Plant Cell Reports
Plant Cell Reports (1987) 6: 326 - 328
© Springer-Verlag 1987
Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.) Darasinh Sihachakr and Georges Ducreux Laboratoire de Morphog6n~se V6g6tale Exp6rimentale, associ6 au CNRS (UA115), B~t 360, Universit6 de Paris XI, Centre d'Orsay, F-91405 Orsay, France Received April 13, 1987 / Revised version received August 8, 1987 - Communicated by A. M. Boudet
ABSTRACT T h i s is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI)of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with m e r i s t e m a t i c areas were induced in the medium supplemented w i t h 2mgl -I zeatin, and plant regeneration occurred when these calli were transferred_~nto the medium with zeatin level reduced t o 0.25mgl . Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI. ABBREVIATIONS. MS: Murashige and Skoog basal medium; IAA: Indol-3acetic acid; NAA: naphthaleneacetic acid; 2,4-D: dichlorophenoxyacetic acid; Mes : 2- (N-morpholino)-ethanesulfonic acid; Cpw: cell and protoplast washing solution.
stems were used for protoplast production. Protoplasts were prepared as described by Sihachakr and Ducreux (1987a), with the following modifications: the enzyme solution (1.5% (w/v) Cellulase R-10, 0.5% (w/v) Maeerozyme R-10 (Yakult, Co Ltd, Japan)) contained 19% (w/v) sucrose as osmoticum, and it was buffered with 0.05%(w/v) Mes. After an incubation period of 1618h at 27°C in the dark, the protoplasts were separated f r o m undigested materials by sieving (100~m sieves). The resulting suspension was diluted with an equal volume of a Cpw (Frearson et al. 1973) containing 21% (w/v) sucrose, then centrifuged at 120 x g for I0 minutes. The floating protoplasts were further washed 3 times by centrifugation at 55 x g for 3 minutes in the same Cpw solution where sucrose was substituted with 0.25M mannitol and 0.125M NaCI. Table I: Various steps of experiments and sequences of media for the transfer of protoplast-derived calli.
INTRODUCTION Few studies have been carried out on sweet potato protoplasts. Bidney and Shepard (1980) isolated protoplasts from petioles, since mesophyll tissues resisted the enzyme digestion. These protoplasts were induced to form callus from which roots were produced. Similar results were obtained from petioles and stems by Sihachakr and Ducreux (1987a) who furthermore showed the influence of organs and genotypes on the development of protoplasts in culture. Culture of protoplasts isolated from high anthocyanin-producing callus was also investigated (Nishimaki and Nozue 1985). But up to now there has not been any report on successful effort to regenerate plants from protoplast culture of sweet potato. In this communication, we report o u r success for the first time in regenerating plants from protoplasts of this plant species.
Time Day 0
7th day 21st day
49th day 77th day
Steps Isolation and culture of protop!~sts in KM8p medium ~upplemented with 0.~mgl 2,4-D, 0.5mgl- zeati~ and 0.5mgl- NAA, at a density of 3 x 10~ protoplasts/ml (5ml of suspension per dish); The culture was fed with the same medium as for initial protoplast culture; Protoplast-deriyed calli were either fed with KMSp+2mgl zeatin or + imgl 2,4-D, or directly transferred into the high zeatin medium; Resulting calli wereltransferred onto the MS solid medium + 2mgl zeatin; Calli were than tran{ferred onto a low zeatin medium (0.25mgl -).
