Plant Cell Reports
Plant Cell Reports (1990) 9:77-79
9 Springer-Verlag1990
Somatic embryogenesis and shoot organogenesis from interspecific hybrid embryos of Vigna glabrescens and V. radiata H. K. Chen, M. C. Mok, and D. W. S. Mok Department of Horticulture and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331, USA Received December 21, 1989/Revised version received April 9, 1990 - Communicated by A. R. Gould
ABSTRACT
MATERIALS AND METHODS
Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vign~ glabrescens x V. radiata crosses and cultured on Murashige and Skoog (MS) medium without growth regulators. The frequency of 15-17 day old embryos that gave rise to somatic embryos increased from 8% to 29% by application a mixture of i00 mg/l gibberellic acid, 25 mg/l e-naphthaleneacetic acid (NAA) and 5 mg/1 kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N6-benzyladenine and 0.05 mg/l NAA. Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.
Plant materials. Seeds of V. glabrescens VII60 and V. radiata VCI973A were obtained from the Asian Vegetable Research and Development Center (AVRDC). Conditions for growth and flower induction and methods of pollination were as described previously (Chen et al., 1989).
INTRODUCTION The difficulty in redifferentiating plants from tissue cultures of large-seeded legumes has limited genetic transformation and other in vitro manipulations of this important group of plants. However, in recent years somatic embryos and plants have been generated from cotyledons and protoplast cultures of soybean (Glycine max (L.) Merr.) (Christianson et al., 1983; Lazzeri et al., 1985; Li et al., 1985; Ranch et al., 1985; Barwale et al., 1986). Plant formation via somatic embryogenesis is also possible from immature embryos and shoot apices of Pisum sativum L. (Kysely et al., 1987), cell suspensions of Phaseolus acutifolius L. (Kumar et al., 1988) and protoplasts of V. aconitifolia Jarq. (Shekhawat and Galston, 1983). In spite of these important advances, many of the Vigna and Phaseolus species are still not responsive to similar regeneration procedures. In an attempt to transfer pest resistance from V_t. ~labrescens M. M. and S. (2n=4x=44) to V. radiata (L.) Wilczek (mungbean; 2n=2x=22), interspeeific hybrids were generated (Chen et al., 1989). Unexpectedly, hybrid embryo cultures produced plants, either via embryogenesis or organogenesis. We report here results suggesting that the ability to regenerate plants is associated with interspecific hybridity.
Offprint requests to: D. W. S. Mok
Growth regulator treatment. In order to delay pod abscission, a solution containing i00 mg/l gibberellie acid (GA3) , 25 mg/l a-naphthaleneacetic acid (NAA) and 5 mg/l kinetin was applied to run-off to the pedicels of developing pods (Gosal and Bajaj, 1983). The treatment began the second day after pollination and was repeated daily until ten days after pollination. Culture methods. Pods resulting from V. glabrescens x V. radiata crosses were collected from 15 to 21 days after pollination, and those of the reciprocal cross 5 to ii days after pollination. Selfed pods of the entire age range (5 to 21 days) were used as controls. Pods were surface sterilized for 1 min in 70% ethanol, followed by I0 min in 20% Clorox, and rinsed in sterile water. Embryos were excised from the pods and cultured on Murashige and Skoog (1962) mineral nutrients supplemented with 30 g/l sucrose, 100 mg/l myo-inositol, i00 mg/l glutamine, 5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 1 mg/l thiamine and 8 g/l Difco bacto-agar (MS medium). The medium was adjusted to pH 5.7 prior to autoclaving and dispensed in screw-cap jars (20 ml/jar). Cultures were incubated at 25~ and a 16h light period at 3000 ix. Somatic embryos formed on immature zygotic embryos of V_~.$1abrescens x V. radiata were germinated on MS medium containing 0.5 mg/l kinetin. Callus tissues which formed on immature zygotic embryos of __V" radiata x V__~.glabrescens were maintained on MS medium for 4 passages of 3-4 weeks and then transferred to MS medium supplemented with 40 mg/l adenine sulfate, 170 mg/l sodium phosphate, 2 mg/l N6-benzyladenine (BAP) and 0.05 mg/l NAA to induce shoot formation. Shoots were transferred to MS medium containing one-half strength minerals and supplemented with 0.01 mg/l NAA to induce rooting. RESULTS Hybrid embryos of V. glabrescens x V. radiata developed to the co tyle~-onar~ stage hut ~ 4 not mature. These embryos possessed uneven, sponge-like cotyledons and an elongated axis. Immaturh embryos
