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A glutamine synthetase gene (GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC3) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC 3 -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N2 fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC 3 -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N2 fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM $$ {\rm NO}_3^ - $$ treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.

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 β-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%.

Mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) variations were examined in six cultivars of Lens culinaris ssp. culinaris and two (mtDNA) or one (nuDNA) accession(s) of L. culinaris ssp. orientalis. Total leaf DNA was digested with up to 15 restriction endonucleases, separated by agarose gel electrophoresis and trasferred to nylon membranes. To examine mtDNA variation, blots were probed with mtDNA coding for cytochrome c oxidase I (coxI) and ATPase 6 (atp6) of both wheat and maize as well as apocytochrome b (cob) and Orf25 (orf25) of wheat. Sixteen combinations of mtDNA probes and restriction enzymes revealed 34 fragments that discriminated between at least two lentil accessions. For nuDNA analysis, probes from cDNA and genomic DNA clones of lentil were used to probe the same blots, and identified 46 diagnostic fragments from 19 probe/enzyme combinations. Each lentil accession could be unequivocably distinguished from all others on the basis of both mitochondrial and nuclear DNA fragment patterns. The mitochondrial restriction fragment similarities ranged from 0.944 to 0.989, with a mean of 0.970 but nuclear restriction fragment similarities varied from 0.582 to 0.987, with a mean of 0.743. The apparent genetic relationships among accessions differed according to the source of DNA examined, although the commercial varieties Laird, Brewer and Redchief showed similarly high levels of mean similarity with both nuclear (0.982) and mitochondrial DNA (0.983). Key words: Lens culinaris Medik., genetic variation, mitochondrial, nuclear, DNA, lentil

A soybean cytosolic glutamine synthetase gene ( GS15 ) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter ( LBC 3 ), or a putative root-specific promoter ( rolD ) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies (Lines 1, 7, 8 and 9) and four lines with two copies each of GS15 (Lines 2, 4, 6 and 11) were compared to the wild-type (WT) parental line for levels of cytosolic glutamine synthetase (GS1), glutamine synthetase (GS) activity, N accumulation, N derived form the atmosphere (NDFA), and biomass of plants grown on 0.0, 0.1, 1.0 or 10.0 mM NH 4 + . Enhanced levels of GS1 were detected in leaves of one of the two lines transformed with the 35S-GS15 construct, and all three lines containing the rolD-GS15 construct. All three lines containing the LBC 3 -GS15 construct had increased levels of GS1 in nodules. Despite the increased levels of GS1 in many transformants, only the roots of lines containing the rolD-GS15 construct consistently demonstrated enhanced levels of GS activity (up to 12-fold). Positive responses in plant N content, NDFA, and biomass were rare, but increases in plant biomass and N content of up to 17% and 54%, respectively, occurred in some of the rolD-GS15 lines at certain levels of ammonium. In general, GS15 copy number did not seem to differentially affect phenotype of the transformants, and transformants respond to ammonium concentrations in similar patterns to that previously observed with nitrate. Despite the fact that the rolD-GS15 transformants consistently resulted in increased GS activity in roots and resulted in some occurrences of increases in biomass and plant N content, the lack of consistent positive growth effect across all transformants indicates that the generalized overexpression of GS1 in tissues holds little potential for positive growth responses in pea.

A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both beta-glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.

