Susanne Stirn and Hans-Jorg Jacobsen. Institut fur Genetik der Universitat Bonn. Kirschallee 1, 5300 Bonn 1, FRG. SUMMARY. Plant regeneration via somatic ...
Production and Characterization of Monoclonal Antibodies against Marker Proteins for Somatic Embryogenesis in Pea (Pisum sativum L.)
Susanne Stirn and Hans-Jorg Jacobsen Institut fur Genetik der Universitat Bonn Kirschallee 1, 5300 Bonn 1, FRG
SUMMARY
Plant regeneration via somatic embryogenesis is now possible in pea both from immature somatic embryos, shoot apices as well as from protoplast derived calli (1,2). Pea callus cultures initiated from leaves or epicotyls cannot be routinely regenerated up to now. Therefore. we started to investigate specific biochemical aspects of induction and development of somatic embryos. We identified marker proteins only specific of embryogenic tissues by raising monoclonal antibodies against total cytosolic proteins of embryos and embryogenic calli. Antibodies were selected which react only or preferentially with proteins from embryos or embryogenic calli. Two hybridoma cell clones have been identified producing antibodies which recognize proteins in embryogenic callus ( 50000 D, IIIK1-7C5) and/or somatic embryos (20000 D, IIIK1-12A2). The monoclonal antibody IIIK1-7C5 also recognizes a polypeptide with a molecular weight of 50000 in early globular somatic embryos of Daucus carota.
INTRODUCTION Plant tissue culture techniques provide new possibilities for in vitro propagation and manipulation of plants (3, 4). The use of the in vitro techniques for plant breeding depends on the regeneration of plants from callus or suspension cultures. Plant regeneration via somatic embryogenesis can be efficiently achieved only in a few plant species like carrot, tobacco or caraway (5,6), but the agronomically important pulse crops behaved recalcitrantly for a long period of time. Recently, regeneration protocols for soybean and pea were described (7,1). In these cases. immature zygotic embryos or shoot apices were the explant source for the successful I induction and regeneration of somatic embryos. Pea callus cultures initiated from leaves or epicotyls cannot be routinely regenerated. Since variations in nutritional compounds and culture conditions have had only limited success, we started to study the biochemical events of induction and development of pea somatic embryos. 460 H. J. J. Nijkamp et al. (eds.), Progress in Plant Cellular and Molecular Biology © Kluwer Academic Publishers 1990
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Therefore, we were looking for marker proteins only specific of tissues capable of plant regeneration, e.g. the embryogenic calli and raised monoclonal antibodies against them. Since the yield of somatic embryos was low when we started these experiments, we decided to raise antibodies against total cytosolic proteins of embryos and embryogenic calli. Antibodies against the marker proteins were identified in two differential screenings of the hybridoma supernatants against proteins of non-embryogenic calli. MATERIAL AND METHODS Immature zygotic embryos as well as shoot apices of 5-day old pea seedlings were used as explants for the induction of somatic embryo formation. On MS-medium with high auxin (4 !-1M 2,40), embryogenic calli developing 1-5 somatic embryos ("04") are formed within 4-5 weeks (1). After subculturing the calli on a medium with 4.4 IlM Kinetin, the calli continue to form somatic embryo ("K1") as well as meristematic centers beneath the surface of the calli (embryogenic callus "C"). As control, epicotyl segments of 5-days old pea seedlings were grown in the presence of 4.4 IlM 2,40 and 11.1 uM Kinetin. Then a non-embryogenic callus without any meristematic centers is formed (N)'
zygotic embryo or shoot apex
1
4-5 weeks 4 !-1M 2,40
callus with meristematic centers and embryos "04"-embryos
1
4-5 weeks 4.4 IlM K1
epicotyl segments 8-10 weeks 4 IlM 2,40 and 11.1 IlM K1
1
callus without meristematic centers and embryos
-1-
"N"
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non-embryogenic callus
callus with meristematic centers and embryos - "BA"-embryos
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"C"- embryogenic callus Somatic embryos of Oaucus carota were induced as described by (6). After 5 and 14 days in suspension culture, early globular and cotyledonary somatic embryos were harvested, respectively. Tips (''1''), leaves ("L"), epicotyls ("E") and roots ("R") of 10-days old, pea seedlings were harvested and used for control experiments. From these tissues soluble proteins were extracted. Embryos and tissues of young pea seedlings were ground in an agate mortar, calli were extracted in a Warring blendor (extraction buffer: 0.2 M boric aCid, 2 mM borax, 0.25 M KCI, 20 mM TRIS/HCI, 4 mM diethyldithiocarbamate, 2.5% (w/v) polyclar AT, pH 6.8) (8). Both extracts were
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centrifu ged at 148000 x g for 2 hou rs at 4 ' C. If nessecary. the supernatants were desalted and concentrated by ultrafiltration. The protein content was determined according to Bradford (9).
IMMUNIZATION
60-days old female balb-c mice were immunized with 100 I-Ig protein (from embryos "04" and "K1" and embryogenic callus "C") in 100 ul complete Freund's Adjuvant subcutaneously ("prime"). After 4 weeks immunization was repeated by intraperitoneal injection of 100 Ilg protein in 100 III incomplete Freund's Adjuvant for at least three times. Before fusion of mice spleen cells with myeloma cells (SP2/0). the mice were boosted with 100 I-Ig pure protein intraperitoneally. The fusion was performed according to Pratt (10) with polyethyleneglycole and the selection of hybridomas was performed in HATmedium.