78 cultured on MS medium without growth regulators germinated and hybrid plants were obtained routinely. Occasionally adventitious embryos were formed either directly on hybrid cotyledons or on callus derived from cotyledonary tissues (Fig. i). The frequency of embryogenesis was low and influenced by the age of the zygotic embryos (Table I). Embryos isolated less than 18 days after pollination were more embryogenic than older embryos. Application of growth regulators to the developing pods enhanced pod retention from 55% to 75% and increased the frequency of embryogenesis from cotyledons for both age groups (Table I). Some of the somatic embryo s developed into plantlets when transferred to medium with kinetin (Fig. 2). In contrast, no somatic embryos were observed on cotyledons of selfed embryos of either parent. Embryos of the reciprocal cross developed only to the heart stage, and the pods usually abscised 9-11 days after pollination. Therefore, experiments were performed with embryos at an earlier stage than those of the reciprocal cross. Embryos recovered from treated pods were also at the heart stage (0.3 0.5 mm), even though treating pods with growth regulators prolonged pod retention (up to 18 days Figure 2. Plantlet from somatic embryo of V. glabrescens x V. radiata cultured on medium with 0.5 mg/l kinetin after pollination). When immature embryos of this hybrid were cultured on MS medium, they did not germinate but some formed callus. The frequencies of callus formation were 24% (of 66 embryos) and 28% (of 96 embryos) respectively, for hybrid embryos obtained from the untreated and treated pods. These callus tissues could be maintained on MS medium. Multiple shoots were formed from hybrid callus after transfer to medium containing 2 mg/l BAP and 0.05 mg/l NAA. As many as 6 shoots were obtained from a single callus tissue. However, the frequency of organogenic callus was low (7%) and was not increased by treatment of pods with growth regulators. Selfed embryos cultured on the same media as hybrid embryos did not give rise to organogenic callus although 13 of 75 embryos of V. radiata formed callus. DISCUSSION Figure i. Somatic embryos formed on a cultured cotyledon of a~zygotic embryo of V. $1abrescens x V__t. radiata Table i. Effects of pod treatment and embryo age on the frequency of embryogenic cotyledons of V. glabrescens x V. radiata. ....................................................
Embryo age __!~z~
....... ~
Pod ~
No. _
_
~
!
~
% _
~
~
....
15-17
-
60
8
15-17
+
78
29
18-21
-
88
1
18-21
+
92
11
a
Application of a solution containing i00 mg/l GA3, 25 mg/l NAA and 5 mg/l kinetin daily from 2 to i0 days after pollination
The ability to regenerate via somatic embryogenesis or shoot organogenesis appears to be a unique property of interspecific embryos of V. glabrescens and V. radiata. Selfed embryos of the parental species were unable to undergo regeneration under identical conditions. Redifferentiation potentials seem to be associated with the unique developmental features of the interspecific embryos. The loose, sponge-like cotyledons of V. glabrescens x V. radiata embryos tended to become embryogenic, even without hormonal treatments of the pods. Similarly, the heart-stage embryos derived from the reciprocal cross gave rise to callus tissues which were in some cases intrinsically organogenic. At present, it is not yet possible to distinguish genetic and developmental effects on regeneration. However, backcross progeny of the interspecific hybrid (to V_2_~ radiata) had normal and abnormal embryos (Chen et al. 1989). The ability of these embryos to regenerate will be examined in order to assess the relative influence of genotype vs. abnormal embryo development on embryogenesis and organogenesis in Vigna. In order to increase pod retention, a combination of growth regulators was applied to the pods of V__~.glabrescens x V. radiata crosses. The
79 treatment not only enhanced pod retention, but also stimulated embryogenesison cultured cotyledons. However, culturing of selfed and hybrid embryos directly on medium containing the three growth regulators did not result in the formation of somatic embryos or organogenic callus (unpublished data). It is possible that uptake of the growth regulators during embryo development in vivo is of greater influence on redifferentiation in vitro than treatment after excision. The results presented here may be of interest with regards to other large seeded legumes, such as Phaseolus, where redifferentiation from immature embryos or cell cultures has not been possible. We have studied interspecific hybridization of Phaseolus extensively (Mok et al. 1978, 1986). As in Vigna, the interspecific embryos of Phaseolus are developmentallyabnormal. The possible variation in regeneration ability of these embryos has not been explored. We will examine the possibility of regeneration in interspecific combinations in Phaseolus based on the approaches applied to Vigna. ACKNOWLEDGEMENT Research was supported by a grant (87-CRSR-2-3185) from IARC Special Constraints Research Grants Program of AID/USDA/CSRS, and by the Oregon Agricultural Experiment Station. This is technical paper No. 9095 of the Oregon Agricultural Experiment Station.
