Plant Cell Reports (1986) 5 : 329-331
lnterspecific hybridization of Phaseolus vulgaris L. and Phaseolus angustissimus A. Gray using in vitro embryo culture
Plant Cell Reports © Springer-Verlag 1986
T e l e m a c h o s B e l i v a n i s 1,, and C l a i r e D o r 6 2 1 Fonctionnaire du Ministbre de l'Agriculture de Grbce, en stage ~ la Station de G6n6tique et d'Am61ioration des Plantes, INRA, F-78000 Versailles, France z INRA, Station de G6n6tique et d'Am61ioration des Plantes, route de Saint-Cyr, F-78000 Versailles, France Received March 11, 1986 / Revised version received July 7, 1986 - Communicated by A. M. Boudet
ABSTRACT The introgression of new desirable characters is very necessary in common bean (Phaseolus vulgaris L.) Interspecific hybridization of P. vulgaris L. with P. angustissimus A. Gray, a wild species with narrow leaves, could be successfully achieved for the first time using embryo culEure. Sixteen to twenty-three-day old embryos could grow to plantlets when they were cultivated on modified Monnier's medium. The leaf shape of the hybrid plants obtained showed intermediary traits between the two parents. A chromosome stock doubling could be achieved for one embryo. INTRODUCTION
Five different heterozygous ~. vulgaris L. plants with male sterile dominant genes were pollinated with P. angustissimus A. Gray (NI 788) ~. Numerous crosses were made in Summer. Pods were collected between 13 and 23 days after pollination.
2) In vitro culture Pods were desinfected by immersion in a 2 % calcium hypochlorite solution for 5 minutes and rinced twice in sterilized water. They were then aseptically dissected under the binocular and embryos without maternal teguments placed in Petri dishes. The culture media used were : - modified Monnier's culture media (Monnier, 1973), the sucrose concentration being 80 g/l for M8 and 40 g/l for M18 ; a MS basal medium (Murashige and Skoog, -
Common bean (Phaseolus vulgaris L.) is a very important crop species in which the introgression of tolerances to various diseases and of desirable physiological traits is very necessary. Since the first successful intercrossing Phaseolus vulgaris L. x P. coecinens Lam. was established in 1866 by Mendel (Mendel, 1866), numerous hybridizations within the genus Phaseolus itself have been attempted or achieved, as recently reviewed by Hucl et Scoles (1985). Within a range of interspecific hybridizations in the Bean breeding programme of INRA in Versailles, we used the wild species P. angustissimus A. Gray originating from Texas, Arizona and New Mexico area and considered by Mar~chal et al. (1978) as a species related to P. filiformis Bentham. One of the most important traits of this species is the narrow shape of its leaves. Interspecific hybridization usually leads to sterile material and recovering fertility is often obtained by doubling the chromosome stock (Prendota et al. ]983 , and Thomas and Waines 1984 ). Therefore colchicine treatments were carried out.
1962) ; a N30K basal medium (Margara, 1978). The culture media were adjusted to pH = 5.8 and sterilized during 20 min at 114 ° C in the autoclave. The embryo and plantlet cultures were conducted in a growth cabinet regulated at a 18 ° C night temperature and a 23 ° C day temperature and a 2000 lux light with 16 hour photoperiod. Plantlets were then transferred to soil within "mini-serres" (Ets Bouillard Fr~res SA - F-71370 Saint-Germain-du-Plain) which were gradually opened to provide acclimatization. Plantlets were then grown in the greenhouse. -
3) Chromosome stock doubling After 26 h in vitro growing embryos were dipped in a filtrated sterilized colchicine solution at a concentration of 7.5 x 10 -4 within a Petri dish which was placed under vacuum for two hours. They were then transferred back to the culture medium in Petri dishes.
MATERIAL AND METHOD I) Plant culture Parent plants provided by Dr. H. Bannerot and Dr. Mar~chal, were grown at the Station de G~n~tique et d'Am~lioration des Plantes of Versailles, in a greenhouse using Phytoclaude 400 lights and a trickle irrigation.
