Plant Science, 83 (1992) 195-203 195 Elsevier Scientific Publishers Ireland Ltd.

Isolation and characterization of three monoclonal antibodies specific for pistil and stamen tissues of tobacco

(Nicotiana tabacum L.)

L u i s C a l m s a a n d R u s s e l l M a l m b e r g b

aInstituto de Agroquimica y Tecnologia de Alimentos, Unidad de Biologia Molecular y Celular de Plantas. C/ Jaime Roig, 11, 46010 Valencia (Spain) and bUniversity of Georgia, Botany Department, Athens, GA 30602 (USA)

(Received January 21st, 1992; revision received March 5th, 1992; accepted March 5th, 1992)

Two immunization procedures were compared for their ability to yield monoclonal antibodies that react to antigens in the different flower parts (sepals, petals, stamens and pistil) and avoid immunodominance of some pistil-specific components in the extracts. The hybridomas were screened previously by ELISA against extracts from the different flower parts and later by in situ immunolocaliza- tion on floral sections and Western blotting. Three monoclonal antibodies displaying specificity for style (5F9) and anther (8F2 and 8G1) tissues were selected and purified by affinity chromatography. The antibody 8GI is periodate-sensitive, implying that the epitope is a carbohydrate, while the 5F9 and 8F2 were insensitive. Western blot analysis showed that the antibodies selected react to specific bands of the different organ extracts. Immunolocalization assays showed that the antibody 8F2 recognizes an antigen that is localized in the endothecium cells of the tobacco anther while the 8G1 antigen is detectable in the stomium cells. Monoclonal 5F9 recognizes an antigen localized in a very limited region of the style tube.

Key words: anther; pistil; Nicotiana tabacum; monoclonal antibodies

Introduction

The use of monoclonal antibodies as develop- mental markers can be compared with a similar use of nucleic acid probes. There are advantages to each approach. Antibodies can detect a variety of types of molecules; they can thus be used to measure the types of biochemical differentiation between cells and tissues. If two tissues differ in their production of an active enzyme or com- pound, then the developmental regulation could occur at a variety of steps; monoclonal antibodies will measure events later on this chain than cDNA probes would. Antibodies thus provide a chance to detect developmental events that are not directly

Correspondence to: Dr. Luis Canas, Instituto de Agroquimica y Tecnologia de Alimentos (CSIC), Unidad de Biologia Molecular y Celular de Plantas, C/ Jaime Roig, 11, 46010 Valencia, Spain.

due to differences in mRNA abundance. Im- munolocalization techniques can also have ex- traordinary resolution, allowing detection of the antigen at specific locations within a cell, as well as between cells.

Tobacco (Nicotiana tabacum L.) is a favorable system for investigating many aspects of floral development; the flowers are complete, large, easi- ly manipulated and there exist a variety of mutants with abnormal flowers [1]. In addition, it is possi- ble to regenerate tobacco flowers from epidermal explants using the thin cell layer technique [2].

Our approach has been to inject relatively crude preparations into mice, let the murine immune systems sort out the antigens, and then subse- quently detect the hybridomas that are useful as cell- and tissue-specific markers. The antibodies selected can be used in a large variety of experi- ments to elucidate flower initiation and differ-

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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entiation, for the identification of genes involved in these steps and also they are valuable tools for the analysis of floral mutants [3].

The isolation and characterization of cell- and tissue-specific genes through differential hybri- dization of nucleic acids are certainly important steps in increasing our understanding of plant dif- ferentiation and development, but molecules of fundamental importance other than polypeptides may be overlooked and these may be detected through hybridoma technology. However, these techniques may not detect compounds of low anti- genicity or those masked by immunodominance of others. Arabinogalactans have been reported to be a particular problem [4]. To remove the problems of immunodominance, several strategies have been proposed [5,6]. We report here the use of a co- inoculation strategy to eliminate a series of glycosylated molecules present in the pistil and in the other flower parts and then direct the im- munoresponse towards the production of anti- bodies more specific for a particular floral organ. In our case, we were particularly interested to pro- duce monoclonals specific for the different parts of the tobacco anther.