MATERIALS AND METHODS Two cultivars of Ipomoea batatas, Guyana and Duclos XI, were maintained under conditions of in vitro culture according to the methods of Sihachakr (1982). Briefly, single node cuttings of stems were regularly subcultured at 4-week intervals on the MS basal medium (Murashige and Skoog 1962) containing v}tamins accorI ding to Morel and Wetmore (1951), 20~i'- sucrose, imglIAA or NAA, and solidified with 7gl- agar. The cultures were placed in a gro~thlchamber at 27°C with 12h/day illumination (62~Em- s- ), and 60% relative humidity. Shoots 4cm long with 4-5nodes were taken from 4-weekold cultures. Lamina were then r e m o v e d s i n c e they resisted the enzyme digestion, and only petioles and
Offprint requests to: D. Sihachakr
Prot~plas[s were initially cultured at a density of 3 x i0 ml- in KM8p medi}m (Kao and Mich~yluk 1975) supplem_~ nted with 0.2mgl-- 2,4-D 0.5mgl-- zeatin, 0.5mgl NAA, 0.05% (w/v) Mes and 0.35M glucose as osmoticum. Five ml of the protoplast suspension were plated in 60 x 15 m m special tissue culture Petri dishes. The cultures were incubated at 27°C in the dark for the first27 ~ays, followed by illumination of 12h/day (62~Em- s- ) and one addition of one ml of similar medium to each of culture dishes. At the 21st day of culture, two treatments were applied to the cultures. In treatment i, each dish of the cultures was fed with one ml of a medium containing
327 similar salt components as in the in![ial one and a single growth regulator, either 2mgl zeatin or lmgl -I 2,4-D. In treatment 2, protoplast-derived calli were separated from the liquid medium by centrifugation at 55 x g for 5 minutes, and re~uspended in 5ml'of a medium supplemented with 2mgl zeatin. At the end of the 4th week of treatment period, cell suspensions and calli derived respectively from treatment i and 2 were transferred onto 90 x 20 m m Petri d£shes filled with 25mi of MS solid m e d i u m containing the same elements as that for plant propagation except for growth regulators replaced by 2mglzeatin. Four weeks later, the resulting cavil were transferred onto a low zeatin medium (0.25mgl -). Various steps of experiments are summarized in Table i. RESULTS AND DISCUSSION In vitro plants provided yields comparable to those from plants grown under greenhouse conditions (Sihachakr and Ducreux 1987a). The highest mean yield w a s obtained for cultivar Guyana (Table 2). The protoplasts differred in their cultural behavior according to plant origins. Most protoplasts from in vitro plants were rich in cytoplasm and they had fallen to the bottom of the culture dishes, while our previous work reported more than 90% of protoplasts from greenhouse plants to be less dense and consequently either suspended in the culture m e d i u m or floated on the surface. Table 2: Means and SE of the protoplast yields (number of purified protoplasts per gram of tissue), the percentage of dividing cells after 7 days as a percentage of the total number of protoplasts obtained from 3 independent experiments, and the percentage of plant regeneration on the base of 80 calli.
Cultivars
YielJs x i0protoplasts
Guyana Duclos XI
1,041.6~145.8 539.7~i04.8
% dividing cells 29.6~5.6 25.0t4.1
% plant regeneration 0 5.0
Cell wall formation was achieved within 24-48h, and first division occurred within 72h in most small and medium-sized protoplasts, as previously described with plant material from greenhouse (Sihachakr and Ducreux 1987a), but a relatively high division frequency was obtained when in vitro plants were used as the source of protoplasts. The highest percentage of division was obtained for cultivar Guyana (Table 2). Most dividing cells gave rise to micro-calli consisting of 10-15 cells at the day 21. At this time, significant morphological changes were observed, when zeatin or 2,4-D-supplemented medium was added to the culture. Both zeatin and 2,4-D induced the same morphogenetic response for the two cultivars. Three weeks after the addition of these media, most cells separated from the clusters, leading to the occurrence of single cells which gave rise to elongated bodies with 3-6 cells resembling