Micropropagation of Potato: Evaluation of Closed, Diffusive and Forced Ventilation on Growth and Tuberization

Different types of ventilation of the culture vessel headspace,each with and without the ethylene inhibitor AgNO₃(3.0 µM)or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid(ACC) (2.0 µM) in the culture medium, were investigatedin terms of their effects on the growth of potato cuttings (Solanumtuberosum L. ‘cara’). Concentrations of CO₂ , O₂andethylene in the culture vessel headspaces were monitored duringthe study. Growth was substantially enhanced and vitrification(stunting and epinasty of leaves and hooking of stem apices)was reduced by increasing the efficiency of ventilation, theeffects being greatest with forced ventilation. In the conventionaldiffusive treatment (via a polypropylene membrane), leaf epinastyoccurred but the leaves were not stunted unless ACC had beenadded. AgNO₃prevented vitrification in the latter case and reducedit in the sealed treatment. On the other hand, with all forcedventilation treatments, even with the addition of ACC, the plantletsgrew well and some of the growth parameters exceeded those inthe diffusive + AgNO₃treatment. Ethylene removal was clearlyan important factor contributing to the better growth foundwith diffusive and especially with the forced ventilation treatment;with the latter, ethylene concentrations in the culture vesselswere virtually zero. In addition, enhanced CO₂supply probablycontributed to the better performance under forced ventilationcompared to diffusive ventilation. Callus developed on the stembases in all sealed (airtight) and diffusive treatments exceptwhere AgNO₃was used. No callus was observed in any treatmentwhere forced ventilation was applied and in vitro tuberization(tuber size) was considerably improved by this treatment. Copyright2001 Annals of Botany Company Callus, ethylene, potato, tuberization, vitrification     

Enzymic Maceration of Citrus Callus and the Regeneration of Plants from Single Cells

The maceration medium comprised a basal nutrient medium (BM)containing an optimum concentration of 3% (w/v) sucrose. Mannitoland sorbitol were inferior osmotica. Addition of potassium dextransulphate adversely affected maceration. ‘Macerozyme’was not as effective as ‘Macerase’ in the productionof single cells. The optimal concentration of ‘Macerase’was found to be 2–3% (w/v). Single cells obtained by filtering the macerate were rinsedwith BM and cultured in, and on, agar media comprising: BM;BM + 500 mg 1^–1 malt extract (ME); and BM + 10% (v/v)coconut milk (CM). No growth or organization was observed incultures where cells were mixed in with warm medium prior togelling. When spread on the surface of gelled media supplementedwith ME and CM, proliferation and organization occurred. Manymicroscopic globular proembryoids developed within 3 weeks onthe supplemented media. Microscopic torpedo-shaped embryoidswere frequently observed on BM + CM, rarely on BM + ME, andnot at all on unsupplemented BM. The high frequency of microscopic globular proembryoids, andlater of macroscopic pseudo-bulbils, formed on BM + ME leadsus to postulate that pseudobulbils are derived from globularproembryoids in which polarity is not established by the 16to 32-cell stage. Microscopic torpedo-shaped embryoids probablygive rise to macroscopic heart-shaped embryoids which developinto plantlets. The technique reported in this article provides an ideal systemfor examining embryogenesis per se and for studying the effectsof various treatments on embryogenesis and organ differentiationin vitro. It also affords excellent opportunities for the breedingof solid mutant plants.     

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

In vitro-grown shoot tips of five tetraploid potato (SolanumtuberosumL.) clones were cryopreserved by vitrification. Excisedshoot tips (0.5–0.7 mm) were pre-cultured on filter paperdiscs over half strength liquid Murashige and Skoog (MS) mediumsupplemented with 8.7 µMGA₃and different combinationsof sucrose (0.3, 0.5 and 0.7M) plus mannitol (0, 0.2 and 0.4M)for 2 d under a 16 h photoperiod at 24 °C. The pre-culturedshoot tips were either successively loaded with 20 and 60% PVS2 solutions or directly exposed to concentrated vitrificationsolution before physical vitrification during liquid nitrogentreatment. The vitrified shoot tips were warmed rapidly andtreated with dilution mixture (MS+1.2Msucrose) for 30 min beforeplating on regrowth medium. Addition of mannitol to the pre-culturemedium improved survival of vitrified shoot tips. Direct dehydrationof pre-cultured shoot tips with concentrated PVS 2 was detrimentalto survival of vitrified shoot tips. Shoot tips pre-culturedon medium containing 0.3Msucrose plus 0.2Mmannitol, and loadedwith 20% PVS 2 for 30 min followed by 15 min incubation in 60%PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resultedin up to 54% survival after vitrification. About 50% of vitrifiedand warmed shoot tips formed shoots directly. Post-thaw culturingof vitrified shoot tips on medium containing an elevated levelof sucrose (0.2M) under diffuse light for the first week enhancedthe survival rate. Continuous culturing of vitrified shoot tipson high-sucrose medium induced multiple shoot formation.Copyright1998 Annals of Botany Company Solanum tuberosumL., potato, cryopreservation, germplasm conservation,in vitroconservation, meristems, shoot tips, tissue culture, vitrification.     