Transgenic pea plants can be produced by Agrobacterium-mediated transformation of thin slices from developing embryo axes. To determine if the method is effective for different pea genotypes, seven pea breeding lines adapted to western Canadian growing conditions were tested, using three different Agrobacterium tumefaciens transformation vectors. All vectors contained the gus (uidA) gene coding for the b-glucuronidase (GUS) protein, but with different chemical selection genes. In total, 323 transgenic plants were recovered from 39 independent transformation events. Transgenic plants were recovered from each genotype and each selection system, but not from all combinations. GUS-positive explants were obtained from seeds harvested between 24 and 31 days after flowering. The mean time from Agrobacterium treatment to planting into soil averaged 186 days. Based on the initial number of seeds used, the transformation frequency was 0.6% (i.e. six independent transgenic events per 1000 axes sliced). The inserted genes were functional and inherited in a Mendelian fashion. Although more plants were recovered by selection on chlorsulfuron, GUS activity was generally greater in plants selected on kanamycin. GUS activity in the leaves of the original plants varied, but GUS activity in the second generation was correlated with that of the original transformants. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Four polyubiquitin genes, PUB1, PUB2, PUB3 and PUB4, were isolated from a pea (Pisum sativum L. cv Alaska) genomic library and completely sequenced. They represent all of the four polyubiquitin genes of the ubiquitin gene family in pea. The coding regions of the genes contain five or six coding units arranged as tandem repeats. The different coding repeats of the four genes share homologies between 75 and 97%, encoding the same protein of 76 amino acids identical to those from other higher plants. The open reading frames of PUB1, PUB2 and PUB4 terminate in the additional amino acid, phenylalanine (F), and PUB3 terminates in isoleucine (I). The polyubiquitin genes all contain intron sequences ranging from 584 to 1114 bp immediately 5' to the ATG initiation codon of the first coding sequence. Of the four genes, three are associated with long AT-rich (85.4-89.4% A+T) sequences ranging from about 331 to 478 bp at their 5' or 3' ends. The PUB4 gene was found to be linked to a moderate to highly repetitive DNA at its 5' flanking sequence. The greater sequence homology between different genes than among individual repeating units of a gene suggests that the polyubiquitin genes may have arisen by gene duplication of a single gene sequence.

Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of β-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 μF; and using 125 μg of calf thymus carrier DNA and 75 μ of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.

The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was studied during greening in 12Pisum sativum L. genotypes. The proportion of mRNA coding for the small subunit of Rubisco (SSU mRNA) was also monitored by hybridization with a cDNA (complementary DNA) probe from one of the nuclear genes coding for SSU (rbcS). Both the SSU mRNA and Rubisco (in g·FW)(-1)) contents rapidly increased in all genotypes on illumination of dark-grown seedlings. Natural genetic variability was found in the amounts of SSU mRNA, Rubisco· (g FW)(-1), total RNA·(g FW)(-1) and mRNA·(μg total RNA)(-1). Differences among genotypes in SSU mRNA were apparently the result of differences in the rate of light-induced accumulation of therbcS gene transcripts. Genotype means for SSU mRNA and Rubisco· (g FW)(-1) amounts during greening were significantly correlated (r=0.788;P

Genotypic variability in photosynthesis per unit of leaf area may be useful in increasing gross productivity of crop plants, but only if it can be demonstrated to be measurable and related to growth in field stands and if it is not physiologically linked to compensating differences in leaf area production. To examine these issues, six genotypes of pea (Pisum sativum L.), selected for differences in photosynthetic CO₂ exchange per unit of leaf area (CER), were planted in replicate field plots at densities of 25 and 100 seeds m⁻². Mean CER, leaf area index (LAI), and relative growth rate (RGR) were determined for each genotype ✕ density combination over six weeks of growth in two years. At the higher planting density, LAI increased by 72%, but CER and RGR decreased by 22 and 24%, respectively. Genotypes differed in all three traits, but the variability in LAI was related to a twofold difference between two developmentally distinct groups that did not differ in either CER or RGR. Although CER of all genotypes decreased at high density, the magnitude of the CER response to increased LAI differed. In both years, mean RGRs of plants in the twelve genotype ✕ density treatments were significantly correlated (P < 0.01) with mean CERs, and the genotype CER and RGR means from all data were also correlated (P < 0.05). That these correlated differences were not due to differences in photosynthetic area was shown by the superiority in CER (38%) and RGR (34%) of genotype PI 269810 over genotype PI 269770, even though the two genotypes did not differ in mean LAI.

