Plant Cell, lissue and Organ Culture 39: 219-223, 1994. Q 1994KluwerAcademicPublishers. Printedin the Netherlands.
Effect of abscisic acid and cytokinins on the development of somatic embryos in Hevea brasiliensis P. Veisseire, L. Linossier & A. C o u d r e t Laboratoire de Physiologie et Biotechnologies V~g~tales, 4 rue Ledru, 63038 Clermont-Ferrand Cedex 1, France
Received4 August1993;acceptedin revisedform 10 May 1994 Key words: abscisic acid, embryogenic cell suspension, rubber, somatic embryogenesis
Abstract Addition of liquid medium, conditioned by an embryogenic suspension, to MH1 solid medium (3,4dichlorophenoxyacetic acid 9 IxM, 6-benzyladenine 9 taM) permitted the frequent induction of highly embryogenic calli from slices of internal integument of immature seeds of Hevea brasiliensis M011. Arg. The proliferation of embryogenic cell clusters was achieved in MH1 liquid medium. Abscisic acid (ABA), cytokinins and adenine were tested for their ability to affect development of somatic embryos to plantlets. The transfer of embryogenic cell clusters on auxin-free solid medium with 10-SM ABA for 2 months stimulated embryo development. When torpedo-shaped embryos were transferred to medium with adenine or cytokinins they turned green in 1 month. Green embryos produced secondary embryos when they were collected and placed on medium without growth regulators. Abbreviations: ABA - abscisic acid, BA - 6-benzyladenine, IBA - indole-3-butyric acid, NOA - fl-
naphthoxyacetic acid, 2iP - 2-iso-pentenyladenine, 3,4-D - 3,4-dichlorophenoxyacetic acid
Introduction The multiplication of Hevea brasiliensis, the main source of natural rubber, is achieved only by grafting and can lead to a loss of production due to an incompatibility between the grafted bud and the understock. Tissue culture would solve this problem by making it possible to propagate selected clones. Micropropagation was investigated (Enjalric & Carron 1982) and led to the production of several hundreds of plantlets by microcutting, but there still existed problems of juvenility and root formation. Later, Carron & Enjalric (1985) reported obtaining somatic embryos of Hevea brasiliensis on a solid medium. The correlation between high polyamine content (El Hadrami et al. 1989) or callus water status (Etienne et al. 1991) and the occurrence of somatic embryogenesis have been demonstrated. However, somatic embryogenesis of Hevea brasiliensis remains difficult because calli obtained from the internal integument of immature fruits frequently display browning (necrosis)
leading to tissue degeneration and a loss of embryogenic competence (Housti et al. 1991). Recently, a new procedure has been developed to induce somatic embryogenesis of Hevea brasiliensis using liquid medium conditioned by an embryogenic suspension (Guerrier 1992). This procedure greatly increased the percentage of calli showing somatic embryos. In this case calli were white, friable and could be cultivated in liquid medium. A method for cryopreservation of embryogenic cell suspensions was also achieved (Veisseire et al. 1993). Abscisic acid was identified as an important media component for maturation of somatic embryos in several conifer species (Hakman & von Arnold 1985, 1988; Durzan & Gupta 1987; Boulay et al. 1988; Dunstan et al. 1988). In Hevea brasiliensis, Etienne et al. (1993) showed that both a decrease of 3,4-D and BA supply in the solid culture medium and the addition of ABA stimulated embryo induction. In this paper we report the effects of ABA and cytokinins on development of
220 MH1 1600 1400 o
A
1200 1000
1"
MH2 1600
[
~E
~
['~IGLOBULAR
1400
w
r-'/-JHEART
1 200
I,
o
TORPEOO
1000 o
8oo
,=.,
800
600
>-
600
400
=
400
o.
200
200 m
0
0
10-7
10-6
10-5
10-4
~
r~,
0
0
z
10-7
10-6
10-5 10-4
ABA CONCENTRATIONS (M)
ABA CONCENTRATIONS (U)
XMH1
XMH2
1600
1 600
1400
1400 o
1200
D
1 200
o
1000
I
1000
800
~
800
o
600
600 =
400
200
400
200
0
0
z
10-7
10-6
10-5
10-4
AIBA CONCENTRATIONS ( M )
~ z
0
0
10-7
10-6
10-5
10-4
ABA CONCENTRATIONS (M)
Fig. 1. Effects of media composition, culture methods and ABA concentrations on the development of somatic embryos. Cells treated with ABA were obtained in MH1 (A), MH2 (B) liquid medium or by alternate subculture in MHI and MH2 liquid medium. When the last four subcultures were made in MHI the multiplication condition was denoted XMHI (C) and when the last four subcultures were made in MH2 it was denoted XMH2 (D). The evaluation of the different shaped embryos was done after 2 months culture. Empty bars, globular-shaped embryos; hatched bars, heart-shaped embryos; solid bars, torpedo-shaped embryos. Error bars indicate standard deviations.
