Effect of Pulp Cell Number and Assimilate Availability on Dry Matter Accumulation Rate in a Banana Fruit [ Musa sp. AAA group 'Grande Naine' (Cavendish subgroup)]

Fruit position on the bunch (inflorescence) is an importantpart of variability in banana fruit weight at harvest, as fruitsat the bottom of the bunch (distal fruits) are approx. 40% smallerthan those at the top (proximal fruits). In this study, therespective roles of cell number and cell filling rate in thedevelopment of pulp dry weight are estimated. To this end, thesource/sink ratio in the plant was altered at different stagesof fruit development. Leaf shading (reducing resource availability),bunch bagging (increasing sink activity by increasing fruittemperature), and bunch trimming (decreasing sink size by fruitpruning), applied once cell division had finished, showed thatthe pulp filling rate depends on resource availability. Bunchbagging and bunch trimming were also carried out before theend of cell division to estimate the role of pulp cell numberin the development of pulp dry weight. A sampling method wascalibrated to evaluate pulp cell number from the digestion ofa fixed portion of the pulp in a solution of chromic and nitricacids. A relationship was found between pulp cell number andfruit length at the end of cell division. It was observed thatpulp cell number is a determining factor in pulp dry weightvariability within a bunch. On the other hand, the cell fillingrate was identical for all fruits in the bunch and was influencedby the source/sink ratio. A Michaelis-Menten relationship wasinvoked to relate the cell filling rate in a bunch to the source/sinkratio during bunch filling. Copyright 2001 Annals of BotanyCompany Banana fruit, Musa sp., fruit growth, cell number, cell filling rate, source/sink ratio, temperature     

复方壮果素对香蕉果实生长发育和产量的效应

多年多点试验结果表明,在香蕉断蕾5～7天喷施复方壮果素,对促进香蕉果指生长和提高产量都有明显的效果;喷施壮果素的单指鲜重、果肉重以及果肉中的千物质和淀粉积累基本上能与果指长度、粗度同步增加,且不影响香蕉果实外观和品质。     

Effect of Post-anthesis Drought on Cell Division and Starch Accumulation in Developing Wheat Grains

Wheat plants (Triticum aestivum L., cv. Warigal) were subjectedto 20 d of water deficit during the period of endosperm celldivision. Drought accentuated the differences in final grainweight between spikelets and between grains within spikelets.The distal grains of top spikelets were most affected by drought.The maximum number of endosperm cells was, respectively, 30and 40 per cent lower in basal grains and distal grains of draughtedplants. In basal grains of middle spikelets, the number of largestarch granules per cell was unaffected but the number of smallstarch granules per cell was 45 per cent lower in grains ofdraughted plants. The initiation of small starch granules wasmore affected than cell division because severe water deficitoccurred earlier during the former process than the latter.Final dry weight appeared to correlate well with the maximumnumber of endosperm cells, but depended also on the number ofstarch granules per cell. Consequently, the amount of dry matterper cell was not constant in both treatments. The concentration of sucrose per endosperm cell was lower onlyin the droughted distal grains of top spikelets. The supplyof sucrose to endosperm cells did not regulate the initiationof small starch granules. Triticum aestivum L., wheat, drought, grain growth, cell division, starch     

Do Genetic Make-up and Growth Manipulation Affect Tomato Fruit Size by Cell Number,or Cell Size and DNA Endoreduplication?