bipolar structures (Fig. I-2), but they failed to further develop into organized ones. When subcultured on the MS medium free of growth regulators, these elongated bodies finally died, meanwhile in the MS medium containing 2mgl zeatin they developed into calli, but none of the latter regenerated plants. Similar morphological changes also occurred in the protoplast cultures of Solanum nigrum (Sihachakr and Ducreux 1987b), and S. melongena (Gleddie et al. 1986) after an addition of medium containing high level of zeatin or 2,4-D, and these changes were followed by further development into organized structures and somatic embryos. Transfer of the protoplast-derived calli~ 21 days -1 oldginto the KM8p liquid medium supplemented with 2mgl
zeatin promoted their profuse growth resulting in the formation of green compact calli. Some portions of these calli differentiated meristematic areas consisting of very small green cells (Fig. 3). The meristematic areas were apparently identical in their structure for the two genotypes. They increased in size and number, particularly in cultivar Duclos XI, when the call!lwere subcultured on the MS medium containing 2mgl zeatin. Plant regeneration occurred within 8 weeks after a second transfer of these calli onto the MS medium with zeatin level reduced to 0.25mgl . These results are in accordance with previous works in sweet potato which reported that texture of plant explantderived calli was important factor in plant regeneration. Soft and friable calli did not give rise to any organized structures, while compact ones were shown to be organogenic (Bouhassan 1984) or embryogenic (Liu and Cantliffe 1984) depending upon hormone supply. Genotypes are well known to influence further plant regeneration from protoplasts of several plant species (Nicotiana, Banks and Evans 1976; Lycopersicon, Morgan and Cocking 1982; herbaceous species of dicotyledoneae class, Binding et al. 1981). In this study, of 80 ealli of cultivar Duclos XI 4 regenerated plants, while none of that of cultivar Guyana did (Table 2). One to two shoots were developed from meristematic areas in the case of cultivar Duclos XI (Fig. 4). Sufficiently developed shoots were then excised from organogenic calli, and transferred onto an IAA-supplemented medium for plant propagation according to the methods of Sihachakr (1982). The organogenic calli continually regenerated shoo~s, when subcultured on the low zeatin medium ( 0 . 2 5 m g l - ) ( F i g . 5). Actually, more than 30 plants were regenerated from 4 organogenic calli of cultivar Duclos XI. The results in this study reconfirmed once more the effective role of zeatin in the improvement of plant regeneration from protoplast cultures, as reported by Sihachakr and Ducreux in Solanum nigrum (1987b) and S. melongena (1987c). Moreover, the successful regeneration of plants in this study may also be due to the use of in vitro plants as the source of protoplasts, since in vitro conditions provided homogeneous and juvenile plants which displayed high potential of totipotency (Nozeran and Bancilhon 1972; Nozeran et al. 1982). ACKNOWLEGDMENTS We wish to thank Mrs Servaes for her excellent technical assistance, M. Froger for the photography and M. Harran for revising the english text. REFERENCES Banks MS, Evans PK (1976) Plant Sci. Lett. 7:409-416 Bidney DL, Shepard JF (1980) Plant Sci. Lett. 18:335342 Binding H, Nehls R, Knock R, Finger J, Mordhorst G (1981) Z. Pflanze~physiol. 101:119-130 Bouhassan A (1984) Th~se 3~ cycle, Univ. Paris XI, Orsay, France Erearson EM, Power JB, Cocking EC (1973) Dev. Biol. 33:130-137 Gleddie S, Keller WA, Setterfield G (1986) Can. J. Bot. 64:355-361 Kao KN, Michayluk MR (1975) Planta 126:105-110 Liu JR, Cantliffe DJ (1984) Plant Cell Rep. 3:112-115 Morel G, Wetmore RH (1951) Amer. J. Bot. 38:141-143 Morgan A, Cocking EC (1982) Z. Pflanzenphysiol. 106: 97-104 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-479 Nishimaki T, Nozue M (1985) Plant Cell Rep. 4:248-251 Nozeran R, Bancilhon L. (1972) Ann. Am41io. Plantes 22:167-185 Nozeran R., Ducreux G., Rossignol-Bancilhon L. (1982) Bull. Soc. Bot. Fr. 129:107-130
328
Fig.
