An Analysis of Seed Development in Pisum sativum L.

Growth analysis has been performed on developing seeds and seedcomponents of six contrasting genotypes of Pisum sativum. Seeddevelopment has been divided into three phases of high growthrate separated by two ‘lag’ phases, or phases oflow growth rate. It is suggested that the timing of these growthphases may not be determined by the developing seed, since thereappeared to be no consistent correlation with particular physiologicalstages of seed development. The relationship between the absolutegrowth rate of the embryo sac and of the embryo as determinedfrom changes in volume is reflected in the accumulation andabsorption of the endosperm. The relative growth rate of theembryo volume was invariably higher than that of the embryosac although the difference between these two relative ratesvaried with genotype and may account in part for the differencein seed phenotype. It is suggested that the testa and embryo are sinks which bothcompete for the endosperm which may act as a common source,and that this relationship accounts for variation in endospermvolume. It is concluded that seed development is dependent on threelevels of plant organization, the maternal parent, interactionsbetween the components of the seed and the genetic constitutionof the embryo. Pisum sativum L., garden pea, seed development, growth analysis     

An Analysis of Seed Development in Pisum sativum XVII. The Effect of the rb Locus Alone and in Combination with r on the Growth and Development of the Seed

Near-isogenic lines have been produced in pea to examine theeffects of genes at the r and rb loci on the growth and developmentof the seed. The rb alleles were found to have an effect onthe growth of the testa as well as on that of the embryo incontrast to those at the r locus which only affect the latter.The relationship between the fresh and dry weights of the embryosof rb isoline and the double mutant differed from that of theround-seeded wild-type line. Furthermore, for this relationship,the double mutant line very closely resembled the rr isoline.Copyright1994, 1999 Academic Press Embryo, growth, pea, Pisum sativum L., rugosus loci, seed development, testa, wrinkled-seeded     

An Analysis of Seed Development in Pisum sativum: VI. ABSCISIC ACID ACCUMULATION

The abscisic acid (ABA) content of wrinkled (rr) pea seed tissueshas been quantified during development using multiple-ion-monitoringcombined gas chromatography-mass spectrometry and a deuteratedinternal standard. The level of ABA in the embryo generallyincreased with increasing cotyledon fresh weight while thatin the testa showed a distinct maximum at the time of maximumendosperm volume and the slowing in the growth of the testa.Pods contained relatively little ABA on a fresh weight basis.The total seed ABA content showed a biphasic distribution, thefirst maximum following the maximum growth rate of the testaand the second that of the embryo. The biphasic distributionof ABA in the pea seed was confirmed using a second pea genotype,near-isogenic to the first except for the r locus, and by theanalysis of individual seeds using a radioimmunoassay for ABA.The first maximum was composed mainly of a testa component andthe second mainly of an embryo component. When plants were grownin different environments, wrinkled seeds were found to containslightly more ABA than round (RR) but this was only significantlate in development. Immature seeds were capable of metabolizing17'-deoxy ABA to ABA, as determined by incorporation of either³H or ²H, and the metabolite was present mainly in the testa.The production of ABA in pea seeds is discussed in relationto the development of the different seed tissues. Key words: Abscisic acid, peas, seed development     

An Analysis of Seed Development in Pisum sativum II. The Effect of the r-Locus on the Growth and Development of the Seed

The r-locus is one of the few genetic loci known to affect thestorage product composition and the morphology of pea (Pisumsativum) seeds. Lines which are near-isogenic except for ther-locus have been developed to study the effects of this locuson seed development. The plant phenotypes of these lines werevery similar except for those characteristics previously attributedto the r-locus. The seed development of the two lines followedsimilar patterns until the endosperm was absorbed by the embryo.The fresh weight of the rr line then increased more rapidlydue to the overall effect of a higher rate of water uptake anda lower rate of dry weight increase of the rr embryos comparedwith embryos homozygous for the R-allele. The effect of the r-locus on the relationship between embryofresh weight and dry weight suggests that the alleles may beaffecting the osmotic regulation of the developing embryo. Pisum sativum, pea, seed development, r-locus, genetic variation, growth analysis     

An Analysis of Seed Development in Pisum sativum: IV. COTYLEDON CELL POPULATIONS IN VIVO AND IN VITRO