This work investigated the link between genetic and developmental controls of fruit size and composition. On two isogenic lines (CF12-C and CF14-L), differing by fruit weight and sugar content quantitative trait loci (QTLs) identified previously, basal and tip fruits were characterized at anthesis and at maturity through their growth, dry matter and sugar content, number and size of cells and nuclei DNA content. The influence of competition was assessed by removing either basal or tip ovaries at anthesis. On an intact inflorescence, CF12-C fruits grew less than CF14-L fruits, with 1.67 fewer cell layers and similar cell size, suggesting that genes controlling cell division may be responsible for this fruit size variation. Truss thinning masked the QTL effect on fruit size, mainly by reducing the difference in cell number between the two lines and by promoting cell expansion in tip fruits, so that fruit growth was similar at both positions and for both lines. Thus, in these lines, cell number exerts a control on final fruit size only when there is competition among fruits. Different responses of basal and tip fruits after flower removal suggested that this treatment induced changes in hormonal relationships within the truss. No fixed relationship between DNA endoreduplication and cell size was found, as while cell size and dry matter and sugar contents differed with tomato lines, fruit position and truss size, endoreduplication patterns were the same. CF12-C fruits had a higher dry matter (+0.3% of fresh weight) and carbohydrates (+8% of dry matter) content than CF14-L fruits. The percentage dry matter was independent of truss size but decreased slightly from basal to tip fruits.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.     

Effect of Fruit Load on Partitioning of Dry Matter and Energy in Cantaloupe (Cucumis melo L.)

Cantaloupe (Cucumis melo L.) plants set groups of fruits whichgenerate large variations in the reproductive:vegetative dryweight balance. We studied the influence of fruit number onthe partitioning of dry matter and energy between the vegetativeand reproductive organs and among the seeds and the variousfruit tissues during the development of the first fruits. Over2 years and on two Charentais cantaloupe cultivars, fruit numberwas either limited to one or left unrestricted, which led tothe setting of two to six fruits. Because of the high lipidcontent in seeds, the distribution of assimilates was studiedin terms of energy equivalent as well as dry weight. Measureddry weights were converted into energy equivalents by calculatingthe construction cost of tissues from their elemental composition.Seeds differed from other tissues in showing an increase inconstruction cost, from 1.1 to 1.8 g CH₂O g^-1d. wt between 10and 30 d after pollination. For this reason, during the secondhalf of fruit development on plants with unrestricted fruitload, they made up to 31% of the fruit and 12% of the aerialpart of the whole plant in terms of dry weight, but 39 and 18%in terms of energy (glucose equivalents). The fraction of assimilatesallocated to the fruits showed a saturation-type response tothe number of fruits per plant. It did not increase in cultivarTalma above two fruits per plant, which could be due to a decreasingsink strength with fruit rank, whereas cultivar Galoubet maintaineda more homogeneous fruit size within plants. At a similar fruitload, the reproductive:vegetative dry weight balance differedbetween the 2 years of the experiment, probably because of variationin the fruit sink strength. Copyright 1999 Annals of BotanyCompany Charentais cantaloupe, Cucumis melo L., assimilate distribution, construction cost, development, dry matter partitioning, fruit load, seeds, sink strength.     

香蕉果实成熟软化时果皮和果肉中α-L-阿拉伯呋喃糖苷酶活性的变化

阿拉伯糖是果实软化过程中变化最明显的细胞壁糖残基之一,α-L-阿拉伯呋喃糖苷酶是导致细胞壁多糖中阿拉伯糖残基降解的主要糖苷酶。为阐明该酶在香蕉果实成熟软化中的作用,实验对香蕉贮藏过程中果皮和果肉中该酶活性以及果实硬度、呼吸强度和乙烯释放量的变化进行了研究。结果表明:α-L-阿拉伯呋喃糖苷酶在果实初期的变化很小,到果实硬度开始急剧下降时达到最大,增加量达10倍以上,且果肉中的酶活性大于果皮中;乙烯吸收剂处理延缓了香蕉果实呼吸和乙烯高峰的出现时间,降低了果实硬度、果皮和果肉中α-L-阿拉伯呋喃糖苷酶活性变化的速度和幅度。以上结果表明α-L-阿拉伯呋喃糖苷酶起诱导香蕉果实成熟的作用,在果实的软化中起着十分重要的作用,且其活性受乙烯的调节。     

