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     

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     

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     

Reversible Structural Changes Associated With Callus Formation and Plantlet Development from Aseptically Cultured Shoots of Taro (Colocasia esculenta var antiquorum)

Taro callus maintained on Knop's medium with 2, 0·2 or0·02 mg l^–1 2,4,5-trichiorophenoxyacetic acid (2,4,5-T)or Linsmaier-Skoog (LS) containing 1 mgl^–1 of the cytokininadenine-N-benzyl-9-tetrahydro-2H- pyran-2-yl (SD8339) or 6 dimethylaininopurineand 0·1 mgl^–1 -naphthaleneacetic acid underwenta transition to a stable organized growth form which is referredto as a calloid. On transfer to LS medium th 0·2 mgl^–12,4,5-T in the absence of cytokinin the calloid reverts backto callus. Colocasia esculenta(L.)Schott, taro, callus, calloid, in vitro selection, histology, micropropagation, tissue culture, cytokinin     

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     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

In Vitro Micropropagation of Passiflora edulis (Purple Passionfruit)

A technique for the micropropagation of Passiflora edulis fromseedling explants is described. A multiplication rate of 4.5per month can be achieved in an Murashige and Skoog medium supplementedwith 2 % sucrose and 2 mg l^–1 BAP. Using this technique,no callus is produced. Problems of chlorosis and a method foravoiding subsequent death after prolonged culture are discussed. Passionfruit, Passiflora edulis, micropropagation, tissue culture, chlorosis     

Effects of Na2SO4, K2SO4 and KCl on Growth and Ion Uptake of Callus Cultures of Vaccinium corymbosum L. cv. Blue Crop

Non-selected and Na₂SO-, K₂SO₄- or KCl-selected callus culturesof Vaccinium corymbosum L. cv. Blue Crop were grown on mediasupplemented with 0, 25 and 50 mM Na₂SO₄ (non-selected and Na₂SO(-selectedonly), 0, 25 and 50mMK₂SO₄ (non-selected and K₂SO₄-selectedonly) or 0, 50 and 100 mM KCl (non-selected and KCl-selectedonly). On all media, growth of selected callus (on a fresh-weightor dry-weight basis) was greater than that of non-selected callus,and selected callus grew optimally on the level and type ofsalt on which it was selected. Selected callus was friable andmaintained a higher f. wt:d. wt ratio. Tissue water potentialin selected callus was more negative than in non-selected callus. Flame photometry and chloridometry showed Na⁺, K⁺ and Cl^–accumulated in callus to concentrations equal to or greaterthan the initial concentration in the medium. Turbidometry showedthat tissue SO₄^2- concentration was lower than the concentrationin the medium. In most cases selected callus accumulated moreNa⁺, K^sup, SO₄^2– or Cl^– than non-selected callus.Vacuolar ion concentration was measured by electronprobe X-raymicroanalysis, and on most media selected callus had highervacuolar ion concentrations than non-selected callus. SO₄^2–and Cl^– were accumulated in the vacuoles at concentrationshigher than the external medium, but vacuolar Na⁺ concentrationdid not reach external concentration on Na₂SO₄ and on potassiumsalts was maintained between 12 and 17 mM. Vacuolar K⁺ concentration(approx. 142–191 mM on no salt) decreased on Na₂SO₄ andincreased on K₂SO₄ and KCl. There was no precise correlation between total or specific ionaccumulation (Na⁺, K⁺, SO₄^2– and Cl^– and fresh-weightyield. Results suggest that selection results in adaptationin response to decreased water potential of the medium. Vaccinium corymbosum, blueberry, electronprobe X-ray microanalysis, callus, in vitro selection, salt tolerance, KCl, K₂SO₄, Na₂SO₄     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Effect of Light and NaCI Salinity on the Growth of Callus Cultures of Ipomoea pes-caprae (L.) R. Brown and Ipomoea batatas (L.) Lam

Callus cultures of Ipomoea pes-caprae and I. batatas were establishedon MS medium containing 10^–5 M 2,4-D and 10^–8 Mbenzyladenine. Ipomoea pes-caprae calli exhibited green pigmentationand grew better in the light than in darkness. Callus tissuesof I. batatas showed a pale-yellow colouration and they grewat the same rate in light as in dark conditions. I. pes-capraeand I. batatas callus cultures were sensitive to the presenceof 60 mM NaCl in the culture medium, the growth of the formerbeing more sensitive in light than in darkness. The significanceof the responses of I. pes-caprae callus cultures in relationto the mechanism of salt tolerance is discussed. Ipomoea batatas, Ipomoea pes-caprae, sweet potato, railroad vine, callus cultures, salinity, light     

