Insertional polymorphism and antiquity of PDR1 retrotransposon insertions in pisum species

Sequences flanking 73 insertions of the retrotransposon PDR1 have been characterized, together with an additional 270 flanking regions from one side alone, from a diverse collection of Pisum germ plasm. Most of the identified flanking sequences are repetitious DNAs but more than expected (7%) lie within nuclear gene protein-coding regions. The approximate age of 52 of the PDR1 insertions has been determined by measuring sequence divergence among LTR pairs. These data show that PDR1 transpositions occurred within the last 5 MY, with a peak at 1-2.5 MYA. The insertional polymorphism of 68 insertions has been assessed across 47 selected Pisum accessions, representing the diversity of the genus. None of the insertions are fixed, showing that PDR1 insertions can persist in a polymorphic state for millions of years in Pisum. The insertional polymorphism data have been compared with the age estimations to ask what rules control the proliferation of PDR1 insertions in Pisum. Relatively recent insertions (< approximately 1.5 MYA) tend to be found in small subsets of the Pisum accessions set, "middle-aged" insertions (between approximately 1.5 and 2.5 MYA) vary greatly in their occurrence, and older insertions (> approximately 2.5 MYA) are mostly found in small subsets of Pisum. Finally, the average age estimate for PDR1 insertions, together with an existing data set for PDR1 retrotransposon SSAP markers, has been used to derive an estimate of the effective population size for Pisum of approximately 7.5 x 10(5).     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Field Assessment of Outcrossing from Transgenic Pea (Pisum Sativum L.) Plants

The frequency of outcrossing from a transgenic line of peas into three cultivars ('Carneval', 'Montana', 'Tipu') was studied in the field in 1997 and 1999. Two dominant traits, normal leaf form and a highly-expressed -glucuronidase (gusA) gene, were used as markers of pollen transfer. Because of heterogeneity in the commercial seed sources, leaf form alone was unreliable for assessing pollen migration into 'trap' plants. Of approximately 9000 offspring tested, only five plants scored positive for the presence of both markers. All five were located in 'trap' plots situated near the transgenic line. This represents a mean outcrossing rate of 0.07%.     

Transformation of pea (Pisum sativum L.) byAgrobacterium tumefaciens

Explants fromPisum sativum shoot cultures and epicotyls were transformed by cocultivation withAgrobacterium tumefaciens vectors carrying plant selectable markers and transformants could be selected on a medium containing kanamycin. Transformants could also be obtained at a low frequency by cocultivating small protoplast-derived colonies. The transformed nature of the calli obtained from selection was confirmed by opine assay and DNA analysis. In addition five cultivars of pea were tested for their response to seven differentAgrobacterium tumefaciens strains. The response pattern coincided largely between the different pea cultivars, being more dependent on the bacterial strain than the cultivar used.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - Km kanamycin - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT neomycin phosphotransferase - OCS octopine synthase     

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts

Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.     

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.     

Genetic control of fasciation in pea (Pisum sativum L.)

The inheritance and manifestation of fasciation character in three fasciated lines of common pea Pisum sativum L. were investigated. All studied forms are characterized by abnormal enlargement of stem apical meristem leading to distortions in shoot structure. It was estimated that fasciation in mutant Shtambovyi is connected with recessive mutation in gene FAS, which was localized in linkage group III using morphological and molecular markers. It was demonstrated that fasciation in cultivar Rosacrone and line Lupinoid is caused by recessive mutation of the same gene (FA). The peculiar architecture of inflorescence in the Lupinoid line is a result of interaction of two recessive mutations (det fa). Investigation of interaction of mutations fa and fas revealed that genes FA and FAS control consequential stages of apical meristem specialization. Data on incomplete penetrance and varying expressivity were confirmed for the mutant allele fa studied.     