A computer-controlled automated gas-exchange system was developed to perform repetitive measurements of physiological activities in intact plants. Shoot and root temperatures, flow rates, gas pressures, rates of leaf CO2 and H2O exchange, and rates of CO2 exchange, H2 evolution, and C2H2 reduction by the roots can be determined for four plants in a single experiment. The controlling computer operates 32 switches that control gas flows, sampling devices, lighting, and irrigation and it records and processes analog data from 15 measuring instruments. Rates of physiological activities are calculated automatically and results are printed and stored on diskettes for later plotting or analysis. A wide variety of experimental protocols are possible with little need for computer programming, because the system is controlled by a master program and an interactively developed file of experimental variables. The values recorded from the measuring instruments are proportional (r2 > 0.999) to either independent determinations or to calibration standards and the rates of physiological processes, measured with the system, are reproducible and within expected ranges.

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H2 evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H2 or 3H2 in the presence of acetylene. In all cases, the rate of H2 evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H2 evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup− strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H2 evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup− strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H2 evolution by Hup+ strains were similar in magnitude to their H2-saturated rates of Hup activity. The curvilinear relationship between H2 evolution and acetylene reduction for a representative Hup+ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.

The possibility that breeding programs may be reaching a point of limited progress because of the lack of genetic diversity has often been raised. However, there is also an often-expressed feeling that considerable genetic variability may exist in fundamental physiological processes. In this review, an attempt is made to examine whether there is evidence of genetic variability in quantitative physiological characters, and if so, whether such variability is likely to be useful to crop improvement programs. The results, presented from the literature and the author’s laboratory, indicate that physiological characters demonstrate considerable genotypic variability in expression. Moreover, in cases where genotype performance has been studied over a range of environments or where crossing studies have been carried out, physiological characters often have relatively high heritabilities. This suggests that, at least from a genetic standpoint, improvements in the expression of physiological processes may be possible. On the other hand, the problems of environmental sensitivity and its resultant error variability, combined with cumbersome methodology and complex physiological interactions, make it difficult to relate individual physiological characters to agronomic benefits, and make large genetic studies difficult. The exploitation of physiological diversity remains a major challenge to plant scientists.Key words: Genetic heritability, physiology of yield

Pea plants (Pisum sativum L. cv. Trapper) were inoculated and grown in controlled-environment chambers at two irradiance levels. Shoot and root dry weights and nitrogen contents, total leaf and stipule areas, and rates of C2H2 reduction were determined during growth in different treatments of NH4NO3 addition. Although overall growth increased with irradiance, the growth responses to combined nitrogen addition were similar at both light levels. Two phases of early vegetative growth were identified by their different responses to combined nitrogen. During the first phase, low levels of NH4NO3 greatly increased the relative growth rate, the growth per unit leaf area, and the percentage of nitrogen in the tissues. This indicated a period of nitrogen stress which lasted only until the 3rd week. Over the next 2 weeks, combined nitrogen increased the relative growth rates to a lesser extent, primarily through an increased partitioning of assimilates to shoot development. This distribution effect was rapidly reversible on changing nutrient conditions. The early stimulation of leaf area development by addition of combined nitrogen during these two growth phases resulted in greater capacity for symbiotic fixation after NH4NO3 was removed.

Inoculated pea plants (Pisum sativum L.) were grown with N-free nutrients in a controlled environment room and rates of respiratory CO₂ evolution and C₂H₂ reduction by the intact nodulated roots were determined. Experiments followed changes related to diurnal cycles, light and dark treatments, partial defoliation, aging of plants and NH₄NO₃ addition. In all experiments, changes in C₂H₂ reduction were associated with parallel changes in the respiration rate, although in all but the defoliation experiment there was a basal level of respiration which was independent of the rate of C₂H₂ reduction. In conditions which affected growth or plant size as well as C₂H₂ reduction, respiration changed by an average of 0.42 mg CO₂ (μmol C₂H₂ reduced)⁻¹. However, some treatments decreased C₂H₂ reduction without greatly changing the growth and in these conditions respiration was decreased by an average of 0.27 mg CO₂ (μmol C₂H₂ reduced)⁻¹. While this value may also include some respiration associated with other processes, it is proposed that it more closely estimates respiration directly associated with energy utilization for acetylene reduction; whereas the higher value includes respiration related to maintenance and growth processes as well.