Callus Culture of Sainfoin (Onobrychis viciifolia) and Plant Regeneration through Somatic Embryogenesis

Callus of sainfoin (Onobrychis viciifolia Scop.) was initiatedfrom stem and root explants which were obtained from seedlingsgrowing in vitro, on Linsmaier Skoog (LS) medium supplementedwith 1 mg l^–1 2, 4-D and 1 mg l^–1 BA or only 1 mgl^–1 BA, and the Vacin and Went medium without hormones.Somatic embryos were formed on LS medium containing 1 m l^–1BA. Embryos developed into complete plants on filter paper saturatedwith hormone-free LS medium. Onobrychis viciifolia, somatic embryogenesis, callus culture, plant regeneration     

Somatic Embryogenesis and Its Hormonal Regulation in Tissue Cultures of Freesia refracta

Somatic embryogenesis can be induced in tissue cultures of Freesiarefracta either directly from the epidermal cells of explants,or indirectly via intervening callus. These two pathways ofsomatic embryogenesis can be controlled and regulated by varyingthe combinations and levels of exogenous hormones. When younginflorescence segments were cultured in vitro on modified N₄(MN₄) medium supplemented with 2 mg l^–1 indoleacetic acid(IAA) and 3 mg l^–1 6-benzylaminopurine (BAP), some ofthe epidermal cells began to exhibit the features of embryogeniccells. These cells produced embryoids and developed into newplants through direct somatic embryogenesis. If the same explantswere placed on Murashige and Skoog's (MS) medium containing2 mg l^–1 IAA, 05 mg l^–1 BAP and 05 mg l^–1naphthaleneacetic acid (NAA), pale-yellow translucent nodularcalluses appeared on the surface of the explants. When thiskind of callus was transferred to MN6 medium with 2 mg l^–1IAA and 3 mg l^–1 BAP, embryoids formed which further developedinto plantlets. The regenerated plants were morphologicallynormal and possessed the normal diploid chromosome number of2n = 22. A similar result has also been obtained with youngleaf explants of this plant. The early segmentations of embryogeniccells and the development of embryoids were studied using histologicaland scanning electron microscopic techniques, and the resultshave been discussed in association with the ontogeny and originof the embryoids. Freesia refracta Klatt, somatic embryogenesis, plant regeneration, exogenous hormones     

Initiation and Growth of Callus and Cell Suspensions of Theobroma cacao L

Callus and suspension cultures of Theobroma cacao L., initiatedfrom immature cotyledons of beans from pods harvested 120–130days after pollination were established. A modified B-5 or Murashige—Skoogagar medium sustained growth of callus without loss of vigourafter each sub-culture. A 15-fold weight increase occurred duringthe 4 week culture periods at 30 ± 1 °C. Coconutwater improved callus growth substantially. The optimum hormonalconcentrations for growth of suspensions were 0.5 mg 1^–1of 2, 4-dichlorophenoxyacetic acid and 0.1 mg I^–1 of kinetinin a Murashige—Skoog basal medium liquid medium. The optimumtemperature for growth of suspensions was 25–30 °C.The cell number and cell mass of suspensions increased 20-foldin 14 days. No organogenesis or embryogenesis was observed. Theobroma cacao L., acao, cell culture, suspension culture, tissue culture.     

Effect of External Supply of Growth Substances on Axillary Proliferation and Development in Dioscorea bulbifera

Bulbil development in cultured nodes of D. bulbifera proceededin the absence of growth substances from the medium. When IAAwas incorporated into the medium at the concentrations of 5mg l^–1 and 10 mg l^–1 the cultured nodes producedlarger bulbils than in its absences. When the concentrationof IAA was increased to 15 mg l^–1, however, the culturednodes produced a callus instead of a properly organized bulbil.The dry weight of bulbils increased when kinetin was added tothe medium at the concentrations of 0.05, 0.5, and 2.5 mg l^–1.The greatest increase was with 0.5 mg l^–1 kinetin. Onincreasing the concentration of kinetin in the medium to 5.0mg l^–1 the tissue produced had smaller dry weight thanthose produced in the absence of growth substances. Additionof different combinations of IAA and kinetin to the basal mediumresulted in the production of normal bulbils, roots, and shootsin some instances (suitable combinations) and in the productionof callus and abnormal shoots in others (non suitable combinations).     

