Plant Cell Reports
P l a n t Cell R e p o r t s (1993) 1 3 : 9 9 - 1 0 2
9 Springer-Verlag 1993
High efficiency plant regeneration from petiole explants of Carica papaya L. through organogenesis M. Hossain, S . M . Rahman, R. Islam, and O.I. Joarder Department of Botany, Rajshahi University, Rajshahi-6205, Bangladesh Received 24 November 1992/Revised version received 31 August 1993 - Communicated by G.C. Phillips
Abstract. Callus cultures were obrained from petiole explants of Carica p a p a y a on MS medium containing 0.5-10.5 gM ~t-naphthaleneacetic acid (NAA) in combination with 0.5-5 gM benzyladenine (BA). Hardgreen calli were transferred to MS medium containing I00 mg1-1 casein hydrolysate (CH) with specific BANAA fon'nulation, where they developed adventitious buds within 2 weeks of culture. Maximum number of adventitious buds were obtained in 2 gM BA and 0.1 gM NAA. Shoot regeneration occurred from these adventitious buds by the end of the 4th week. Regenerated shoots were elongated in hormone-free medium and rooted in half-strength MS fortified with 3 gM NAA and 0.5 gM gibberellic acid (GA3). The regenerants were transfelled to soil after acclimatization.
Materials and Methods Proliferating shoot cultures were raised from shoot apices of a 5month-old field grown female papaya plant cv "Rajshahi-red' using the procedures reported earlier (Hossain et al., 1991) Petiole explants ~(5-6 m m in length) were excised from these proliferated shoots and induced to form callus on MS medium (Murashige and Skoog, 1962) supplemented with different concentrations and combinations of BA and NAA. The prohferated calli were subcultured in MS medium containing casein hydrolysate (100 mgl-1) with BAP alone or in combination with N A A for adventitious shoot induction. The adventitious shoots were excised and elongated in hormone-free MS medium. The regenerated shoots were rooted in half strength MS medium with 3 g M NAA and 0.5 p~M GA3. The pH of the media was adjusted to 5.8 before sterilization by autoclaving, and solidified with 0.7% Difco Bacto-agar. The cultures were maintained at 25+2~ with 16 h photoperiod. All experiments were repeated twice and mean confidence intervals were given for P = 0.05 using the t-test.
Results and Discussion Introduction Papaya (Carica papaya L.) fruit is economically useful for its food value and for its valuable proteolytic enzymes, papain and chymopapain (Medora et aI., 1979). Papaya, being essentially cross-pollinated and seed propagated, shows great variability and, therefore, vegetative propagation from selected clones is highly desirable. Rapid clonal propagation of desffed genotypes is one of many applications of plant tissue culture. Successful propagation of fruit species by this method is limited. Papaya plants have been regenerated from seedling apices (Yie and Liaw, 1977) and from apical buds (Litz and Conover, 1980; Litz et at., 1983) and lateral buds (Drew, 1988; Purnima, 1988; Rajeevan and Pandey, 1986) of mature ta'ees. However, in vitro adventitious regeneration may give a higher rate of shoot production with more potential for mass propagation than does multiplication from axillary buds. The present paper describes a technique for production of multiple shoots through organogenesis from petiole explants derived from aseptically grown shoots of C a r i c a papaya. Correspondence to: O.J. J o a r d e r
A number of preliminary experiments revealed that phytohonnones are essential for induction of callus from petiole explants and no callus was induced by MS basal medium alone. Irrespective of growth regulator concentrations used, when cultured on media containing BA and NAA the explants swelled within one week and callus formation started from the cut ends within 2 weeks of incubation. It was noticed that BA alone failed to induce any callus and the explants died within a short period, except at 2 and 5 gM BA where the explants increased in size by lateral swelling (Table 1). Media supplemented with BA (0.5-5 gM) and a high concentration (2.5-10 gM) of NAA yielded moderate to high amounts of callus, which was crystalline and transparent in appearance and white or brown in colour. The callus appeared as loosely-packed friable regions of vacuolated cells, which produced more callus after subculture to shoot induction medium. No shoot bud formation or nodular growth was observed even after 5 weeks of subculture. On the other hand, media containing BA (0.5-5 ~tM) and a low concentration (0.5 BM) of NAA induced a lesser amount of callus, which was translucent to opaque in appearance and light green in
100 colour. This callus appeared as hard, compact regions of non-vacuolated cells, which produced nodular structures within 5 weeks of culture initiation. These nodular structures developed into shoot buds when subcultured in appropriate media.
