Bean lectins

Summary Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F₂ plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.     

Purification of a seed glycoprotein: N-terminal and deglycosylation analysis of phaseolin

Phaseolin, the major storage protein of the French bean Phaseolus vulgaris cv. Tendergreen, has been isolated and purified by either ion-exchange chromatography or reversed-phase HPLC. These purification procedures were used to fractionate the native protein aggregate into its characteristic subunit components. Amino-terminal sequence analysis was performed on the intact peptide subunits. Native phaseolin was chemically cleaved at a unique tryptophan residue which is proximal to the N-terminal region of the protein with BNPS-skatole and the resulting peptide fragments were isolated via reversed-phase HPLC. Chemical and enzymatic sequence results obtained from these peptide fragments are in full agreement with the results obtained for the full length peptide subunits. These N-terminal analyses show that the signal peptide cleavage process is somewhat random resulting in the phaseolin polypeptides having possibly three or four different N-termini. Native phaseolin and purified subunits were chemically deglycosylated with trifluoromethanesulphonic acid in the presence of an anisole scavenger. One-dimensional SDS-PAGE analysis of the deglycosylated products show that differential glycosylation is largely responsible for much of the observed molecular weight heterogeneity found among phaseolin polypeptides.     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

Expression,glycosylation and secretion of phaseolin in a baculovirus system

In this report, we describe the efficient expression and glycosylation, in insect cells, of -phaseolin polypeptides (M _r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M _r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/10⁶ cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Esterase as a Marker to Study the Genetic Fidelity of Micropropagated Banana

Isozymic profiles of different micropropagated banana (Musa spp.) cultivars (Giant Governor, Dwarf Cavendish, Robusta, Champa, Kachakel and Chatim) of West Bengal, India were assessed at different subcultural passages. Variation with respect to the banding pattern was noticed only in esterase but not in peroxidase and acid phosphatase. Of the six cultivars, four showed variation both at isozymic and yield level. Two cultivars (Kachakel and Chatim) maintained their esterase profile and genetic stability even after twenty subcultural passages. This revised version was published online in July 2006 with corrections to the Cover Date.     

Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.     

Selection for increased percentage phaseolin in common bean

Summary Two selection methods were compared to determine which was more efficient for increasing percentage phaseolin in the common bean (Phaseolus vulgaris L.). A base population consisting of families segregating for six seed protein alleles (Phas ^S , Phas ^C , Phas ^T , phas ^-, lec^-, and Arcl ⁺), all of which have measurable effects on percentage phaseolin, was subjected to either three cycles of S₁ family recurrent selection for increased percentage phaseolin (PPS), or one cycle of selection for combinations of the protein alleles (PAS) known to have positive effects on phaseolin accumulation. One cycle of PAS resulted in an increase in percentage phaseolin that was equivalent to three cycles of PPS. Selection under both methods produced increases in several correlated traits including percentage total protein, phaseolin as a percent of total protein, mg protein/seed, and mg phaseolin/seed. The amount of nonphaseolin protein per seed decreased, while seed yield was unaffected by either selection procedure. By selecting for favorable seed protein alleles identified by electrophoresis, it was possible to rapidly increase percentage phaseolin without the need for field evaluation.     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Identification of broccoli and cauliflower cultivars with RAPD markers

Summary RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.     

Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a β-1,3-glucanase. The content and properties of the latter enzyme differ between lutoid-body fractions from four different rubber clones (cultivars). While the enzyme from clone GT.1 is a glycoprotein with carbohydrate attached to two glycosylation sites, the enzymes from other clones contain little or no carbohydrate. Latex from clone GT.1 has a higher β-1,3-glucanase content than those from the other three clones, but with a significantly lower specific activity. The enzyme exhibits a pH optimum at 4.5, but there is a second one at 6.7. Peptides isolated from β-1,3-glucanase of clone GT.1 showed that the enzyme is heterogeneous at the C-terminus, probably as a result of removal of a vacuolar targeting sequence by an endopeptidase, followed by further removal of C-terminal residues by a carboxypeptidase-like activity. This incomplete digestion can be related to glycosylation at the extreme C-terminus of the mature enzyme. Non-glycosylated Hevea β-1,3-glucanases exhibit less C-terminal heterogeneity. A homologue of the antifungal protein osmotin was isolated from rubber clones which are less susceptible to fungal diseases. Another identified protein is identical to a citrate binding protein (CBP), already sequenced as cDNA, but with cleaved-off N-terminal signal and C-terminal vacuolar targeting peptides. Four C-terminal propeptides of vacuolar proteins in Hevea are positively identified, which is a valuable contribution to previously known examples of this type of processing.     

Phaseolin-protein Variability in Wild Forms and Landraces of the Common Bean(Phaseolus vulgaris): Evidence for Multiple Centers of Domestication

