Embryo Rescue in Wide Crosses in Arachis. 2. Embryo Development in Cultured Peg Tips of Arachis hypogaea

Embryo rescue techniques in Arachis are potentially importantfor recovering interspecific hybrids which have the propensityto abort. Pegs are commonly produced in interspecific crosses,but either they fail to reach the soil because growth is arrested,or pods are produced but embryo development is never re-initiated.Peg tips, with the ovule and embryo, of A. hypogaea L. cv. ‘NC6’, were used to determine whether peg tips can be usedas nurse tissue for in vitro culture of embryos. Tissues werecollected 1, 2, 3 and 4 d after self-pollination, after whichpeg meristems were removed from half the pegs, and culturedon five media combinations. Continued reproductive developmentwas observed for embryos cultured at all four collection days;however, the highest frequency of growth was observed in 1-d-oldtissues. Evidence is presented that meristematic activity mayrestrict embryo growth in the 2- to 4-d-old embryos and, oncethe sequence of events is initiated to slow embryo growth, itis not easily reversed in vitro. Achievements of embryo growthto multicellular, globular stages (stages 1–1 or 1–2)encourage the development of methods to recover very young embryosthrough tissue-culture techniques. Embryo culture, morphology, interspecific hybridization, Arachis hypogaea, comparative light and scanning electron microscopy, peanuts, groundnuts     

Early Reproductive Ontogeny in Interspecific Crosses of Arachis hypogaea and Section Arachis Species

Wild Arachis species have been recognized as sources of resistanceto pests and pathogens that infect A. hypogaea L. and causesubstantial yield losses. However, utilization of these geneticresources for crop improvement has been difficult. This studywas conducted to (a) understand the processes of early embryogrowth and development in four Arachis species, two A. hypogaeacultivars and their hybrids and (b) identify parental compatibilitiesin reciprocal crosses of A. hypogaea. The results indicatedthat delayed fertilization beyond 24 h, coupled with slow proembryogrowth, leads to embryo abortion in many interspecific crosses.For example, in female A. cardenasii crosses, lack of or delayedfertilization leads to failure to obtain hybrids. When A. batizocoiwas used as a female parent, delayed fertilization and the inabilityof quiescent proembryos to resume growth after soil penetrationcaused abortion. Embryos of A. hypogaea x A. glandulifera crossesdeveloped normally during the first 21 d after fertilization,but then aborted at a later time. In this study, A. hypogaeawas always a better female parent than the wild Arachis species.Increasing the number of pollinations per cross, using the cultivatedspecies as the female parent, utilizing different A. hypogaeavarieties, and embryo rescue techniques are suggested to improvethe probability of obtaining interspecific hybrids in Arachis.Copyright1995, 1999 Academic Press Peanut, interspecific hybrids, Arachis, wild species, incompatibilities     

Development of Photosynthetic Structure and Function during Light-Induced Greening in Photomixotrophic Cells of Arachis hypogaea L.

The synthesis of chlorophyll and development of photochemicalactivities were complete within 70–80 h in greening leaves,whereas these processes continued for 8 days with an initiallag of 8 h for pigment synthesis in greening Arachis hypogaeaL cells. The activity of photosystem I in cultured Arachis cellswas detected earlier (24–36 h after illumination) thanthat of photosystem II (42–54 h after illumination) andthe development of the latter coincided with the synthesis ofa 46,000 dalton polypeptide of the thylakoid membranes. Experimentalstudies with cultured cells have the advantage in that the temporalsequence of the assembly of membrane components and associatedfunctions are determined easily because of longer developmentalperiod of chloroplast. (Received January 29, 1982; Accepted April 13, 1983)     

Comparative Embryo Sac Morphology at Anthesis of Cultivated and Wild Species of Arachis

Introgression of germplasm from wild to cultivated species ofArachis is severely impeded because abortion processes oftenoccur as a prepeg-, peg-(gynophore), or postpeg-elongation event.A comparative study of embryo sac morphology at anthesis wasundertaken to determine if observable differences were presentthat could possibly explain abortion prior to peg tip swellingfollowing soil penetration. Two wild Arachis species (A. duranensisand A. stenosperma) plus A. hypogaea cultivars NC 6 and Argentinewere studied. Differences in starch grain concentration andcytoplasmic stranding organization were observed between A.hypogaea cultivars and the wild Arachis species. These differencesprobably have a significant impact on energy availability atsyngamy and the subsequent early cell division of the embryo.An improper energy balance could contribute to the onset ofabortion in interspecific hybrids. Modification in egg apparatusorganization among all species was also observed which may accountfor low percentages of seed recovery resulting from interspecifichybridization attempts. Embryo sac morphology, interspecific hybridization, Arachis hypogaea L., Arachis species, light microscopy, scanning electron microscopy, embryo abortion, fertilization incompatibility, peanuts, groundnut     

Geotropic Responses and Pod Development in Gynophore Explants of Peanut (Arachis hypogaea L.) Cultured In Vitro

Peanut gynophore explants cultured in vitro on a defined mediumshow a positive geotropic response in both light and dark whenplanted either horizontally, or vertically with the tip pointingupwards. The growth following the initial curvature dependedon age of the gynophores and on the levels of growth substancesin the medium. In the dark and in presence of 0·01–0·1p.p.m. kinetin, naphthalene acetic acid at concentrations of0·1 p.p.m. and lower promoted gynophore elongation. Athigher concentrations elongation was promoted to a lesser extentin younger explants, caused enlargement of the ovary and formationof pods. Young explants generally elongated more than olderones and pod formation took place inside the medium, while inolder ones it took place above the medium. In the light, theinitial positive geotropic response was followed by elongationbut without any enlargement of the ovary. Decapitation of gynophores1·5–2·0 mm below their tip, removing theovary but leaving most of the intercalary meristem, had no effecton the geotropic response and elongation. The initial geotropicresponse and elongations of explants in vitro was not dependenton the presence of the ovary but on the meristem proximal toit. Changes in growth substances balance during gynophore developmentseem to affect geotropic response, elongation and pod formationin the peanut.     

Comparison of Embryo Development in Wild and Cultivated Arachis Species

Embryo development following selfing was investigated in twowild diploid peanut species, Arachis batizocoi Krap. et Greg.(coll. K 9484) (2n = 20) and A. duranensis Krap. et Greg. nom.nud. (coll. K 7988) (2n = 20), and one cultivated tetraploidspecies, A. hypogaea L. NC-Ac 18000 (2n = 40). Rates of pegelongation and sequences of embryo development for each specieswere compared. Peg elongation rates were similar for the twowild species, but for A. hypogaea it was only one-third to one-halfthat of the diploid species. Embryos in A. hypogaea showed slightlymore rapid cell division than in the wild species. The observedvariation in reproductive development between the wild and cultivatedspecies indicate that different control mechanisms may governdevelopment in the different species and may be at least partiallyresponsible for failure to produce viable interspecific hybridsat various ploidy levels. The observations are also importantfor determining the time at which embryos of different speciesof Arachis will reach the appropriate stage of development forsuccessful culture on an artificial medium during embryo rescueprocedures. Peanut, Arachis hypogaea, wild species, embryo, peg     

The Morphology and Anatomy of Ovule and Fruit Development in Arachis hypogaea L

The vascular system of the monocarpellary gynoecium with tenwell differentiated traces and a few cross links probably representsa precocious development of the post-fertilization vasculatureof the fruit wall. The restriction of the two integuments ofthe ovule to the micropylar half, and the endothecial natureof the chalazal cells adjoining the embryo sac appear to indicatea pathway of derivation of the unitegmic tenumucellate ovulefrom the bitegmic crassinucellate one. During double fertilization,a dark staining refractive body appears in the nucleolus ofthe egg as well as the fusion product of the polar nuclei. The peg that carries the ovary into the soil after fertilizationgrows by the activity of a rib meristem at the basal solid partof the gynoecium. During sub-soil fruit development, the ovarywall develops a prominent spongy inner zone which finally disappears,and a peripheral zone that forms the mature fruit wall. Theabinitio nuclear endosperm is much reduced and degenerates afterproducing a few cell layers in the chalazal half alone. Seeddevelopment is pachychalazal. The main vascular supply of theseed branches at the chalaza into eight to ten strands in theseed coat. All seeds that have a vascular ramification in theseed coat are probably pachychalazal. In the variety Valencia, diminutive fruits with viable seedmay develop aerially from pegs that fail to grow long enoughto reach the soil from the higher nodes. Arachis hypogaea L., groundnut, fruit development, seed development, carpel vasculature, seed vasculature, pachychalaza     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

Initiation and Morphogenesis of Groundnut (Arachis hypogaeaL.) Pods in Solution Culture