T a b l e 1. Effectofdifferent concentrations a n d combinations of BA a n d NAA in MS m e d i u m on callus induction fi:om petiole explants of p a p a y a . T h e r e were 15-20 explants per trealment a n d data were recorded alter 5 weeks of culture initiation. G r o w t h r e g u l a t o r s in g M
BA
NAA
0.5 2.0 5.0 9.0
0.5
2.0
5.0
Nature
and
degree
of callusing S S
0.5 2.5 5.0 10.5
HB+ PW+++ PW+++ PW+++
0.5 2.5 5.0
HG+ PB++ PW+++
10.5
PW+++
0.5 2.5 5.0 10.5
ItG+ PW++ PW+++ PW+++
S, s w e l l i n g ; H G , h a r d - g r e e n ; PB, s p o n g y - b r o w n , P W , s p o n g y - w h i t e , +, s m a l l c a l l u s g r o w t h , ++, m o d e r a t e c a l l u s g r o w t h a n d +++, a b u n d a n t c a l l u s g r o w t h .
The 5-week-old, hard, compact in'egularly shaped calli induced on 0.5 gM NAA and 2 pM BA were used to study morphogenesis. When transferred to MS basal medium without any exogenous hormones the calli died within a short p e r i o d ; however, when they were transferred to medium containing BA (2 gM) they developed multiple shoots but at a low frequency. In order to increase the frequency of regeneration, different concentrations and combinations of BA (0.5-5 gM) and N A A (0.05-0.5 gM) were tested. The results are summarized in Table 2. Under appropriate conditions of auxin and cytokinin, the 5 week-old calli showed the formation of a number of adventitious shoot buds in compact masses (Fig. 1A). Later, many of these buds developed into shoots (Fig. 1B). However, the degree of shoot formation was different on different media. NAA at greater than 0.1 gM and BA at greater than 2 BM showed significant inhibitory effects on the induction of shoots. The best treatment in terms of number of shoots per inoculum (81.97 _+ 9.56) was 2 gM BA + 0.1 BM NAA+CH (Table 2).
Table 2. Effect of BA and NAA in MS m e d i u m + CH on adventitious bud proliferation from hard-green callus obtained from initial culture (MS + 2 BM BA + 0.5 p.M NAA). There were 10-25 calli per treatment and data were recorded after 10 weeks of culture (5 weeks after subculture) Growth regulator concentration in subcul-ture media (gM) BA NAA 0.5
% of inocu l u m with shoot buds
Number of shoots/inoculum (X+S.E.)
--
2.0 5.0
4.20 d
4.85+0.36 e
0.5
0.05 0.1 0.5
14.60 b 8.40 c
38.19+ 4.25 b 12.63-+3.91 d
0.2
0.05 0.1 0.5
12.90 b 26.50 a
19.90+5.16 c 81.97_+9.56 a
5.0
0.05 0.1 0.5
In each column, means followed by the same letter are not significantly different at P = 0.05 by Duncan's Multiple Range Test.