To determine the best combination for potential use in transformation of Pisum sativum L., 13 genotypes were inoculated with wild-type Agrobacterium tumefaciens strains A281, C58 and Ach5. A281 appeared to be the most virulent strain, as determined by size and number of tumours, followed by C58 and Ach5. Genotypes differed considerably in their response to inoculation and genotype x strain interaction was evident. Genotypes also responded differently to in vivo or in vitro inoculation. Axenic calli from tumours could be grown on hormone-free medium and the presence of the specific opines for each strain in the callus indicated successful transfer and expression of T-DNA. Southern blot analysis of DNA from callus of A281-inoculated material showed that both TR and TL T-DNA had been incorporated into the pea genome.

Pisum sativum L. cv. Trapper plants were inoculated and grown in a controlled environment on N-free nutrient solution. After 4 weeks N was supplied to treatment plants as NH(4)NO(3), KNO(3), or NH(4)Cl and rates of C(2)H(2) reduction, root + nodule respiration, and leaf photosynthesis were determined 1 week later. The increase in respiration per unit of C(2)H(2) reduction was not affected by either the form of N added or the light conditions during growth, although the basal respiration rate with no C(2)H(2) reduction increased with irradiance level. The mean regression coefficient from plots of respiration versus C(2)H(2) reduction was 0.23 + 0.04 (P [unk] .01) mg of CO(2) (mumol of C(2)H(2) reduced)(-1) which was very similar to the value for the coefficient of respiration associated with nitrogenase activity estimated by subtracting growth and maintenance respiration. Since the rate of N accumulation in N-free nutrient conditions was proportional to the rate of C(2)H(2) reduction, it appears that the method gives a true estimate of the energy requirements for N fixation which for these conditions was equivalent to 17 grams of carbohydrate consumed per gram of N fixed.

Estimated values for the respiration associated with symbiotic nitrogen fixation in Pisum sativum L. were independent of irradiance, temperature, plant age, and CO(2) concentration, despite large variation in the total rates of C(2)H(2) reduction and root + nodule respiration. Similar values were also found in Phaseolus vulgaris L., Vicia faba L. and Glycine max (L.) Merr. Among all combinations of four Pisum cultivars with four Rhizobium leguminosarum inoculants only the plant genotype significantly affected the fixation-linked respiration, although both plant and bacterial types significantly influenced the total rate of C(2)H(2) reduction. On the basis of measured rates of H(2) evolution and C(2)H(2) reduction, or total nitrogen gain in the same system, the least respiration per unit of ammonia produced symbiotically was estimated as 4.8 to 6.9 moles CO(2) (mole NH(3))(-1) in Laxton's Progress and the greatest as 9.3 to 13.3 moles CO(2) (mole NH(3))(-1) in an Indian cultivar, as compared to a theoretical minimum respiration requirement of 4.7 moles CO(2) (mole NH(3))(-1) in peas.

The genetic improvement of photosynthesis has often been proposed as a route to increased crop productivity. However, although there is extensive genotypic variability in photosynthesis per unit of leaf area or leaf weight in many crop species, the exploitation of this variability remains difficult. Not only is photosynthesis very sensitive to environmental factors, but there are reports of considerable genotype by environment and genotype by developmental stage interactions. Under such conditions, selection progress would be difficult and large numbers of measurements would be needed to assess phenotypic expression.

The Marquillo x Kenya Farmer 1 "grass-clump" dwarf selection of Triticum aestivum L. was grown under continuous 2000 foot candle light and several regimes of alternating 16 degrees and 26 degrees temperatures combined in total cycle lengths of 6, 12, 24, or 48 hr. Plants at 26 degrees grew as normal wheat. Those exposed to 0.25 to 2 hr of 16 degrees per cycle showed typical "grass-clump" dwarf characteristics which were independent of the cycle length. Treatments with 16 degrees exposures of 4 to 8 hr per 24 hr and 12 to 16 hr per 48 hr exhibited vegetative "grass-clump" dwarfness for 40 days but later displayed extensive reproductive development. Longer 16 degrees treatments killed the plants at a very early stage of vegetative development before floral initiation. The data supported an hypothesis that all 4 growth habits were related to the temperature sensitivity of the vegetative meristem. The cessation of meristem development was possibly due to the accumulation of a stable inhibitory substance produced at low temperatures.