Plant Breeding 101, 173-1~0 (1988)@ 1988 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0179-9541

School of Biological Sciences, University of Birmingham (U. K.

Attempts at Interspecific HybridizationBetween Phaseolus vulgaris L. and P. acutifolius A. GrayUsing Embryo Rescue

J.

A. ANDRADE-AGUILAR and M. T. JACKSON

With one figure and 3 tables

Received June 3, 1988/ Accepted July 12, 1988

AbstractAttempts at interspecific hybridization were madebetween Phaseolus vulgaris (common bean) andP. acutifolius (tepary bean) using a new pollinationmethod and embryo rescue. The success of interspe-cific hybridization depended upon the pollinationtechnique, the species and individual genotypes usedas female and male parents, and the growth condi-tions. A high hybridization efficiency was achievedwhen P. vulgaris was used as female parent. Whenpods were left on the maternal plants hybrid seedswere obtained which developed into abnormal seed-lings. These died at an early stage of growth. Hybridplants were grown to maturity following embryorescue, but all showed different developmental ab-normalities. The growth of these was determinatelike the female parent, P. vulgaris, but leaf morpho-logy was closer to the male parent, P. acutifolius.The utilization of P. acutifolius germpl~sm for theimprovement of common bean remains limited, andfurther studies are needed to develop promising em-bryo rescue protocols in such wide crossing pro-grammes.

Key words: Phaseolus vulgaris -Phaseolusacutifolius -common bean -tepary bean -inter-specific hybridization -embryo rescue -in vitroembryo culture

Successful hybridization techniques whichmanipulate reproductive systems are importantfor the utilization of the genetic diversity ofPhaseD/us gene pools. In the past many at-tempts have been made to incorporate the fourmost important PhaseD/us bean species, name-ly P. vulgaris (common bean), P. coccineus

(runner bean), P. acutifolius (tepary bean), andP. lunatus (lima bean) into a common genepool using conventional techniques (HONMA1956, HONMA and HEECKT 1958, 1959, KEDARand BEMIS 1960, COYNE 1964, THOMAS 1964,AL- Y ASIRI and COYNE 1966, SMARTT 1970, HAQand SMARTT 1978). It has been suggested thatthis would enable plant breeders to improvethese crops for high yield alongside pest anddisease resistance and drought and/or heat re-sistance amongst other characteristics. How-ever, distinct barriers to intra- and interspecifichybridization exist between these PhaseD/usspecies (THOMAS 1964, AL- Y ASIRI and COYNE1966, SMARTT and HAQ 1972, MOK et al. 1978,SMARTT 1979, HUCL and SCOLES 1985).

The grouping of these four most importantPhaseolus bean species according to the genepool concept of HARLAN and DE WET (1971) isconvenient for the study and understanding ofbarriers to hybridization between thesespecies. SMARTT (1981, 1984, 1985 and 1986)has defined the primary, secondary and terti-ary gene pools for P. vulgaris. The primarygene pool consists of domesticated (GP1A)and wild populations (GP1B)j the secondarygene pool of P. vulgaris consists mainly ofP. coccineus (GP2) and the tertiary gene poolconsists of P. acutifolius (GP3A) andP. lunatus (GP3B).

The success to intra- and interspecific hy-bridization depends on the relationship be-tween several factors such as species and/orgenotypes used as male and female parents in

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174 ANDRADE-AGUILAR and JACKSON

Embryo rescue has recently been used toobtain some hybrids between these Phaseolusspecies, but they were self-sterile or only par-tially fertile (PRENDOTA et al. 1982, THOMAS andWAINES 1984, PRATr et al. 1985, PARKER andMICHAELS 1986). Some level of fertility hasbeen gradually recovered only by backcrossingto either parent, but mainly to P. vulgaris.