Adventitious shoots were excised from shoot clumps and subcultured individually onto hormone-free MS medium for elongation. The clumps were cut into four segments and transfen'ed to fresh medium after removing dead and discoloured tissues, for further initiation of adventitious shoots to increase the proliferation. Following this procedure the stock cultures were maintained for more than 14 months (12 subcultures) without decrease in multiplication rate (data not shown). The average multiplication rate was 9-fold per subculture. After 4 weeks of culture in holanone free-medium, the elongated shoots were transfelxed to rooting medium for root induction (Fig. 1C). Root initiatiation was observed within 2 weeks. The percentage of rooted shoots reached above 65% within 3 weeks. Regenerated plantlets were transferred to pots containing soil mixed with compost. During the initial one week, the potted plantlets were covered with glass beakers to provide high humidity. Transplantation survival was 60%. Plantlets were subsequently transferred to larger pots and gradually acclimated to outdoor conditions (Fig. 1D). Plant regeneration in papaya through adventitious shoot organogenesis from stem (Arora and Singh, 1978; Medora et al., 1979) and cotyledon (Litz et aI., 1983) cultures has been reported. Medom et al. (1979) described optimum conditions for initiation and growth of callus from papaya seedling stem explants, and they suggested that 2.4-diclorophenoxyaceticacid was suitable for this purpose. They reported that BA was inhibitory to callus growth, although this observation conflicts with the r e s u l t s o b t a i n e d b y A f t r a and S i n g (1978), Yie and Liaw (1977), and the results of our present study where a low ratio of BA relative to NAA was used to form hard, green, nodular callus. Adventitious shoot formation from petiole callus was achieved here by
101
Fig.1. Plant regeneration from petiole explants of Carica papaya through organogenesis. A Development of adventitious buds from hard-green callus after 2 weeks of subculture in MS+CH+2.0 p-M BA+0.1 gM NAA, B. Development of adventitious shoots in MS+CH+2,0 I-tM BA+0.1 gM NAA after 5 weeks culture. C, Development of adventitious roots on micropropagated shoot in half-strength MS+0.3 gM NAA+0.5 gM GA3 after 4 weeks culture. D. Two-month-old plantlet establised in earthen pot.
subculturing callus onto medium with high cytokininauxin ratio. Similar observations were made in cotyledon (Litz et aL, 1983) and stem (DeBruijne, 1974) cultures, except that we used CH which was found to be effective for shoot bud induction (data not shown). We obtained more than 65% rooting success in viU'o of regenerated shoots using half-strength MS medium with 0.3 BM NAA and 0.5 gM GA 3. In conU'ast, less than 10% in vitro rooting on NAA was obtained with shoots originated from shoot apex cultures (Winnaar, 1988). This difference may be related to the difference of genotype used in each case. In the present investigation, more than 80 shoots per inoculum were obtained from the primary established culture after 10 weeks, and the subsequent multiplication rate of adventitious shoots from shoot clumps was 9-fold per monthly subculture. In viu'o regenerated shoots were successfully established in soil. Tissue cultured papaya plants are susceptible to shoot and root rots in the initiall establishment stage (Drew, 1988). Treatment of soil withl a fungicide, Agrason, before planting was found
here to be most beneficial for satisfactory survival of plants in the soil. While these plants might not be clonal and may possibly be off-types, the shoot proliferation and conversion rates are much higher than the previous reports (Drew, 1988; Purnima, 1988; Rajeevan and Pandey, 1986; Winnaar, 1988). The procedure we described for adventitious regeneration of shoots fi-om petiole explants of papaya is simple, rapid and reproducible, and may be ideally suited to use in transformation studies and for mass progation if the frequency of off-types is low.