The Dynamics of Growth and Dry Matter Distribution in Cucumber

The dynamics of growth and proportional dry matter distributionbetween the vegetative parts and fruits of cucumber were studieddaily during a growing season. Most of the changes in dailyintegral of total solar radiation were reflected by changesin plant growth rate. Sometimes a time lag of a few days occurred,indicating the plants were adapting to radiation. The proportional dry matter distribution between fruits andvegetative parts showed a cyclic pattern. The daily proportionaldry matter distribution to the fruits varied between 40 and90% of the total dry matter. However, the cumulative dry weightof the fruits was fairly constant at 60% of the cumulative plantdry weight. The daily proportional dry matter distribution did not seemto be linked directly to the climate conditions (temperature,CO₂ concentration, relative humidity or daily light integral).The proportional distribution to the fruits showed a clear positivecorrelation with the fruit load (number and weight of fruits)on a plant. The number of fruits on a plant changed considerablyduring the growing season. This number was limited not by theformation of new fruits but by abortion of fruits within about10 d after flowering. The number of young fruits that did notabort appeared to correlate positively with the growth rateof the vegetative parts. Cucumis sativus (L.), cucumber, biomass allocation, partitioning, vegetative-generative growth, fruit growth, fruit abortion     

Characterization of multiple forms of α-glucan phosphorylase from Musa paradisiaca fruits

Three forms of α-glucan phosphorylase from mature banana fruit pulp separated by ammonium sulfate fractionation and DEAE-cellulose chromatography were anodic at pH 8·6 on starch gel electrophoresis. The three forms differed in sensitivity to the phenolics extracted from immature and mature banana fruit pulp. Only two forms of the enzyme were detected in immature banana fruit pulp.     

Vegetative Parthenocarpy in the Cactus Pear Opuntia ficus-indica (L.) Mill.

The dependence of fruit development on fertilization was studiedin two clones of Opuntia ficus-indica, Ofer and BSI. Fruitsof the clone Ofer bear fully developed seeds, whereas fruitsof the clone BSI contain only degenerated seeds and it was suspectedthat BSI is parthenocarpic. The two clones differed in theirpattern of fruit development. The increase in fruit fresh weightin Ofer was a result of both peel and pulp growth, whereas inBSI fruit growth was mainly due to pulp growth and the peelhad reached its final weight almost at anthesis. Pulp growthin BSI commenced earlier and was faster than in Ofer, but thefinal pulp weight in BSI was only 64% of that in Ofer. Seedgrowth in BSI was limited to the development of semilignifiedand lignified seed coats, whereas in Ofer 43·5% of theseeds were completely developed, the remainder being similarto those found in BSI. Fruits bearing well-developed seeds werealso found in a 'mutant' of BSI that, like Ofer, contained smallerovules at anthesis than flower of the regular BSI genotype.Germination in vivo and in vitro were similar in BSI and Ofer,but pollen tubes failed to reach the ovules in the regular BSIgenotype, while penetrating the ovules in Ofer and the small-ovuledgenotype of BSI. Good-quality fruits similar to Ofer fruitsin weight but with a higher peel to pulp ratio developed inBSI after flower-sterilization in the spring and in the autumn.In Ofer, sterilized flowers failed to develop fruits in springand partially set fruit in the autumn; the fruits consistedalmost exclusively of peel tissue. It was concluded that BSIis a vegetative parthenocarpic clone, i.e., that pollinationis not required for fruit set and development.Copyright 1993,1999 Academic Press Opuntia ficus-indica, cactus pear, fruit development, parthenocarpy, pollen-tube-growth, fluorescence microscopy     