In this paper the results of attempts arereported to produce hybrids between P. vul-garis and P. acutifolius using a new pollinationmethod and embryo rescue.

the reciprocal crosses, environmental condi-tions and pollination techniques. Reciprocaldifferences have been found in successful inter-specific crosses, P. vulgaris being in generalmore successful when used as female parent(HONMA 1956, AL- Y ASIRI and COYNE 1966,SMARTT 1970, MOK et al. 1978). Nevertheless,THOMAS and WAINES (1984) reported no recip-rocal differences between P. vulgaris andP. acutifolius.

Apparently, no pre-fertilization barriersseem to exist for the interspecific reciprocalcrosses between P. vulgaris and P. acutifolius(MOK et al. 1978, RABAKOARIHANTA et al. 1979).However, post-fertilization barriers to embryodevelopment have been suggested to be themajor difficulty in obtaining interspecific hy-brids between these two species where normalfertilization can be achieved. Embryos com-monly abort between three and 24 days afterpollination (HONMA 1956, THOMAS 1964,SMARTT 1970, MOK et al. 1978, PRENDOTA et al.1982, THOMAS and WAINES 1984). A delay inendosperm and embryo divisions (RA-BAKOARIHANTA et al. 1979) and further failure ofendosperm development appear to be the causeof embryo abortion and subsequent collapse ofthe pods (AL-YASIRI and COYNE 1964, 1966).Pods appeared initially to develop normallybut 16 days after pollination they began tocollapse and by 22 days all pods had collapsed.

Embryo rescue and culture techniques are apromising field of research to overcome post-fertilization barriers for the successful growthof interspecific progeny between Phaseolusbean species. These would enable plant breed-ers to transfer useful desirable traits from onespecies into another (PRATT 1983). The firstreported interspecific hybrids between P. vul-garis and P. acutifolius were obtained usingartificial culture medium (HONMA 1955, 1956).From several hundred embryos 14 to 20 daysafter pollination only four putative hybridplants were grown to maturity. The morphol-ogy of these plants was similar to that of thefemale parent, P. vulgaris, leading SMARTT(1970, 1980) to question their hybrid status,based on results of crosses which he carried outbetween these species, as well as the high fertil-ity of the plants reported by HONMA. Nosuccess was obtained in the reciprocal crossesusing P. acutifolius as female.

Materials and MethodsGenetic material: Sixteen accessions of P. vulgarisand 20 of P. acutifolius from Centro Internacional deAgricultura Tropical (CIAT) were used in this study(Table 1). Plants were grown under varying condi-tions of temperature and daylength in an attempt toobtain synchronization in flowering between thetwo species.

Hybridization techniques: Initially several hybri-dization techniques were tried, but with littlesuccess. A new method, namely "The InsertionMethod" (ANDRADE-AGUILAR and JACKSON 1988)was developed and used with success for mostcrosses. Essentially this pollination method consistsin placing the keel containing the style-stigma andstamens from the male parent over the style-stigmaof the female parent after emasculation. The advan-tage with this method of pollination is that it keepsthe anthers and the pollen bearing style-stigma of themale flower in close contact with the stigma of thefemale flower over a period long enough to ensurefertilization, and reduces the problem of desiccationof the female and male reproductive organs. Hy-bridization efficiency was scored as the number ofsuccessful pollinations/total number of pollinationsx 100. A successful pollination was considered asone in which pod growth was normal on the mater-nal plant just before removal for embryo rescue(2-3 weeks after pollination), or left on the mater-nal plant for further development.

Embryo culture: Two artificial culture media wereused, namely B5 (GAMBORG et al. 1968) and MS(MURASHIGE and SKOOG 1962). Both media weresupplemented with sucrose 30 g 1-1 (3 %) and agar7 g 1-1 (0.7 %).

