Regeneration of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) by a cyclic organogenic system

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars (Espace, Classic, Solara, and Puget). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and -naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.     

Development and Study of SCAR Markers in Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> 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 origin.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Mapping of the <Emphasis Type="Italic">multifoliate pinna</Emphasis> (<Emphasis Type="Italic">mfp</Emphasis>) leaf-blade morphology mutation in grain pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

The multifoliate pinna (mfp) mutation alters the leaf-blade architecture of pea, such that simple tendril pinnae of distal domain are replaced by compound pinna blades of tendrilled leaflets in mfp homozygotes. The MFP locus was mapped with reference to DNA markers using F₂ and F_2:5 RIL as mapping populations. Among 205 RAPD, 27 ISSR and 35 SSR markers that demonstrated polymorphism between the parents of mapping populations, three RAPD markers were found linked to the MFP locus by bulk segregant analyses on mfp/mfp and MFP/MFP bulks assembled from the F_2:5 population. The segregational analysis of mfp and 267 DNA markers on 96 F₂ plants allowed placement of 26 DNA markers with reference to MFP on a linkage group. The existence of common markers on reference genetic maps and MFP linkage group developed here showed that MFP is located on linkage group IV of the consensus genetic map of pea.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Water Stress and Apical Dominance in <Emphasis Type="Italic">Pisum sativum</Emphasis>

A recent study of apical dominance in isolated rhizomes of Agropyron repens L. Beauv. suggested that inhibition of the lateral buds by the rhizome apex largely depends on the supply of water, nitrogen and carbohydrate, any of which could act as a limiting factor and thus determine the degree of inhibition¹. To test this hypothesis, further experiments were conducted with peas (Pisum sativum, variety ‘Alaska’), which exhibit strong apical dominance and which are widely used in the study of this phenomenon². The results agreed well with the concept of limiting nutritional factors and suggest that for this species water stress may be particularly significant.     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

Study of dominant symbiotic mutants of pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Phenogenetic studies of four symbiotic hypernodulating mutants of pea (Pisum sativum L.) induced from seeds of cultivar Rodno by chemical mutagen EMS were conducted. All mutants have improved symbiotic traits, i.e., an increased number of root nitrogen fixating nodules and high activity of nitrogenase. Symbiotic traits were shown to be inherited dominantly. Mutants grown in the field or in a greenhouse showed superiority over the original cultivar in productivity. An important feature of hypernodulating mutants was found that is responsible for the appearance of high-height productive plants in F₂ after crossing mutants and the original cultivar. Constant lines retaining the ability for high-level production up to the F₅ generation were created based on individual plants.     

Genetic control of fasciation in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> 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.     

Mapping of quantitative trait loci for resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in <Emphasis Type="Italic">Pisum sativum</Emphasis> subsp. <Emphasis Type="Italic">syriacum</Emphasis>

Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings. The wild Pisum sativum subsp. syriacum accession P665 shows good levels of resistance to this pathogen. Knowledge of the genetic factors controlling resistance to M. pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M. pinodes in pea have never included this wild species. The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M. pinodes in P. sativum subsp. syriacum and to compare these with QTLs previously described for the same trait in P. sativum. A population formed by 111 F_6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers. The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes. Six QTLs associated with resistance to M. pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait. While QTLs MpIII.1 and MpIII.2 were detected both for seedlings and field resistance, MpV.1 and MpII.1 were specific for growth chamber conditions and MpIII.3 and MpIV.1 for field resistance. Quantitative trait loci MpIII.1, MpII.1, MpIII.2 and MpIII.3 may coincide with other QTLs associated with resistance to M. pinodes previously described in P. sativum. Four QTLs associated with earliness of flowering were also identified. While dfIII.2 and dfVI.1, may correspond with other genes and QTLs controlling earliness in P. sativum, dfIII.1 and dfII.1 may be specific to P. sativum subsp. syriacum. Flowering date and growth habit were strongly associated with resistance to M. pinodes in the field evaluations. The relation observed between earliness, growth habit and resistance to M. pinodes is discussed.     