The Growth and Yield Responses ofFragaria ananassato Elevated CO2and N Supply

Strawberry plants (Fragaria ananassaDuchesne var. Elsanta) weregrown in pots at two concentrations of carbon dioxide (partialpressures of 39 and 56 Pa) and with three rates of nitrogensupply (0.04, 0.4 and 4 mMas nutrient solution) to study theirindividual and interactive effects on plant growth and fruityield. Nitrogen deficiency reduced total dry biomass and relativegrowth rate (RGR), mainly through reductions in leaf area ratio(LAR) and plant N concentration (PNC), although both the netassimilation rate (NAR) and root weight ratio (RWR) increased.Elevated CO₂increased the N productivity (NP) but reduced theLAR. High CO₂increased the fruit yield by 42% at high N supplyand by 17% at low N supply. The CO₂yield enhancement occurredthrough an increase in the flower and fruit number of individualplants. This resulted in an increase in the fruit weight ratio(FWR) of plants at high CO₂. Nitrogen deficiency reduced thefruit yield by about 50% through decreases in fruit size, fruitset and the number of fruits. However, N deficiency increasedthe proportion of total plant dry biomass allocated to fruits.There were no significant interactions between CO₂and N supplyon yield.Copyright 1998 Annals of Botany Company Nitrogen; carbon dioxide; strawberry (Fragaria ananassaDuchesne); fruit yield.     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)     

Some Morphological and Chemical Characteristics of Developing Fruits of Raphia hookeri

Ndon, B. A. 1985. Some morphological and chemical characteristicsof developing fruits of Raphia hookeri.—J. exp. Bot. 36:1817–1830. Fruits which were at different stages of development were randomlysampled from different inflorescences of Raphia hookeri palms.The morphological characteristics and chemical (the dry matter,lipid and carbohydrate) contents of the exocarp and seeds weredetermined. The results showed that the seed length, circumferenceand volume were optimal at 24 months after pollination whichindicates that Raphia seeds attained maximum size at that period.The seed endosperm was liquid or semi-liquid between 6–18months after pollination but became solid with a prominent embryoat 24 months. The seed dry matter was low at the early stagesof development but there was a rapid increase in seed dry weightat 18–33 months after pollination. The seeds were physiologicallymatured at 30–33 months after pollination, while the exocarpmatured at 24–30 months after pollination. The Raphia seeds were low in lipid (about 2%) compared to theexocarp which had 30–40% lipid at full maturity. Maximumamount of lipid was accumulated within the exocarp at 36–42months after pollination and this period indicates the timefor harvesting Raphia fruits for maximum oil which is probablythe most economic part of the fruit. The total sugar concentration increased in the exocarp withincrease in maturity. Conversely the concentration of sugarsdecreased within the seeds as the fruit matured. Maximum totalsugar concentration (about 309 mg g^–1 dry fat free sample)was found in the exocarp at 36–42 months after pollination.Mature seeds at 48 months after pollination had about 50 mgof total sugars per g of fat free sample. There was insignificantaccumulation of starch in the exocarp. The mature seeds werelow in starch (5–10% of the dry weight). Key words: Raphia hookeri, development, fruit     

Fine mapping of QTLs of chromosome 2 affecting the fruit architecture and composition of tomato

Negative correlations between quality traits and fruit size may hamper the breeding of fresh market tomato varieties for better organoleptic qualities. In a recent QTL analysis, QTLs with large effects on fruit weight, locule number and several quality traits were detected in the distal 50 cM of chromosome 2, but favorable alleles for fruit weight and locule number were unfavorable to quality traits. Substitution mapping was undertaken to determine whether the effects were due to a single QTL or to several tightly linked QTLs. Several chromosomal segments were characterized using near-isogenic lines. Five of them appeared to be involved in one or several traits. Considering the five segments from the top to the bottom of the region, the QTLs detected in each segment controlled the variation of: (1) fruit weight, (2) soluble solids content and dry matter weight, (3) fruit weight, (4) locule number and (5) fruit weight, dry matter weight, total sugars, titratable acidity and soluble solids content. This last cluster illustrates an antagonism between fruit weight and four quality traits, as favorable alleles are not conferred by the same parent in both cases. Nevertheless, several antagonistic QTLs were separated from each other in the first four segments, holding the promise for marker-assisted improvement of fruit quality traits without compromising the fruit size.     

Effects of Seed Number on Competition and Dominance among Fruits in Capsicum annuum L.