A sample of 106 wild forms and 99 landraces of common bean (Thaseolus vulgaris) from Middle America and the Andean region of South America were screened for variability in phaseolin seed protein using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and two-dimensional isoelectric focusing SDS/PAGE. The Middle American wild forms exhibited phaseolin patterns similar to the ‘S’ pattern described previously in cultivated forms, as well as a wide variety of additional banding patterns—‘M’ (Middle America) types—not encountered among common bean cultivars. The Andean wild forms showed only the ‘T’ phaseolin pattern, also described previously among cultivated forms. Landraces from Middle America showed ‘S’ or ‘S’-like patterns with the exception of 2 lines with ‘T’ phaseolin. In Andean South America, a majority of landraces had the ‘T’ phaseolin. Additional types represented in that region were (in decreasing order of frequency) the ‘S’ and ‘C’ types (already described among cultivated forms) as well as the ‘H’ (Huevo de huanchaco) and ‘A’ (Ayacucho), (new patterns previously undescribed among wild and cultivated beans). In each region—Middle America and Andean South America—the seeds of landraces with ‘T’ phaseolin were significantly larger than those of landraces with ‘S’ phaseolin. No significant differences in seed size were observed among landraces with ‘T,’ ‘C,’ ‘H,’ and ‘A’ phaseolin types of the Andean region. Our data favor 2 primary areas of domestication, one in Middle America leading to small-seeded cultivars with ‘S’ phaseolin patterns and the other in the Andes giving rise to large-seeded cultivars with ‘T’ (and possibly ‘C,’ ‘H,’ and ‘A’) phaseolin patterns.     

Enhanced available methionine concentration associated with higher phaseolin levels in common bean seeds

Summary The relationship between available methionine concentration and the levels of phaseolin — the major seed storage proteins of the common bean — was studied using three groups of genetic materials: First, the F₂ progenies of interspecific crosses between P. vulgaris cultivars and aP. coccineus subsp. coccineus line (cv. Mexican Red Runner) having no detectable phaseolin; second, the F₂ progenies and segregating F₃ families of crosses between cultivated P. vulgaris lines and a Mexican wild bean accession (PI 325690-3) carrying a gene producing a reduction in phaseolin content; third, two inbred backcross populations: SanilacxBush Blue Lake 240 (population 2) and Sanilacx15R 148 (population 6). Total seed N levels were determined by micro-Kjeldahl, phaseolin levels by rocket immunoelectrophoresis and available methionine levels by the Streptococcus zymogenes bioassay. Our results indicate that in all the genetic materials studied, with the exception of population 6, higher phaseolin levels lead to increased available methionine concentration. Although phaseolin has a low methionine concentration, it is actually a major source of available methionine in common bean seeds, because it represents a large part of total seed nitrogen and because limited differences exist between the methionine concentrations of the different protein fractions. This contrasts with the situation in cereals such as maize, barley and sorghum, where increased levels of the major limiting amino acid (lysine) can be achieved through a decrease in the amounts of the main seed storage protein fraction (prolamines). In population 6, no relationship was observed between available methionine and phaseolin content. Other factors, such as additional methionine-rich polypeptides or the presence of tannins, might obscure the positive relationship between phaseolin and available methionine content in population 6.     

Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride–pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.     

An N-band marker for gene Lr18 for resistance to leaf rust in wheat

The leaf rust resistance gene, Lr18, of common wheat cultivars has been derived from Triticum timopheevi and is located on chromosome arm 5BL. Chromosome banding (N-banding) analyses revealed that in the wheat cultivars carrying Lr18 that were examined, which had been bred in 6 different countries, chromosome arm 5BL possessed a specific terminal band not carried by their susceptible parental cultivars. It was suggested that this terminal N-band was introduced from T. timopheevi together with Lr18. N-banding analysis of a T. timopheevi strain showed that one of two timopheevi chromosomes had provided Japanese wheat lines containing Lr18 with the terminal band.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Direct amplification of minisatellite-region DNA with VNTR core sequences in the genus Oryza

 A polymerase chain reaction (PCR) application, involving the directed amplification of minisatellite-region DNA (DAMD) with several minisatellite core sequences as primers, was used to detect genetic variation in 17 species of the genus Oryza and several rice cultivars (O. sativa L.). The electrophoretic analysis of DAMD-PCR products showed high levels of variation between different species and little variation between different cultivars of O. sativa. Polymorphisms were also found between accessions within a species, and between individual plants within an accession of several wild species. The DAMD-PCR yielded genome-specific banding patterns for the species studied. Several DAMD-PCR-generated DNA fragments were cloned and characterized. One clone was capable of detecting multiple fragments and revealed individual-specific hybridization banding patterns using genomic DNA from wild species as well as rice cultivars. A second clone detected only a single polymorphic locus, while a third clone expressed a strong genome specificity by Southern analysis. The results demonstrated that DAMD-PCR is potentially useful for species and genome identification in Oryza. The DAMD-PCR technique also allows for the isolation of informative molecular probes to be utilized in DNA fingerprinting and genome identification in rice. Received: 1 October 1996 / Accepted: 25 April 1997     

Bruchid resistance of common bean lines having an altered seed protein composition

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) are toxic to one of the most damaging pests of bean seeds, Zabrotes subfasciatus (Boheman), but they appear to have little effect on another important bean pest, Acanthoscelides obtectus (Say), when introduced into standard cultivars by backcrossing. With the goal of increasing arcelin concentration to improve resistance, we modified seed-protein composition by introducing a null allele for the major seed protein, phaseolin, into lines (SMARC1, 2 and 4) or three phytohemagglutinin types (SMPHA lines). These lines were tested for resistance to both insects by measuring percentage insect emergence (%E) and days-to-adult emergence (DAE). For SMARC lines, arcelin type was the most important factor in resistance levels, with SMARC1 lines being most resistant, SMARC2 lines intermediate, and SMARC4 lines the least resistant to both bruchids. Additionally, the absence of phaseolin was a significant factor in the resistance of SMARC lines to A. obtectus. SMARC1 lines without phaseolin had half the percentage insect emergence of lines with phaseolin. SMARC1 lines with an altered seed composition had the highest levels of resistance to both bruchids of any large-seeded line reported to-date. Received: 2 April 1997 / Accepted: 20 May 1997     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.