1 and.2: Elongated bod}es induced by the addition of one ml of medium containing either 2mgl -I zeatin or Imgl- 2,4-D to the protoplast culture Fig. 3: Callus with meristematic areas (arrows) Fig. 4: Regenerated plant Fig. 5: Organogenic calli continually regenerated shoots, when subcultured on low zeatin medium Sihachakr D (1982) Agro. Trop. (Paris) 37:142-151 Sihachakr D, Duereux G ( 1 9 8 7 a ) C a n . J. Bot. 65:192-197 $ihachakr D, Ducreux G (1987b) Plant Science (in
press) Sihachakr D, Ducreux G (1987c) Plant Cell Tissue Organ Culture (submitted)
Plant Cell Reports (1987) 6: 326 - 328
© Springer-Verlag 1987
Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.) Darasinh Sihachakr and Georges Ducreux Laboratoire de Morphog6n~se V6g6tale Exp6rimentale, associ6 au CNRS (UA115), B~t 360, Universit6 de Paris XI, Centre d'Orsay, F-91405 Orsay, France Received April 13, 1987 / Revised version received August 8, 1987 - Communicated by A. M. Boudet
ABSTRACT T h i s is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI)of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with m e r i s t e m a t i c areas were induced in the medium supplemented w i t h 2mgl -I zeatin, and plant regeneration occurred when these calli were transferred_~nto the medium with zeatin level reduced t o 0.25mgl . Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI. ABBREVIATIONS. MS: Murashige and Skoog basal medium; IAA: Indol-3acetic acid; NAA: naphthaleneacetic acid; 2,4-D: dichlorophenoxyacetic acid; Mes : 2- (N-morpholino)-ethanesulfonic acid; Cpw: cell and protoplast washing solution.
stems were used for protoplast production. Protoplasts were prepared as described by Sihachakr and Ducreux (1987a), with the following modifications: the enzyme solution (1.5% (w/v) Cellulase R-10, 0.5% (w/v) Maeerozyme R-10 (Yakult, Co Ltd, Japan)) contained 19% (w/v) sucrose as osmoticum, and it was buffered with 0.05%(w/v) Mes. After an incubation period of 1618h at 27°C in the dark, the protoplasts were separated f r o m undigested materials by sieving (100~m sieves). The resulting suspension was diluted with an equal volume of a Cpw (Frearson et al. 1973) containing 21% (w/v) sucrose, then centrifuged at 120 x g for I0 minutes. The floating protoplasts were further washed 3 times by centrifugation at 55 x g for 3 minutes in the same Cpw solution where sucrose was substituted with 0.25M mannitol and 0.125M NaCI. Table I: Various steps of experiments and sequences of media for the transfer of protoplast-derived calli.
INTRODUCTION Few studies have been carried out on sweet potato protoplasts. Bidney and Shepard (1980) isolated protoplasts from petioles, since mesophyll tissues resisted the enzyme digestion. These protoplasts were induced to form callus from which roots were produced. Similar results were obtained from petioles and stems by Sihachakr and Ducreux (1987a) who furthermore showed the influence of organs and genotypes on the development of protoplasts in culture. Culture of protoplasts isolated from high anthocyanin-producing callus was also investigated (Nishimaki and Nozue 1985). But up to now there has not been any report on successful effort to regenerate plants from protoplast culture of sweet potato. In this communication, we report o u r success for the first time in regenerating plants from protoplasts of this plant species.
Time Day 0
7th day 21st day
49th day 77th day
Steps Isolation and culture of protop!~sts in KM8p medium ~upplemented with 0.~mgl 2,4-D, 0.5mgl- zeati~ and 0.5mgl- NAA, at a density of 3 x 10~ protoplasts/ml (5ml of suspension per dish); The culture was fed with the same medium as for initial protoplast culture; Protoplast-deriyed calli were either fed with KMSp+2mgl zeatin or + imgl 2,4-D, or directly transferred into the high zeatin medium; Resulting calli wereltransferred onto the MS solid medium + 2mgl zeatin; Calli were than tran{ferred onto a low zeatin medium (0.25mgl -).