The effects of seed number on set, development and growth ofa fruit, and on inhibition of later-developed fruits were studiedby varying the pollen load on the stigma of sweet pepper flowers(Capsicum annuum L.). Despite much variation, a linear increasein individual fruit weight with seed number could be observed.Seed number affected the growth rate rather than the growingperiod of fruit. When seed numbers were low, the probabilityof fruit setting was positively related to seed number. However,a relatively low seed number (50–100 seeds/fruit: 20–30%of the maximum seed number) was sufficient for maximal fruitset. An increase in seed number increased the inhibitory effect ofa fruit on set and growth of later-developing fruits. As a result,when pollination treatments were applied to all the flowersof a plant, results could be quite different to those obtainedwhen only a limited number of flowers were treated. Fruit setof the second fruit was reduced by the application of a highpollen load to the first flower, even when the first fruit abortedbefore it had accumulated much dry matter. Our results suggestthat growth inhibition of the second fruit by seed number ofthe first fruit is controlled both by competition for limitedassimilates, as well as by dominance due to the production ofplant growth regulators by the developing fruit. Sweet pepper; Capsicum annuum L.; pollination; fruit set; abortion; abscission; fruit growth; first-fruit dominance; sink strength     

The effect of assimilate supply on fruit growth and hormone levels in tomato plants

Rates of dry matter accumulation and contents of starch, sugars and abscisic acid (ABA) of tomato fruits differed significantly during development at three positions (proximal, middle and distal) on a truss. Proximal fruits, which accumulated dry matter most rapidly during early development, generally had least ABA (per g DW).Partial defoliation reduced carbon accumulation by all fruits but increased ABA, especially in distal fruit, and indoleacetic acid (IAA), particularly in proximal fruits. The ABA content of leaves in partially defoliated plants was similar to that of leaves on non-defoliated plants.Removal of distal fruits on a truss enhanced carbon movement to the remaining proximal fruits and also increased their ABA content early in development but did not affect their IAA content. On the other hand, when proximal fruit were removed there was no large or lasting increased accumulation of carbon by the remaining distal fruits and they contained less ABA and IAA than fruits on plants without fruit thinning. Leaf carbon and ABA levels showed no marked trend in response to fruit thinning.The amount of carbon in the stems was increased by fruit thinning but decreased by partial defoliation.The possible roles of ABA and IAA in regulating fruit growth are discussed.Part of this work has been presented to a Symposium on Phloem loading and related processes at Bad Grund/Oberhar, W. Germany, July 1979.     

Maximum Fruit Growth Potential and Seasonal Patterns of Resource Dynamics During Peach Growth

Maximum fruit growth potential, the growth attained by fruitswhen they are grown under optimal environmental conditions inthe presence of a non-limiting supply of resources, was estimatedfor two peach [Prunus persica (L.) Batsch] cultivars that differin the timing of resource demand for reproductive growth. Maximumpotential fruit growth was estimated on trees that were heavilythinned at bloom. On these trees, resource availability exceededresource demand for fruit growth. For both cultivars, the mean dry weights of fruits grown onunthinned trees were approximately half the mean dry weightsof fruits grown on trees that were heavily thinned at bloom,indicating that fruit growth was source-limited on unthinnedtrees. Comparison of the seasonal patterns of relative growthrate of fruits on unthinned and heavily thinned trees indicatedthe source-limited fruit growth occurred during distinct periodsof the growing season. On the early maturing cultivar, source-limitedfruit growth occurred from 300 degree-days after bloom untilharvest (4·5-10 weeks after bloom). On the late maturingcultivar, source-limited fruit growth occurred from 200-900and 1600-1900 degree-days (3·5-12 and 18-20 weeks) afterbloom. Although the final dry weight of fruits on the early maturingcultivar was only half that of fruits on the late maturing cultivar,the potential net sink strength of fruits was significantlyhigher on the early than the late maturing cultivar throughoutthe entire growth period of the early maturing cultivar. Resourceavailability for fruit growth was similar on the early and latematuring cultivars, indicating that selection for early maturingfruits has not changed the patterns of resource availabilityfor fruit growth.Copyright 1995, 1999 Academic Press Maximum fruit growth potential, carbon economy, partitioning, resource availability, resource limitation, source-limited growth, sink activity, sink strength, growth analysis, relative growth rate, Prunus persica (L.) Batsch, peach     