Effects of growth medium, temperature, salinity and seawater source on the growth of Gymnodinium catenatum (Dinophyceae) from Bahia Concepcion, Gulf of California, Mexico

Laboratory studies were performed to determine the effect oftemperature, salinity, seawater sources and culture media onthe vegetative growth of clonal cultures of Gymnodinium catenatumisolated from Bahía Concepción, Mexico. Theseisolates were heterothallic and isogamous. Exponential growthrates of G. catenatum in f/2 with different selenium concentrationsand soil extract and GSe media were moderate. Maximum cell yieldswere obtained in GSe and f/2 media with selenium (10^–8and 10^–7 M), while in f/2 medium with soil extract cellyields were considerably lower. The highest percentage of longchains was found in f/2 media supplied with selenium (10^–8M). The optimal temperature range for growth was 11.5–30°C,with the highest growth rates between 21 and 29°C. The rangeof salinity tolerated by G. catenatum changed with seawatersource. With seawater from Vineyard Sound (Massachusetts, USA),G. catenatum grew at salinities from 15 to 36, with an optimalgrowth rate obtained at salinities between 26 and 30. With seawaterfrom Bahía Concepción, this species toleratedsalinities from 25 to 40, with optimal growth at salinitiesbetween 28 and 38. Ecophysiological measurements reported hereare consistent with the environment of the bay, which has limitedinput of humic materials from runoff and high salinity and temperature.These data, when viewed with data from studies of globally distributedG. catenatum, demonstrate the ability of this species to livein a broad array of habitats.     

The In Vitro Culture of Immature Barley Embryos on Different Culture Media

Immature barley embryos (Hordeum distichum var. Julia) of between0•20 and 0•80 mm in length, were isolated from thedeveloping grain and cultured in vitro on various culture media.The subsequent development of the embryos was followed overa period of weeks, and where germination ensued the growth rateof shoot and root meristems was compared with in vivo germinationrates. Various growth media were assessed for their abilityto support normal development of immature embryos. A numberof published media failed to support satisfactory developmentof young embryos. The addition of 1–15 per cent coconutmilk to Norstog's Medium I (mineral + vitamin solns) enhancedembryo development and lowered the threshold of viability fromembryos of 0•50 mm in length to 0•35 mm. Althoughin many cases germination ensued, embryo development was largelyabnormal. A slightly greater enhancement of growth was achievedwith 0•05–0•30 per cent casein hydrolysate asthe growth medium supplement, although abnormal developmentwas not eliminated. A further lowering of the viability thresholdto include embryos of 0•25 mm in length was obtained bycombining 2•7 mM glutamine with the casein hydrolysatesupplement. Normal development and germination of embryos assmall as O25 mm was however obtained on Norstog's Medium JJand the results were reproduced in four additional if . distichumvarieties. In each case the critical threshold of viabilitywas found to lie in embryos of 0•20–0•30 mmin length.     

Studies on Pyricularia oryzae Cav. in Sierra Leone: Morphological and Physiological Variability of Some Isolates

The morphological and physiological variability of six isolatesof Pyricularia oryzae, the causal organism of rice blast disease,were investigated. The rate of growth, colony characters, andtime of sporulation were found to vary with the different isolatesthough not by appreciable amounts. Each of the isolates showedconsistently better growth on Takahashi's B medium than on Czapek-Dox'smedium although the growth trend was the same in both media.The colony characters developed by each isolate are not dependenton the medium on which it is growing—a pointer to thefact that such characters may be genetically controlled. Germinationwas faster in distilled water than in 2 per cent agar. All theisolates produced appresoria in vivo and in vitro; those producedin vivo were, however, considerably larger than those producedin vitro. On the basis of appresorial types, the isolates werefound to fall into two physiological races—smooth-walledand rough-walled. Each isolate produced consistently only oneappresorial type in vitro from the apical or basal cell of theconidium. The utilization of carbon and nitrogen compounds variedfrom one isolate to the other, carbon compounds being generallybetter utilized than nitrogen compounds. Pyricularia oryzaecan metabolize a wide range of carbon compounds. However, mannose,sucrose, glucose, fructose, and maltose proved to be most suitablecarbon sources. The variations in the utilization of the variouscarbon and nitrogen compounds seem to reflect inherent biochemicaland physiological differences among the isolates.     

Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

An efficient vitrification procedure was developed and successfullyapplied to cryopreserve six endangered West Australian species(family Haemodoraceae: Anigozanthos humilis ssp. chrysanthusHopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectansHopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopperand C. wonganensis Hopper). Species were initially evaluatedfor cryostorage using a basic vitrification protocol involving:culturing plantlets in vitro for 21 d; excision of shoot apices;preculture of apical tips on 0.4 M sorbitol for 2 d, followedby incubation in PVS2 (plant vitrification solution 2) for 25min at 0 °C, then direct immersion in liquid nitrogen (LN).Warming of retrieved material was for 1 min in a 40 °C waterbath. Using this protocol five of the six species exhibitedlow post-storage survival, while the sixth species, A. viridisssp. terraspectans posted higher survival (61.1%). Using A.viridis ssp. terraspectans as an indicator species, the initialprotocol was modified to include: 3 d preculture on 0.80 M glycerol,loading treatment with 2.0 M glycerol plus 0.4 M sucrose solutionfor 20 min, followed by 25 min exposure to a modified PVS2.Survival was significantly improved in the test species, andin further experiments three other species also showed significantimprovements with the new protocol. Key findings include: effectivenessof glycerol in the preculture medium; the effect of precultureduration; the importance of a loading stage for these species;and the successful use of modified PVS2 solutions with reducedor zero dimethyl sulfoxide (DMSO). Copyright 2001 Annals ofBotany Company A. humilis ssp. chrysanthus, A. kalbarriensis, A. viridis ssp.terraspectans , Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, kangaroo paws, Haemodoraceae, vitrification, cryopreservation, rare and endangered, conservation     

'Vitrified' Dianthus--Teratomata in vitro due to Growth Factor Imbalance

Cultured shoot tips of Dianthus caryophyllus often develop asabnormal, ‘vitrified’ plants that have the characteristicsof teratomata: stable, unlimited development with fasciatedshoot apices, a bush habit, reduced stem elongation and succulentleaves. High concentrations of NAA in the culture medium increasedand benzylaminopurine decreased the proportion of shoot-tipsthat developed as abnormal plants. The requirements of abnormaland normal plants for continued development differed. Duringthe initial stages of development abnormal plants did not requireauxin and they were able to continue their growth in the absenceof roots. It is suggested that the abnormal plants are teratomata,similar to those induced by Crown Gall bacteria, and their inductionand abnormal traits could be due to imbalance of auxins andcytokinins. Dianthus caryophyllus L., carnation, abnormal plant growth, habituation, vitrification, teratoma (plant)     

Morphological Response of Witchweed (Striga asiatica) to In Vitro Culture

Excised sorghum root segments (5–10 mm in length) werecultured for 50 d in four different liquid media containingmineral salts, vitamins, amino acids, glucose, and IAA. Theroots were removed and the remaining medium was solidified withan equal volume of warm 1–6% water agar. Dry unconditionedor conditioned Striga asiatica seeds were transferred to themedium. Some of the seeds germinated and developed into parasitic-typeseedlings. These seedlings had haustoria, tubercles, dense roothairs, branched shoots, and multiple shoot-borne adventitiousroots. The plumule pole developed into a shoot, but the radiclepole displayed only rudimentary development. On the same media,but which had not previously been used to grow sorghum roots,the seedlings displayed a well-developed radicle-derived rootsystem, but the plumule did not grow. Shoots began to appearon the roots only after 35–50 d of culture. These seedlingshad no haustoria, no tubercles, few or scattered root hairs,no shoot-borne adventitious roots and few shoot branches, andappeared to be non-parasitic-type seedlings. Shoots grew ina medium supplemented with IAA and kinetin, but did not in amedium containing NAA plus IBA. On replacement of glucose andIAA with sucrose and 2,4-D, respectively, Striga seeds germinated,and the heart-shaped embryos dedifferentiated into calli. Thecalli have been maintained by subculturing for over 9 months.The results demonstrated that a host signal, in addition tothose for germination and haustorium formation, is requiredfor further development. Moreover, morphogenesis of culturedS. asiatica is influenced by exogenous growth regulators. Key words: Striga asiatica, parasitic weeds, haustoria, Sorghum bicolor, in vitro culture     

The Effects of Modifying Sucrose Concentration on the Development of Maize Kernels Grown in vitro

Intact Zea mays L. kernels attached to cob tissue develop tomaturity when grown in vitro. This experiment was designed todetermine if it is possible to prolong kernel growth by refreshingthe culture medium. Blocks of maize kernels were grown in vitroon media containing several concentrations of sucrose. Kernels,at all concentrations of sucrose, developed to maturity at 30–35d post-pollination, indicating that it is not possible to extendthe kernel growth phase by supplying a carbohydrate source.Kernels grown on media containing 80 g 1^–1 or higher sucroseconcentration had a significantly greater percentage of kernelsthat developed to maturity, and had greater weight and starchcontent per seed. Zea mays, kernel culture, seed development, starch     

Effect of Method of Agar Addition on Post-autoclave pH of the Tissue Culture Media

Tissue culture media (MS, GB-5 and N6) were gelled with agarby employing different methods When agar was dissolved directlyin media by autoclaving at 120 °C for 1 mm followed by sterilization,the media pH dropped markedly — more so at the lower concentrationsof agar On the other hand, when agar was first dissolved indistilled water and added to the culture media the pH loss wasminimized considerably pH measurements carried out as a functionof time showed a gradual decline in media pH The media supplementedwith Panax ginseng callus became more acidic than the mediawith no callus Tentative reasons for the post autoclave pH fallassociated with various methods of agar addition are described Culture media, agar addition, post-autoclave pH, Panax gingseng     

In Vitro Acclimatization of Chrysanthemum and Sugar Beet Plantlets by Treatment with Paclobutrazol and Exposure to Reduced Humidity

Methods are described to promote in vitro acclimatization ofchrysanthemum and sugar beet. Plants were cultured at relativehumidity levels of 58%, 81%, and 100% or with alternating combinationsof these levels. The effect on plant growth of the additionto growth media of the anti-gibberellin paclobutrazol was alsoinvestigated. Leaves which were initiated and which developedon plants at relative humidity levels below 100%, or on plantscultured on growth media supplemented with 10.0µM paclobutrazol,displayed increased epicuticular wax, improved stomatal functioning,and reduced leaf dehydration. Reduced humidity levels also increasedtrichome density in chrysanthemum and decreased the incidenceof vitrification in sugar beet. Plant growth for both specieswas optimal at 81% relative humidity. Theaddition of paclobutrazolto the growth medium resulted in a reduction in plant height.     

Influence of Genotype and Environment on Growth of Barley Embryos in vitro

Isolated embryos of three contrasting barley genotypes werecultured in vitro on a range of media comprising 16 combinationsof sucrose (3–12 per cent) and 2, 4-D (0–8 mg 1^–1)concentration. Cultures were incubated at a range of temperaturesfrom 5 to 25°C and were examined after 21 days when shootlengths as well as fresh and dry weights were recorded. Therelative influence on growth of increasing concentrations ofsucrose and 2, 4-D was investigated, as was their interactionwith the incubation temperature. The genotypes were found todiffer markedly in their response to these two media components,with each parameter of growth differentially affected. Resultsare discussed in relation to the known dormancy characteristicsof these genotypes. Hordeum vulgare L., barley, embryo, dormancy, 2, 4-dichlorophenoxy acetic acid, sucrose     