References
Arora IK, Singh RN (1978) Sci Hort 8:357-361 DeBruijne F, DeLanghe E, van Rijk R (1974) Int Symp Fytofman Fytiat 26:637-645 Drew RA (1988) Hort Science 23:609-611 Hossain M, Rahman SM, Joarder OI (1991) Plant Tiss Cult 1:29-25
102 Litz RE, Conover RA (1980) Hort Science 15:733-735 Litz RE, O'Hair SK, Conover RA (1983) Hort Science 19:287-293 Medora RS, Hildarback DE, Mell GP (1979) Z Pflanzenphysiol 91:79-82 Murashige T, Skoog F (1962) Physiol Plant 15: 473497 Purnima, SB (1988) Bitechnology Centre, Indian Agric Res Inst, New Delhi 110012, India
Rajeevan MS, Pandey RM (1986) Plant Cell Tiss Org Cult 6:181-188 Winnaar WD (1988) Plant Cell Tiss Org Cult 12: 305310 Yie ST, Liaw SI (1977) In Vitro 13:564-568
P l a n t Cell R e p o r t s (1993) 1 3 : 9 9 - 1 0 2
9 Springer-Verlag 1993
High efficiency plant regeneration from petiole explants of Carica papaya L. through organogenesis M. Hossain, S . M . Rahman, R. Islam, and O.I. Joarder Department of Botany, Rajshahi University, Rajshahi-6205, Bangladesh Received 24 November 1992/Revised version received 31 August 1993 - Communicated by G.C. Phillips
Abstract. Callus cultures were obrained from petiole explants of Carica p a p a y a on MS medium containing 0.5-10.5 gM ~t-naphthaleneacetic acid (NAA) in combination with 0.5-5 gM benzyladenine (BA). Hardgreen calli were transferred to MS medium containing I00 mg1-1 casein hydrolysate (CH) with specific BANAA fon'nulation, where they developed adventitious buds within 2 weeks of culture. Maximum number of adventitious buds were obtained in 2 gM BA and 0.1 gM NAA. Shoot regeneration occurred from these adventitious buds by the end of the 4th week. Regenerated shoots were elongated in hormone-free medium and rooted in half-strength MS fortified with 3 gM NAA and 0.5 gM gibberellic acid (GA3). The regenerants were transfelled to soil after acclimatization.
Materials and Methods Proliferating shoot cultures were raised from shoot apices of a 5month-old field grown female papaya plant cv "Rajshahi-red' using the procedures reported earlier (Hossain et al., 1991) Petiole explants ~(5-6 m m in length) were excised from these proliferated shoots and induced to form callus on MS medium (Murashige and Skoog, 1962) supplemented with different concentrations and combinations of BA and NAA. The prohferated calli were subcultured in MS medium containing casein hydrolysate (100 mgl-1) with BAP alone or in combination with N A A for adventitious shoot induction. The adventitious shoots were excised and elongated in hormone-free MS medium. The regenerated shoots were rooted in half strength MS medium with 3 g M NAA and 0.5 p~M GA3. The pH of the media was adjusted to 5.8 before sterilization by autoclaving, and solidified with 0.7% Difco Bacto-agar. The cultures were maintained at 25+2~ with 16 h photoperiod. All experiments were repeated twice and mean confidence intervals were given for P = 0.05 using the t-test.
Results and Discussion Introduction Papaya (Carica papaya L.) fruit is economically useful for its food value and for its valuable proteolytic enzymes, papain and chymopapain (Medora et aI., 1979). Papaya, being essentially cross-pollinated and seed propagated, shows great variability and, therefore, vegetative propagation from selected clones is highly desirable. Rapid clonal propagation of desffed genotypes is one of many applications of plant tissue culture. Successful propagation of fruit species by this method is limited. Papaya plants have been regenerated from seedling apices (Yie and Liaw, 1977) and from apical buds (Litz and Conover, 1980; Litz et at., 1983) and lateral buds (Drew, 1988; Purnima, 1988; Rajeevan and Pandey, 1986) of mature ta'ees. However, in vitro adventitious regeneration may give a higher rate of shoot production with more potential for mass propagation than does multiplication from axillary buds. The present paper describes a technique for production of multiple shoots through organogenesis from petiole explants derived from aseptically grown shoots of C a r i c a papaya. Correspondence to: O.J. J o a r d e r
A number of preliminary experiments revealed that phytohonnones are essential for induction of callus from petiole explants and no callus was induced by MS basal medium alone. Irrespective of growth regulator concentrations used, when cultured on media containing BA and NAA the explants swelled within one week and callus formation started from the cut ends within 2 weeks of incubation. It was noticed that BA alone failed to induce any callus and the explants died within a short period, except at 2 and 5 gM BA where the explants increased in size by lateral swelling (Table 1). Media supplemented with BA (0.5-5 gM) and a high concentration (2.5-10 gM) of NAA yielded moderate to high amounts of callus, which was crystalline and transparent in appearance and white or brown in colour. The callus appeared as loosely-packed friable regions of vacuolated cells, which produced more callus after subculture to shoot induction medium. No shoot bud formation or nodular growth was observed even after 5 weeks of subculture. On the other hand, media containing BA (0.5-5 ~tM) and a low concentration (0.5 BM) of NAA induced a lesser amount of callus, which was translucent to opaque in appearance and light green in
100 colour. This callus appeared as hard, compact regions of non-vacuolated cells, which produced nodular structures within 5 weeks of culture initiation. These nodular structures developed into shoot buds when subcultured in appropriate media.