Material and Methods

Plant material Tobacco plants (Nicotiana tabacum L. cv. Xan-

thi) harvested for protein preparation were greenhouse-grown under natural lighting con- ditions.

Preparation of tissue extracts Whole flowers ranging in development from 1

mm length to 1 day pre-anthesis and sepals, petals, stamens and pistils from pre-anthesis flowers of mixed sizes, were ground to a fine powder under liquid nitrogen. Grinding buffer (100 mM Tris, pH 7.4, 5 mM EDTA, 20 mM fl-mercaptoethanol, 0.1% SDS, 5 mM PMSF and 10/~g/ml leupeptin) was added (2 ml per g of tissue), followed by fur- ther grinding with mortar and pestle. The homogenate was incubated at 37°C for 3 rain, chilled on ice for 2 -3 min, filtered through Miracloth and centrifuged at 12 000 × g for 15 min at 4°C. The pellet was discarded and the

supernatant precipitated at 4°C with ammonium sulfate (90% saturation), centrifuged again at 12 000 × g for 15 min and the pellet resuspended in grinding buffer was dialyzed overnight (20 mM Hepes, pH 7.4, 0.1 mM EDTA, 5 mM /3- mercaptoethanol and 50 mM NaC1) in a cold chamber. Finally the dialyzed extract was cen- trifuged at 1500 × g for 10 rain and the superna- tant stored at -20°C. Protein concentrations in the extracts were estimated by the method of Bradford [7] using BSA as a standard.

Immunizations Two immunization procedures were compared

for their ability to yield monoclonal antibodies which react with a specific flower organ. In the first method, BALB/c mice were injected with 100 /~1 of extract at a protein concentration of approx. 1 mg/ml. Initial immunizations were in complete Freund's adjuvant and subsequent injections with incomplete adjuvant were made at 2-week inter- vals, the last being 3 days prior to the spleen harvest and fusion. Hybridomas were prepared by fusion to the SP2/O-Agl4 myeloma cell line using standard techniques [8]. In the second method, a first mouse was injected three times with 100/~1 of a 1:1 mixture of leaf and pistil extracts with a pro- tein concentration of approx. 1 mg/ml at 2-week intervals. The mouse was bled from the eye and the polyclonal serum obtained (0.1-0.3 ml) was co- injected into a second mouse mixed l: l with an organ extract (sepal, petal or stamen), to avoid im- munodominance of common proteins between pistil and the other flower parts.

Enzyme-linked immunosorbent assays These reactions were performed using minor

modifications of standard protocols [9]. Each well was coated with 100/zl of antigen (50/zl/ml in 50 mM sodium carbonate, pH 9.6) and adsorbed onto 96-well microtiter plates overnight at 4°C. Plates were washed three times with ELISA wash buffer (20 mM Tris, 0.9% NaC1, 0.1% BSA, pH 7.2), then blocked with 3% BSA in wash buffer for 1 h. The plates were washed again and hybridoma super- natants were added for a 2-h incubation at room temperature. Following three rinses with wash buffer, wells were incubated for 2 h at room

temperature with a goat anti-mouse IgG-alkaline phosphatase conjugate at 1:5000 dilution. Follow- ing three rinses with wash buffer, 100 #1 of 1 mg/ml p-nitrophenyl phosphate in 10% diethylamine (pH 9.8) was added to each well. After 30 min, 100 #1 per well of 3 M NaOH was added to stop the reac- tions; absorbance at 400 nm was measured.

Isotyping of antibodies Isotyping was performed by ELISA using

polyclonal anti-isotype antibodies. Goat anti- mouse immunoglobulins at 100 /zg/ml were ad- sorbed onto 96-well microtiter plates for 2 h at room temperature. Wells were then blocked with 3% BSA for 1 h, rinsed with ELISA wash buffer and 100 #1 of hybridoma supernatant was added per well. Two hours later the wells were rinsed three times with ELISA wash buffer and goat anti- mouse isotypes conjugated to alkaline phos- phatase were added at 0.2/~g/ml and incubated for an additional 2 h. Following three rinses with ELISA wash buffer, the plates were developed with p-nitrophenyl phosphate and handled as with the other ELISAs.