A recently-developed solution culture technique was used tostudy the effects of aeration and calcium (Ca) on groundnut(ArachishypogaeaL.) pod development. Two experiments were conductedwith seven groundnut lines, TMV-2, Chico and A116L4 (Spanish),CBRR4 (Valencia), A125L25 (ValenciaxSpanish), and Shulamit Strain1 (SH-1) and Virginia Brunch Strain 1 (VB-1) (Virginia). Plantswere grown in a potting mix, and the attached gynophores culturedin darkened polycarbonate jars containing nutrient solution.Non-aeration of solution prevented pod development, but podsand seeds of all lines developed in aerated, darkened nutrientsolutions (ionic strength approx. 9 mM). Normal pods and seedswere produced by TMV-2, Chico and CBRR4, but constricted podsdeveloped in SH-1 and VB-1. A secondary gynophore developedbetween the basal and apical seed compartments in A116L4 andA125L25, and in VB-1 at high Ca (500–2500 µM) insolution. The secondary gynophores were similar to those producedin otherArachisspp. but not usually found in cultivated formsofA. hypogaea.Septate and non-septate hairs developed on submergedgynophores and pods, but were sparse on those of SH-1 and VB-1.The magnitude of the effects of aeration and Ca concentrationon pod initiation and morphogenesis differed in experimentsconducted in summer and winter and among the lines tested.Copyright1998 Annals of Botany Company Arachis hypogaeaL., calcium, groundnut, pod morphology, secondary gynophore, solution culture.     

Morphogenic Potential of Cultured Explants of Crocus chrysanthus Herbert. cv. E. P. Bowles

Explants of leaves, basal plates, petals, anthers and ovariesof young growing corms of Crocus chrysanthus var. E. P. Bowleswere cultured on MS basal media with 20 different combinationsof either kinetin and NAA or BAP and 2, 4-D in the dark. Nomajor change was observed except on ovary explants. The ovaryexplants produced callus at 5.0 mg 1^–1 and 10 mg^–1BAP and subsequently stigma-like structures formed on the surfaceof the callus. Transfer to light resulted in the stigma-likestructures developing a yellow pigmentation whereupon they cameto resemble the naturally-grown stigmas. Corm formation andshoot regeneration was obtained from the callus when the ovaryexplants were cultured on media containing 5.0 and 10 mg I^–1BAP with 0.5 mg 1^–1 2, 4-D. Increasing the level of 2,4-D markedly reduced the number of shoots produced per explant. Key words: Crocus chrysanthus, callus, ovary explants     

Photomorphogenesis of the Gynophore, Pod and Embryo in Peanut, Arachis hypogaea L.

Darkened excized gynophores ceased to elongate after 8–10days in vitro and started to form a pod. Gynophore elongationwas inhibited to a greater extent in total darkness than underlow irradiance, while pod and embryo growth was stimulated indarkness only. Intact gynophores, enclosed in transparent vials containingglass beads, continued to elongate in both light and darkness.In light the elongating gynophores thickened as they penetratedbetween the glass beads, forming a seedless pod at the bottomof the vials. In the dark the elongating gynophores producedsmall pods in which the seeds had started to grow. Excized gynophores elongated in vitro under continuous whitelight at a rate similar to that of intact exposed gynophores.The rate of elongation in vitro, was lower under continuousblue or red-enriched light, than under white light, and wasfurther reduced under continuous far-red irradiation. Pods didnot form during any of the continuous irradiation treatmentsbut only after transfer to darkness, the largest pods formingafter continuous far-red irradiation. As little as 10 min daily exposure to red or far-red irradiancehad the same effect on gynophore elongation as continuous irradiation.Pods formed only when the daily periods of far-red irradiationwere 30 min or less. Reducing the daily exposures to 2 min decreasedthe time to onset of pod formation from 30 to 16 days. Far-redfollowing red irradiation was effective in inhibiting gynophoreelongation stimulated by red irradiation. Pod formation in red/far-redirradiation was only 50 per cent of that observed in far-redirradiation. The involvement of light in continual gynophoreelongation and in the concomitant inhibition of proembryo growthis discussed. Arachis hypogaea L., peanut, gynophore, photomorphogenesis, embryo development, pod development, proembryo     

Responses to Saturation Deficit in a Stand of Groundnut (Arachis hypogaea L.). 2. Growth and Development