Cytology The counting of chromosomes was carried out on root meristem tips collected on growing plantlets and stained by a schiff reagent solution. The morphology of pollen was observed using the Alexander staining method (Alexander, 1969).
* P ~ s e n t addre~: Direction de l'Agriculture, Pr6~cture Lassithiou, Saint-Nicolaos, Cr~te, Or6ce Offp~nt requests to: C. Dor6
330 RESULTS Crosses were carried out during 1985 Summer on about 40 flowers. In many cases pods began to develop but within four weeks they turned yellow and finally dried. No mature seed containing viable embryo could be ever found. Pods contained seeds with shrivelled teguments and very small brownish embryos. First experiments were achieved on the M8 medium containing 80 g/l sucrose. The embryo growth was rather unorganized with a tendency to develop calli. Therefore we tried to grow the embryos on a medium with a reduced sucrose concentration such as M18. In this case, embryos developped normally and within eight days they reached a stage where cotyledons were well developped and the first leaves visible. On this M18 medium,embryos collected ]6 to 23 days after pollination could be rescued. Nineteen days after pollination seemed to be the optimal age for a successful culture (40 % of embryos could then be rescued) whereas older embryos tended to abortion and could be hardly or not even rescued. After eight days on the M18 medium young plantlets were transferred to two different media : N30K and MS (without hormones like the previous ones). Both allowed a balanced development of stems and roots (Fig. |). Plantlets could be easily transferred to soil as soon as the root system was branched.
Fig. I : Young rooted interspecific plantlet on MS medium. We tried to apply directly the colchicine on young embryos in vitro for chromosome stock doubling. Our previous studies on cell division rate in apical meristem of P. vulgaris L. x P. filiformis Bentham interspecific hybrid embryo showed that the maximal cell division rate obtained occured between 26 hours and 36 hours after in vitro culture of the embryo (unpublished data). Therefore, we applied colchicine on embryos 26 hours after plating and could obtain one chromosome stock-doubled hybrid plant with 44 chromosomes (Fig. 2).
Fig. 2 : 44 c h r o m o s o m e s ~ e t a p h a s ~ of the stock-doubled hybrid plant, Seventeen hybrid plants have been already obtained with embryo culture. Their phenotypes are intermediary between the two parents and there is a great variability between the different hybrids, as shown by leaf morphology (Fig. 3).
Fig. 3 : Morphological aspects of the leaves at the same physiological age : I = Phaseolus vulgaris L. (2n=22) 2 = Phaseolus angustissimus A. Gray (2n=22) 3,4,5 = F 1 interspecific hybrid between P. vulgaris x P. angustissimus (2n=22) 6 = Colchicine doubled F I interspecific hybrid between P. vulgaris x P. angustissimus(4n=4~
331 Twenty-two chromosome hybrid plants are sterile. However stamens are present and contain a few pollen grains. With Alexander's method, we observed that only a very small number of these pollen grains was heterogenally stainable because of their disturbed cellular cytoplasm.
DISCUSSION AND CONCLUSION As far as we know, for the first time hybrids of P. vulgaris L. with P. angustissimus A. Gray, were successfully obtained. Observations show that fertilization and early development of the hybrid embryo naturally occur in situ. However gradually the embryo degenerates and is irremediably damaged if it stays in situ more than 23 days after pollination. The in vitro culture is thus indispensable for embryo rescuing and hybrid plant obtention. Monnier's medium with reduced sucrose concentration appears to be very efficient because of its specificity for embryo culture. It is probably the key of our results. On the other hand, rates of embryo rescue are rather high probably because of the heterozygous state of P. vulgaris L. female plants as also demonstrated in case of P_. vulgaris L. x P. acutifolius A. Gray hybridization (Pratt et al. 1985). In spite of plants partial sterility trials are now on course with a back crossing programme with the two parents. Meiosis and other observations about fertility will be developped in a complementary paper.