REFERENCES Barwale UB, Kerns HR, Widholm JM (1986) Planta 167:473-481 Chen HK, Mok MC, 8hanmugasundaram S, Mok DWS (1989) Theor Appl Genet 78:641-647 Christianson ML, Warnick DA, Carlson PS (1983) Science 222:632-634 Gosal SS, Bajaj YPS (1983) Euphytica 32:129-137 Kumar AS, Gamborg OL, Nabors MW (1988) Plant Cell Reports 7:322-325 Kysely W, Myers JR, Lazzeri PA, Collins GB, Jacobsen H-J (1987) Plant Cell Reports 6:305-308 Lazzeri PA, Hildebrand DF, Collins GB (1985) Plant Mol Biol Rep 3:160-168 Li BJ, Langridge WHR, Szalay AA (1985) Plant Cell Reports 4:344-347 Mok DWS, Mok MC, Rabakoarl~hanta A (1978) Theor Appl Genet 52:209-215 Mok DWS, Mok MC, Rabakoarihanta A, Shii CT (1986) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, Vol 2: Crops I. Springer-Verlag, Berlin Heidelberg, pp 309-318 Murashige T, 8koog F (1962) Physiol Plant 15:473-497 Ranch JP, Oglesby LO, Zielinski AC (1985) In Vitro 21:653-658 Shekhawat NS, Galston AW (1983) Plant Sci Lett 32:43-51
Plant Cell Reports (1990) 9:77-79
9 Springer-Verlag1990
Somatic embryogenesis and shoot organogenesis from interspecific hybrid embryos of Vigna glabrescens and V. radiata H. K. Chen, M. C. Mok, and D. W. S. Mok Department of Horticulture and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331, USA Received December 21, 1989/Revised version received April 9, 1990 - Communicated by A. R. Gould
ABSTRACT
MATERIALS AND METHODS
Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vign~ glabrescens x V. radiata crosses and cultured on Murashige and Skoog (MS) medium without growth regulators. The frequency of 15-17 day old embryos that gave rise to somatic embryos increased from 8% to 29% by application a mixture of i00 mg/l gibberellic acid, 25 mg/l e-naphthaleneacetic acid (NAA) and 5 mg/1 kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N6-benzyladenine and 0.05 mg/l NAA. Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.
Plant materials. Seeds of V. glabrescens VII60 and V. radiata VCI973A were obtained from the Asian Vegetable Research and Development Center (AVRDC). Conditions for growth and flower induction and methods of pollination were as described previously (Chen et al., 1989).
INTRODUCTION The difficulty in redifferentiating plants from tissue cultures of large-seeded legumes has limited genetic transformation and other in vitro manipulations of this important group of plants. However, in recent years somatic embryos and plants have been generated from cotyledons and protoplast cultures of soybean (Glycine max (L.) Merr.) (Christianson et al., 1983; Lazzeri et al., 1985; Li et al., 1985; Ranch et al., 1985; Barwale et al., 1986). Plant formation via somatic embryogenesis is also possible from immature embryos and shoot apices of Pisum sativum L. (Kysely et al., 1987), cell suspensions of Phaseolus acutifolius L. (Kumar et al., 1988) and protoplasts of V. aconitifolia Jarq. (Shekhawat and Galston, 1983). In spite of these important advances, many of the Vigna and Phaseolus species are still not responsive to similar regeneration procedures. In an attempt to transfer pest resistance from V_t. ~labrescens M. M. and S. (2n=4x=44) to V. radiata (L.) Wilczek (mungbean; 2n=2x=22), interspeeific hybrids were generated (Chen et al., 1989). Unexpectedly, hybrid embryo cultures produced plants, either via embryogenesis or organogenesis. We report here results suggesting that the ability to regenerate plants is associated with interspecific hybridity.