Isolation and Characterization of Sodium Chloride-Resistant Callus Culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were initiated from seedling root segments ofmungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851on modified PC-L2 basal medium. Growing cells were exposed toincreasing concentrations of NaCl in the medium. A concentrationof 300 mol m^–3 NaCl proved completely inhibitory to growthof the calli. On incubation for 25 d, cells which could toleratethis concentration of NaCl grew to form cell clones. Selectedclones were characterized with regard to their growth behaviour,K⁺, Na⁺ and free proline content when grown under stress aswell as on normal media and were compared with the normal sensitivecallus. The selected callus was capable of growing on mediumcontaining NaCl at the inhibitory concentration. The K⁺ contentof the selected callus was lower in the case of the NaCl mediumthan for the normal medium. However, the selected clones maintainedhigher K⁺ and Na⁺ levels, with increased salinization comparedwith the wild-type cells. Salt-selected cells accumulated higherlevels of free proline under NaCl stress compared to wild-typecells. Under normal conditions, however, the amounts of freeproline in selected and non-selected calli were comparable. Key words: Vigna radiata, callus culture, NaCl stress     

The production of microbial-regulatory materials by isolated aspen tissue

Isolated aspen (Populus tremuloides) callus tissue was foundto secrete materials which resulted in an initial stimulation,followed by an inhibition, of the growth of an Agrobacteriumspecies. Isolated aspen tissue also produced starch digestionmaterials which were not identical to the antimicrobial materials.The antimicrobial material was primarily bactericidal. A progressiverelease of antimicrobial materials was found as the weight ofthe tissue increased. The presence of dimethylsulfoxide in theculture medium did not influence the secretion of antimicrobialmaterials under the conditions employed. Attempts to isolateand characterize the antimicrobial materials have been unsuccessful. ¹Supported in part by the Pioneering Research Program of theBoard of Trustees of the Institute of Paper Chemistry, Appleton,Wisconsin, acting on behalf of a group of sponsoring pulp andpaper companies. ²Portions of this study were presented in partial fullfillmentof the requirements of the Master of Arts degree. Present address:Department of Biological Sciences, University of California,Santa Barbara, California, U.S.A. (Received December 10, 1970; )     

NAA-Induced Organogenesis and Embryogenesis in Hypocotyl Callus of Solarium melongena L.

Hypocotyl explants of S. melongena showed three types of regenerationthrough callus formation depending on the concentration of NAAin the medium. At 0.8 mg l^–1, only callus was produced.Lower concentrations resulted in callus, adventitious roots(optimum, 0.016 mg 1^–1 NAA), and adventitious shoots (noNAA). Roots and shoots developed during the early stages ofculture. Higher concentrations of NAA depressed callus growthand stimulated embryoid formation (optimum 8.0 mg 1^–1NAA), Embryoids were identifiable after about 6 weeks as greenspots on the surface of callus: Addition of 6-BA enhanced shootproduction but inhibited both root and embryoid production.Whole plants were obtained from embryogenic callus after transferto NAA free medium. Genotypic differences in response were observed. In general,the potential for embryogenesis was independent of or inverselyrelated to the potential for organogenesis.     

In vitro Regeneration of Mustard Plants (Brassica juncea var. RAI-5) on Cotyledon Explants from Non-irradiated, Irradiated and Mutagen-treated seed

Shoot formation in cotyledon explants of mustard (Brassica junceavar. Rai-5) was observed on Murashige and Skoog's medium supplementedwith NAA^* (1 mg l–1) and BA (1 mg l–1). Hypocotylsegments failed to differentiate shoots. Complete plants wereobtained when shoots were rooted in MS medium with NAA (1 mgl–1). EMS, a chemical mutagen, had an inhibitory effecton shoot regeneration. Gamma rays in doses above 2 kR suppressedshoot regeneration but stimulated callus growth. Brassica juncea, mustard, regeneration, tissue culture     

Laticifer Differentiation of Calotropis gigantea R. Br. ex Ait. in Cultures

The initiation and subsequent development of laticifers werestudied in callus tissues of Calotropis gigantea grown on MS(Murashige and Skoog) medium supplemented with 1 mg l^–1IAA. Laticifer development was related to the age of the cultureand could be preserved by repeated subculturing on similar mediumwith IAA. Our previous work had established that cardenolidebiosynthesis was related to rhizogenesis and here we reportthe culture system of laticifer, long term preservation anddifferentiation of this hydrocarbon producing energy plant. Calotropis gigantea, Asclepiadaceae, laticifer, differentiation, cardenolides, tissue culture     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa