Growth of Carnation Meristems in vitro: Anatomical Structure of Abnormal Plantlets and the Effect of Agar Concentration in the Medium on their Formation

Carnation meristems cultured in vitro grow into shoots of threetypes: normal, translucent and succulent. The apical meristemof succulent shoots was of the mantle-core type and it lackedpro-vascular tissue. The leaf had large vacuolated mesophyllcells, fewer stomata (often plugged), and no plate meristem.A higher agar concentration in the medium increased the percentageof normal shoots developing. This supports other indicationsthat the water potential of the medium affects morphogenesis. carnation, Dianthus caryophyllus L, meristem culture, abnormal plantlets, shoot meristem     

Effects of 2,3,5-Triiodobenzoic Acid on Plantlet Formation from Cultured Tissues of Taro, Colocasia esculenta (L) Schott (Araceae)

Cultures of a Californian cultivar of Colocasia esculenta varantiquorum, UCI Runner, produced abnormal structures in additionto plantlets on Linsmaier-Skoog (LS) medium supplemented with1 0 mg 1^–1 adenine-N-benzyl-tetrahydro-2H-pyran-2-yl or6-dimethylaminopurine and 0 1 mg 1^–1 napthaleneaceticacid after at least 32 weeks of culture A number of substitutionsand combinations of growth regulators were tested in an attemptto stimulate normal plantlet development These included trialswith 2,4,5-trichlorophenoxyacetic acid, aminocyclopropane-1-carboxylicacid, and 2,3,5-triodobenzoic acid (TIBA) When tissues werecultured on LS medium without hormones, and supplemented with1 mg 1^–1 TIBA, plantlet production occurred in 2 to 4weeks and the number of abnormal structures was reduced Auxin, calloid, callus culture, cytokinins, micropropagation, development     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

A Method for in Vitro Storage of Lolium multiflorum Lam

A means of storing Lolium multiflorum Lam. stock plants in vitrohas been developed over a period of three years. Variousexplantsand cultureconditions were examined. Shoot tips 0.3–0.9mm long were found to be best for establishing storage culturesbecause they gave a high yield of plantlets in culture and importantpathogens are eliminated. For sub-culturing, after each annualcycle of storage at 2–4 °C, shoot tips, tiller buds,tiller bases and nodes can be used. Tiller buds were most abundantand best for increasing the number of stored plantlets whennecessary. There was no advantage of including an auxin in theculture medium for shoot tips and Murashige and Skoog's mediumwith 0.2 mg l^–1 kinetin has been adopted for initiating,sub-culturing and storing cultures. The optimum temperaturerange for regenerating plantlets was 20–25 °C. Lightwas necessary for a high rate of plantlet regeneration and lightquality and intensity affected their growth. Lolium multiflorum Lam., ryegrass, storage in vitro, tissue culture     

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     

Effect of Light Intensity, Carbon Dioxide Concentration, and Leaf Temperature on Gas Exchange of Spray Carnation Plants

The rates of CO₂ assimilation by potted spray carnation plants(cv. Cerise Royalette) were determined over a wide range oflight intensities (45–450 W m^–2 PAR), CO₂ concentrations(200–3100 vpm), and leaf temperatures (5–35 °C).Assimilation rates varied with these factors in a way similarto the response of single leaves of other temperate crops, althoughthe absolute values were lower. The optimal temperature forCO₂ assimilation was between 5 and 10 °C at 45 W m^–2PAR but it increased progressively with increasing light intensityand CO₂ concentration up to 27 °C at 450 W m^–2 PARand 3100 vpm CO₂ as expressed by the equation TOpt = –6.47-h 2.336 In G + 0.031951 where C is CO₂ concentration in vpmand I is photo-synthetically active radiation in W m^–2.CO₂ enrichment also increased stomatal resistance, especiallyat high light intensities. The influence of these results on optimalization of temperaturesand CO₂ concentrations for carnation crops subjected to dailylight variation, and the discrepancy between optimal temperaturesfor growth and net photosynthesis, are discussed briefly     

In vitro Organogenesis and Plantlet Formation in Cashew (Anacardium occidentale L.)

Rapid induction of multiple plantlets of Anacardium occidentalewas obtained from cotyledonary explants. Lin and Staba medium,containing 05 mg 1^–1 of both IAA and KN, promoted directorganogenesis and plantlet formation. Plantlets developed froman organized hemispherical mass of meristematic tissue arisingfrom single epidermal cells. Bipolar differentiation resultedin the formation of shoot and root primordia in a sequentialmanner Anacardium occidentale L., cashew, cotyledon explant, organogenesis, plantlet formation     

Callus Growth and Plantlet Regeneration in Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae)

Explants of taro cultivars belonging to Colocasia esculentavar. esculenta have been nearly impossible to culture untilrecently. Here, we describe a method which induces callus formationfrom bud explants of Colocasia esculenta var. esculenta cv.Akalomamale, brings about shoot and root production, and leadsto plantlet regeneration. The medium used is half-strength Murashige-Skoog(HMS) solution containing 25 ml taro tuber extract (TE), 2 mg2, 4, 5-trichlorophenoxyacetic acid and 200 mg glutamine 1^–1.TE is an important requirement for bud explants and callus tissues.Root induction on callus-derived shoots (i.e. plantlet formation)occurs on HMS containing only 25 ml TE and 100 ml coconut water1^–1. Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae), coconut water, micropropagation, plantlet regeneration, root formation, taro extract, tissue culture     

Callus Induction and Plant Regeneration in Alstroemeria

Callus was induced from mature embryos of Alstroemeria cv. ‘Butterfly’cultured on MS medium supplemented with 2·0 or 4·0mg dm^–3 2,4–D or picloram and incubated at 25°Cin the dark. The effect of auxin concentration and precultureof embryos was studied. Callus was capable of regeneration aftertransfer to MS medium containing 4.0 mg dm°3 BAP. Shootsand whole plantlets were regenerated. The effect of growth regulators,used in the callus induction medium and the regeneration medium,on plant regeneration was studied Key words: Alstroemeria, callus, plant regeneration.     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration     

Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Three cultivars of M. sativa and one cultivar of O. viciifoliawere evaluated for their response to inoculation with A. rhizogenesstrain A4T (containing pRiA4b). A cultivar-dependent responsewas observed in M. sativa with 94%, 25%, and 4% of infectedstem explants producing transformed roots in the cultivars Vertus,Regen-S, and Rangelander, respectively. In O. viciifolia cv.Hampshire Giant, an explant-dependent response was observedwith 78% and 50% of seedling cotyledon and hypocotyl explantsresponding, respectively. Leaf explants failed to produce transformedroots. Transformed roots showed plagiotropic and negativelygeotropic growth on hormone-free agar MS medium. Productionof transgenic shoots from O. viciifolia root cultures occurredspontaneously. Recovery of transgenic plants from M. salivacv. Rangelander was achieved by transfer of callus (inducedon UM medium containing 2·0mg dm^–3 2,4-D and 0·25mg dm^–3 kinetin) to MS medium containing 0·5 ingdm^–3 BAP and 0·05 mg dm^–3 NAA. Cultured rootsof both species synthesized opines (agropine and mannopine).Extensive morphological variation was observed in plants ofM. sativa (clone Al) and O. viciifolia (clone A4Tl) establishedin the glasshouse. DNA sequences homologous to TL-DNA and TR-DNAwere present in root clones and regenerated plants. Key words: Agrobacterium rhizogenes, Medicago sativa, Onobrychis viciifolia, transformed roots, transgenic plants     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Abscisic Acid and the Regulation of Water Loss in Plantlets of Brassica oleracea L. var. botrytisRegenerated Through Apical Meristem Culture

Water loss was studied in regenerated plantlets of Brassicaoleracea var. botrytis cv. Currawong derived through apicalmeristem culture. Hardening of plantlets was eliminated by asingle application of a polyvinyl resin (S600) sprayed immediatelyafter transplanting. Plantlets sprayed with S600 had highercuticular resistances than unsprayed plantlets; this treatmenthad no effect on stomatal resistance. Leaves formed during theculture period showed very little wax formation and using markedleaves it was found that only reduced levels of wax formed onthese leaves even after transplanting. New leaves formed aftertransplanting, showed typical wax formation compared to seedgrown plants. Abscisic acid (ABA) at 10^–4 M applied as a leaf sprayto transplants did not cause a substantial increase in stomatalresistance in leaves which had been initiated during the cultureperiod. Leaves of seed-grown plants as well as leaves of plantletsformed after transplanting did respond to a leaf spray of ABAat 10^–4 M by a large increase in stomatal resistance. Relative concentrations of K, Na, Ca, P, S and Mg in guard cellswere calculated for each leaf type by X-ray micro-probe analysis.K/Na values decreased in the order: seedling > leaves formedafter transplanting > leaves intiated during culture. A highpositive correlation was also found between K/Na and K/P forthe three leaf types. K:Mg and K:Ca ratios for leaves formedduring culture were low in comparison to the values obtainedfor leaves formed after transplanting and seedlings for whichthe values were similar. Brassica oleracea var. botrytis, cauliflower, regenerated plantlets, meristem culture, stomatal resistance, water loss, abscisic acid, X-ray micro-probe analysis     