ACKNOWLEDGEMENTS We are thankful to Dr. H. Bannerot who is the initiator of this project and to Dr. S. Essad for his contribution in cytology. We want also gratefully acknowledge the technical assistance by J.C. Lescure and F. Charlot, the correction of the English by E. Drevet.
REFERENCES Alexander MP (]969). Differential staining of aborted and non aborted pollen. Stain Technology 44, 3 : I]7-122. Hucl P, Scoles GJ (]985). Interspecific hybridization in the common bean : a review. Hortscienee, 20, 3 : 352-357. Marechal R, Mascherpa JM, Stainier F (]978). Etude taxonomique d'un groupe complexe d'esp~ces des genres Phaseolus et Vigna (Papilionacese) sur la base de donnges morphologiques et polliniques, traitges par l'analyse informatique, Bolssiera, 28 : p.141. Margara J. (]978). Mise au point d'une gamme de milieux pour les conditions de la culture in vitro C.R. Aead. Agr. Fr. : 654-661. Mendel G (]866). Versuche uber Pflanzenhybriden Verhandl.d.Naturforsch.Ver. Brun, 4 : 3-47. Monnier M (1973). Croissance et dgveloppement des embryons globulaires de C apsella bursa-pastoris cultiv~s in vitro dans un milieu g base d'une nouvelle so-olution min~rale. Soe. bot. Fr. Mem. Coil. Morphologie, 179-]94. Mnrashige T, Skoog F (1962). A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plantarum, ]5 : 473-477. Pratt RC, Bressan RA, Hasegawa PM (]985). Genotypic diversity enhances recovery of hybrids and fertile backcrosses of Phaseolus vulgaris L. x P. acutifolius A. Gray. Euphytica, 34 : 329-344. Prendota K, Baudouin JP, Margchal R (]983). Fertile allopolyploids from the cross P. Acutifolius x P. vulgaris. Bull. Rech. Agron. Gembloux, 17, 2 : 177-190. Thomas CV, Waines JG (1984). Fertile backcross and allotetraploid plants from crosses between tepary beans and common beans. J. Hered., 75 : 93-98.
lnterspecific hybridization of Phaseolus vulgaris L. and Phaseolus angustissimus A. Gray using in vitro embryo culture
Plant Cell Reports © Springer-Verlag 1986
T e l e m a c h o s B e l i v a n i s 1,, and C l a i r e D o r 6 2 1 Fonctionnaire du Ministbre de l'Agriculture de Grbce, en stage ~ la Station de G6n6tique et d'Am61ioration des Plantes, INRA, F-78000 Versailles, France z INRA, Station de G6n6tique et d'Am61ioration des Plantes, route de Saint-Cyr, F-78000 Versailles, France Received March 11, 1986 / Revised version received July 7, 1986 - Communicated by A. M. Boudet
ABSTRACT The introgression of new desirable characters is very necessary in common bean (Phaseolus vulgaris L.) Interspecific hybridization of P. vulgaris L. with P. angustissimus A. Gray, a wild species with narrow leaves, could be successfully achieved for the first time using embryo culEure. Sixteen to twenty-three-day old embryos could grow to plantlets when they were cultivated on modified Monnier's medium. The leaf shape of the hybrid plants obtained showed intermediary traits between the two parents. A chromosome stock doubling could be achieved for one embryo. INTRODUCTION
Five different heterozygous ~. vulgaris L. plants with male sterile dominant genes were pollinated with P. angustissimus A. Gray (NI 788) ~. Numerous crosses were made in Summer. Pods were collected between 13 and 23 days after pollination.