Flower Formation from Citrus unshiu Buds Cultured In Vitro

Flowers were formed in vitro when buds of Satsuma mandarin (Citrusunshiu Mare) from the summer flush of growth harvested duringthe winter rest period before the onset of flower initiation,were grown on a solid Murashige and Skoog medium supplementedwith sucrose and a cytokinin. Flower development was dependentupon illumination, and was enhanced when a piece of stem wasattached to the bud. The percentage of flowering explants wasalways lower than the percentage of naturally flowering budsin spring, but treatments such as ringing which increase floweringin vitro, increased the number of explants flowering int vitroas well. Citrus unshiu Marc, ‘Satsuma’ mandarin, in vitro flowering, ringing     

Fungal Assciations: II. Cultural Studies on Rhizoctonia solani Kuhn, Fusariutn solani Snyder and Hansen, and Other Fungi, and Their Interactions

Several types of interaction between Rhizoctonia solani, Fusariumsolani, and Phoma foveata were found when these fungi were grownon potato-dextrose agar. After being used by Rhizoctonia a potatomash medium gave better growth of Rizoctonia and Fusarium thanit did when the medium was initially used by Fusarium; and thiswas so whether the reaction of the spent medium was readjustedor not. It is suggested that potato mash medium used by Fusariumcontains a thermostable factor(s) affecting the subsequent growthof Rhizoctonia or Fusarium. The range of pH values suitable for Rhizoctonia growth was narrowerthan that for Fusarium, optimum values being approximately 5•9for the former and 7•8 for the latter. In mixed culturesof the two fungi on potato-dextrose agar adjusted to differentpH values, the fungus for which the reaction of the medium wasmore suitable usually became visually predominant after sometime. A study of various carbon sources showed that poor growth ofRhizoctonia was obtained when pectin was used as the sole sourceof carbon. On a pectin-agar medium, the rate of growth of aRhizoctonia colony increased on the sector which lay towardsan adjacent Fusarium colony; also, after the two fungi camein contact, there was more rapid growth of Rhizoctonia roundthe Fusarium colony than elsewhere. On a synthetic liquid mediumwith pectin as the carbon source better Rhizoctonia growth wasobtained when Fusarium-spent medium was added to it than whenRMzoctoma-spent medium was added. Rhizoctonia showed partial deficiencies in thiamine, biotin,and inositol. Both the extract of Fusarium mycelium, grown onvitamin-free medium, and the Fusarium-spent medium, stimulatedthe growth of Rhizoctonia on vitamin-free medium.     

Studies on Fat Accumulation in Nitzschia palea Kutz

Fat accumulation in Nitzschia palea Kütz. starts perceptiblyat the end of the exponential phase of growth and reaches amaximum during the stationary phase of growth when the wholecell is filled with fat. Filtrates from old cultures enhancedfat synthesis probably by the production of an autotoxin whichinhibits cell growth but at the same time favours fat synthesis.The presence of an inhibitory autotoxin has not been provedbut indirect evidence suggests its presence. The nitrogen content of the culture medium also influences theamount of fat formed, more fat being accumulated in nitrogen-deficientmedia than in media provided with adequate nitrate supply. Theoptimum temperature for the growth of N. palea was found tobe about 30 °C while that for fat synthesis was around 35°C. Below 15 °C and above 40 °C, fat synthesis wasdrastically reduced. Blue and red light independently favoured fat synthesis whereasa combination of both did not. This is probably due to the selectiveinfluence of these wavelengths on the production of fat precursorsor intermediates.     

Studies on the Growth in Culture of Plant Cells: XI. THE INFLUENCE OF SHAKING RATE ON THE GROWTH OF SUSPENSION CULTUEES

The influence of shaking rates (expressed as revolutions permin) on orbital shaking platforms (1 in (2.54 cm) diam. rotarymotion) on the growth of cell suspension cultures of Acer pseudoplatanusL. and Atropa belladonna cultivar lutea Döll are described.By following cell growth and respiration and the levels of oxygenand carbon dioxide in the media during the progress of incubationit is concluded that the reduction of growth at sub-optimalshaking rates is not due to oxygen deficiency or toxic accumulationof carbon dioxide. The growth of the Atropa cell suspensionin ‘closed systems’ has been studied by the developmentof modified culture vessels and evidence obtained that the reducedgrowth in the systems is due to the formation by the culturesof an unidentified volatile growth inhibitor and not to eitheroxygen depletion or toxic accumulation of either carbon dioxideor ethylene. It is suggested that the reduced growth in ‘opensystems’ cultures at sub-optimal shaking speeds is eitherdue to retention of this volatile inhibitor or to restrictionof nutrient uptake by the existence of a stationary liquid-phaseboundary to the cells.     