Pods of different ages and/or sizes from apparentinterspecific crosses were removed from the maternalplants and placed in water. The pods were surfacesterilized in 70 % ethanol for 3-5 min, immersed in10 % sodium hypochlorite (plus one or two drops of

Table 3. The mean age and size of pods, and the number and mean size of embryos rescued following crossesbetween P. vulgaris X P. acutifolius and reciprocal. Sizes of pods and embryos based on dimensions of lengthand breadth

Acc. No. Mean age(days)

EmbryosNumber Size (mm)cultured L B

PodsSize (mm)

L BCulturemedium

P. vulgarisG-O197G-1594G-4906G-4979G-5169G-5307G-5343G-5974G-7339G-7418G-7537G-7624'Red Kidney'

P. acutifolius

G-40040G-40066AG-40110G-40120G-40123

2023211822

22

2222

2122

222021

829385

105102104

13511782

11011296

107

129

1011119

14117

11108

11

121224

20

26121433

13198

1.5

5.8

1.6

1.6

2.6

3.6

3.9

2.4

2.6

3.9

2.7

5.5

1.6

0.9

2.7

0.9

1.0

1.7

2.0

2.4

1.5

1.6

2.3

1.6

2.8

0.9

MMMMMMMMMMMMM

2020182021

5155476852

11989

10

0.81.50.52.10.9

MSMSMSMS, B5MS

412

224

0.61.00.51.30.8

ing, and consequently was the most widelyused either as female or male parent. Theseresults indicate that it is potentially usefulgermplasm for interspecific hybridization pro-grammes.

tings. The only plant which grew to maturityresembled the maternal parent in its vegetativeand floral morphology. Furthermore, pollenstainability was rather high (79.5 %) and itprod~ced seed which resembled that of thefemale parent in size, shape and colour. Thesefindings strongly suggest that it was derivedfrom a self pollinatIon.

Interspecific hybrid seed

From the 122 pods left on the maternalplants only 12 produced seed; the others wereempty. Five of these pods produced 13 seedson P. vulgaris as female parent and seven podsproduced also 13 seeds on P. acutifolius asfemale parent. Most seeds were abnormal, andthey were usually small, shrunken, shrivelledand had a broken and incomplete testa. Theseseeds were sown but only five seedlingsemerged. The seedlings showed developmentalabnormalities such as absence of one or both ofthe primary leaves, variegated leaves and slowgrowth. Four of these plants died at an earlyvegetative stage of development, although at-tempts were made to propagate them by cut-

Embryo rescue

During summer 1987, 89 pods which de-veloped after interspecific crosses, were re-moved from the maternal plants from which148 embryos of different sizes were rescued(Table 3). In some developing ovules and ap-parent hybrid embryos signs of abortion wereclearly noted. Ovules were shrunken and ces-sation of growth was evident. Cessation ofgrowth of the embryos was also evident and anuneven development of the cotyledons wasapparent. One cotyledon stopped its develop-ment while the other continued for further

is;

B5is, B5isisis:S, B5:S

~Sis:S

:S, B5:S:S

Fig. 1. Leaf morphology of Phaseolus vulgaris (left) and P. acutifolius (male, right), and the hybrid betweenthem (middle)

nique and by the genotype of each species usedas female and male parents. It was observedthat during the emasculation and pollination ofP. acutifolius some damage was caused, be-cause flowers are smaller and more delicate anddifficult to manage than those of P. vulgaris.BUISHAND (1956) obtained 30 and 70 % successin crossing using the rubbing and hookingmethods, respectively. In the breeding pro-gramme at CIAT 50 % efficiency has beenreported using a combination of the rubbingand hooking methods under controlled condi-tions (18 to 27 °C) (CIAT 1982). In this work,the "Insertion Method" gave an average of68 % efficiency when using P. vulgaris as thefemale parent. Two other factors affecting hy-bridization success were: 1. the conditions ofgrowth, and 2. the physiological, phenologicaland morphological stage of the plants used asfemale parents. The successful crosses underconstant conditions of 25 °C produced podswhich were apparently normal and which grewfaster compared with those produced underother conditions, but which were neverthelessempty when left on the maternal plant. Also,many pollinations were performed onto both

growth. Seventy-three embryos died at differ-ent stages of development and the rest did notsurvive in the petri dish to be transferred intoindividual tubes. Only 63 embryos were trans-planted to soil in the glasshouse which de-veloped into plantlets. From these, only twosurvived to maturity, and they showed differ-ent developmental abnormalities, such as var-iegated and crinkled leaves, and uneven de-velopment of the three leaflets. Some leaveshad the terminal leaflet longer or shorter thanthe lateral leaflets whereas the converse wastrue for others. The growth habit was determi-nate as in the female parent, P. vulgaris, butleaf morphology was similar to that ofP. acutifolius, clearly suggesting their hybridstatus (Fig. 1). Flower development was lim-ited, even after 120 days. Small flower budsoften dropped off the plants before pollenfertility could be ascertained or floral mor-phology determined.