Offprint requests to: D. W. S. Mok
Growth regulator treatment. In order to delay pod abscission, a solution containing i00 mg/l gibberellie acid (GA3) , 25 mg/l a-naphthaleneacetic acid (NAA) and 5 mg/l kinetin was applied to run-off to the pedicels of developing pods (Gosal and Bajaj, 1983). The treatment began the second day after pollination and was repeated daily until ten days after pollination. Culture methods. Pods resulting from V. glabrescens x V. radiata crosses were collected from 15 to 21 days after pollination, and those of the reciprocal cross 5 to ii days after pollination. Selfed pods of the entire age range (5 to 21 days) were used as controls. Pods were surface sterilized for 1 min in 70% ethanol, followed by I0 min in 20% Clorox, and rinsed in sterile water. Embryos were excised from the pods and cultured on Murashige and Skoog (1962) mineral nutrients supplemented with 30 g/l sucrose, 100 mg/l myo-inositol, i00 mg/l glutamine, 5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 1 mg/l thiamine and 8 g/l Difco bacto-agar (MS medium). The medium was adjusted to pH 5.7 prior to autoclaving and dispensed in screw-cap jars (20 ml/jar). Cultures were incubated at 25~ and a 16h light period at 3000 ix. Somatic embryos formed on immature zygotic embryos of V_~.$1abrescens x V. radiata were germinated on MS medium containing 0.5 mg/l kinetin. Callus tissues which formed on immature zygotic embryos of __V" radiata x V__~.glabrescens were maintained on MS medium for 4 passages of 3-4 weeks and then transferred to MS medium supplemented with 40 mg/l adenine sulfate, 170 mg/l sodium phosphate, 2 mg/l N6-benzyladenine (BAP) and 0.05 mg/l NAA to induce shoot formation. Shoots were transferred to MS medium containing one-half strength minerals and supplemented with 0.01 mg/l NAA to induce rooting. RESULTS Hybrid embryos of V. glabrescens x V. radiata developed to the co tyle~-onar~ stage hut ~ 4 not mature. These embryos possessed uneven, sponge-like cotyledons and an elongated axis. Immaturh embryos
78 cultured on MS medium without growth regulators germinated and hybrid plants were obtained routinely. Occasionally adventitious embryos were formed either directly on hybrid cotyledons or on callus derived from cotyledonary tissues (Fig. i). The frequency of embryogenesis was low and influenced by the age of the zygotic embryos (Table I). Embryos isolated less than 18 days after pollination were more embryogenic than older embryos. Application of growth regulators to the developing pods enhanced pod retention from 55% to 75% and increased the frequency of embryogenesis from cotyledons for both age groups (Table I). Some of the somatic embryo s developed into plantlets when transferred to medium with kinetin (Fig. 2). In contrast, no somatic embryos were observed on cotyledons of selfed embryos of either parent. Embryos of the reciprocal cross developed only to the heart stage, and the pods usually abscised 9-11 days after pollination. Therefore, experiments were performed with embryos at an earlier stage than those of the reciprocal cross. Embryos recovered from treated pods were also at the heart stage (0.3 0.5 mm), even though treating pods with growth regulators prolonged pod retention (up to 18 days Figure 2. Plantlet from somatic embryo of V. glabrescens x V. radiata cultured on medium with 0.5 mg/l kinetin after pollination). When immature embryos of this hybrid were cultured on MS medium, they did not germinate but some formed callus. The frequencies of callus formation were 24% (of 66 embryos) and 28% (of 96 embryos) respectively, for hybrid embryos obtained from the untreated and treated pods. These callus tissues could be maintained on MS medium. Multiple shoots were formed from hybrid callus after transfer to medium containing 2 mg/l BAP and 0.05 mg/l NAA. As many as 6 shoots were obtained from a single callus tissue. However, the frequency of organogenic callus was low (7%) and was not increased by treatment of pods with growth regulators. Selfed embryos cultured on the same media as hybrid embryos did not give rise to organogenic callus although 13 of 75 embryos of V. radiata formed callus. DISCUSSION Figure i. Somatic embryos formed on a cultured cotyledon of a~zygotic embryo of V. $1abrescens x V__t. radiata Table i. Effects of pod treatment and embryo age on the frequency of embryogenic cotyledons of V. glabrescens x V. radiata. ....................................................