Periodate sensitivity of antibody reactions For the periodate sensitivity test [10,11], antigen

at 50/zg/ml and 100/~1 per well was adsorbed onto 96-well microtiter plates overnight at 4°C. The plates were then rinsed three times with 50 mM sodium acetate (pH 4.5) and were incubated in acetate buffer (SAB) at 25°C for 1 h in the dark. For periodate treatment, wells were treated in the same way except for the addition of fresh periodic acid (0.1-20 mM) in SAB. All wells were then blocked with 1% glycine in SAB for 1 h followed by three rinses with ELISA wash buffer. The wells were next blocked in 3% BSA and subsequently handled according to the standard ELISA protocol.

Purification of the antibodies The monoclonal antibodies were purified from

hybridoma medium with an anti-mouse IgG- agarose affinity column. Purified antibody was assayed by absorbance at 280 nm, then resuspen- ded at 0.1 mg/ml in PBS-3% BSA and stored at -80°C (5F9:M.026, 8F2:M.027 and 8Gl:M.028).

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Electrophoresis and blotting Protein samples for Western blots were

solubilized in buffer containing (final concentra- tions) 0.13 M Tris (pH 6.8), 5% glycerol, 1% SDS, 0.36 /3-mercaptoethanol, 0.001% bromophenol blue. The mixture was boiled for 2 min and then resolved using the system of Laemmli [12], on 7-15% polyacrylamide slab gels. The proteins were transferred from the gel to Immobilon PVDF membranes using a Trans-Blot Cell. The transfer was done in 25 mM Tris (pH 8.3), 192 mM glycine, 20% methanol at 100 V for 1 h. To visualize the transferred proteins and molecular weight markers, vertical strips were stained with 0.1% Amido black 10B, 45% methanol and 10% acetic acid and destained in 2% acetic acid, 90% methanol. The rest of the blot was cut into strips for testing with antibodies. The basic medium was TBS buffer (0.15 M NaC1 and 20 mM Tris-HC1 pH 8.0) containing 2% nonfat dried milk and either 0.05% (blocking and antibody dilutions) or 0.5% (washes) Tween 20. The membrane strips were blocked overnight at 4°C and incubated for 1 h at room temperature with purified antibodies (2 #g/ml). In some experiments, spent hybridoma supernatants were used instead of purified anti- body solutions with the same result. Following in- cubation with primary antibody, strips were washed three times (10 min each) and incubated for 1 h with goat anti-mouse IgG coupled to alkaline phosphatase diluted 1:2000. The strips were washed as above, incubated for 5 min in 0.1 M Tris (pH 9.0), 0.5 mM MgC12, 0.1 mM 5-bromo-4-chloro-3-indoylphosphate and 0.1 mM nitrobluetetrazolium; a purple color indicates anti- body binding.

Immunolocalizations Flower buds and dissected organs in different

development stages [13] were fixed in 5% parafor- maldehyde, dehydrated in ethanol/tert-butanol series and embedded in paraffin (Paraplast Plus). Specimens were mounted on a microtome and sec- tions of 8 t~m thick were transferred to glass slides coated with 0.4% gelatin + 0.04% CrK(SO4) 2 • 12H20 and heated to 45°C for 30 min. Sections on slides were washed for 10 min in PBS (0.01 M sodium phosphate and 0.15 M NaC1 pH 7.2) to

198

Table I. Results of five immunizations of BALB/c mice with different tobacco flower extracts. NS, number of spleens harvested; NH, number of hybridomas screened; NP, number of initial positives, ELISAs against YFB extract; NC, number of clones initially retained, ELISAs against floral organ extracts; MAb, monoclonal antibody selected; Ig, antibody isotype; PS, periodate sensitivity; YFB, young flower buds; S, sepal; P, petal; A, anther; C, carpel; pcsL/C, polyclonal serum leaf/carpel; +, periodate sensitive; - - , periodate insensitive; nd, not determined.