Immunolocalization of lipoxygenase in pea (Pisum sativum L.) carpels

Summary Polyclonal antibodies against a part of pea (Pisum sativum L.) LOXG protein have been raised to study the pattern of distribution of related lipoxygenases in pea carpels. The antiserum recognized three lipoxygenase polypeptides in carpels. One of them became undetectable 24 hours after fruit development induction, suggesting that it may correspond to the protein derived from loxg cDNA. Immunolocalization experiments showed that lipoxygenase protein was present only in pod tissues: it was abundant in the mesocarp and, from the day of anthesis, in the endocarp layers. Lipoxygenase distribution is regulated throughout development. The association of lipoxygenase with cells in which processes of expansion and growth will potentially take place support a role in pod growth and development.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IgG immunoglobulin G - GA₃ gibberellic acid - LOX lipoxygenase - PAGE polyacrylamide gel - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulfate - Tris 2-amino-2-hydroxymethyl-1,3-propanediol     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Inheritance and mapping of isoenzymes in pea (Pisum sativum L.)

Summary Three isoenzyme systems (amylase, esterase and glutamate oxaloacetate transaminase) were examined in seeds of pea (Pisum sativum L.) and shown to give clear variation in their band patterns on gel electrophoresis between different lines. The inheritance of these isoenzyme systems, and the location of their genes on the pea genome was investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase was found to be on chromosome 2, linked to the loci k and wb (wb ... 9 ... k ... 25 ... Amy). The gene for esterase was found to be linked with the gene Br (chromosome 4) but the exact location is uncertain because of the lack of the morphological markers involved in the cross. The gene for glutamate oxaloacetate transaminase was found to be on chromosome 1 and linked with the loci a and d (a... 24... Got... 41 ... d).     

Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.)

Whole plant regeneration via somatic embryogenesis was obtained in pea (Pisum sativum L.) using explants from immature embryos or shoot apex segments. The induction of somatic embryos required picloram or 2,4-D. Germination of fully-developed embryos was accomplished by subculture on medium with only cytokinin and then on medium supplemented with cytokinins in combination with a reduced auxin concentration. Plantlets obtained from both zygotic embryos and shoot apices were transferred to soil and were grown to maturity. Nine plants were examined cytologically, revealing three tetraploids (2n=4x=28) and six diploids (2n=2x=14).Abbreviations Picloram 4-amino-3,5,6-trichloropicolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - IBA indole-3-butyric acid KAES Journal Article No. 87-3-4     

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.)

This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.Electronic Supplementary Material Supplementary material is available for this article at     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Aflatoxin formation and varietal difference of cow pea (Vigna unguiculata (L.) Walp.) and garden pea (Pisum sativum L.) cultivars

Different cultivars of cow pea and garden pea seeds were surveyed for susceptibility or resistance towards the toxigenic and aflatoxin-producing mould (Aspergillus flavus IMI 102135). The results show that aflatoxin production varied among the different cultivars of both cow pea and garden pea. Morphological and histological characters of the different cultivars tested did not show any relation between colour, shape and size of seeds and the amount of aflatoxin produced. The chemical analysis of the different constituents obtained from both seed coats and seed kernels with susceptible, partially resistant and resistant cow pea and garden pea cultivars revealed that the resistant cultivars of cow pea (namely: Balady cultivar) and garden pea (namely: Melting Sugar cultivar) contained lower levels of sodium and higher levels of phosphate and potassium.     

Pathways of auxin transport in the intact pea seedling (Pisum sativum L.)

Summary When small colonies of the pea aphid [Acyrthosiphon pisum (Harris)] were established on the stem of Meteor Dwarf Pea seedlings (Pisum sativum L.), ¹⁴C was found in the honeydew 4.5 h after applying IAA-1-¹⁴C to a fully-expanded foliage leaf. In contrast, no activity was found in the honeydew or aphids 4.5 h after the application of IAA-1-¹⁴C to the intact apical bud even though the internode upon which the aphids were feeding contained high levels of ¹⁴C. The lack of radio-activity in aphids feeding on stems to which IAA-1-¹⁴C was applied via the apical bud was found not to be influenced by the internode position or by the transport interval allowed (up to 24 h).Radioactivity derived from either foliar or apical applications of IAA-1-¹⁴C was not transported through stem tissues killed by heat treatment. Xylem function was shown not to be impared by the heat treatment employed.It was concluded that the long-distance transport of IAA from the apical bud of intact pea seedlings does not take place in the phloem sieve tubes involved in the transport of metabolites from foliage leaves, or in the non-living tissues of the xylem.     

Development and study of SCAR markers in pea (Pisum sativum L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F1 and F2. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism type.     

Interaction of pea (Pisum sativum L.) lectins with rhizobial strains

Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.