Development of SCAR markers linked to <Emphasis Type="Italic">sin</Emphasis>-<Emphasis Type="Italic">2</Emphasis>, the stringless pod trait in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

With increasing consumer demand for vegetables, edible-podded peas have become more popular. Stringlessness is one of most important traits for snap peas. A single recessive gene, sin-2, controls this trait. Because pollen carrying the stringless gene is less competitive than pollen carrying the stringy gene, there are fewer than expected stringless plants recovered in segregating generations. Marker-assisted selection (MAS) is a valuable tool to identify plants with the traits of interest at an early stage in the breeding process. The objective of this study was to identify robust, user-friendly molecular markers tightly linked to sin-2. A total of 144 target region amplification polymorphism (TRAP) primer combinations were used to screen four DNA bulks, which were constructed from 32 pea breeding lines based on their phenotypes. Sixty polymorphic TRAP primer combinations were identified between bulks of stringless and stringy pods. Five primer combinations, F6_Trap03_168, F6_SA12_145, F10_ODD8_130, F11_GA5_850, and F12_SA12_190, showed more than 90 % association with the stringless phenotype in 32 pea breeding lines. Two of the TRAP markers, F10_ODD8_130 and F12_SA12_190, were cloned, sequenced, and successfully converted to sequence characterized amplified region (SCAR) markers. These two SCAR markers were validated using 20 F₅ recombinant inbred lines derived from a cross between Bohatyr (a dry pea variety with strings) and S1188 (a stringless snap pea variety) and showed strong marker-trait association. The results will have direct application in MAS of stringless edible-podded peas.     

Linkage of a RAPD marker with powdery mildew resistance <Emphasis Type="Italic">er-1</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

The aim of this study was to investigate the inheritance of powdery mildew disease and to tag it with a DNA marker to utilize for the marker-assisted selection (MAS) breeding program. The powdery mildew resistant genotype Fallon ^er and susceptible genotype 11760-3 ^ER were selected from 177 genotypes by heavy infestation of germplasm with Erysiphe pisi through artificial inoculation The F₁ plants of the cross Fallon/11760-3 indicated the dominance of the susceptible allele, while F₂ plants segregated in 3: 1 ratio (susceptible: resistant) that fit for goodness of fitness by χ² (P > 0.07), indicating monogenic recessive inheritance for powdery mildew resistance in Pisum sativum. A novel RAPD marker OPB18 (5′-CCACAGCAGT-3′) was linked to the er-1 gene with 83% probability with a LOD score of 4.13, and was located at a distance of 11.2 cM from the er-1 gene.     

Antioxidative Responses and Morpho-anatomical Alterations for Coping with Flood-Induced Hypoxic Stress in Grass Pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.) in Comparison with Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Flooding stress constrains crop growth and yield because most agricultural species are flood-sensitive. However, many of the plant species that live in permanently or temporarily flooded habitats have evolved specific traits to cope with these harsh conditions. Grass pea (Lathyrus sativus L.) is a legume that tolerates stresses such as drought, diseases, and pests; however, it is unclear whether grass pea has a tolerance mechanism for flooding stress. To understand if grass pea tolerates hypoxia and how it deals with hypoxic stress, the effects of hypoxia on root tip death, physiological, and morpho-anatomical alterations in grass pea and pea (Pisum sativum), which is sensitive to hypoxia, were compared. The results showed that activities of antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, and glutathione content in grass pea were greater than in pea during hypoxia, which protected the root tip from oxidative damage and reduced ion leakage, which helped maintain membrane integrity. Furthermore, aerenchyma and lateral root development accompanied by ethylene production, moderate ROS accumulation-mediated cell death, and Ca²⁺ spatial-temporal heterogeneity developed well in grass pea compared to pea, which may not only facilitate internal gas diffusion but also promote removal of toxic by-products under hypoxic conditions. These results demonstrate that grass pea is more tolerant to hypoxic stress induced by flooding than garden pea seedlings. This discovery not only provides significant information for understanding the hypoxia-tolerant mechanisms in plants, but also promotes the usability of grass pea in flood-prone areas.     

Influence of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain on the production of transgenic peas (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures.Communicated by P. Debergh     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

RAPD and ISSR analyses of regenerated pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L. plants

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R₀). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7–5.3% in R-9 regenerants, and 10–15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 71–77.Original Russian Text Copyright © 2005 by Kuznetsova, Ash, Hartina, Gostimskij.     

Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.