n i t i a t e d m a i n l y from the base of the shoot tip, and at the cut end of stem segments. Those e x p l a n t s w i t h o u t callus p r o l i f e r a t i o n were u s u a l l y s w o l l e n p r e s u m a b l y due to e x p a n sion growth of tissue. Roots were o c c a s i o n a l l y from the vein region of s w o l l e n leaf explants. C a l l u s i n d u c t i o n also varied with d i f f e r e n t cultivars; the e x p l a n t s from 'solo' p r o d u c e d more callus than the e x p l a n t s from 'Sunrise' on m e d i u m c o n t a i n i n g 1.0 mg/l NAA.
E m b r y o g e n e s i s o c c u r r e d only on call± from root culture. Stem and shoot tip e x p l a n t s showed a b u n d a n t callus p r o l i f e r a t i o n , however, no e m b r y o g e n e s i s was o b s e r v e d (Table i). A l t h o u g h a small q u a n t i t y of callus was i n d u c e d from root e x p l a n t s after one month; some of the b r o w n i n g e x p l a n t s p r o d u c e d callus later on (Fig. i). S o m a t i c embryo f o r m a t i o n from root callus occurred (Fig. 2) after three m o n t h s when c o m p a c t and hard s t r u c t u r e s were seen on the s u r f a c e of the callus. D i f f e r e n t stages of d e v e l o p i n g e m b r y o s were observed on the s u r f a c e of y e l l o w callus (Fig. 3-6). The e m b r y o g e n i c callus r e g e n e r a t e d more than one h u n d r e d s o m a t i c e m b r y o s on each e x p l a n t when s u b c u l t u r e d on the medium. For both c u l t i v a r s of papaya the o p t i m a l m e d i u m for e m b r y o g e n e s i s was 1/2 MS m o d i f i e d m e d i u m with 1.0 mg/l NAA, 0.5 mg/l kinetin, and 1.0 mg/l GA3, in w h i c h 30% of the root c u l t u r e s of 'Sunrise' and 20% of the root c u l t u r e s of 'Solo' r e g e n e r a t e d embryos. C a l l u s and somatic embryo f o r m a t i o n was s u s t a i n e d for more than two years by s u b c u l t u r i n g p o r t i o n s of callus with s e v e r a l somatic embryos. During the s u b c u l t u r e of e m b r y o g e n i c callus and somatic embryos, new callus and e m b r y o i d s were formed, a few s o m a t i c e m b r y o s d e v e l o p e d h y p o c o t y l , t r i - l o b e d leaves (Fig. 7) and roots; roots were generally formed d i r e c t l y from the callus and not from s o m a t i c embryos. The shoots p r o d u c e d roots after they were i s o l a t e d and t r a n s f e r e d into NAA c o n t a i n ing m e d i u m for one m o n t h (Fig. 8). More than forty rooted plantlets were successfully t r a n s f e r r e d to pots and c u l t u r e d under greenh o u s e c o n d i t i o n s (Fig. 9).
Table I. E f f e c t s of growth r e g u l a t o r s on c a l l u s i n g ( a ) and somatic e m b r y o g e n e s i s (E) in tissue c u l t u r e of two p a p a y a c u l t i v a r s (Solo and Sunrise). The 1/2 MS m e d i u m (1962) was used as the basal medium. Data were c o l l e c t e d after 1 m o n t h c u l t u r i n g in 16 h light (2000 lux) and 8 h d a r k n e s s at 25 ± 2°C.
NAA
Explants Shoot tip Stem Leaf Cotyledon Root
(a)
1.0 mg/l
Solo/Sunrise ++/+ ++/+ ++/0 0/+ ++(E)/+
degree of c a l l u s i n g : +++, abundant.
O, not visible;
NAA Kinetin
1.0 0.5
Solo/Sunrise +++/+++ +++/+++ +/++ 0/++ +/+
+, limited,
NAA 1.0 GA 3 1.0
NAA Kinetin GA 3
Solo/Sunrise
Solo/Sunrise
++/++ 0/+ 0/0 0/0 O(E)/O(E)
/++ +/++ 0/0 0/0 O(E)/+(E)
u s u a l l y at the cut surface;
1.0 0.5 1.0
++, l o c a l i z e d ;
350
Figs. i-9. C a l l u s induction, s o m a t i c e m b r y o g e n e s i s and plant r e g e n e r a t i o n from root e x p l a n t s of papaya. i. C a l l u s (C) induced from the browning root e x p l a n t after l m o n t h of culture. 2. E m b r y o g e n i c callus (EC) a p p e a r e d on root callus after 3 m o n t h of culture. 3. S o m a t i c e m b r y o s (SE) on the surface of root callus. 4. B i p o l a r t o r p e d o - s h a p e d embryo (SE).