DiscussionThe success of interspecific hybridization be-tween P. vulgaris and P. acutifolius appearedto be highly influenced by the pollination tech-

179Attempts at Interspecific Hybridization Between PhaseDlus vulgaris L: and P. acutifDlius A. Gray

This relationship may be a useful marker todistinguish and verify hybrid embryos at anearly stage of development, but it was notedonly in particular crosses and not as a generalpattern of hybrid embryos.

In many respects the present work has con-firmed the findings of earlier workers. It isclear that hybridization between P. vulgarisand P. acutifolius remains problematical, evenwith the help of embryo rescue. In terms ofgermplasm utilization, the level of success isextremely low, and will continue to presentproblems for the plant breeder wishing toutilize P. acutifolius for its drought tolerantproperties, and pest and disease resistance, un-less protocols can be developed which promisea high level of success.

species as female parents using old plants sincesynchronization in flowering between acces-sions of both species was difficult. It is knownthat considerable flower abscission occurs to-wards the end of the flowering period as anatural phenomenon (CIAT 1982). Also thegrowth habit seemed to affect this fact, since itis correlated to the flowering period as well asto the branching pattern. Pollinated flowers onthe old lateral branches of P. acutifolius gener-ally failed to develop into pods with rescuable

embryos.No true interspecific hybrid plants could be

raised from the F1 seeds. From the 26 seedsproduced (13 seeds on each species) only fivegerminated, and the single plant which sur-vived appeared to be a selfed plant as indicatedby its morphological characteristics and pollenstainability, even though it showed abnormaldevelopment. Furthermore, it set selfed seedwhich resembled the female parent. One ex-ception is that reported by SMARTT (1970) whoobtained hybrid seeds which produced threeviable plants from the cross P. vulgaris(female) x P. acutifolius (male) and six fromthe reciprocal cross using one particular geno-type of each species. However, no furtherprogeny were obtained from these plants. Asecond exception is that reported by PRENDOTAet al. (1982). From over 3500 pollinations, theyobtained 13 seeds from the cross P. vulgaris(female) x P. acutifolius (male) but only threeplants grew to flower, and from almost 1400pollinations, 23 seeds were obtained from thereciprocal cross of which 21 plants were raisedto flower. However they do not report furtherprogeny. They concluded that P. acutifoliuswas more successful as a female parent in pro-ducing the hybrid seeds.

Failure of endosperm development appearsto be the cause of embryo abortion and subse-quent collapse of the pods (AL- YASIRI and COy-NE 1964, 1966). MoK et al. (1978) found that adistinct characteristic of the hybrid embryoswas the uneven development of the two cotyle-dons. The rate of growth and final size of thesehybrid embryos seemed to be influenced bythe genotypes of both parents. This fact maybe due to the gradual failure of endospermdevelopment as suggested by RABAKOARIHANTAet al. (1979). Moreover, in the present work, arelationship was observed between the unevendevelopment of the cotyledons and the unevendevelopment of the primary unifoliate leaves.~

ZusammenfassungHerstellung yon Artbastarden zwischenPhaseolus vulgaris L. 'und P. acutifolius A.Gray mittels Embryokultur