Immunogen NS NH NP NC MAb Ig PS

YFB 2 176 22 2 5F9 M-kap - - S 1 216 78 12 9H1 nd nd P 1 134 41 7 7Eli nd nd A 1 93 27 2 8F2 M-kap - - C 1 288 82 23 9G2 IgG3 +

YFB + pcsL/C 1 1259 172 42 a 8G 1 M-kap +

aNumber of clones retained against stamen extracts.

hydrate the sections. All washes and incubation steps were done at room temperature by overlay- ing tissue sections with the corresponding solu- tions. The basic medium was PBS or TBS buffer containing 2% nonfat dried milk and either 0.05% (blocking and antibody dilutions) or 0.5% (washes) Tween 20. Sections were blocked by 30 min incubation in basic medium. Purified monoclonal antibodies or hybridoma medium were diluted (2/~g/ml) and incubated 2 h with the sections. Control slides were treated with unused hybridoma medium. After 3 × 10 min washes, second antibody conjugated with alkaline phos- phatase (see above) was diluted 1:100 and in- cubated 2 h with the sections. After 3 × 10 min washes, the reaction of alkaline phosphatase was developed (see above) for 30 min; a purple precipitate forms at the site of localisation. A Nikon Diaphot microscope was used for sample visualization and photography.

Results

Isolation and purification of monoclonal antibodies Hybridomas were prepared by standard techni-

ques from mice immunized with crude extracts of tobacco flowers and screened for antibodies that were specific for a floral organ or tissue (sepal, petal, stamen or pistil). The first screening was an ELISA test against the extract used in the ira-

munization protocol. Positives were screened a second time with identically prepared extracts of leaves, sepals, petals, stamens and pistils, adjusted to equal protein concentrations. Hybridomas showing differential reactivity for a determinate

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8F2 Fig. 1. ELISA patterns from 5F9 and 8F2 monoclonal anti- body lines with crude extracts from the different flower organs (L, leaf; S, sepal; P, petal; A, anther; C, carpel; ST, stigma/style). Absorbance at 400 nm was measured.

floral organ were retained for cloning and further testing.

The results from five immunizations using this method are summarized in Table I. Using young floral bud extracts as immunogen, we identified 22 cell lines showing specificity for the immunogen after the first screening by ELISA, but only 13 lines were retained as a result of a second ELISA against the different organ extracts. Two clones were chosen after analysis by immunolocalization on floral sections: 5F9, stigma/style specific, see Fig. 1 and 7E4, showing very uniform reactivity, retained to be used as positive control in im- munolocalization assays. A great number of lines screened were pistil-specific, this fact may reflect either immunodominance of some pistil-specific components in the extracts, or it may indicate that pistils exhibit a much more distinctive biochemical differentiation than do other floral tissues. The results using separate floral organs to immunize mice were very similar and we retained only one line (8F2) showing specificity mainly for stamen extracts (Fig. 1). The other cell lines initially re- tained were discarded after immunolocalization analysis because they reacted with more than one part of the flower in a manner that is consistent with the patterns obtained from the ELISA reac- tions (not shown).

To avoid immunodominance of some pistil- specific components and increase the production of antibodies against the other floral organs, a sec- ond immunization procedure was assayed. Our in- terest was to produce monoclonals specific for the different parts of the tobacco anther. To this end, we immunized a mouse (#1, Fig. 2) with a mixture of leaf and carpel extracts and the polyclonal serum obtained was incubated with an anther ex- tract and injected in a second mouse (#2, Fig. 2). The difficulty to produce monoclonal antibodies specific for the different flower organs is probably due to the presence of large amounts of proteins in common to all flower parts and the immuno- dominance of some pistil-specific components in the flower extracts. The use of several immunosub- tractive strategies, like the co-inoculation method, it would be useful to alleviate this problem and may help to recover antibodies to antigens of in- terest. In fact, this second method resulted in a

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Fig. 2. ELISA patterns from mouse #1 immunized with a mix- ture of leaf/carpel extracts and mouse #2 immunized with a mixture of polyclonal serum from mouse #! and anther extract (C, carpel; A, anther; P, petal; S, sepal; L, leaf; L/C, leaf/carpel extract).

great number of cell lines producing antibodies against anther extracts (see results from one of the immunizations assayed in Table I and Fig. 2). After immunolocalization assays we retained some interesting lines; one of these produces the anti- body 8G1 which recognizes specific patches of cells in the stomium region of the anther, a tissue involved in anther maturation.