MATERIALS AND METHODS Two cultivars of Ipomoea batatas, Guyana and Duclos XI, were maintained under conditions of in vitro culture according to the methods of Sihachakr (1982). Briefly, single node cuttings of stems were regularly subcultured at 4-week intervals on the MS basal medium (Murashige and Skoog 1962) containing v}tamins accorI ding to Morel and Wetmore (1951), 20~i'- sucrose, imglIAA or NAA, and solidified with 7gl- agar. The cultures were placed in a gro~thlchamber at 27°C with 12h/day illumination (62~Em- s- ), and 60% relative humidity. Shoots 4cm long with 4-5nodes were taken from 4-weekold cultures. Lamina were then r e m o v e d s i n c e they resisted the enzyme digestion, and only petioles and
Offprint requests to: D. Sihachakr
Prot~plas[s were initially cultured at a density of 3 x i0 ml- in KM8p medi}m (Kao and Mich~yluk 1975) supplem_~ nted with 0.2mgl-- 2,4-D 0.5mgl-- zeatin, 0.5mgl NAA, 0.05% (w/v) Mes and 0.35M glucose as osmoticum. Five ml of the protoplast suspension were plated in 60 x 15 m m special tissue culture Petri dishes. The cultures were incubated at 27°C in the dark for the first27 ~ays, followed by illumination of 12h/day (62~Em- s- ) and one addition of one ml of similar medium to each of culture dishes. At the 21st day of culture, two treatments were applied to the cultures. In treatment i, each dish of the cultures was fed with one ml of a medium containing
327 similar salt components as in the in![ial one and a single growth regulator, either 2mgl zeatin or lmgl -I 2,4-D. In treatment 2, protoplast-derived calli were separated from the liquid medium by centrifugation at 55 x g for 5 minutes, and re~uspended in 5ml'of a medium supplemented with 2mgl zeatin. At the end of the 4th week of treatment period, cell suspensions and calli derived respectively from treatment i and 2 were transferred onto 90 x 20 m m Petri d£shes filled with 25mi of MS solid m e d i u m containing the same elements as that for plant propagation except for growth regulators replaced by 2mglzeatin. Four weeks later, the resulting cavil were transferred onto a low zeatin medium (0.25mgl -). Various steps of experiments are summarized in Table i. RESULTS AND DISCUSSION In vitro plants provided yields comparable to those from plants grown under greenhouse conditions (Sihachakr and Ducreux 1987a). The highest mean yield w a s obtained for cultivar Guyana (Table 2). The protoplasts differred in their cultural behavior according to plant origins. Most protoplasts from in vitro plants were rich in cytoplasm and they had fallen to the bottom of the culture dishes, while our previous work reported more than 90% of protoplasts from greenhouse plants to be less dense and consequently either suspended in the culture m e d i u m or floated on the surface. Table 2: Means and SE of the protoplast yields (number of purified protoplasts per gram of tissue), the percentage of dividing cells after 7 days as a percentage of the total number of protoplasts obtained from 3 independent experiments, and the percentage of plant regeneration on the base of 80 calli.
Cultivars
YielJs x i0protoplasts
Guyana Duclos XI
1,041.6~145.8 539.7~i04.8
% dividing cells 29.6~5.6 25.0t4.1
% plant regeneration 0 5.0
Cell wall formation was achieved within 24-48h, and first division occurred within 72h in most small and medium-sized protoplasts, as previously described with plant material from greenhouse (Sihachakr and Ducreux 1987a), but a relatively high division frequency was obtained when in vitro plants were used as the source of protoplasts. The highest percentage of division was obtained for cultivar Guyana (Table 2). Most dividing cells gave rise to micro-calli consisting of 10-15 cells at the day 21. At this time, significant morphological changes were observed, when zeatin or 2,4-D-supplemented medium was added to the culture. Both zeatin and 2,4-D induced the same morphogenetic response for the two cultivars. Three weeks after the addition of these media, most cells separated from the clusters, leading to the occurrence of single cells which gave rise to elongated bodies with 3-6 cells resembling bipolar structures (Fig. I-2), but they failed to further develop into organized ones. When subcultured on the MS medium free of growth regulators, these elongated bodies finally died, meanwhile in the MS medium containing 2mgl zeatin they developed into calli, but none of the latter regenerated plants. Similar morphological changes also occurred in the protoplast cultures of Solanum nigrum (Sihachakr and Ducreux 1987b), and S. melongena (Gleddie et al. 1986) after an addition of medium containing high level of zeatin or 2,4-D, and these changes were followed by further development into organized structures and somatic embryos. Transfer of the protoplast-derived calli~ 21 days -1 oldginto the KM8p liquid medium supplemented with 2mgl
