Transformation of peas (Pisum sativum L.) using immature cotyledons

A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 mg/l phosphinothricin. The nptII and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the Buster (=Basta) leaf-paint test and the phosphinothricin acetyl transferase enzyme assay.Abbreviations BA 6-benzylaminopurine     

Quantitative trait loci for lodging resistance,plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.)

With the development of genetic maps and the identification of the most-likely positions of quantitative trait loci (QTLs) on these maps, molecular markers for lodging resistance can be identified. Consequently, marker-assisted selection (MAS) has the potential to improve the efficiency of selection for lodging resistance in a breeding program. This study was conducted to identify genetic loci associated with lodging resistance, plant height and reaction to mycosphaerella blight in pea. A population consisting of 88 recombinant inbred lines (RILs) was developed from a cross between Carneval and MP1401. The RILs were evaluated in 11 environments across the provinces of Manitoba, Saskatchewan and Alberta, Canada in 1998, 1999 and 2000. One hundred and ninety two amplified fragment length polymorphism (AFLP) markers, 13 random amplified polymorphic DNA (RAPD) markers and one sequence tagged site (STS) marker were assigned to ten linkage groups (LGs) that covered 1,274 centi Morgans (cM) of the pea genome. Six of these LGs were aligned with the previous pea map. Two QTLs were identified for lodging resistance that collectively explained 58% of the total phenotypic variation in the mean environment. Three QTLs were identified each for plant height and resistance to mycosphaerella blight, which accounted for 65% and 36% of the total phenotypic variation, respectively, in the mean environment. These QTLs were relatively consistent across environments. The AFLP marker that was associated with the major locus for lodging resistance was converted into the sequence-characterized amplified-region (SCAR) marker. The presence or absence of the SCAR marker corresponded well with the lodging reaction of 50 commercial pea varieties.Communicated by H. F. Linskens     

Inheritance of seed yield and quality traits in peas (Pisum sativum L.)

Summary A half diallel analysis involving nine cultivars showed that additive as well as non-additive gene effects were important for the inheritance of seed yield per plant, 100-seed weights, protein content and potassium per cent. For remaining traits non-additive genetic components were important. Overdominance was observed for all traits except for 100-seed weight, which expressed partial dominance. Parents PMR-T10, EC21857, EC109182, T163 and EC109189 were good general combiners for seed yield, seed weight and quality traits. In general there was a good relationship between per se performance and the gca effects of the parents for all traits. Cross combinations such as LMR8 x EC109182,LMR8 x PMR-T10,LMR8 x EC21857,PMRT10 x EC21857 and P23 x EC21857 were found promising. The seed yield was positively correlated with other quality traits. Protein had a positive correlation with methionine and phosphorus. All the values of correlation co-efficients were non-significant except for yield with potassium, 100-seed weight and protein with methionine, indicating that yield and quality attributes can be improved simultaneously by simple selection procedures.     

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).     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Mechanism of uptake and translocation of zinc by pea plants (Pisum sativum L.)

Summary Radiotracer studies revealed that the mechanism of zinc uptake was not purely non-metabolic. The over all uptake of Zn by intact plant ofPisum sativum L. (Var. T-163) occurred through an initial nonmetabolic phase followed by a metabolically mediated absorption. The translocation of zinc from plant roots to shoot was more sensitive to metabolic inhibitors and thus was a metabolic function.Research Publication No. 1541/116/0 through Experiment Station, G. B. Pant University, Pantnagar-263145, India.     

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.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

The effect of surface soil consolidation on the early root development of pea (Pisum sativum L.)

Cores of repacked soil were consolidated with a compressive strength testing machine, after peas had been planted in the centre of the core. The number that emerged were counted and root and shoot lengths and diameters were measured. Consolidation had no effect on emergence, root length or root diameter of the peas grown in a loamy sand, whereas emergence, root length and root diameter were affected by a small increase in load in a clay loam.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.)

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F₂ progeny of a cross between two cultivated types (Primo and OSU442-15) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (Slow) and a P. sativum ssp. humile accession (JI1794). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the Primo x OSU442-15 cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the JI1794 x Slow cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the Primo x OSU442-15 and JI1794 x Slow crosses.     

Possible control sites of polysaccharide synthesis during cell growth and wall expansion of pea seedlings (Pisum sativum L.)

The activities of the enzymes of uridine diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) were measured by using enzymic preparations (protein precipitated between 40–65% (NH₄)₂SO₄ saturation) isolated from segments at different stages of elongation of the third internode of pea seedlings. All enzymic activities increased from dividing and non-elongated cells to fully elongated cells. At all stages of growth, the specific activity or the activity per cell of UDP-D-glucose dehydrogenase was much lower than that of UDP-D-glucuronate decarboxylase and this may represent a controlling step in the formation of UDP-D-xylose. During elongation, changes were also found in the activities of the epimerases. These could be correlated with the corresponding variations which occur in the chemical structure and physical properties of pectins during cell wall extension. However, the high levels of the epimerases present in cells which have completed elongation growth suggest that pectin synthesis is mainly controlled at the sites of the synthetase reactions.     

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     

Effects of pod infection by Mycosphaerella pinodes on yield components of pea (Pisum sativum)

Yield reduction of pea (Pisum sativum) due to various types of infections by Mycosphaerella pinodes on pods was assessed. A range of disease severities was created on pods of pea plants grown in the glasshouse, by painting the pods with different concentrations of spore suspensions, at three different pod development stages: lag phase, the beginning of seed filling (BSF) and mid-filling of the seeds. Seed number at harvest was reduced only if the pods were infected before BSF, as shown previously for whole plant infections. Pod infections led to individual seed weight (ISW) losses from zero (for late infections, at mid-filling) to 20% (for earlier infections and severe disease). Infection during the lag phase affected ISW by reducing seed growth rate, whereas infection at BSF tended to reduce the duration of seed filling. There was a linear relationship between the area under the disease progress curve and the percentage decrease in ISW. This model should be complemented by the effect of leaves and stem infections, in order to predict ISW losses in diseased crop conditions, in which epidemics occur on all aerial parts of the pea plant.