Table 1. Growth regulator and sucrose content of media used for culture ofHevea brasiliensis somatic embryos. Growth regulator
Sucrose
0tM)
(raM)
Medium
3,4-D
NOA
BA
MHO MH1 MH2
0
0
0
9 0
0 2.25
9 2.25
58 234 58
embryogenic cell clusters obtained in different culture conditions.
Materials and methods
Initiation of callus culture Calli were induced from the internal integument of clone PB 235 seeds taken from fruits 8 to 10 weeks after pollination. Fruits were surface disinfested in 1.25% sodium hypochlorite solution for 30 min and washed three times with sterilized water. Slices of internal integument were cultivated in petri dishes (100 × 15 mm) on MH1 solid medium (Carron & Enjalric 1985) containing 234 mM sucrose, 9 ~tM BA and 9 IxM 3,4-D. The pH was adjusted to 5.8 before autoclaving at 120 °C and 100 kPa for 20 min. The medium was
221 solidified with 6.8 g 1- l agar (Sigma A 1296). Cultures were incubated in the dark at 28 ~ I°C. Three weeks after starting the culture, calli were transferred to fresh MH1 solid medium supplemented with conditioned medium (Guerrier 1992).
Cell suspension culture An embryogenic cell suspension ofHevea brasiliensis was obtained by inoculating embryogenic white and friable calli that appeared 9 weeks after cultivation on MH1 solid medium. Two grams of embryogenic callus were suspended in 30 ml MH1 liquid medium in a 100 ml flask closed with cellophane and allowed to grow on a rotary shaker (110 rpm) at 28 + I°C in darkness. After 1 month of culture, suspensions were filtered and subcultured every 10 days. About 0.5 g of embryogenic cell clusters between 100 and 500 Ixm in diameter were transferred into new media after washing. Maintaining the suspension culture was performed in two different ways; continuously in MH1 or MH2 liquid medium (Carron & Enjalric 1985), and alternately four subcultures in MH1 liquid medium and four subcultures in MH2 liquid medium to decrease culture time in presence of high auxin concentration. When the last four subcultures were made in MH1, the multiplication condition of the culture was designated XMH1. It was designated XMH2 when the last four subcultures were made in MH2. Concentrations of sucrose and plant growth regulator in the different media are shown in Table 1.
Assay procedures Cell materials from the different culture conditions were filtered, washed and 0.5 g of embryogenic cell clusters over 500 ~tm diameter were placed on sterile filter paper covering MH0 solid medium (MH0=MH2 medium lacking NOA and BA) containing 58 mM sucrose and supplemented with filter-sterilized ABA (0, 10 -7, 10 -6, 10 -5, 10 -4 M). Each treatment consisted of three petri dishes. Experiments were repeated twice. Counting of embryo types was carried out 2 months after the transfer on solid medium. The content of each dish was resuspended in 20 ml MH0 liquid medium. Samples of 1 ml of the resuspended cell masses were observed using a microscope. Results were expressed as number of somatic embryos per gram of plant materials treated by the different ABA concentrations.
Fig. 2. Growthof embryogeniccell clusters of Hevea brasiliensis obtained in MH1 liquid medium. Photo was taken 6 weeks after transfer on medium without growth regulator. Bar on photograph represents2 mm.
Fig. 3. Developmentof Hevea brasiliensissomaticembryosfrom embryogeniccell clusters obtained in MHI liquid medium. Photo was taken 6 weeks after t~-afJsferon mediumwith 10-5 M abscisic acid. Bar on photographtep~.~sents2 mm.
Torpedo-shaped embryos, obtained on medium with 10- 5 M ABA, were placed on MH0 solid medium for 1 month (five petri dishes with 10 embryos for each condition) with adenine or cytokinins. The effects of cytokinins and adenine were compared at concentrations of 0.4, 2.2 and 4.4 ~tM for BA; 2.5 and 4.9 pM for 2iP; 2.3 and 4.6 ~tM for kinetin and 0.7, 3.7 and 7.4 p.M for adenine. Cultures were incubated at 28 + 1 ° C with a 12 h photoperiod (50 ~tmol m -2 s - l ; the light source is cool white fluorescent tubes (Mazda TF 65, 58 W)). Only embryos showing both elongation of the root and development of a green shoot were considered as germinated. Results were expressed as percentage
222 Table 2. Effects of different concentrations of cytokinins and adenine on torpedo-shaped embryos. Before transfer of torpedo-shaped embryos to MH0 solid medium, the percentage of germination was estimated. It was indicated as percentage of the number of embryos showing both an elongation of the root apex and a development of the green shoot to the total number of embryos examined in each treatment. Mean + standard deviation for 100 embryos per treatment. Growth substance
Concentration (btM)
Germination (%)
BA
0.4 2.2 4.4 2.5 4.9 2.3 4.6 0.7 3.7 7.0
9 -4- 4 18 4- 6 27 -l- 8 68 4- 7 43 4- 7 30 4- 3 36 4- 10 26 + 2 32 4- 8 74 4- 10
2iP Kinetin Adenine
of embryos placed on solid medium. Green embryos were then individually collected and placed in glass tubes (150 x 20 mm) containing MH0 medium with 29 mM sucrose and closed with plastic caps. Effect of activated charcoal (Merck 2183) was tested at 5 g 1-l.