Embryo age __!~z~
....... ~
Pod ~
No. _
_
~
!
~
% _
~
~
....
15-17
-
60
8
15-17
+
78
29
18-21
-
88
1
18-21
+
92
11
a
Application of a solution containing i00 mg/l GA3, 25 mg/l NAA and 5 mg/l kinetin daily from 2 to i0 days after pollination
The ability to regenerate via somatic embryogenesis or shoot organogenesis appears to be a unique property of interspecific embryos of V. glabrescens and V. radiata. Selfed embryos of the parental species were unable to undergo regeneration under identical conditions. Redifferentiation potentials seem to be associated with the unique developmental features of the interspecific embryos. The loose, sponge-like cotyledons of V. glabrescens x V. radiata embryos tended to become embryogenic, even without hormonal treatments of the pods. Similarly, the heart-stage embryos derived from the reciprocal cross gave rise to callus tissues which were in some cases intrinsically organogenic. At present, it is not yet possible to distinguish genetic and developmental effects on regeneration. However, backcross progeny of the interspecific hybrid (to V_2_~ radiata) had normal and abnormal embryos (Chen et al. 1989). The ability of these embryos to regenerate will be examined in order to assess the relative influence of genotype vs. abnormal embryo development on embryogenesis and organogenesis in Vigna. In order to increase pod retention, a combination of growth regulators was applied to the pods of V__~.glabrescens x V. radiata crosses. The
79 treatment not only enhanced pod retention, but also stimulated embryogenesison cultured cotyledons. However, culturing of selfed and hybrid embryos directly on medium containing the three growth regulators did not result in the formation of somatic embryos or organogenic callus (unpublished data). It is possible that uptake of the growth regulators during embryo development in vivo is of greater influence on redifferentiation in vitro than treatment after excision. The results presented here may be of interest with regards to other large seeded legumes, such as Phaseolus, where redifferentiation from immature embryos or cell cultures has not been possible. We have studied interspecific hybridization of Phaseolus extensively (Mok et al. 1978, 1986). As in Vigna, the interspecific embryos of Phaseolus are developmentallyabnormal. The possible variation in regeneration ability of these embryos has not been explored. We will examine the possibility of regeneration in interspecific combinations in Phaseolus based on the approaches applied to Vigna. ACKNOWLEDGEMENT Research was supported by a grant (87-CRSR-2-3185) from IARC Special Constraints Research Grants Program of AID/USDA/CSRS, and by the Oregon Agricultural Experiment Station. This is technical paper No. 9095 of the Oregon Agricultural Experiment Station.
REFERENCES Barwale UB, Kerns HR, Widholm JM (1986) Planta 167:473-481 Chen HK, Mok MC, 8hanmugasundaram S, Mok DWS (1989) Theor Appl Genet 78:641-647 Christianson ML, Warnick DA, Carlson PS (1983) Science 222:632-634 Gosal SS, Bajaj YPS (1983) Euphytica 32:129-137 Kumar AS, Gamborg OL, Nabors MW (1988) Plant Cell Reports 7:322-325 Kysely W, Myers JR, Lazzeri PA, Collins GB, Jacobsen H-J (1987) Plant Cell Reports 6:305-308 Lazzeri PA, Hildebrand DF, Collins GB (1985) Plant Mol Biol Rep 3:160-168 Li BJ, Langridge WHR, Szalay AA (1985) Plant Cell Reports 4:344-347 Mok DWS, Mok MC, Rabakoarl~hanta A (1978) Theor Appl Genet 52:209-215 Mok DWS, Mok MC, Rabakoarihanta A, Shii CT (1986) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, Vol 2: Crops I. Springer-Verlag, Berlin Heidelberg, pp 309-318 Murashige T, 8koog F (1962) Physiol Plant 15:473-497 Ranch JP, Oglesby LO, Zielinski AC (1985) In Vitro 21:653-658 Shekhawat NS, Galston AW (1983) Plant Sci Lett 32:43-51