2) In vitro culture Pods were desinfected by immersion in a 2 % calcium hypochlorite solution for 5 minutes and rinced twice in sterilized water. They were then aseptically dissected under the binocular and embryos without maternal teguments placed in Petri dishes. The culture media used were : - modified Monnier's culture media (Monnier, 1973), the sucrose concentration being 80 g/l for M8 and 40 g/l for M18 ; a MS basal medium (Murashige and Skoog, -
Common bean (Phaseolus vulgaris L.) is a very important crop species in which the introgression of tolerances to various diseases and of desirable physiological traits is very necessary. Since the first successful intercrossing Phaseolus vulgaris L. x P. coecinens Lam. was established in 1866 by Mendel (Mendel, 1866), numerous hybridizations within the genus Phaseolus itself have been attempted or achieved, as recently reviewed by Hucl et Scoles (1985). Within a range of interspecific hybridizations in the Bean breeding programme of INRA in Versailles, we used the wild species P. angustissimus A. Gray originating from Texas, Arizona and New Mexico area and considered by Mar~chal et al. (1978) as a species related to P. filiformis Bentham. One of the most important traits of this species is the narrow shape of its leaves. Interspecific hybridization usually leads to sterile material and recovering fertility is often obtained by doubling the chromosome stock (Prendota et al. ]983 , and Thomas and Waines 1984 ). Therefore colchicine treatments were carried out.
1962) ; a N30K basal medium (Margara, 1978). The culture media were adjusted to pH = 5.8 and sterilized during 20 min at 114 ° C in the autoclave. The embryo and plantlet cultures were conducted in a growth cabinet regulated at a 18 ° C night temperature and a 23 ° C day temperature and a 2000 lux light with 16 hour photoperiod. Plantlets were then transferred to soil within "mini-serres" (Ets Bouillard Fr~res SA - F-71370 Saint-Germain-du-Plain) which were gradually opened to provide acclimatization. Plantlets were then grown in the greenhouse. -
3) Chromosome stock doubling After 26 h in vitro growing embryos were dipped in a filtrated sterilized colchicine solution at a concentration of 7.5 x 10 -4 within a Petri dish which was placed under vacuum for two hours. They were then transferred back to the culture medium in Petri dishes.
MATERIAL AND METHOD I) Plant culture Parent plants provided by Dr. H. Bannerot and Dr. Mar~chal, were grown at the Station de G~n~tique et d'Am~lioration des Plantes of Versailles, in a greenhouse using Phytoclaude 400 lights and a trickle irrigation.
Cytology The counting of chromosomes was carried out on root meristem tips collected on growing plantlets and stained by a schiff reagent solution. The morphology of pollen was observed using the Alexander staining method (Alexander, 1969).
* P ~ s e n t addre~: Direction de l'Agriculture, Pr6~cture Lassithiou, Saint-Nicolaos, Cr~te, Or6ce Offp~nt requests to: C. Dor6
330 RESULTS Crosses were carried out during 1985 Summer on about 40 flowers. In many cases pods began to develop but within four weeks they turned yellow and finally dried. No mature seed containing viable embryo could be ever found. Pods contained seeds with shrivelled teguments and very small brownish embryos. First experiments were achieved on the M8 medium containing 80 g/l sucrose. The embryo growth was rather unorganized with a tendency to develop calli. Therefore we tried to grow the embryos on a medium with a reduced sucrose concentration such as M18. In this case, embryos developped normally and within eight days they reached a stage where cotyledons were well developped and the first leaves visible. On this M18 medium,embryos collected ]6 to 23 days after pollination could be rescued. Nineteen days after pollination seemed to be the optimal age for a successful culture (40 % of embryos could then be rescued) whereas older embryos tended to abortion and could be hardly or not even rescued. After eight days on the M18 medium young plantlets were transferred to two different media : N30K and MS (without hormones like the previous ones). Both allowed a balanced development of stems and roots (Fig. |). Plantlets could be easily transferred to soil as soon as the root system was branched.
Fig. I : Young rooted interspecific plantlet on MS medium. We tried to apply directly the colchicine on young embryos in vitro for chromosome stock doubling. Our previous studies on cell division rate in apical meristem of P. vulgaris L. x P. filiformis Bentham interspecific hybrid embryo showed that the maximal cell division rate obtained occured between 26 hours and 36 hours after in vitro culture of the embryo (unpublished data). Therefore, we applied colchicine on embryos 26 hours after plating and could obtain one chromosome stock-doubled hybrid plant with 44 chromosomes (Fig. 2).