um was MH1. The MH1 medium greatly increased the number of embryos obtained and many were developed into torpedo stage. When MH1 medium was used to multiply embryogenic cell clusters, a few somatic embryos at torpedo stage were also observed on medium with 10 -4 M ABA. After 2 month's growth on media containing 10 -5 M ABA, the embryos transferred to a medium without growth regulator did not turn green. In contrast embryos cultivated in the presence of cytokinins or adenine rapidly turned green (Table 2). The best results were obtained with 7 btM adenine. In this case 74% of the torpedo-shaped embryos had green cotyledons and developed white roots. Other embryos showed secondary embryogenesis and recallusing. The same phenomenon occurred when adenine was replaced by 2iP or kinetin, but the number of embryos that recallused in I month increased. With BA, torpedo-shaped embryos turned green, but their cotyledons showed extensive enlargement before forming secondary embryos. In each case, green embryos were transferred on MH0 medium where they callused again and gave embryogenic calli after 1 month of culture. The addition of activated charcoal to adsorb plant growth regulators resulted in delay of the process of secondary embryogenesis but did not stop it.
Discussion Results The maintaining of embryogenic competence of a An embryogenic cell line of Hevea brasiliensis was efficiently multiplied either in MH1 or MH2 liquid media or by subculturing alternately in MH1 and MH2. In these culture conditions, the cell suspension did not develop beyond embryogenic cell clusters. Little development occurred when embryogenic cell clusters were transferred to a solid medium without growth regulators (Fig. 1). Under these conditions the plant material grew (Fig. 2) and a few little globular embryos were observed in some petri dishes. Embryos at heart or torpedo-shaped stages were never observed. Use of ABA-containing media at different concentrations gave a positive response on embryo development (Figs 1, 3). After 2 months culture, the optimal level of abscisic acid for obtaining torpedo-shaped embryos was 10 -5 M whatever the multiplication medium we used. The sensitivity of the cell line to ABA varied with the composition of the medium used to multiply embryogenic cell clusters. The effect of abscisic acid was more pronounced when the multiplication medi-
Hevea brasiliensis cell line in different liquid culture
media made it possible to investigate maturation and germination of somatic embryos. Maturation appeared as a major problem. As for Picea abies (Boulay et al. 1988), treatment with ABA was necessary for the formation of individual embryos and elongation of globular embryos. Moreover abscisic acid increased the frequency of normal embryos in different species (Ammirato 1977; Attree & Fowke 1991). This growth regulator had a promotive effect on the accumulation of storage lipids and proteins in conifer somatic embryos (Feirer et al. 1989; Roberts et al. 1990). In Hevea brasiliensis, Etienne et al. (1993) reported that the decrease of 3,4-D and BA and the addition of ABA in solid medium stimulated induction of embryogenic cells. These authors showed that the two operations resulted in a decrease in endogenous ABA in embryogenic callus. In our study, ABA was added without other growth regulators but cells were obtained from liquid medium containing auxins and cytokinins. Esti-
223 mation of endogenous A B A content in embryogenic cell clusters and somatic embryos should enable us to determine the impact of exogenous A B A treatment on the endogenous level. The best results were obtained when multiplication medium was MH1. This finding is in agreement with the report of Boulay et al. (1988). The longer Picea abies suspension cultures were in liquid medium with 2,4-D (5 ~tM), B A and kinetin (2 gtM each), the sooner somatic embryos appeared in the abscisic acid maturation medium. These data showed that a long-time culture with high concentrations of auxins and cytokinins before the treatment with A B A was beneficial in some species. Torpedo-shaped embryos treated with abscisic acid did not turn green when exposed to light on a medium without growth regulators. This result might be due to an inhibitory effect o f A B A on the greening. If cytokinins are effective in completely reversing abscisic acid inhibition in axes of Phaseolus vulgaris (Sussex et al. 1975), in Hevea brasiliensis their effect was transient. Future large-scale industrial applications require recovery of plantlets directly from liquid cultures. For this goal, application of A B A and cytokinins in liquid medium has been tested also (data not shown). When green plantlets were obtained in liquid medium, they also callused again when they were collected and placed on solid medium without growth regulators. These observations confirm the idea that the interaction and balance of all the plant growth regulators are important in the control of embryo growth and differentiation. They suggest studying growth regulator combinations such as A B A and auxins or A B A and cytokinins to improve maturation and germination of H e v e a brasiliensis somatic embryos. If plantlets could be regenerated from embryogenic suspension cultures o f Hevea brasiliensis, much work still would need to be done to determine the influence of exogenous plant growth regulators on the pool of endogenous hormones in somatic embryos. This knowledge would further allow the control of the levels of endogenous growth regulators (especially abscisic acid).