zeatin promoted their profuse growth resulting in the formation of green compact calli. Some portions of these calli differentiated meristematic areas consisting of very small green cells (Fig. 3). The meristematic areas were apparently identical in their structure for the two genotypes. They increased in size and number, particularly in cultivar Duclos XI, when the call!lwere subcultured on the MS medium containing 2mgl zeatin. Plant regeneration occurred within 8 weeks after a second transfer of these calli onto the MS medium with zeatin level reduced to 0.25mgl . These results are in accordance with previous works in sweet potato which reported that texture of plant explantderived calli was important factor in plant regeneration. Soft and friable calli did not give rise to any organized structures, while compact ones were shown to be organogenic (Bouhassan 1984) or embryogenic (Liu and Cantliffe 1984) depending upon hormone supply. Genotypes are well known to influence further plant regeneration from protoplasts of several plant species (Nicotiana, Banks and Evans 1976; Lycopersicon, Morgan and Cocking 1982; herbaceous species of dicotyledoneae class, Binding et al. 1981). In this study, of 80 ealli of cultivar Duclos XI 4 regenerated plants, while none of that of cultivar Guyana did (Table 2). One to two shoots were developed from meristematic areas in the case of cultivar Duclos XI (Fig. 4). Sufficiently developed shoots were then excised from organogenic calli, and transferred onto an IAA-supplemented medium for plant propagation according to the methods of Sihachakr (1982). The organogenic calli continually regenerated shoo~s, when subcultured on the low zeatin medium ( 0 . 2 5 m g l - ) ( F i g . 5). Actually, more than 30 plants were regenerated from 4 organogenic calli of cultivar Duclos XI. The results in this study reconfirmed once more the effective role of zeatin in the improvement of plant regeneration from protoplast cultures, as reported by Sihachakr and Ducreux in Solanum nigrum (1987b) and S. melongena (1987c). Moreover, the successful regeneration of plants in this study may also be due to the use of in vitro plants as the source of protoplasts, since in vitro conditions provided homogeneous and juvenile plants which displayed high potential of totipotency (Nozeran and Bancilhon 1972; Nozeran et al. 1982). ACKNOWLEGDMENTS We wish to thank Mrs Servaes for her excellent technical assistance, M. Froger for the photography and M. Harran for revising the english text. REFERENCES Banks MS, Evans PK (1976) Plant Sci. Lett. 7:409-416 Bidney DL, Shepard JF (1980) Plant Sci. Lett. 18:335342 Binding H, Nehls R, Knock R, Finger J, Mordhorst G (1981) Z. Pflanze~physiol. 101:119-130 Bouhassan A (1984) Th~se 3~ cycle, Univ. Paris XI, Orsay, France Erearson EM, Power JB, Cocking EC (1973) Dev. Biol. 33:130-137 Gleddie S, Keller WA, Setterfield G (1986) Can. J. Bot. 64:355-361 Kao KN, Michayluk MR (1975) Planta 126:105-110 Liu JR, Cantliffe DJ (1984) Plant Cell Rep. 3:112-115 Morel G, Wetmore RH (1951) Amer. J. Bot. 38:141-143 Morgan A, Cocking EC (1982) Z. Pflanzenphysiol. 106: 97-104 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-479 Nishimaki T, Nozue M (1985) Plant Cell Rep. 4:248-251 Nozeran R, Bancilhon L. (1972) Ann. Am41io. Plantes 22:167-185 Nozeran R., Ducreux G., Rossignol-Bancilhon L. (1982) Bull. Soc. Bot. Fr. 129:107-130
328
Fig.
1 and.2: Elongated bod}es induced by the addition of one ml of medium containing either 2mgl -I zeatin or Imgl- 2,4-D to the protoplast culture Fig. 3: Callus with meristematic areas (arrows) Fig. 4: Regenerated plant Fig. 5: Organogenic calli continually regenerated shoots, when subcultured on low zeatin medium Sihachakr D (1982) Agro. Trop. (Paris) 37:142-151 Sihachakr D, Duereux G ( 1 9 8 7 a ) C a n . J. Bot. 65:192-197 $ihachakr D, Ducreux G (1987b) Plant Science (in
press) Sihachakr D, Ducreux G (1987c) Plant Cell Tissue Organ Culture (submitted)
![Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tips by vitrification.](/assets/img/hold.png)