Effects of Gibberellic Acid on Floral Development in vivo and in vitro in the Cleistogamous Species, Lamium amplexicaule L.

Using established developmental markers in the corolla and androeciumof Lamium amplexicaule L., we investigated the apparent inductionof open (chasmogamous — CH) from closed (cleistogamous— CL) flowers after application of GA₃ (0.1 mM). In vivotreatment of potentially CL flower primordia caused cell expansionbut not the increased cell division in anthers and corolla necessaryto convert a CL into a CH flower. Floral primordia that appearedto be of undetermined floral type were grown in vitro on a basalmedium supplemented with kinetin (0.1 p.p.m.) and grew to maturityas CL flowers. On media additionally supplemented with GA₃,flowers underwent anthesis, but no true CH flower was produced.Gibberellins appear to be directly responsible for anthesisin the CH flower; but additional, as yet unknown growth factorsare involved in the switch from CL to CH floral form in a developinginflorescence. floral morphogenesis, Lamium amplexicaule L, henbit, cleistogamy, gibberellin, kinetin, anthesis     

Responses of Marchantia in Aseptic Culture to Well-known Auxins and Antiauxins

Responses of young thalli of Marchantia nepalensis to ten well-knownauxins and antiauxins in aseptic culture are described. Effectsof one concentration of an auxin or an antiauxin were studiedin both liquid and solid cultures. The normal growth of inoculated thalli was inhibited but callus-liketissue was produced on them by the highest concentration (I.Omg./I.) of almost all the growth substances except MH, TIBA,and 2, 4^–DNP. At a later period callus-like tissue producedby IAA, IBA, and IPA differentiated into new thalli but thatproduced by NAA, NOA, 2,4^–D, and TCPA only did so on beingtransferred to control medium. At lower concentrations inoculated thalli developed normallybut they were soon overgrown by daughter thalli. No protonemalphase comparable to that of mosses could be observed in thedevelopmental stage of a regenerating thallus. The early stageis characterized by a stable filamentous structure, normallyconsisting of two to three cells. Filamentous structures ofvarying number of cells were however produced in some treatments. Profuse rhizoids were produced by the highest concentrationof NOA, 2,4^–D, TCPA, IBA, and IPA. Certain growth substancesinhibited the formation of tuberculate but not of smooth rhizoidsin liquid but not in solid cultures. Also tuberculate rhizoidswith feebly developed pegs were produced in certain liquid culturesonly and even in control liquid culture. More gemma-cups wereproduced in liquid cultures. Germinated gemmae with rhizoidswere invariably present in solid but not in corresponding liquidcultures. Germinated gemmae within gemma-cups were frequentlyfound. The significance of these results is discussed.     

Embryo Rescue in Wide Crosses in Arachis. 1. Culture of Ovules in Peg Tips of Arachis hypogaea

Interspecific hybridization in Arachis is restricted by earlyembryo abortion for many cross-combinations. Rescue of youngembryos in vitro within a week after fertilization is necessaryto recover these embryos before they abort. Peg tips, with theovule and embryo tissues, of A. hypogaea L. cv. ‘NC 6’,were cultured to compare ovule growth, callus production andpeg elongation. Tissues were collected 1, 2, 3 and 4 d afterself-pollination, after which peg meristems were removed fromhalf the pegs and cultured on five media combinations. One-day-oldpegs had significantly (P = 0.01) more ovule growth than oldertissues. Presence of the meristem had a greater inhibition toovule growth for 2- to 4-d pegs than for 1-d-old pegs. Significantlymore callus was produced on 4-d pegs than younger tissues, andkinetin had the greatest stimulatory effect on callus. Elongationof pegs with the meristem attached was observed most often inmedia with high sucrose levels. The observations indicate thatvery young ovules can be grown in vitro, and techniques maybe applicable to rescue of young embryonic tissues of Arachis. Ovule culture, interspecific hybridization, Arachis hypogaea, peanuts