Plant Cell, Tissue and Organ Culture 9:167-171 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Short communication

Somatic embryogenesis in Viola faba L.

MIROSLAV GRIGA t*, MARIE KUBAL,AKOV,~ 2 & EVA TEJKLOV,~ 1 10SEVA - Plant Breeding and Seed Corporation, Research and Breeding Institute of Technical Crops and Legumes, CS-787 O1 ,~umperk - Temenice (* addressee for correspondence); 2Czechoslovak Academy of Sciences, Institute of Experimental Botany, Sokolovsk6 6, CS-772 O00lomouc, Czechoslovakia

Received 21 October 1986; accepted in revised form 4 March 1987

Key words: Viciafaba L., callus culture, suspension culture, somatic embryogenesis

Abstract. The paper describes a method of somatic embryo induction in callus and suspension cultures of Viciafaba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichloro- phenoxyacetic acid. The medium with 2.5#M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.

Introduction

In vitro regeneration of grain legume crops through somatic embryogenesis has been an intractable task until recently. Somatic embryo induction in Glycine sp., Pisum sativum and Phaseolus vulgaris was first reported in the 80s (Glyeine sp. [1, 3, 5, 7, 8, 9, 13]; Pisum sativum [6]; Phaseolus vulgaris [10]).

The present paper deals with somatic embryogenesis induced in another economically important legume crop - horse bean (Viciafaba L.).

Materials and methods

Horse bean plants (Viciafaba L. var. minor), white-flowering breeding lines 6601 and 6602 were grown on the experimental plots of Plant Breeding Station Horn/Mo]t6nice - Star~i Ves. Pods of 6-8 cm in size at the stage of

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green seed maturity were removed from the 1 st and 2nd fruiting canopy in the first half of July.

Pods bearing a remainder of pedicels were surface sterilized in 2.5% w/v Chloramin B (N-chlorobenzene-sulfonamide, sodium salt; Lachema Bohumin) for 30 min, and afterwards washed in 5 changes of sterile distilled water. Immature cotyledons of 4-8 mm in length were isolated from the pods. A part of the cotyledon with adjacent axillary meristem was removed so as not to mistake proliferation of that for de novo regeneration from callus.

The cotyledons were placed on the basal medium L2 [12] containing 1% sucrose, 0.7% Difco agar and supplemented with 0, 0.5, 2.5, 5.0, 10 and 20/~M 2,4-D. All media were adjusted to pH 5.8 and autoclaved at 100 kPa for 15 min. The cotyledons were placed in 250 ml Erlenmeyer flasks with 50 ml culture medium each, and incubated under 16 h photoperiod. There were 6-7 cotyledons per flask and 30-35 per variant of the culture medium. In the course of the culture the temperature regime responded to the techni- cal equipment available at the breeding station and was as follows:

- - first three weeks: day/night temperature of 29 _ 2 °C/25 ___ 2 °C (gradual temperature fall and rise; in the course of 2 h the temperature was lowered from 29 °C to 25 °C and vice versa, i.e. 2 °C/1 h),

- - next seven weeks: day/night temperature of 26 _ 2 °C/22 _ 2 °C (gradual temperature fall and rise as mentioned above),

- - afterwards constant temperature of 20 _ 2 °C was maintained.

In the 3rd subculture calluses were put onto the same media as mentioned above but containing 2.5% sucrose. In the course of the 4th subculture the calluses were transferred into liquid medium with 2.5% sucrose, without 2,4-D or with 2.5 #M 2,4-D. They were then incubated on a roller under 16 h photoperiod at the temperature of 23 + 2 °C.

For histological studies the embryogenic calli were fixed in Navashin's fluid [11] and embedded in paraffin. Serial sections were cut at 10/~M and stained with saturated basic fuchsin and picroindigocarmine [11].

R e s u l t s a n d d i s c u s s i o n

The process of callus formation on explanted cotyledons was rather slow. On 2,4-D free medium the cotyledons kept their green colour for a long period without callus formation. On the contrary, 2,4-D rich media contri- buted to yellow-green callus formation of friable structure. The cotyledons were losing their chlorophyll. The first subculture was performed after

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Fig. 1. Embryogenic callus of Vicia faba L. (3rd subculture) on the solidified L2 medium supplemented with 2.5% sucrose and 2.5 #M 2,4-D. The bar represents 1 cm.

Figs. 2 & 3. Various developmental stages of somatic embryos in liquid L2 medium. The bar represents 0.5 ram.

Fig. 4. Meristematic centre in callus culture (derived from horse bean immature cotyledons) resulting in globular somatic embryo. The bar represents 10 #M.

Fig. 5. Globular stage of horse bean somatic embryo. The bar represents 100/~M.

Fig. 6. Early heart-like stage of horse bean somatic embryo. The bar represents 100#M.

8-week incubat ion and the whole explants were t ransferred on to the fresh medium. In the 2nd subculture, which t ook place 4 weeks later, the callus

was separated f rom the explant. Dur ing the course o f the 3rd subculture there was an increase in callus fo rmat ion owing to elevated content o f sucrose ( f rom 1% up to 2.5%). At this stage the med ium with 2.5 # M 2,4-D

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was chosen because it was most favourable for callus growth and its friable structure in view of its anticipated transfer into liquid medium. The callus was yellow-green in colour, its structure being nodular with a tendency to spontaneous disintegration into small aggregates (Fig. 1). The callus produced on the media with 0.5 and 5.0 #M 2,4-D had a similar character. There was a decrease in callus mass growth on the media with 10 and 20 #M 2,4-D, the callus being more compact, and creamy to yellow-white in colour.

