Phloem translocation in regenerating in vitro- heterografts of different compatibility

Phloem translocation of [¹⁴C]-sucrose and 5/6-carboxyfluorescein(CF) from scion into the stock was studied in in vitro-heterograftsof Lycopersicon on Solanum (L/S) and Vicia on Helianthus (V/H)at various stages of regeneration. Autografts of all partnersserved as controls. Corresponding with the translocation experimentsnewly formed sieve-tube connections between the graft partnerswere counted. ¹⁴C-translocation experiments with [¹⁴C]-sucrose revealed anage-dependent increase of radioactivity in the stock of allcombinations. In L/S and all autografts the major increase of¹⁴C-label in the stock occurred 5–10 d after grafting.In V/H, however, import of label into the stock remained lowthroughout the regeneration period. In L/S grafts, increasesin the numbers of sieve-tube connections parallel the increasingrate of ¹⁴C-transport, indicating functioning sieve-tube connectionsin the graft union. In contrast, V/H grafts did not show thisstrong correlation between structure and function of wound repairphloem. This suggested the existence of non-transporting sieve-tubesbetween the graft partners. Similar results were obtained withCF-transport, showing that effective phloem translocation acrossthe graft interface occurred in L/S, but not in V/H grafts.The observed differences in phloem translocation are discussedwith regard to compatibility/incompatibility phenomena in heterografts. Key words: Compatibility/incompatibility, in-vitro-heterografts, phloem transport ([¹⁴C]-sucrose, carboxyfluorescein), wound phloem     

Prevention of auxin-induced vascular differentiation in wound callus by surface-to-surface adhesion between calluses of stock and scion in cactus grafts

The relationship between formation of CVB, which connects vascularcut ends of stock and scion, and the condition of tissue whereCVB is expected to be induced was studied using a cactus graftcombination of Notocactus submammulosus var. pampeanus youngseedlings on Hylocereus trigonus plants. When a scion with its basal part removed by a transverse cutwas put onto a transverse cut surface of a stock with a distanceof 1 mm between the vascular cut ends of both graft components,CVB was formed in only 13% of the grafts in the callus massmade by adhesion between calluses of stock and scion. The percentagearose to ca. 100% when 100 ppm NAA was applied to the scionapices. This promotive effect of NAA decreased with increasingtime from grafting to NAA application. When wound calluses of stock and scion were prevented from contactonly at the portion between the vascular cut ends of stock andscion by introducing a piece of aluminium foil into the graftunion, CVB was formed in 80% of the grafts in the callus ofthe stock without NAA application. In the case where the scionwas grafted at right angle onto an additional longitudinal cutsurface made 1 mm from the vascular bundle of the stock withremaining intact parenchyma between this cut surface and thevascular bundle of the stock, CVB was not formed even when NAAwas applied. These results suggested that wound response wasa prerequisite for CVB differentiation and that surface-to-surfaceadhesion of calluses inhibited CVB formation by making calluscells differentiate into quiescent parenchyma cells rapidly.The mechanism of this phenomenon is discussed. (Received September 29, 1977; )     

Scion Control of Genotypic Differences in Mineral Salts Accumulation in Soyabean (Glycine max L. Merr.) Seed

Stem-root grafts of seedling plants were used to ascertain thatgenotypic differences in P, Mg, Mn, and B accumulation in soyabean(Glycine max L. Merr.) seeds are controlled by the scion ofthe plant. The effect of the graft per se on mineral accumulationwas negligible. These results are similar to those reportedfor Sr and Ca accumulation in soyabeans. Mechanisms which couldaccount for these observations are briefly discussed.     

Graft formation in Solanum pennellii (Solanaceae)

Three phases of cohesion were observable during the development of compatible autografts in Solanum pennellii. Phase I cohesion 1) lasted 4–5 d after grafting, 2) was characterized by an average increase in tensile strength of 4 g breaking weight (BW) mm^–2 graft area (GA) d^–1, and 3) correlated positively with cellular interdigitation at the graft interface. The fresh weight of the scion increased by approximately 5% d^–1 during the first 2 d after grafting. Phase II cohesion occurred 5–15 d after grafting, during which time 1) the tensile strength of the graft union increased by 14 g BW mm^–2 GA d^–1, 2) vascular differentiation across the graft interface was completed, and 3) the fresh weight of the scion increased by 9% d^–1. Phase III cohesion occurred subsequent to 15 d after grafting, during which time 1) the tensile strength of the graft union leveled off at a value similar to that of an ungrafted internode, and 2) the fresh weight of the scion increased by 14% d^–1. These results are discussed relative to mechanisms underlying the formation of compatible grafts.     