Acknowledgement This research was performed at the Blaise Pascal University o f Clermont-Ferrand (France) and was supported by a grant from M I C H E L I N Tyre Company,
References AmmiratoPV (1977) Hormonalcontrol of somatic embryo development from cultured cells of caraway.Plant Physiol. 59:579-586 Attree SM & Fowke LC (1991)Micropropagation through somatic embryogenesisin conifers. In: Bajaj YPS (Ed) Biotechnologyin Agriculture and Forestry: High-tech and Micropropagation, Vol 17, (pp 53-70) Springer-Veflag,Berlin Boulay MR Gupta PK, Krogstrup P & Durzan DJ (1988) Development of somaticembryos from cell suspensioncultures of Norway spruce (Picea abies Karst). Plant Cell Rep. 7:134--137 Carron MP & Enjalric F (1985) Embryogen~se somatique/t partir du tegument interne de la graine d'Hevea brasiliensis (Kunth) Miill-Arg.C.R. Acad. Sci. Paris 300 (III) 17:653-658 Dunstan DI, Bekkaoui E Pilon M, Fowke LC & Abrams SR (1988) Effects of ABA and analogues on the maturation of white spruce (Picea gluca) somatic embryos. Plant Sci. 58:77-84 Durzan DJ & Gupta PK (1987) Somatic embryogenesisand polyembryogenesisin Douglas-fircell suspensioncultures. Plant Sci. 52: 229-235 El Hadrami I, Carton MP & d'Auzac J (1989) Variabilit6clonale du potentielembryog~nechez Hevea brasiliensis; relations avec les polyamines (PA) et les peroxydases (PO) des cals. C.R. Acad. Sci. Paris 308 (III): 299-305 Enjalric F & Carron MP (1982) Microbouturage in vitro de jeunes plants d'Hevea brasiliensis (Kunth. Mtill-Arg). C.R. Acad. Sci. Paris 295 (III) 3:259-264 Etienne H, Berger A & Carron MP (1991) Waterstatus of callus from Hevea brasiliensis during induction of somatic embryogenesis. Physiol. Plant. 82:213-218 Etienne H, Sotta B, Montoro P, Miginiac E & Carton MP (1993) Relationsbetween exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in expression of somatic embryogenesisin Hevea brasiliensis (M011. Arg.). Plant Sci. 88: 91-96 Feirer RP, Conkey JH & Verhagen SA (1989) Triglycerides in embryogenic conifer calli: a comparison with zygotic embryos. Plant Cell Rep. 8:207-209 Guerrier J (1992) Embryogen~se somatique chez Hevea brasiliensis. Induction ta partir de graines imrnatures. Etude des /3carboxylations pendant la multiplication et le d6veloppement des amas embryog~nes. Tbese d'Universit6, Univ. Blaise Pascal, Clermont-Ferrand,85 pp Hakman I &von Arnold S (1985) Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce). J. Plant Physiol. 121:149-158 Hakman ! & von Arnold S ( 1988) Somaticembryogenesisand regeneration from suspensioncultures of Picea glauca (white spruce). Physiol. Plant. 72:579-587 Housti E Coupe M & d'Auzac J (1991) Facteurs enzymatiques du brunissement in vitro et capacit6 embryog~ne des cals d'Hevea brasiliensis. C.R. Acad. Sci. Paris (III) 313:461--466 Roberts DR, Flinn BS, Webb DT, WebsterFB & Sutton BCS (1990) Abscisic acid and indole-3-butyricacid regulation of maturation and accumulationof storage proteins in somatic embryos of interior spruce. Physiol. Plant. 78:355-360 Sussex I, Clutter M & Walbot V (1975) Benzyladenine reversal of abscisic acid inhibition of growth and RNA synthesis in germination bean axes. Plant Physiol. 56:575-578 Veisseire P, Guerrier J & Coudret A (1993) Cryopreservation of embryogeniccell suspension ofHevea brasiliensis. Cryo-Letters 14:295-302