Mit Hilfe finer neuen Bestaubungstechnik undanschlieBender Embryokultur wurde versucht,Phaseolus vulgaris (gewohnliche Gartenbohne)unci P. acutifolius (Texas-Bohne) miteinanderzu kreuzen. Der Bastardierungserfolg warvan der Bestaubungstechnik, van den als Mut-ter oder Vater verwendeten Arten unci Geno-typen sowie van den Wachstumsbedingungenabhangig. Die Kreuzungen gelangen am be-sten, wenn P. vulgaris als Mutter verwendetwurde. BelieB man die Hiilsen an den Mutter-pflanzen, erhielt man Bastardsamen, aus denensich anomale Keimpflanzen entwickelten, diebereits im friihen Stadium ihrer Entwicklungabstarben. Uber Embryokultur hingegen ge-lang es, Pflanzen bis zur Reife heranzuziehen,die allerdings auch verschiedenartige Anoma-lien aufwiesen. Das Wachstum dieser Pflanzenwar, wie bei clem miitterlichen Elter P. vulga-ris, terminiertj aber die Morphologie der Blat-ter war der des Vaters, P. acutifolius, sehr ahn-lich. Eine Nutzung von Genen aus P. acutifo-lius zur Verbesserung der Gartenbohne bleibtbegrenzt. Es sind daher weitere Untersuchun-gen notig, geeignete Techniken der Embryo-kultur zu entwickeln, urn diese in solch weitenKreuzungen einsetzen zu konnen.

The senior author thanks the International Board forPlant Genetic Resources for a fellowship throughwhich this study was carried out, and to CIA T forthe seeds of Phaseolus species.

PARKER, J. P., and T. E. MICHAELS, 1986: Simplegenetic control of hybrid plant development ininterspecific crosses between PhaseD/us vulgaris L.and P. acutifo/ius A. Gray. Plant Breeding 97,315-323.

PRATr, R. C., 1983: Gene transfer between teparyand common beans. Desert Plants 5, 57-63.

--, R. A. BRESSAN, and P. M. HASEGAWA, 1985:Genotypic diversity enhances recovery of hybridsand fertile backcrosses of PhaseD/us vulgaris L. XP. acutifo/ius A. Gray. Euphytica 34, 329-344.

PRENDOTA, K., J. P. BAUDOIN, and R. MARECHAL,1982: Fertile allopolyploids from the crossPhaseD/us acutifo/ius X PhaseD/us vulgaris. Bull.Rech. Agron. Gembloux 17, 177-190.

RABAKOARIHANTA, A., D. W. S. MOK, and M. C.MOK, 1979: Fertilization and early embryo de-velopment in reciprocal interspecific crosses ofPhaseD/us. Theor. Appl. Genet. 54, 55-59,.

SMARTr, J., 1970: Interspecific hybridization be-tween cultivated American species of the genusPhaseD/us. Euphytica 19, 48G-489.

--, 1979: Interspecific hybridization in the grainlegumes -a review. Econ. Bot. 33, 329-337.

--, 1980: Evolution and evolutionary problemsin food legumes. Econ. Bot. 34, 219-235.

~-, 1981: Gene pools in PhaseD/us and Vignacultigens. Euphytica 30, 445-449.

--, 1984: Gene pools in grain legumes. Econ.Bot. 38, 24-35.

--, 1985: Evolution of grain legumes. IV. Pulsesin the genus PhaseD/us. Expl. Agric. 21,193-207.

--, 1986: Evolution of grain legumes. VI. Thefuture -the exploitation of evolutionary know-ledge. Expl. Agri. 22, 39-58.

--, and N. HAQ, 1972: Fertility and segregationof the amphidiploid PhaseD/us vulgaris L. X P. coc-cineus L. and its behaviour in backcrosses. Euphy-tica 21, 496-501.

THOMAS, H., 1964: Investigations into the inter-relationships of PhaseD/us vulgaris L. and P. coc-cineus Lam. Genetica 35, 59-74.

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Authors' addresses: j. A. ANDRADE-AGUILAR, Cen-tro Regional para Estudios de Zonas Aridas y Semi-aridas -Colegio de Postgraduados, Iturbide 73,Salinas de Hidalgo, San Luis Potosi, 78600 Mexico(Mexico); M. T. JACKSON, Plant Genetics Group,School of Biological Sciences, University ofBirmingham, P.O. Box 363, Birmingham B15 2Tr(U.K.).

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