The effect of applied heat on graft union formation in dormant Picea sitchensis (Bong.) Carr

The effect of application of heat on the development of graftunions in dormant Picea sitchensis (Bong.) Carr (Sitka spruce)was observed at monthly intervals from October to March. Noadvantage over controls was gained in terms of final graft successrates. It is suggested that this may have been a result of themild winter conditions during the course of the experiment,which enabled callus formation to occur in unheated controls.On the other hand, scions of grafts which had been heated for3 weeks, and then returned to ambient conditions showed considerablygreater growth than unheated controls during the following springand summer. The amount of extension depended on how late inthe winter the graft had been prepared and heated, with leastgrowth by grafts made in October, and most by grafts made inMarch. Microscopical examination showed that callus formationwas more rapid in heated grafts than in controls, although callusformation occurred in all control grafts examined. Heat alsoinduced cambial reactivation and tracheid formation, particularlyin the vicinity of needle or bud traces in the scion. In keepingwith this, it was found that the presence of needles and budswas essential for cambial reactivation in normal dormant stemsand that reactivation was restricted to the heated region. Key words: Grafting, Sitka spruce, callus, dormancy, differentiation     

The Role of Direct Cellular Contact in the Formation of Compatible Autografts inSedum telephoides

In order to determine the importance of direct cellular contactfor the formation of a compatible graft, I have monitored thedevelopment of compatible autografts in Sedum telephoides inwhich porous membrane filters were inserted between the graftingsurfaces. The tensile strengths of these grafts levelled offat approximately 10 g breaking weight per square millimetregraft area baween 2 and 5 d after grafting. Callus proliferationoccurd at both grafting surface by 5 d after grafting. By 21d after grafting, vascular redifferentiation across the graftinterface was complete. These results indicate that direct cellularcontact is not necessary for the (I) cohesion of the stock andscion, (2) callus proliferation or (3) vascular redifferentiationthat occur during the formation of a compatible graft. Sedum telephoides, Crassulaceae, grafting, vascular redifferentiation, compatibility     

Root Water Uptake, Leaf Water Storage and Gas Exchange of a Desert Succulent: Implications for Root System Redundancy

A technique used for hydroponics was adapted to measure instantaneousroot water uptake from the soil for a leaf succulent CAM species,Agave deserti. Comparisons were made to previously modelledwater fluxes for A. deserti and to Encelia farinosa, a non-succulentC₃species. Net CO₂uptake and transpiration forA. deserti underwell-watered conditions occurred primarily at night whereasroot water uptake was relatively constant over 24 h. Leaf thicknessdecreased when transpiration commenced and then increased whenrecharge from the stem and soil occurred, consistent with previousmodels. A drought of 90 d eliminated net CO₂uptake and transpirationand reduced the water content of leaves by 62%. Rewetting theentire root system for 7 d led to a full recovery of leaf waterstorage but only 56% of maximal net CO₂uptake. Root water uptakewas maximal immediately after rewetting, which replenished rootwater content, and decreased to a steady rate by 14 d. Whenonly the distal 50% of the root system was rewetted, the timefor net CO₂uptake and leaf water storage to recover increased,but by 30 d gas exchange and leaf water storage were similarto 100% rewetting. Rewetting 10 or 20% of the root system resultedin much less water uptake; these plants did not recover leafwater storage or gas exchange by 30 d after rewetting. A redundancyin the root system of A. deserti apparently exists for dailywater uptake requirements under wet conditions but the entireroot system is required for rapid recovery from drought.Copyright1999 Annals of Botany Company Agave deserti Engelm., desert, drought, gas exchange, rewetting, roots, succulent, water uptake.     