Fig. 2 : 44 c h r o m o s o m e s ~ e t a p h a s ~ of the stock-doubled hybrid plant, Seventeen hybrid plants have been already obtained with embryo culture. Their phenotypes are intermediary between the two parents and there is a great variability between the different hybrids, as shown by leaf morphology (Fig. 3).
Fig. 3 : Morphological aspects of the leaves at the same physiological age : I = Phaseolus vulgaris L. (2n=22) 2 = Phaseolus angustissimus A. Gray (2n=22) 3,4,5 = F 1 interspecific hybrid between P. vulgaris x P. angustissimus (2n=22) 6 = Colchicine doubled F I interspecific hybrid between P. vulgaris x P. angustissimus(4n=4~
331 Twenty-two chromosome hybrid plants are sterile. However stamens are present and contain a few pollen grains. With Alexander's method, we observed that only a very small number of these pollen grains was heterogenally stainable because of their disturbed cellular cytoplasm.
DISCUSSION AND CONCLUSION As far as we know, for the first time hybrids of P. vulgaris L. with P. angustissimus A. Gray, were successfully obtained. Observations show that fertilization and early development of the hybrid embryo naturally occur in situ. However gradually the embryo degenerates and is irremediably damaged if it stays in situ more than 23 days after pollination. The in vitro culture is thus indispensable for embryo rescuing and hybrid plant obtention. Monnier's medium with reduced sucrose concentration appears to be very efficient because of its specificity for embryo culture. It is probably the key of our results. On the other hand, rates of embryo rescue are rather high probably because of the heterozygous state of P. vulgaris L. female plants as also demonstrated in case of P_. vulgaris L. x P. acutifolius A. Gray hybridization (Pratt et al. 1985). In spite of plants partial sterility trials are now on course with a back crossing programme with the two parents. Meiosis and other observations about fertility will be developped in a complementary paper.
ACKNOWLEDGEMENTS We are thankful to Dr. H. Bannerot who is the initiator of this project and to Dr. S. Essad for his contribution in cytology. We want also gratefully acknowledge the technical assistance by J.C. Lescure and F. Charlot, the correction of the English by E. Drevet.
REFERENCES Alexander MP (]969). Differential staining of aborted and non aborted pollen. Stain Technology 44, 3 : I]7-122. Hucl P, Scoles GJ (]985). Interspecific hybridization in the common bean : a review. Hortscienee, 20, 3 : 352-357. Marechal R, Mascherpa JM, Stainier F (]978). Etude taxonomique d'un groupe complexe d'esp~ces des genres Phaseolus et Vigna (Papilionacese) sur la base de donnges morphologiques et polliniques, traitges par l'analyse informatique, Bolssiera, 28 : p.141. Margara J. (]978). Mise au point d'une gamme de milieux pour les conditions de la culture in vitro C.R. Aead. Agr. Fr. : 654-661. Mendel G (]866). Versuche uber Pflanzenhybriden Verhandl.d.Naturforsch.Ver. Brun, 4 : 3-47. Monnier M (1973). Croissance et dgveloppement des embryons globulaires de C apsella bursa-pastoris cultiv~s in vitro dans un milieu g base d'une nouvelle so-olution min~rale. Soe. bot. Fr. Mem. Coil. Morphologie, 179-]94. Mnrashige T, Skoog F (1962). A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plantarum, ]5 : 473-477. Pratt RC, Bressan RA, Hasegawa PM (]985). Genotypic diversity enhances recovery of hybrids and fertile backcrosses of Phaseolus vulgaris L. x P. acutifolius A. Gray. Euphytica, 34 : 329-344. Prendota K, Baudouin JP, Margchal R (]983). Fertile allopolyploids from the cross P. Acutifolius x P. vulgaris. Bull. Rech. Agron. Gembloux, 17, 2 : 177-190. Thomas CV, Waines JG (1984). Fertile backcross and allotetraploid plants from crosses between tepary beans and common beans. J. Hered., 75 : 93-98.