Zinc-induced Vacuolation in Root Meristematic Cells of Cereals

In the absence of Zn, vacuolar volume fractions of root meristematiccells of Secale cereale L. cv. K2, Triticum aestivum L. cv.Chinese Spring and Oryza sativa L. cv. IR34 were 5.64 x 10^–2,2.17 x 10^–2 and 1.63 x 10^–2 µm³ vacuole µm^–3tissue, respectively. A 4-d exposure to a subtoxic concentrationof zine (0.2 µg Zn cm^–3) induced a 2.93-fold anda 6.78-fold increase in the total vacuolar volume fraction inOryza and Triticum, respectively, whereas no significant increasewas observed for Secale. It is proposed that this Zn-inducedvacuolation represents a compartmentalization mechanism. Theinitial total vacuolar volume fraction in Secale was greaterthan that for Oryza and Triticum and this may enable compartmentalizationof the metal soon after the onset of treatment so reducing itscytotoxic effects. These findings are similar to those observedin contrasting cultivars of Festuca rubra L. Triticum aestivum L, Secale cereale L, Oryza sativa L, zinc, root meristem, vacuolation     

Boron Nutrition of Cultured Tobacco BY-2 Cells: I. Requirement for and Intracellular Localization of Boron and Selection of Cells that Tolerate Low Levels of Boron

Cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow2) grown under the standard culture conditions (1 mg boron liter^–1medium as boric acid) contained boron at a concentration of2.26 mg boron kg^–1 oven-dried cells and the protoplastcontained 1.26% of the boron in the cells. The cells requiredboron for growth and the half-maximum growth rate was obtainedwith 0.056 mg of boron liter^–1 medium. Subculturing thecells in media with lower concentrations of boron allowed selectionof cells that can grow even in the presence of 1 µg boronliter^–1 medium. Cell walls of the selected cells seemedto be thicker than those of the control cells and Golgi bodieswere accompanied by more secretory vesicles than those in thecontrol cells. (Received May 25, 1992; Accepted September 10, 1992)     

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     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Effects of Sucrose Concentration on the Photosynthetic Ability of Rose Shoots In Vitro

Reducing the concentration of sucrose in the culture mediumover successive subcultures has been tested as a method forincreasing the ability of rose shoots grown in vitro (Rosa cvsIceberg and Peace) to take up CO₂. Shoots maintained on ‘constant’10, 20 and 40 g I^–1 sucrose showed decreased levels ofCO² uptake at higher sucrose concentrations, although cv. Peacegrew least at 10 g l^–1 and showed correspondingly lowamounts of CO₂ uptake compared with 20 and 40 g l^–1. Bothcultivars died when sucrose was omitted from the medium. Assucrose concentration was reduced in the medium, so CO₂ uptakeof shoots initially cultured on 20 and 40 g l^–1 sucrosewas found to increase, although a concentration of 10 gl ^–1sucrose seemed to be limiting, below which the growth and chlorophylllevels of shoots declined. Rosa hybrid, rose, shoot culture in vitro, photosynthetic ability, sucrose, infra-red gas analysis     

Simulating the Mineral Environment of Aluminium Toxic Soils in Plant Cell Culture

The development of a medium for studying aluminium toxicityin plant cell cultures is described. To prevent the precipitationof Al added to the standard cell culture medium, it was necessaryto lower the phosphate concentration from 1250 mmol m^–3to10 mmol m^–3, and the pH from 5.8 to 4-0. Two additionalmodifications were the use of unchelated iron and a reductionin the calcium concentration from 3.0 mol m^–3 to 0.1 molm^–3. Since the gelling properties of agar are inhibitedat pH 4.0, cells were cultured on filter paper supported bypolyurethane foam sturated with liquid medium. The only limitationto the growth of plated Nicotiana plumbaginifolia Viv. cellson the modified medium was the reduced phosphate concentration.This was partly overcome by ‘preloading’ the cellswith phosphate prior to each experiment. In addition, the filterpaper with adhering cells was transferred to fresh medium everysecond day to replenish phosphate, and to re-establish the initialpH of4.0 (which otherwise drifts upward). With the modifiedmedium, Al toxicity was observed in plated N. plumbaginifoliacells at both 200 mmol m^–3 and 400 mmol m^–3 Al.There was no toxicity at these Al concentrations when the normalphosphate concentration or pH were restored to the modifiedmedium. Partial alleviation of Al toxicity occurred with restorationof the normal calcium concentration or chelated iron. Chelationof Al with citrate or EDTA also mitigated Al toxicity. In additonto Al toxicity, the modified medium should also prove usefulfor studying other metal toxicities in plant cell culture. Key words: Al toxicity, Cell culture, Nicotiana plumbaginifolia     

In Vitro Propagation of Potato (Solanum tuberosum L.)

Potato shoots were propagated in vitro by placing nodes fromsprouted tubers on Murashige and Skoog type medium without hormones.The vigour of growth and the rate of node production increasedwith both day-length and temperature over the ranges 8–24h and 15–25 °C respectively. Propagation rates ofup to x 10 per month were obtained. In vitro plantlets spontaneouslyformed roots either in agar or liquid cultures. Plantlets leftin the culture jars for 3–4 months eventually senescedand formed small tubers in 16 and 24 h day-lengths. In a day-lengthof 8 h vegetative growth continued by branching and no tuberswere formed. Solanum tuberosum L., potato, tissue culture, propagation, temperature, day-length