5. M a t u r e somatic embryos fully formed c o t y l e d o n s .
with
6. N u m e r o u s callus.
on root
somatic
7. A shoot with hypocotyl. 8. A plant roots. 9. A plant
DISCUSSION Embryogenesis and o r g a n o g e n e s i s have been o b t a i n e d from calli d e r i v e d from p a p a y a seedling petiole (Arora, 1978a) and stem s e g m e n t s (Yie and Liaw, 1977). However, no embryogenesis was o b s e r v e d on s e e d l i n g c o t y l e d o n c u l t u r e (Litz and Conover, 1983). Our r e s u l t s show that it is easier to induce callus on shoot tips and stem s e g m e n t s in the same c u l t u r e m e d i u m and for the root segm e n t s it is easier to induce embryogenesis on GA3 c o n t a i n i n g medium. This shows that the s u c c e s s of callus i n d u c t i o n and e m b r y o g e n e s i s of p a p a y a is d e p e n d e n t on s o u r c e s of the e x p l a n t and the m e d i u m c o m p o n e n t s . O p t i m a l papaya callus f o r m a t i o n from the v a r i o u s e x p l a n t s was o b t a i n e d on a m e d i u m c o n t a i n i n g both NAA and kinetin. The r e s u l t s of Arora and Singh (1978a, b) and Litz and Conover (1983) indicate that a d d i t i o n of c y t o k i n i n to the NAA m e d i u m i n c r e a s e s the g r o w t h of callus. In the r e p o r t s of Yie a n d Liaw (1977) and Arora and S i n g h (1978b), callus d e r i v e d from papaya s e e d l i n g stem s e g m e n t s can give rise to a d v e n t i t i o u s shoots or embryos. But in our study, no r e g e n e r a t i o n was o b s e r v e d on any o f the stem cultures. T h i s may be due to the m e d i u m c o n t a i n i n g high
somatic
with
grown
embryos
tri-lobed
well
leaf and
developed
in soil.
auxin (1.O mg/1), and the lack of t r a n s f e r ring the c a l l u s to m e d i u m with higher concentrations of c y t o k i n i n (i.0 - 2.0 mg/l kinetin) and lower c o n c e n t r a t i o n s of a u x i n (0 - 0.05 mg/l IAA) (Yie and Liaw, 1977), w h i c h is c r i t i c a l for r e g e n e r a t i o n . D e B r u i j n e et al. (1974) s u g g e s t e d that embryogenesis seemed to be d e p e n d e n t on a relatively low s u c r o s e c o n c e n t r a t i o n . Litz and C o n o v e r (1982) have d e m o n s t r a t e d that a m e d i u m c o n t a i n i n g coconut water is n e c e s s a r y for e m b r y o g e n e s i s in ovule callus. The r e s u l t s of the p r e s e n t study show that e m b r y o g e n e s i s of p a p a y a root e x p l a n t can be o b t a i n e d on a d e f i n e d m e d i u m with the usual c o n c e n t r a t i o n (3%) of s u c r o s e and w i t h o u t the a d d i t i o n of c o c o n u t milk. The papaya root c u l t u r e s p r o d u c e d s o m a t i c e m b r y o s on the m e d i u m s u p p l e m e n t e d w i t h NAA only. We c o n s i d e r that auxin is e s s e n t i a l for i n d u c t i o n of e m b r y o g e n i c c a l l u s in p a p a y a as in wild c a r r o t ( H a l p e r i n and W e t h e r e l l , 1964). K i n e t i n is p r o b a b l y not c r i t i c a l for i n d u c t i o n of e m b r y o g e n i c callus, b u t the p r e s e n c e of GA 3 in the m e d i u m i n c r e a s e s e m b r y o g e n e s i s . The result is s i m i l a r to t~hat o b t a i n e d in a l f a l f a (Kao and M i c h a y l u k , 1981) w h i c h shows that g i b b e r e l l i c acid, t o g e t h e r w i t h auxin, plays an e s s e n t i a l role in the d e t e r m i n a t i o n of e m b r y o g e n y .
351 Embryogenesis provides a simple and quick method to obtain a large number of plants. Papaya root culture can be used for rapid p r o p a g a t i o n for rare and special plants. Because there is more than one root in a plant, the seedling can still live after some roots have been excised.
ACKNOWLEDGEMENTS
This work was supported by the of Agriculture, Taiwan, R.O.C., The thank Mr. D. N. Hwang of F e n g - S h e n H o r t i c u l t u r a l E x p e r i m e n t Station for materials.
Council authors Tropical supplying
REFERENCES
Arora
IK, Singh RN (1978a) Scientia Hort. 8: 357-361 Arora IK, Singh RN (1978b) Curt. Sci. 47: 867-868 D e B r u i j n e E, DeLanghe E, Van RR (1974) Int. Symp. Fytofarm. Fytiat. 26: 637-645 Halperin W, W e t h e r e l l DR (1964) Am. J. Bot. 51: 274-283 Kao KN, M i c h a y l u k MR (1981) In Vitro 17: 645-648 Litz RE, Conover RA (1982) Plant 8ci. Lett. 26: 153-158 Litz RE, Conover RA (1983) Scientia Hort. 19: 287-293 Litz RE (1984) In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of Plant Cell Culture, Vol 2, Macmillan, New York, pp 349-368 M u r a s h i g e T, Skoog F (1962) Physiol. Plant. 15: 482-497 Yie ST, Liaw SI (1977) In Vitro 13: 564-567