A Study of the graft union in in vitro micrografted apple

Light microscopy was used to study graft union formation in in vitro micrografts of tissue cultured apple (Malus domestica. Borkh). Micrografts were constructed using horizontal incisions to form the grafting surface, and placing the cut ends of rootstock and scion into sterile silicon tubing to permit graft formation to occur.The outer morphological and histological development was similar for different stock-scion micrograft combinations but graft union formation was slower in heterografts than in autografts. Initial leaf expansion at the scion shoot apex occurred in all micrografted plantlets within 1–4 days and was not indicative of graft success. Progressive scion growth and development could be used as an indication of graft success by ten to fourteen days after grafting and probably was related to establisment of cell to cell contact at the graft interface. Microscopy showed initiation of callus proliferation in the vascular cumbium and the pith ten days after grafting. Differentiation was observed subsequently and this was reflected in scion development. Longitudinally orientated cambial cells began to differentiate between twenty and forty days after grafting, and formed a bridge between the vascular cylinders of scion and rootstock. The scions at this stage had as many as eight newly expanded leaves and micrografts were strong enough to permit silicon sleeve removal without damage. Continuity of new vascular elements in rootstock and scion was established around forty days. New vascular elements curved slightly in towards the pith to form a c shaped bridge across the graft union. Vascular development continued until it reached completion after six months.     

Some Physiological Characteristics of Two Tomato Cultivars, One Tolerant and One Susceptible to Tobacco Mosaic Virus

A comparison was made of physiological activity in the tomatocv. Potentate, susceptible to tobacco mosaic virus (TMV) withthat in cv. Virocross which is tolerant. Growth analysis overa 5-week period showed Potentate to have a higher relative growthrate and a higher net assimilation rate than Virocross. In reciprocalgrafts scion growth, including dry weight, was greater on Potentaterootstocks than on Virocross. Growth responses were studiedin these grafts following foliar sprays of N-6 benzyladenine(BA) and gibberellic acid (GA). The response of the scion toGA was similar in both cultivars and there was no interactionbetween GA and rootstock for scion growth. An interaction betweenstock and BA was found for leaf growth. Bleeding sap from Virocross contained more gibberellins, morecytokinins, and a lower concentration of amino compounds, especiallynon-protein acids, than Potentate. There were more gibberellinsin the roots of Potentate but no difference between shoots ofthe two cultivars. In reciprocal grafts TMV multiplied more rapidly in Virocrossscions on Potentate rootstock than on their own, but Virocrossstocks did not affect virus multiplication in Potentate scions.Spraying Potentate plants with BA reduced the rate of virusmultiplication. It is suggested that virus multiplication in the shoot may becontrolled by two distinct root factors, viz, the supply ofnative cytokinins and the amount and nature of the nitrogenouscompounds which directly influence the rate of shoot growth.     

Synthesis and Characterization of Nicotiana-Solanum Graft Chimeras

Intergeneric Nicotiana tabacum L./Solanum laciniatum Ait. graftchimeras were produced from decapitated grafts made betweenthese two graft compatible species. Graft unions were treatedwith or without either the auxin, p-chlorophenoxyacetic acid(p-CPA) or the cytokinin, benzylaminopurine (BAP). While BAPwas inhibitory to shoot formation, p-CPA increased the numberof adventitious shoots and raised the frequencies of shoot-forminggrafts obtained. Approximately 9% (14/151) of the shoots producedat p-CPA-treated graft unions were intergeneric chimeras. Theauxin application significantly increased levels of chimeralshoot recovery numbers thus indicating a direct auxin effecton chimeral shoot production. The types and numbers of chimerasproduced were independent of the scion/stock graft combinationemployed. All chimeras appeared to arise initially as mericlinalor sectorial chimeras, with a proportion of the former ones(5/14) stabilizing into periclinal chimeras. The morphologicalcharacteristics of these latter chimeras were compared withthose of the two parental species. The LII layer of speciesdetermined the characteristics of the vegetative and floralcomponents. However, the LI layer modified the qualitative traitsof both components. The LIII layer on the other hand alteredthe quantitative traits of the vegetative organs and the flower.This layer also determined the growth type, breeding behaviourand inflorescence type of synthesized chimeras. These organizationalfunctions of the LIII layer have not been reported previously. Chimeras, intergeneric chimera synthesis, grafting, graft chimeras, Nicotiana tabacum, Solanum aviculare, p-chlorophenoxyacetic acid, peroxidase isoenzymes     