A method for quantifying changes in the cell population of Pisumsativum cotyledons during development is described. The methodis based on determining the frequency distribution for cellarea following the random sampling of a single-cell suspensionof cotyledon cells. The population profile of these cells changedprogressively and systematically from a single population, similarin size to meristematic cells, found in embryos less than 3.0mg in fresh weight, to a bimodal population in embryos greaterthan 100 mg fresh weight. This method was used to compare embryosof similar size from two genotypes near-isogenic except forgenes at the r locus. No significant differences were foundbetween the cell population profiles of embryos up to 30 mgfresh weight. However, a significant difference was found betweenembryos with fresh weights of 100 mg, the wrinkled (rr) linehaving a higher mean and maximum cell area (2 951 µm²and 9 240 µm² respectively) than the round (RR) line (2591µm²and 6470 µm²respectively). Comparisons were alsomade between cotyledon cell populations from round (RR) embryosgrown in vivo and in vitro. The most obvious differences werethe higher mean and maximum cell size of the large cell populationof in vitro grown embryos which were twice those found in vivo.Embryos grown to either 30 mg or 100 mg fresh weight in vitrohad a much greater proportion of large cells in the populationwith a corresponding reduction in total cotyledon cell number,compared with similar sized embryos grown in vivo. These data suggest that comparisons between different genotypes,or, between cultured and in vivoembryos, based on morphologicalsimilarities between embryos, may be invalid and subject tomisinterpretation. Key words: Pea, seed development, cell population     

Photosynthetic and Respiratory Studies During Pod and Seed Development in Pisum sativum L.

The photosynthetic and respiratory potential of Pisum sativumfruit depends on a combination of factors such as fruit age,light intensity and the atmospheric CO₂ concentration. Fruitwere capable of a net CO₂ uptake from the atmosphere only duringthe period of pod extension growth and at light intensitiesgreater than 12 klx. During subsequent development the respiratoryCO₂ evolution attributable to seed growth exceeded the photosyntheticcapacity of the pod. Despite this the extent of fruit CO₂ losswas consistently less in the light than in the dark. An increase in the CO₂ concentration beyond 300 p.p.m. markedlyreduced fruit CO₂ loss and in some instances effected a transitionfrom a net CO₂ output to a net CO₂ uptake. Conversely, a decreasein the CO₂ concentration substantially increased the extentof fruit CO₂ loss. The CO₂ compensation point concentrationincreased with fruit age from 150 p.p.m. to possibly more than550 p.p.m., whereas the corresponding value for the pod (minusseed) remained between 120 and 175 p.p.m. throughout fruit development. A proportion of the CO₂ respired by the seed and pod accumulatedwithin the pod cavity. The CO₂ concentration attained dependedon fruit age and nodal location. In the course of fruit developmentconcentrations within the range 0•1 to 4{dot}3 per centCO₂ occurred during mid-photoperiod. The CO₂ concentration washighest when the enclosed seed had attained approximately 50per cent of their final dry weight.     

豌豆中脱落酸含量、叶片导性与土壤含水量之间的关系

脱落酸与植物水分胁迫的关系已进行了大量的研究,现已明确脱落酸可以抑制气孔的开放(Jones和 Mansfield 1970,Weyers和 Hillman 1979,Henson等 1989),但它的作用机制仍然很不清楚。已有报告指出,当土壤水分亏缺时,ABA可以作为根与地上部间的通讯信号,由很运到地上部并抑制气孔的开放(Blackman和 Davies1985,Gollan等 1986,Zhang和 Davies     

Seed and Pod Wall Development in Pisum sativum, L. in Relation to Extracted and Applied Hormones

Developing fruits of Pisum sativum, L., cv. ‘Alaska’,contain relatively large amounts of hormones, mainly concentratedin the embryos and liquid endosperm. A close relationship canbe demonstrated between changes in extractable amounts of gibberellins(mainly GA₂₀), auxins (methyl 4-chloroindol-3yl acetate andprobably 4-chloroindol-3yl acetic acid), and abscisic acid,and changes in growth rates of both the pod wall and seeds.Growth of the pod wall appears to depend largely on hormonessupplied by the seeds. Marked changes in the germination capacity of the maturing seedsare closely associated with changes in extractable amounts ofmethyl-4-chloroindol-3yl acetate and abscisic acid. It is believedthat high concentrations of these substances in the embryo,rather than any restriction imposed by the testa, may preventprecocious germination of the seeds     

The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro

A highly sensitive immunoassay has been used for the detection of a major storage protein, legumin, in embryos of Pisum sativum L.; with this technique nanogram quantities could be measured. In the two varieties tested, legumin could be detected in embryos in vivo, when they had attained a fresh weight of 2·10^-3 g and 3·10^-3 g, respectively. Contrary to earlier claims, embryos cultured in vitro were shown to be capable of initiating legumin synthesis. This capacity to initiate legumin synthesis was confirmed by two-dimensional isoelectric focusing-electrophoresis and fluorography; embryos harvested before initiation of legumin synthesis and cultured in radioactive medium were shown to have synthesized legumin subunits. The amounts of legumin and total protein synthesized per unit fresh weight were consistently greater in vitro than in equivalent embryos grown in vivo.Abbreviations ELISA Enzyme-linked immunosorbent assay - BSA bovine serum albumin - IgG immunoglobulin - SDS sodium dodecyl sulphate - DSP Pisum cv. Dark Skinned Perfection     

The Photosynthetic and Respiratory Potential of the Fruit in Relation to Seed Yield of Leafless and 'Semi-leafless Mutants of Pisum sativum L.