Stands of groundnut were grown in four glasshouses with themaximum saturation deficit (D) of the air limited to 1.0, 2.0,2.5 or 3.0 kPa. The soil was near field capacity when plantsemerged and no water was applied thereafter. In a fifth glasshouse,a stand was grown at low D on soil irrigated to field capacityevery few days. Developmental processes such as timing of flowering, peggingand pod formation were unaffected by D, but the numbers of branches,flowers and pegs were reduced in the drier treatments. Measurementsduring the first 30 d showed that in the drier treatments leafgrowth was reduced, and the partitioning of dry matter intoroots was enhanced. In the unirrigated stands, dry matter production in shoots wasreduced by 40 per cent as the maximum D increased from 1.0 to3.0 kPa. Growth was affected through reductions both in leafarea (and therefore light interception) and in the productivityper unit of light intercepted. These responses to D and soilwater were linked to changes in bulk water potential of leaves. Productivity per unit of water transpired (q) decreased withincreasing D. The product of q and the mean daytime value ofthe difference in vapour pressure between leaf and air was moreconservative than q, and ranged from 3.1 to 5.6 g kPa kg^–1. Groundnut, Arachis hypogaea L., saturation deficit, growth, development, light interception, water use efficiency     

Effects of Desiccation and Cryopreservation on the In Vitro Viability of Embryos of the Recalcitrant Seed Species Araucaria hunsteinii K. Schum

Embryos excised from fresh seed had a moisture content of about37% (fr. wt.), 7% above that of the whole seed: mainly as aresult of the dryness of the seed coat. When cultured for 14d at 26°C embryo growth ranged from unorganized to fullyorganized. Embryos which had been directly surface-sterilizedexhibited lower fully organized growth than aseptically excisedembryos. During desiccation of the whole seed at 15°C and 15% r.h.moisture loss from the embryo was slow until the embryo reacheda mean critical moisture content for fully organized growthin vitro of about 30%. After this point the rate of moistureloss increased, quickly diminishing the moisture content differentialbetween the embryo and the whole seed. No such shoulder in theembryo desiccation curve was observed when using a low viability(7%) seed batch. When dried below 30% moisture content, suchaseptically excised embryos increasingly exhibited unorganizedgrowth in vitro, with only root meristem survival (46%) at 13%moisture content. In contrast, a much higher level of root meristem survival (84%of control) was observed when aseptically excised embryos wererapidly desiccated to 13% moisture content, using a flow ofsterile air. However, the benefits of rapid drying were lostif the embryos were directly surface-sterilized prior to desiccation.Furthermore, 80% of aseptically excised embryos which survivedrapid desiccation to 20% moisture content exhibited root meristemsurvival following subsequent cryopreservation. These findingshave implications for the in vitro conservation of recalcitrantseed tissue. Key words: Araucaria hunsteinii K. Schum., recalcitrant seed embryo, desiccation, cryopreservation     

Regulation of Stem Abscission and Callus Growth in Shoot Explants of Sweet Orange [Citrus sinensis (L.) Osbeck]

Two-node explants from Sweet Orange cv. St Ives Valencia orangeshoots produced prolific callus and formed secondary abscissionzones within internodes when cultured in vitro with abscisicacid (ABA, 5 µM) or -naphthaleneacetic acid (NAA, 5 µM).Benzyladenine (BA, 1 µm) induced callus but had littleeffect on abscission. Secondary abscission zone formation wasassociated with ABA-induced and auxin-induced ethylene formation.Treatment of explants with inhibitors of ethylene synthesis[aminoethoxyvinyl glycine (AVG), Co²⁺, PO₄^3–] preventedformation of secondary abscission zones but had variable effectson callus formation. Newly made explants contained high concentrationsof endogenous ABA (up to 6000 ng g^–1 f.wt), as measuredby GC/MS/SIM. Long-term subculture of explants (two years) inmedia containing BA (1 µm) led to a reduction in endogenousABA level (40 ng g^–1 f. wt) and to loss of capacity toform extensive callus and secondary abscission zones. Citrus sinensis (L.) Osbeck cv. St Ives Valencia, sweet orange, secondary abscission zones, in vitro, ethylene, endogenous ABA, endogenous IAA     

The Effect of Ethylene and Temperature on ATP Accumulation from Various Substrates in Peanut (Arachis hypogaea L. ) Seeds