Graft Formation in Sitka Spruce: A Scanning Electron Microscope Study

Low-temperature and conventional scanning electron microscopyhave been used to examine the callus formed at the graft interfacein Picea sitchensis (Bong.) Carr. Callus cells are producedby both cambium and ray parenchyma dedifferentiation and redifferentiationin scion and stock. Adhesion between the cells derived fromscion and rootstock is thought to be my means of pectinaceousbeads at the surface of the callus cells, preceding a more generalfusion of cell walls. The cambia of the two graft componentsare prevented from growing towards each other by the presenceof callus. Instead, the differentiation of new cambium withinthe callus, in the vicinity of the cambia exposed at the preparedsurfaces of the scion and rootstock, links them to form a continuouscambial layer around the combined stem. Callus, cambium, differentiation, grafting, Picea sitchensis     

Graft Formation in Kalanchoe blossfeldiana

Three phases of cohesion between the stock and scion are observableduring the formation of compatible autografts in Kalanchoe blossfeldiana.The first phase of cohesion: (a) lasts four to five days, (b)is correlated with an accumulation of dictyosomes along thegraft interface and with callus proliferation in the stock andscion, and (c) is characterized by a tensile strength of approximately5 g breaking weight (BW) mm^–2 graft area (GA) by 5 d aftergrafting. The second phase of cohesion lasts from days 5–20after grafting and is correlated with (a) an interdigitationof callus cells at the graft interface, (b) the differentiationof vascular tissue across the graft interface, and (c) a 20-foldincrease in the tensile strength of the graft union to approximately100 g BW mm^–2 GA by 20 d after grafting. This cohesivestrength is comparable to that of an intact, non-grafted stem.The third phase of cohesion is characterized by a levellingoff of the increase in tensile strength of the graft union withtime at approximately 125 g BW mm^–2 GA. The results ofthis study are discussed relative to other structural studiesof and proposed mechanisms for graft development.     

The Influence of Pre and Post-storage Hydration Treatments on Chromosomal Aberrations, Seedling Abnormalities, and Viability of Lettuce Seeds

A 2 h soaking treatment in distilled water, or in aqueous solutionsof cysteine, potassium iodide, or sodium thiosulphate, had nosignificant effect (P > 0.25) on the subsequent longevityof lettuce seeds (Lactuca sativa L.) in two different storageenvironments. Neither did these treatments influence relationsbetween loss in germination and the frequency of chromosomalaberrations observed during first mitoses after storage. Incontrast partial hydration of lettuce seeds after storage byexposure to moist air (humidification) or to an osmoticum (priming)reversed some of the damage which resulted from ageing. Mostof the benefits occurred during the first 3 d of humidificationduring which seed moisture content rose to 34 per cent, or duringthe first 7 d of priming when seed moisture content increasedto 44 per cent. Both post-storage hydration treatments reducedthe frequency of chromosomal aberrations, increased the rateof root growth, and decreased the frequency of morphologicallyabnormal seedlings. Either treatment could be of practical use,but it is suggested that humidification is more convenient.Consideration should be given to adopting a humidification treatmentas standard practice following long-term seed storage for geneticconservation. Lactuca sativa, lettuce, seed storage, seed viability, chemical pre-treatment, seed longevity, seed humidification (conditioning), seed priming, chromosome repair, seedling abnormalities     

巴西橡胶树嫁接接合区接穗和砧木径向生长差异的研究

采用树皮嫁接后不锯砧和光镜观察的方法,研究了巴西橡胶树(Hevea brasiliensis)嫁接后8个月的接合区接穗和砧木木质部径向生长的差异现象。结果表明,接合区接穗木质部的径向生长普遍地小于砧木,这种生长差异是由接穗和砧木亲本固有生长特性的差异引起的,与嫁接亲和性无关。(1)对于同一无性系,接穗的发育阶段决定其生长能力,幼态接穗新分化的木质部显著地大于老态接穗,而两类接穗旁边的砧木之间没有明显差别。(2)砧木生长势明显地影响接穗木质部的生长,砧木生长势越强,砧木和接穗的生长就越快,两者的径向差异也越大。(3)同一砧木上各品系接穗木质部生长差异取决于接穗自身的生长特性,砧木的生长不受接穗品系的明显影响。显微观察表明橡胶树的嫁接是亲和的,接穗新分化的木质部镶嵌在砧木新分化的木质部中,维管组织如导管上下连接畅通,砧穗树皮厚度一致,愈合良好。     