The photosynthetic and respiratory net CO₂ exchange potentialof the fruit of Pisum sativum was evaluated in one normal andthree mutant genotypes differing widely with respect to foliagearea and morphology: AfAf.StSt, conventional; AfAf.StSt, vestigialstipules; afaf.St.St, all leaflets replaced by tendrils butstipules normal (here termed ‘semi-leafless’) andafaf.stst, tendrils and vestigial stipules (‘leafless’).Agronomically the latter phenotype offered improvement in resistanceto lodging, crop drying, and harvester through-put. On a dry weight basis, fruits of the leafless mutant were consistentlymore active photosynthetically in terms of CO² uptake from theatmosphere during the initial 18 days post-anthesis than werethe corresponding fruits of the other three phenotypes. Duringthe subsequent 16-day period of seed filling there was no markeddifference between phenotypes and the fruit continuously lostCO² to the atmosphere, but significantly less CO² was lost inthe light (40 k lux) than in the dark. That the fruit of theleafless mutant may therefore benefit from the increase in lightavailable to the fruit within a sward canopy comprised of tendrilsin place of leaflets is discussed. However, there was stilla significant reduction in seed yield associated with the leaflessmutant despite a normal complement of ovule initials. The growthcurves and mean dry weights per seed were not significantlydifferent between phenotypes. In fruit of the conventional phenotypethe attainment of maximal pod wall weight coincided with theinitiation of seed fillingonday 15 after anthesis. In a seriesof isogenic lines the gene af delayed maximal wall developmentby 6 days whilst the gene st markedly lowered the maximum wallweight attainable. Possible causes of yield reduction in theleafless mutant are discussed in relation to the observed actionof these genes in the homozygous double recessive condition.     

Gibberellin-Induced Changes in the Water Absorption, Osmotic Potential and Starch Content of Cucumber Hypocotyls

The effects of GA₃ on the water absorption, osmotic potentialand starch content of light-grown cucumber (cv. Aonagajibai)hypocotyl sections were examined. GA₃ (100µM.) stimulatedfresh weight increase as well as elongation. It had no effecton the dry weight change in the presence, or absence, of 50mM sucrose although dry weight increased significantly in thepresence of sucrose. In the absence of sucrose the weight wasunchanged. The osmotic potential of the epidermal cells of sectionsincubated with GA₃ was lower than that of the control, and thedifference between the two values was larger in young seedlings.When sections were incubated with sucrose, the osmotic potentialgreatly decreased. This decrease was more marked in the presenceof GA₃. GA₃ reduced the starch content of the sections bothin the presence and absence of sucrose. The total amount ofstarch, however, was markedly increased in its presence. Thedegradation of starch formed in advance from exogenous sucrosein light was not accelerated by GA₃. We discuss a possible rolefor gibberellin in cell elongation, based on our results, interms of cell water relations. (Received January 18, 1983; Accepted July 19, 1983)     

Carbon Import into Developing Ovules of Pisum sativum: The Role of the Water Relations of the Seed Coat

We tested the hypothesis that the transport of carbon to developingpea ovules is controlled by the water potential of the seedcoat, in both the short-term (minutes to hours) and long-term(days). At 14 d after anthesis, when the embryo just fills theseed coat, the osmotic pressure of seed coat apoplast solutionwas about 1 MPa (equivalent to 400 mOsmol kg^–1). Transportof carbon into perfused attached seed coats at this stage ofdevelopment was monitored with radioactive carbon-11. Aftera small (50 mOsmol kg^–1) increment in the osmotic pressureof the bathing solution, transport of carbon increased abruptly,but after about 100 min it returned towards pretreatment values.Therefore, although osmotic pressure in the sink apoplast initiallyaffected carbon import, as expected from the M     

An analysis of seed development in Pisum sativum. VII. Embryo development and precocious germination in vitro

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.     

Interruption of Water Delivery at Physiological Maturity is Essential for Seed Development, Germination and Seedling Growth in Pea (Pisum sativum L.)