Analysis of monoclonal antibodies: isotyping, periodate sensitivity, Western blotting and im- munolocalization

The monoclonal antibodies initially selected by ELISAs, were further analyzed for isotype and periodate sensitivity. The results are summarized in Table I. The Ig subclass of antibodies 5F9, 8F2 and 8G1 is IgM with kappa chains (/x heavy chains of 80 kDa and k light chains of 25 kDa, see Fig. 3A).

Since carbohydrates are immunogenic and also abundant in plant cells (glycosylations), we per- formed a periodate sensitivity test to check if the antigens recognized by our antibodies might be carbohydrate in nature. The periodate sensitivity of antibody reactions shows that antibodies 5F9 and 8F2 are insensitive while 8G1 and 9G2 are sensitive, indicating that the epitopes recognized by these antibodies are likely to be carbohydrate.

Western blotting analysis of the selected anti-

200

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i!ii!ii!ii!ii!ii!iiiiiiiiii!!!iiiii ~i,~!~iiiiii!~iii~Jii~iiiii~i!i~iiiSii ~III

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Fig. 3. (A) SDS-PAGE of the purified antibodies. Lane 1, low molecular weight standards; lane 2, monoclonal 5F9; lane 3, monoclonal 8F2; lane 4, monoclonal 8G1; lane 5, IgM-k control, monoclonal 2F10 [3]. (B) Western blot analysis of the antigens recognized by the purified antibodies. Lane 1, low molecular weight standards; lane 2, monoclonal 8F2 against anther extract; lane 3, monoclonal 5F9 against stigma/style extract; lane 4, monoclonal 8GI against anther extract.

bodies showed in general that they recognized pro- tein bands of different sizes. Antibody 5F9 recognizes multiple bands against a young flower bud extract, but when we used a stigma/style ex- tract the antibody reacted very strongly with a couple of bands of approx. 34-35 kDa (Fig. 3B, lane 3). Antibodies 8G1 and 8F2 also react with multiple bands but more strongly with a low molecular weight protein of approx. 18 kDa in the case of the 8G1 antibody (Fig. 3B, lane 4) and the 8F2 with a protein of approx. 16 kDa (Fig. 3B, lane 2).

Immunolocalization assays with the purified antibodies showed that the 5F9 recognizes an anti- gen most strongly in the pistil (stage 6). The anti-

gen is present in a very determined region of the upper part of the style (Fig. 4, A and B) from stages 6 to 12 of the tobacco pistil development [13]. Immunocytolocalization of this antigen on transverse sections, across the complete pistil, showed that it is localized in cells of the upper region of the style (mainly in the periphery and vascular bundles) but not in the transmitting tract of the stigma and lower part of the style (Fig. 6, A, B, C, D and E). The antigen recognized by the 8F2 antibody is found in a layer of cells around the wall of the anther (Fig. 4, C, D, E and F). The im- munocytolocalization of this antigen showed that is localized in the endothecium cells from stages 1 to 8 (Fig. 5). The 8G1 recognizes an antigen

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Fig. 4. Immunolocalization with selected antibodies on tobac- co floral sections. (A) Longitudinal section of a tobacco stigma/style (stage 6), negative control. (B) As (A) but treated with 5F9 antibody (x25). (C) Longitudinal section of a tobacco flower (stage 1), negative control. (D) As (C) but treated with antibody 8F2 (x 10). (E) Transverse section of a tobacco anther (stage 3), negative control. (F) As (E) but treated with 8F2 anti- body. (G) As (E) but treated with 8G1 antibody (×25).

201

localized in patches of cells in the stomium region, mainly in stage 3 (Fig. 4, G). Histochemical treat- ment with acetic acid and HCI and visualization of the anther sections under polarizing light, showed that the antigen recognized by this antibody is not a crystal deposit of calcium oxalate (not shown).