Effects of Salinity on Growth, Water Relations and Ion Accumulation of the Subtropical Perennial Halophyte, Atriplex griffithii var. stocksii

The effects of salinity on growth, water relations, glycinebetainecontent, and ion accumulation in the perennial halophyte Atriplexgriffithii var. stocksii were determined. The following questionswere addressed: (1) What effect does salinity have on growthresponses at different ages? (2) Is A. griffithii an ion accumulator?(3) Does A. griffithii accumulate glycinebetaine in responseto salinity? Atriplex griffithii plants were grown in pots at0, 90, 180 and 360  m M NaCl in sand culture in a plantgrowth chamber and plants were harvested after 30, 60 and 90d. Plant total dry weight was significantly inhibited at 360m M NaCl. Root growth showed a substantial promotion at 90 mM NaCl. The water potential and osmotic potential of shootsbecame more negative with increasing salinity and time of growth.The Na⁺and Cl^-content in both shoots and roots increased withincreases in salinity. Increased treatment levels of NaCl induceddecreases in Ca⁺, K⁺and Mg²⁺in plants. Atriplex griffithii accumulateda large quantity of ions, with the ash content reaching 39%of the dry weight in leaves. Inorganic ion accumulation is significantin osmotic adjustment and facilitates water uptake along a soil-plantgradient. Glycinebetaine concentration was low in roots, andin stems it increased with increases in salinity. Total amountsof glycinebetaine in leaves increased with increases in salinity,and its concentration increased substantially at 360 m M NaCl.Copyright 2000 Annals of Botany Company Atriplex griffithii, glycinebetaine, growth, ions, water relations.     

Hydraulic Conductances of the Soil, the Root-Soil Air Gap, and the Root: Changes for Desert Succulents in Drying Soil

Water movement to and from a root depends on the soil hydraulicconductivity coefficient (L^soil), the distance across any root-soilair gap, and the hydraulic conductivity coefficient of the root(L^P). After analytical equations for the effective conductanceof each part of the pathway are developed, the influences ofsoil drying on the soil water potential and L^soil are describedduring a 30 d period for a loamy sand in the field. The influencesof soil drying on L^P for three desert succulents, Agave deserti,Ferocactus acanthodes, and Opuntia ficus-indica, are also describedfor a 30 d period. To quantify the effects of soil drying onthe development of a root-soil air gap, diameters of 6-week-oldroots of the three species were determined at constant watervapour potentials of –1.0 MPa and –10 MPa as wellas with the water vapour potential decreasing at the same rateas soil drying during a 30 d period. The shrinkage observedfor roots initially 2·0 mm in diameter averaged 19% duringthe 30d period. The predominant limiting factor for water movementwas L^P of the root for the first 7 d of soil drying, the root-soilair gap for the next 13 d, and L^soil thereafter. Compared withthe ease of water uptake from a wet soil, the decrease in conductancesduring soil drying, especially the decrease in L^soil causedthe overall conductance to decrease by 3 x 10³-fold during the30 d period for the three species considered, so relativelylittle water was lost to the dry soil. Such rectifier-like behaviourof water movement in the soil-root system resulted primarilyfrom changes in L^soil and, presumably, is a general phenomenonamong plants, preventing water loss during drought but facilitatingwater uptake after rainfall. Key words: Agave deserti, Ferocactus acanthodes, Opuntia ficus-indica, rectification, soil water potential, water movement     

Effect of Matric-Priming Duration and Priming Water Potential on Germination of Four Grasses