ANTIBODY SCREENING
The screening protocol consisted of two subsequent ELISA-tests and one immunostain: In the first ELISA, hybridomas producing antibodies against plant proteins were selected. Therefore. half of the wells of a microtiter plate were coated with proteins used for immunization, the other half with buffer as negative control. Subsequently, the wells were incubated with the hybridoma supernatants and rabbit-anti-mouse antibodies coupled with alkaline phosphatase (10). In the second ELISA, antibodies were selected which reacted preferentially with proteins from embryos or embryogenic callus. Therefore, hybridoma supernatants were tested simultaneously for a reaction with proteins from embryos or embryogenic callus and proteins from non-embryogenic callus. The washing and enhancer steps were the same as in the first ELISA (10). The third selection step was an immunostain: SOS-PAGE was performed with proteins from embryos, embryogenic and non-embryogenic calli ("04", "K1", "C", "N Separated proteins from SOS-gels were transferred to nitrocellulose by semi-dry blot. The nitrocellulose then was incubated with the hybridoma supernatants. The enhancement of the signal was performed with rabbit-anti-mouse antibodies and goatanti-rabbit antibodies coupled with alkaline phosphatase (10). Using this scheme we assayed if the antibodies recognize proteins only specific of embryogenic tissue, e.g. the marker proteins for somatic embryogenesis. U
RESULTS
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1830 hybridoma clones resulting from seven fusions were tested. Up to now two embryo-specific clones have been identified: The monoclonal antibody IIlK1-12A2 recognizes a polypeptide with a molecular weight of 20000 in somatic embryos, but not in embryogenic or non-embryogenic calli (s. fig. 1b).
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The monoclonal antibody I1IK1-7C5 recognizes a polypeptide with a molecular weight of 50000 in somatic embryos and embryogenic calli, but not In non-embryogenic calli. Additionally, three other polypeptides were recognized In somatic (45000 0, 32000 0, 20000 D) (s. fig 1 a). Fig 1: Immunostain with the monoclonal antibodies IIIK1-7C5 (a) and III~1-12A2
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(b)
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K1 somatic embryos; C embryogenic calli; N non-embryogeni4 calli; T tips; L leaves; E epicotyls; R roots; 01 globular somatic embryos of Daucus carota; 02 cotyledonary somatic embryos of Daucus carota
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The monoclonal antibody I1IK1-7C5 is the most interesting one: It recognizes a polypeptide with molecular weight of 50000 in early somatic embryos of Daucus carota as well as in tips of 10-days old pea seedlings (s. fig. la)
DISCUSSION
Using the monoclonal antibody technique to identify unknown :mtlgens, care has to be taken of the negative controls for the differential screening procedures. 3ince marker proteins for early somatic embryogenesis are to be selected, the control should be a non-embryogenic callus, only jiffering in its regenerative capacity. :ulturing the same explants (shoot apices) on 2.4-0 and Kinetin 3imultaneously as negative controls, no somatic embryos appeared ifter 8 weeks in culture. But histological investigations revealed neristematic clusters beneath the surface of the calli (data not ~hown). Therefore, we used epicotyl segments, where no induction of ;omatic embryos could be detected after 8 weeks culturing on 2.4-0 md Kinetin.
464 We succeeded in identifying two polypeptides specific of different states of development: The monoclonal antibody IIIK1-12A2 recognizes a 20000 D polypeptide which appears at the globular state of embryogenesis. IIIK1-7C5 recognizes a 50000 D polypeptide in embryogenic calli of pea, early somatic embryos of carrot and tips of young pea seedlings. Therefore, this polypeptid is specific of highly meristematic tissues. Choi et al. (11) identified two polypeptides (20000 D and 50000 D) specific of globular to heart-stage somatic embryos of carrot. The polypeptide with the molecular weight of 50000 could also be detected in embryogenic tissues of other plant species (12). They did not test pea somatic embryos, but due to the molecular weight and the occurence in highly meristematic tissues, it is very likely a similar marker polypeptide. Using the screening protocol described above. we succeeded in demonstrating the presence of the marker polypeptide immediately after the induction of somatic embryogenesis. The function of this polypeptide is still unknown. It is neither a storage protein (vicilin, MW 50000) nor one of the cytoskeletal proteins actin (MW 43000) and tubulin (MW 50000). This could be demonstrated by immunostains with antibodies against these proteins: The polypeptide patterns recognized by the anti-vicilin, anti-actin and anti-tubulin antibodies were quite different to the one shown in Fig. 1a (data not shown). Both antibodies do not recognize carbohydrate epitopes as was proofed after deglycosylation of the proteins (data not shown). The monoclonal antibodies IIIK1-7C5 will be used: a) to study the time course of marker protein expression during the induction phase of somatic embryogenesis: Therefore, calli will be harvested at daily intervals after transfer to induction medium. The expression of marker proteins will be monitored using the methods of differential ELISA and immunostain. b) to determine the cellular and subcellular localization of the marker proteins in tissue sections using immunogold-Iabelled antibodies. b) to isolate the marker protein by affinity chromatography. With the help of polyclonal antibodies raised against the purified marker protein a c-DNA library will be screened (lambda gt 11) as a first step for the characterization of the marker protein genes. The monoclonal antibody IIIK1-7C5 is a promising tool both: - to elucidate early events during induction of somatic embryogenesis (auxin effects, induction, competency) and - to analyse the regenerative capacity of different genotypes and culture conditions. This might become important in order to shorten the laborious and time-consuming method of "try and error" for the establishment of regeneration protocols used throughout tissue culture today.
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Acknowledgements This work was supported by a DECHEMA-fellowship to S.S. research grant of the BMFT to H.-J.J.
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