Discussion

The characterization of the isolated mono- clonals showed that the three antibodies analyzed are highly specific for a tissue or group of cells as we demonstrate by in situ immunolocalization on floral sections. Specially, the antibody 5F9 (style specific) recognizes an antigen only present in a very limited region of the style tube, but it is not the same as the transmitting tract, which usually extends up into the stigma. In fact, this antigen is not localized in the transmitting tract of the stigma like the 5E3 previously described [3]. The localiza- tion of 5F9 looks different from any other anti- body or cDNA previously described. A gene encoding a stylar-specific self-incompatibility- associated glycoprotein has been isolated by direct screening of nucleic acids (cDNA) from Nicotiana alata [14]. In the case of Nicotiana tabacum, ex- pression of these genes was detected only in the transmitting tissue of the style but not in the stigma. The S13 allele accumulated in the upper region of the transmitting tissue of the style and the $22 allele accumulated throughout the

Fig. 5. Immunocytolocalization on anther (stage 3) transverse sections of the antigen recognized by monoclonal 8F2. C, connective; S, stomium; T, tapetum; Vb, vascular bundle; W, wall; E, epidermis; En, endothecium, × 160 and x 320.

202

B

Fig. 6. Immunocytolocalization on pistil (stage 6) transverse sections of the antigen recognized by monoclonal 5F9. (A) Stigma section, negative. (B) Stigma transmitting tissue, negative; (C) style upper region, positive; (D) ovary upper region, negative (x50). (E) Style transverse section showing antigen localization in peripheral cells and the two vascular bundles (x 100).

transmitting tissue in the outer layers of the placenta [ 15].

Monoclonals 8F2 and 8G1 recognize antigens in the endothecium and stomium region of the an- ther, respectively. The 8G1 antibody looks dif- ferent from the NtF-SB1 previously described [3] which recognizes a protein associated with calcium salt crystals present in the stomium cells that rup- ture during anther maturation. The epitope recognized by this monoclonal antibody is specifically associated with crystal idioblasts and the antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Detection of this antigen only in Solanaceaous plants, but not in other plants

known to contain similar crystals, suggests that the antigen is unique to this family and with a high degree of spatial and temporal regulation [16]. Histochemical treatments with acetic acid and HCI and the visualization of the anther sections under polarizing light, showed that the antigen recog- nized by the antibody 8G1 is not a protein associ- ated with a crystal deposit. G01dberg [17] and Koltunow et al. [13] have reported on the isolation of a large number of cDNA clones for genes that are predominantly expressed in tobacco anthers. One of these clones, the TA56, was found to en- code a thiol endopeptidase and the TA56 gene was expressed in the connective and the stomium cells. It was hypothesized that this enzyme may be directly involved in the process of degeneration of the connective and stomium which leads to anther dehiscence. Further experiments with the purified antibodies will reveal the nature of the antigen recognized by them and the degree of temporal regulation showed by the antigens recognized by monoclonals 5F9 and 8G1.

In this work we also tested a new immunization method to avoid problems of immunodominance from some pistil-specific components (glycosyla- ted molecules) present in the whole flower extracts and most of them also present in the isolated floral organs. Our results using this procedure appear useful to improve the isolation of monoclonals specific for floral organs other than carpel, such as for the production of anther-specific antibodies [18].

An alternative way to identify genes that are predominantly expressed in a floral organ is first to identify proteins, the ultimate products of gene expression, that are detectable only in those organs [19]. In this way, the use of monoclonal antibodies as developmental markers for the iden- tification and purification of antigens expressed during floral organ initiation and differentiation, can help to understand the flowering process. Ad- ditional goals of the use of monoclonal antibodies as tools in the study of floral development will be the analysis of homeotic mutants with developmental switches in the flower, the isolation and characterization of antigens that are expressed very early during flower development and finally the identification of genes controlling the expres- sion of these antigens.

Acknowledgements

This work has been supported by grants from National Science Foundation (NSF) DMB-87- 15799, D.O.E. DE-FG0p-91ER2OO34 and U.S.D.A.-GAM-89-01056. L.A.C. was postdoc- toral fellow of the NATO Science Fellowship Pro- gramme 1988.

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