Peanut (Arachis hypogaea L. ) seed powder accumulated ATP fromAMP and phosphoenolpyruvate (PEP) at a rate of approx. 100 pmolmin^–1mg^– powder at 35° C. When peanut seed powderwas incubated with various substrates, which may result in PEPor AMP (ADP) synthesis, then ATP accumulated. The best substratecombinations examined so far were AMP + succinate, NADH₂, andAMP + malate + NAD, with activities of 33, 12 and 12 pmol min^–mg^–1powder,respectively; AMP + malate showed very low activity. Some combinationsexhibited linear activities with time, while others had an exponential-typeprofile. The temperature dependence of the ATP accumulationdemonstrated by the Ahrrenius plot had a double phase with atransition point at 25° C. The Ea values between 15°C and 25° C were 25 000–50 000 cal/mol, while above25° C the Ea values fluctuated between 6000 and 8000 cal/mol(depending on the substrate). The AMP + PEP combination exhibiteda single-phase profile between 15° C and 40° C, withan Ea value of 22 000 cal/mol. In the presence of some substrates,ethephon (ethylene) had a stimulatory effect and caused an increasein the Ea values at the high temperature phase. A comparisonof seed powder from dormant seeds with that from non-dormantseeds revealed that some substrate combinations accumulate ATPfaster in non-dormant seeds and others do so in dormant seeds. Key words: Arachis hypogaea, ATP, Ethylene, Dormancy, Peanut, Seed     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

Utilisation of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Cross-compatibility of species in section Arachis Krap. et Greg. nom. nud., and chromosome pairing and pollen fertility in their interspecific F₁ hybrids were studied to further understand the phylogenetic relationships among these species. Except those with A. batizocoi Krap. et Greg. nom. nud., hybrids between diploid species have near normal bivalent frequency (9.1–9.8) and moderate to high pollen fertility (60–91%). Hybrids between A. batizocoi and other species have low bivalent frequency (5.2–6.9) and very low pollen fertility (3–7%). These results confirm the earlier separation of these species into two groups based on karyomorphology and Mahalanobis D² calculated on arm ratios. These studies also provide a picture of relative affinities between A. batizocoi, the lone member of one cluster, and the other species, and among the rest of the species. They also indicate that the basic chromosome complement in the two groups of species is the same. Chromosome pairing in triploid hybrids, (A. hypogaea L. X diploid wild species), suggests that A. batizocoi is the closest diploid relative of A. hypogaea. It is closer to A. hypogaea subspecies fastigiata Waldron than to A. hypogaea subspecies hypogaea Krap. et. Rig. Other diploid species of the section Arachis are equidistant from A. hypogaea, and have the same genome which has strong homology to one of the genomes of A. hypogaea. Based on the present results, the two tetraploid species, A. monticola Krap. et Rig. and A. hypogaea can be recognised as two forms of the same species. Breeding implications have been discussed in the light of chromosome behaviour observed in hybrids of A. hypogaea X diploid species, and on the presumptions that A. hypogaea has an AABB genomic constitution, and that among the diploid species, the B genome is present in A. batizocoi while the A genome is common to the other diploid species of section Arachis.Submitted as Journal Article No. 328 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

Histological Analysis of Organogenesis and Somatic Embryogenesis Induced in Immature Tissues of Stylosanthes scabra

A well established protocol for in vitro germination of Stylosanthesscabra zygotic embryos was achieved. The response of S. scabraembryonic tissues cultured in vitro was highly dependent onthe kind of growth regulator used. Organogenesis was obtainedby using BAP, otherwise somatic embryogenesis was induced by2, 4-D. Histological aspects of both methods of regenerationwere evaluated. Endogenous neoformed buds seem to develop fromdeepseated vascular nodule structures into callus tissue. Besides,a direct somatic embryogenesis of a multicellular origin issuggested. Stylosanthes scabra, histology, organogenesis, somatic embryogenesis     

Influence of Temperature and Ageing of Ovules and Pollen on Reproductive Success in Trifolium repens L.

Four clones of Trifolium repens L., originating from Iceland,Denmark and Israel, were cultivated in controlled environmentchambers at 10, 14, 18 and 22°C. Seed set at 10°C was,on average, 45% lower than at 18°C. Generally, seed frequencieswere higher in the stylar ovule positions than in the basal.This polarization became more pronounced at low temperatures.At least 30% of the decrease in seed set was caused by inadequatepollen tube growth. This was further supported by in vitro studieswhich showed that pollen developed significantly shorter pollentubes at low temperatures and a strong positive correlationbetween the capacity of male parents to induce seed set in basalovule positions and the length of pollen tubes in vitro. Femaleage, determined by days relative to anthesis, was also foundto have a significant influence on seed set, i.e. when pollinationwas delayed until the fifth day of flowering, seed set decreasedby an average of 60% when compared to florets pollinated onthe first day of anthesis. Pollen age did not influence seedset. Ageing of pollen decreased in vitro germinability, tubeelongation and the percentage of pollen tubes with sperm cells.Seed set, pollen germinability, pollen tube lengths, and abilityto form sperm cells in vitro varied significantly among clones.Copyright1994, 1999 Academic Press Trifolium repens, white clover, temperature, pollen tube, sperm cells, ovule fertility, seed set