In this study, a matric-potential control system was used todetermine the effect of matric-priming duration and primingwater potential on the germination response of Bouteloua curtipendula(Michx.) Torr., Cenchrus ciliaris L., Eragrostis lehmannianaNees, and Panicum coloratum L. Seeds were primed at water potentialsof –1·5 to –7·7 MPa for up to 14 d.Optimum germination generally occurred in treatments primedat high water potential for the shortest period. Germinationof seeds primed at lower water potential and for longer periodsexhibited a negative germination response relative to the control.Seeds were not redried after the priming treatment. Seed-wateruptake measurements suggest that a reduction in the lag timeof imbibition accounted for at least some germination-rate enhancementin the positive-priming treatments Key words: Germination, matric-priming, imbibition     

The Function of Phloem Connections in Regenerating In Vitro-Grafts*

In order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant-grafts after application of ¹⁴C-sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls. A segmental analysis was used to determine the tissue distribution of ¹⁴C-sucrose in a graft. The ¹⁴C-profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound-phloem bridges (shown as files of aniline-blue-positive sieve plates) traverse the graft interface. In 7-d-old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, ¹⁴C-profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose. Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound-phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro-grafts is directly shown.     

The Effect of a Grafted Bud on Vascularization of the Petiole in Phaseolus, Datura and Lycopersicon

The radially-organized petiole of Phaseolus and the dorsiventralpetioles of Datura and Lycopersicon were used as stocks in budgrafting. Petiolar structure was examined after some weeks'growth of the scion, during which the grafted petiole came tofunction as a stem in supporting normal, vigorous shoot growthand was not abscised even at the end of the growing season.Below the graft union, reactivated petiolar cambium producedmassive amounts of secondary tissue with greatly enlarged vessels.Cambial activity was confined to the existing vascular bundlesexcept for the development of a little inter-fascicular cambiumin young, grafted Phaseolus petioles. Datura petioles woundedbelow the graft union, by a cut into the petiolar are from eitherthe abaxial or the adaxial surface and removal of a 1 cm lengthof tissue, responded by restoring the vascular are (abaxialwounds) or almost completing a vascular ring (adaxial wounds).In grafted, wounded Lycopersicon petioles the presence of deadxylem caused the separation of cambium regenerated from thecut vascular are and that arising in relation to the centralwound surface. A similar response, in which stimulated internalphloem plays an important part, occurred in certain woundedLycopersicon stems. The results are discussed in terms of thegradient induction hypothesis. graft, petiole, wound, cambium, xylem, phloem, Phaseolus multiflorus, Datura stramonium, Lycopersicon esculentum, bean, thornapple, tomato     

Temporal and nutritional factors modulate responses to abscisic acid and osmoticum in their regulation of storage protein synthesis in developing seeds of alfalfa (Medicago sativa L.)

Storage protein synthesis during alfalfa (Medicago sativa L.)seed development displays a temporal pattern and is regulatedboth by ABA and low osmotic potentials. Deposition of the 2S,7S and 11S storage proteins occurs maximally at the mid–to late stages of development. Osmoticum, but not ABA alonecould effectively induce storage protein synthesis at earlystages of development in isolated alfalfa embryos placed onwater for several days. Neither ABA nor osmoticum alone couldmaintain storage protein synthesis in isolated late-stage embryosplaced on water. On the other hand, isolated developing embryoscultured on a nutrient (Murashige and Skoog) medium could maintainstorage protein synthesis in the presence of ABA and osmoticum,alone or in combination, for up to 14 d after being dissectedfrom the pod. Of the components of this medium, the inorganicsalts appeared to be the most important. The response to ABAand osmoticum varied with the time of isolation during developmentwith the greatest enhancement of storage protein synthesis inisolated embryos coinciding with the time of maximum synthesiswithin the seed when in planta. Addition of ABA and osmoticumafter the time of maximum storage protein synthesis did notelevate the amount of synthesis, but rather prolonged the timeover which it could take place. Thus, while the amplitude ofstorage protein synthesis could be modified by ABA or osmoticum,the inherent temporal pattern, with maximum synthesis occurringonly at mid– to late stages of embryo development, couldnot. Key words: Embryogenesis, Medicago sativa L., alfalfa, abscisic acid, osmotic potential, storage protein synthesis, nutrient supply