T a b l e 1. Effectofdifferent concentrations a n d combinations of BA a n d NAA in MS m e d i u m on callus induction fi:om petiole explants of p a p a y a . T h e r e were 15-20 explants per trealment a n d data were recorded alter 5 weeks of culture initiation. G r o w t h r e g u l a t o r s in g M
BA
NAA
0.5 2.0 5.0 9.0
0.5
2.0
5.0
Nature
and
degree
of callusing S S
0.5 2.5 5.0 10.5
HB+ PW+++ PW+++ PW+++
0.5 2.5 5.0
HG+ PB++ PW+++
10.5
PW+++
0.5 2.5 5.0 10.5
ItG+ PW++ PW+++ PW+++
S, s w e l l i n g ; H G , h a r d - g r e e n ; PB, s p o n g y - b r o w n , P W , s p o n g y - w h i t e , +, s m a l l c a l l u s g r o w t h , ++, m o d e r a t e c a l l u s g r o w t h a n d +++, a b u n d a n t c a l l u s g r o w t h .
The 5-week-old, hard, compact in'egularly shaped calli induced on 0.5 gM NAA and 2 pM BA were used to study morphogenesis. When transferred to MS basal medium without any exogenous hormones the calli died within a short p e r i o d ; however, when they were transferred to medium containing BA (2 gM) they developed multiple shoots but at a low frequency. In order to increase the frequency of regeneration, different concentrations and combinations of BA (0.5-5 gM) and N A A (0.05-0.5 gM) were tested. The results are summarized in Table 2. Under appropriate conditions of auxin and cytokinin, the 5 week-old calli showed the formation of a number of adventitious shoot buds in compact masses (Fig. 1A). Later, many of these buds developed into shoots (Fig. 1B). However, the degree of shoot formation was different on different media. NAA at greater than 0.1 gM and BA at greater than 2 BM showed significant inhibitory effects on the induction of shoots. The best treatment in terms of number of shoots per inoculum (81.97 _+ 9.56) was 2 gM BA + 0.1 BM NAA+CH (Table 2).
Table 2. Effect of BA and NAA in MS m e d i u m + CH on adventitious bud proliferation from hard-green callus obtained from initial culture (MS + 2 BM BA + 0.5 p.M NAA). There were 10-25 calli per treatment and data were recorded after 10 weeks of culture (5 weeks after subculture) Growth regulator concentration in subcul-ture media (gM) BA NAA 0.5
% of inocu l u m with shoot buds
Number of shoots/inoculum (X+S.E.)
--
2.0 5.0
4.20 d
4.85+0.36 e
0.5
0.05 0.1 0.5
14.60 b 8.40 c
38.19+ 4.25 b 12.63-+3.91 d
0.2
0.05 0.1 0.5
12.90 b 26.50 a
19.90+5.16 c 81.97_+9.56 a
5.0
0.05 0.1 0.5
In each column, means followed by the same letter are not significantly different at P = 0.05 by Duncan's Multiple Range Test.