In the 4th subculture a portion of the callus maintained on the media with 2.5 and 5.0 pM 2,4-D was transferred onto L2 agar medium and into liquid media with 2.5/IM 2,4-D or without 2,4-D. Within 1 to 2 day incubation of the callus in liquid medium, somatic embryos at various developmental stages appeared (Figs 2, 3). The development of somatic embryos could be also documented in histological sections of cultured callus tissue (Figs 4-6). Direct embryogenesis was also observed in the callus cultured on solidified media and somatic embryos could be isolated mechanically from the callus during the subculture. However, the process was less apparent compared to the culture in liquid media. Regular differentiation of bipolar embryonic structures took place following transfer of the callus cultured on agar media with higher 2,4-D levels (5.0 and 2.5 #M) to those containing lower (2.5/~M) or zero 2,4-D concentrations (from 5.0 to 2.5 or zero; from 2.5 to zero). During further development root-like structures formed in the embryos.

The system herein described is the first report of somatic embryogenesis in Vicia faba L. The previous experimental work on in vitro horse bean regeneration were based on explants containing meristematic tissue, i.e. apical or axillary meristems [2, 4, 14]. In our experiment the proliferation of axillary meristems of the cotyledons was eliminated by removal of them together with a cotyledon part adjacent to the embryonal axis. Consequent- ly, somatic embryo induction took place in pure callus culture. In our opinion the important points underlying in vitro callus formation and subsequent induction of morphogenesis are the type of the primary explant (i.e. immature cotyledons), and choice of a genotype. White-flowering horse bean genotypes with a naturally low content of tannin and related phenolic compounds seem to be most suitable for in vitro culture. The initiated embryogenic callus exhibited no symptoms of necrosis owing to oxidation of polyphenols often observed in horse bean tissue culture [15].

The process of embryogenesis was dependent on a change in the sucrose concentration during the culture (increase in sucrose level from 1% to 2.5%), on a decrease in auxin (2,4-D) content or its complete omission from the media. Somatic embryos were produced in the callus maintained on agar media. After transfer into liquid media the embryos at globular to early- torpedo stages were released but their development was terminated at the late-torpedo stage. Somatic embryos had a tendency either to recallusing or

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to forming root-like structures on their surface as long as they were cultured on the same media (L2 without growth regulators or supplemented with 2.5/aM 2,4-D). The role of other factors (e.g. temperature, photoperiod, illumination) has not been yet clarified.

The objective of our future research is to determine conditions of further somatic embryo development and complete plant regeneration.

Acknowledgement

The authors wish to thank Dr. J. Potomkov~i for translation of the manu- script.

References

1. Barwale UB, Kerns HR, Widholm JM (1986) Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta 167:473-481

2. Busse G (1986) In vitro cultivation of Viciafaba and induction of morphogenesis. Biol Zentralbl 105:97-104

3. Christianson ML, Warnick DA, Carlson PS (1983) A morphogenetically competent soybean suspension culture. Science 222:632-634

4. Galzy R, Hamoui M (1981) Induction de l'organogrnese sur des cals de Viciafaba minor provenant d'apex. Can J Bot 59:203--207

5. Gamborg OL, Davis BP, Stahlhut RW (1983) Somatic embryogenesis in cell cultures of Glycine species. Plant Cell Reports 2:209-212

6. Jacobsen HJ, Kysely W (1984) Induction of somatic embryos in pea, Pisum sativum L. Plant Cell Tissue Organ Cult 3:319-324

7. Kerns HR, Barwale UB, Meyer MM Jr, Widholm JM (1985) Correlation ofcotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.). Plant Cell Reports 5:140--143

8. Li B J, Langridge WHR, Szalay AA (1985) Somatic embryogenesis and plantlet regenera- tion in the soybean Glycine max. Plant Cell Reports 4:344-347

9. Lippmann B, Lippmann G (1984) Induction of somatic embryos in cotyledonary tissue of soybean, Glycine max L. Merr. Plant Cell Reports 3:215-218

10. Martins IS, Sondahl MR (1984) Early stages of somatic embryo differentiation from callus cells of bean (Phaseolus vulgaris L.) grown in liquid medium. J Plant Physiol 117: 97-103

11. Nrmec B (1962) Botanickd mikrotechnika. Praha: Nakl. (~SAV 12. Phillips GC, Collins GB (1979) In vitro tissue culture of selected legumes and plant

regeneration from callus cultures of red dover. Crop Sci 19:59-64 13. Phillips GC, Collins GB (1981) Induction and development of somatic embryos from cell

suspension cultures of soybean. Plant Cell Tissue Organ Cult 1:123-129 14. Schulze S, Grunewald J, Schmidt H (1985) Zur in vitro-Regeneration von Viciafaba L.

Z Pflanzenzucht 94:244-250 15. Tejklovd E, Griga M, Novdk FJ (1984) Hormonal regulation of growth of field bean

(Viciafaba L.) shoot apices in in vitro culture. Rostl V2~r 30:543-550 (in Czech)