As pea plants (Pisum sativum L.) cv. Finale and Solara developin the field, pods located at the second flowering node, increasein fresh and dry weight until 75 d after planting (DAP); thereafter,dry weight and moisture content decrease rapidly. Seed developmentconsists of three phases, all limited by low moisture content(MQ. The first phase (PI) corresponds to the formation of theembryo and surrounding structure (MC is stable at 85%). Duringthe second phase (P2) the cotyledons are filled (MC decreasesfrom 84% to 55%). The third phase (P3) entails desiccation onthe mother plant. Fresh and dry weight increase until 75 DAP(55% MC or physiological maturity, PM) and rapidly decreaseduring the third phase until 14% MC. Leakage conductivity ofimmature seeds reflects sap arrival in the testa and accumulationin the apoplast it is important during PI and the onset of P2,but during P2 it decreases slowly until physiological maturityand rapidly thereafter. Immature seed germination is noted from62 DAP, increases until 67 DAP, then decreases with a maximumat physiological maturity; some days after physiological maturitygermination is complete (up to 100%). Seedling growth from immatureseeds during P2 is low, but slowly increases until physiologicalmaturity. When germination (up to 100%) is attained seedlinggrowth is normal. Hydration of the immature seed indicates that55% MC is a highly specific value at which a transition in thehydration rate is occurring. Moisture loss from pea seeds duringthe P2 filling stage appears to be necessary for seed adaptationto the abrupt desiccation which occurs at physiological maturity.Physiological maturity is attained when disruption of the vascularconnection between the pod and the mother-plant occurs. Key words: Physiological maturity, germination, growth, pea     

Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L

During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin-coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constitutents. A 23,000 M_r polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including α-mannosidase, α-galactosidase, and β-N-acetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development.     

The effect of the r a and r b loci on the lipid content of the seed of Pisum sativum

Summary The lipid content of seed from a set of isogenic lines for the R _a : r _a locus has been determined; the results show that this locus as well as affecting the starch, sugar and storage protein content and composition, also has a marked effect on the lipid content of the seed. Genotypes having different combinations of alleles at the r _a and r _b loci have also been examined; an r _a r _a r _b r _b , genotype had 5.57% purified total lipid in its seed — a more than 2-fold increase over that found in the round-seeded varieties (R _a R _a R _b R _b ) usually grown for the dry sed crop.     

Seed Production in Leafless and Conventional Phenotypes of Pisum sativum L. in Relation to Water Availability Within a Controlled Environment

Water requirements in relation to seed production was studiedin near-isogenic lines of leafless (afafstst) and conventional(++++) pea plants (Pisum sativum). The plants were grown toseed maturity in pots in a controlled environment under conditionsof high, medium and low irrigation levels. When each genotypewas irrigated independently and on demand and the soil moisturecontent maintained at 65–80 per cent of full capacitythere was no significant phenotypic difference in water useefficiency (WUE), defined as g d. wt seed per kg H₂O utilized.There existed genotypically-controlled upper and lower limitsto yield between which the total dry weight of seed per plantcan be determined by water availability. There was no significantdifferential effect of genotype or of irrigation treatment onthe number of pods, number of seed, unit seed dry weight andbiological yield per plant. There was significant interactionon stem length, and leaf area at specified nodes. When the wateractually required in relation to the water available was takeninto account, the leafless phenotype consistently utilized 33–38per cent less water and produced a correspondingly lower totaldry weight of seed than the conventional counterpart. Independentlyof regime the total dry weight seed per phenotype remained anear constant proportion of the above-ground biomass. Pisum sativum L., garden pea, leafless peas, seed production, water availability     

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.

In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.

We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

     

An efficient in vitro regeneration system of fieldpea (Pisum sativum L.) via. shoot organogenesis

In-vitro regeneration in fieldpea was achieved from immature embryonic axes and cotyledonary node explants of six genotypes on modified MS media supplemented with different concentration of plant growth regulators, 6-Benzylamino purine (BAP) and Naphthalene acetic acid (NAA). The best regeneration response, leading to multiple shoot formation efficiency (22.34 shoots/explant) was observed in the medium supplemented with 1.0 mg/L BAP and 0.2 mg/L NAA and best frequency (67.55?±?4.74) was achieved on medium containing 2.0 mg/L BAP and 0.4 mg/L NAA. The shoots were subcultured on a medium supplemented with a combination of 1.0 mg/L GA₃, 2.0 mg/L BAP and 0.4 mg/L NAA, which resulted in elongation of 85 % of shoots. Rooting attempted from the elongated shoots, on half strength MS medium and supplemented with three different auxins IBA, IAA and NAA separately, exhibited similar results. Alternatively, micro-grafting of in vitro regenerated shoots onto pre-germinated root stocks raised in green house facility was attempted with high success rate (75 %). The grafted plants could be successfully hardened, fertigated with Hoagland solution and distilled water in a ratio of (1:10) for acclimatization and further development. All the genotypes tested, produced multiple shoots that could be established to mature fertile plant, hence, the medium combinations used were found to be genotype neutral.