Adventitious shoots were excised from shoot clumps and subcultured individually onto hormone-free MS medium for elongation. The clumps were cut into four segments and transfen'ed to fresh medium after removing dead and discoloured tissues, for further initiation of adventitious shoots to increase the proliferation. Following this procedure the stock cultures were maintained for more than 14 months (12 subcultures) without decrease in multiplication rate (data not shown). The average multiplication rate was 9-fold per subculture. After 4 weeks of culture in holanone free-medium, the elongated shoots were transfelxed to rooting medium for root induction (Fig. 1C). Root initiatiation was observed within 2 weeks. The percentage of rooted shoots reached above 65% within 3 weeks. Regenerated plantlets were transferred to pots containing soil mixed with compost. During the initial one week, the potted plantlets were covered with glass beakers to provide high humidity. Transplantation survival was 60%. Plantlets were subsequently transferred to larger pots and gradually acclimated to outdoor conditions (Fig. 1D). Plant regeneration in papaya through adventitious shoot organogenesis from stem (Arora and Singh, 1978; Medora et al., 1979) and cotyledon (Litz et aI., 1983) cultures has been reported. Medom et al. (1979) described optimum conditions for initiation and growth of callus from papaya seedling stem explants, and they suggested that 2.4-diclorophenoxyaceticacid was suitable for this purpose. They reported that BA was inhibitory to callus growth, although this observation conflicts with the r e s u l t s o b t a i n e d b y A f t r a and S i n g (1978), Yie and Liaw (1977), and the results of our present study where a low ratio of BA relative to NAA was used to form hard, green, nodular callus. Adventitious shoot formation from petiole callus was achieved here by
101
Fig.1. Plant regeneration from petiole explants of Carica papaya through organogenesis. A Development of adventitious buds from hard-green callus after 2 weeks of subculture in MS+CH+2.0 p-M BA+0.1 gM NAA, B. Development of adventitious shoots in MS+CH+2,0 I-tM BA+0.1 gM NAA after 5 weeks culture. C, Development of adventitious roots on micropropagated shoot in half-strength MS+0.3 gM NAA+0.5 gM GA3 after 4 weeks culture. D. Two-month-old plantlet establised in earthen pot.
subculturing callus onto medium with high cytokininauxin ratio. Similar observations were made in cotyledon (Litz et aL, 1983) and stem (DeBruijne, 1974) cultures, except that we used CH which was found to be effective for shoot bud induction (data not shown). We obtained more than 65% rooting success in viU'o of regenerated shoots using half-strength MS medium with 0.3 BM NAA and 0.5 gM GA 3. In conU'ast, less than 10% in vitro rooting on NAA was obtained with shoots originated from shoot apex cultures (Winnaar, 1988). This difference may be related to the difference of genotype used in each case. In the present investigation, more than 80 shoots per inoculum were obtained from the primary established culture after 10 weeks, and the subsequent multiplication rate of adventitious shoots from shoot clumps was 9-fold per monthly subculture. In viu'o regenerated shoots were successfully established in soil. Tissue cultured papaya plants are susceptible to shoot and root rots in the initiall establishment stage (Drew, 1988). Treatment of soil withl a fungicide, Agrason, before planting was found
here to be most beneficial for satisfactory survival of plants in the soil. While these plants might not be clonal and may possibly be off-types, the shoot proliferation and conversion rates are much higher than the previous reports (Drew, 1988; Purnima, 1988; Rajeevan and Pandey, 1986; Winnaar, 1988). The procedure we described for adventitious regeneration of shoots fi-om petiole explants of papaya is simple, rapid and reproducible, and may be ideally suited to use in transformation studies and for mass progation if the frequency of off-types is low.
References
Arora IK, Singh RN (1978) Sci Hort 8:357-361 DeBruijne F, DeLanghe E, van Rijk R (1974) Int Symp Fytofman Fytiat 26:637-645 Drew RA (1988) Hort Science 23:609-611 Hossain M, Rahman SM, Joarder OI (1991) Plant Tiss Cult 1:29-25
102 Litz RE, Conover RA (1980) Hort Science 15:733-735 Litz RE, O'Hair SK, Conover RA (1983) Hort Science 19:287-293 Medora RS, Hildarback DE, Mell GP (1979) Z Pflanzenphysiol 91:79-82 Murashige T, Skoog F (1962) Physiol Plant 15: 473497 Purnima, SB (1988) Bitechnology Centre, Indian Agric Res Inst, New Delhi 110012, India
Rajeevan MS, Pandey RM (1986) Plant Cell Tiss Org Cult 6:181-188 Winnaar WD (1988) Plant Cell Tiss Org Cult 12: 305310 Yie ST, Liaw SI (1977) In Vitro 13:564-568