Seasonal Evolution of the Quality of Fresh Glasshouse Tomatoes under Mediterranean Conditions, as Affected by Air Vapour Pressure Deficit and Plant Fruit Load

Changes in yield and quality of fresh tomatoes in response toair vapour pressure deficit (VPD) and plant fruit load werestudied under Mediterranean summer conditions. Plants thinnedto three or six fruits per truss were grown in two compartments,one at a VPD below 1.5 kPa, the other without VPD control. Theseasonal trend in fruit yield and quality was assessed fromApril to September by weekly measurement of number, fresh weightand dry matter content of harvested fruits, together with theoccurrence of blossom-end-rot (BER) and cracking. On two occasions,in July and September, sugar and acid content was measured atthree ripening stages. The seasonal decrease in fresh yieldwas attenuated at low VPD, because of higher individual fruitfresh weight, especially at low fruit load. Low VPD decreasedoccurrence of BER but like low fruit load, it increased fruitcracking. Fruit dry matter content was lower at low VPD, butwas unaffected by fruit load. Sugar content and the ratio ofsugars:acids was increased at high VPD and low fruit load, withinteractive effects depending on season and ripening stage.The influence of VPD on acid content differed with fruit loadand also changed during ripening and between seasons. Resultsshowed that water was the main limiting factor for growth offruits picked in July; at this time, reducing fruit load topromote mean fruit size had negative effects on BER and cracking.Reducing VPD reduced BER but had a negative effect on crackingand diluted both the dry matter and sugar content. For fruitsharvested later in summer, these negative effects were attenuatedbecause fruit growth was also carbon limited. Copyright 2000Annals of Botany Company Lycopersicon esculentum Mill., tomato, water and carbon stress, yield, quality, dry matter, sugar, acid, BER, volatile composition     

Biocide application for increasing storage periods and maintaining quality and chemical constituents of banana fruits

Banana fruits were treated with biocides formulated from essential oils of anise, coriander or black cumin seeds. Treated and non-treated fruits were stored at temperatures of 5, 10, 15 and 20°C. Samples were stored for periods of 10, 20, 30, 40, 50 and 60 days. The collected samples in each interval were subjected to estimation of decay development and quality degree, in addition to the chemical constituents of starch, sugars, vitamin C of pulp and peel chlorophyll content. Results reveal that non-treated banana fruits decayed continuously by lapse of storage periods. However, this decay was significantly delayed by lowering the storage temperature. Soaking banana fruits in the tested biocides showed a positive potential for interrupting the decay in stored banana fruits and this promising impact was much more pronounced at lower storage temperature. Further, biocide treated banana fruits kept their good quality for longer storage periods compared to non-treated fruits, due to lower ripening rates which promisingly prolonged shelf-life. As the starch content gradually decreased during storage, the total sugar content increased. On the other hand, biocide application retarded the conversion of starch into simple sugars, especially at the low temperature rate. Additionally, the imposed treatments maintained vitamin C in banana pulp and lowered the decline in peel chlorophyll content.     

Experiments on Banana Fruit Development

A number of synthetic auxins and auxin-antagonists were appliedin lanolin emulsions to young banana fruits and their effectson fruit development and seed fertility estimated. Auxins stimulatedthe formation of edible pulp in both seedy and parthenocarpicbananas and induced varying degrees of seed sterility. Of theauxin antagonists tried, coumarin (contrary to a mistaken earlierreport) stimulated parthenocarpy and induced seed sterility,three others were not or but slightly active, and thiourea markedlyinhibited parthenocarpy. Study of the relation between sizeof mature fruit and seed content indicates that developing seedsstimulate the growth of pulp and that the amount of stimulationis roughly proportional to the level of parthenocarpy. Fruitdevelopment in the banana seems to fit in with Luckwill's generalscheme of dual hormonal stimulus.