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     

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     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

Callus, Organogenesis and Plantlet Formation in Tissue Cultures of Clitoria ternatea

Decoated seeds of Clitoria ternatea L. germinated on Murashigeand Skoog (Physiologia Plantarum 1962, 15, 473–97) basalmedium (BM) and differentiated callus and bipolar embryoids(two-step method) in low frequency. Calluses developed on lateralroots [BM+KN(0.1 mg 1^–1)], on roots and hypocotyls [BM+KN(0.5mg 1^–1)], and on roots [BM+KN+IAA (0.5 mg 1^–1 ofeach)]. On basal medium with KN (0.5 mg 1^–1) and withKN+IAA (0.5 mg1^–1 of each), multiple shoot buds and embryoids(one-step method) were differentiated directly on split hypocotylsand roots. In the former, shoot buds developed even on unsplithypocotyls. Rhizogenesis on isolated shoot buds occurred efficientlyin BM+indole butyric acid (IBA 0.1 mg 1^–1) and BM+IAA(0.1 mg 1^–1 and 0.5 mg 1^–1). Profuse direct embryoidsand shoot buds developing on root systems are interesting morphogeneticphenomena rarely reported. Clitoria ternatea L., callus, embryoids, multiple shoot buds, regeneration     

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.     

Mineral nutrition and auxin-induced growth of Triticum coleoptile sections

A revised method has been described for assaying auxin by thegrowth of Triticum coleoptile sections. With additions of Ca(NO₃)₂10^–4 and MgSO₄ 10^–5 mol liter^–1 the sensitivityand accuracy have been increased. This is mainly due to Ca.The coleoptiles obviously suffer from Ca-deficiency. The importanceof a strict time schedule for manipulations is emphasiced. Theduration of the tests is limited to 6 hr.Indole-3-acetic acidcan be determined in concentrations down to 10^–9mol liter^–1;from 10^–10 to 10^–9mol liter^–1 the log-logrelation between concentration and growth is linear. Above 10^–7mol liter^–1 elongation takes place under an abnormal increasein elastic extension, indicating that growth is limited by someunknown wallstabilizing factor. The interrelation between auxin,an anti-auxin and Ca are discussed. (Received May 8, 1973; )     

Callus Initiation and Organized Development from Zamia pumila Embryo Explants

Explants derived from Zamia pumila embryos were cultured ona Murashige and Skoog basal medium supplemented with naphthaleneaceticacid (NAA), N⁴-benzylaminopurine (BAP), or combinations of thetwo at 27 °C in darkness. NAA was invariably required forcallus initiation, and its minimal effective concentration was0.1 mg l^–1. BAP was not always required, and dependingon the explant type and NAA concentration, BAP either enhanced,suppressed, or had little effect on the frequency of callusinitiation. High frequency of callus initiation occurred with1.0 mg l^–1 NAA combined with 0.01 or 1.0 mg l^–1BAP. When the concentration of NAA was high relative to thatof BAP, friable callus was produced. As the relative BAP concentrationwas increased, a more compact callus formed. Compact-nodularcallus developed at equal concentrations of NAA and BAP overa wide range of absolute concentrations. Friable callus formedroots only. Compact-nodular callus formed roots, shoots andembryo-like structures. Root and shoot formation predominatedand were of nearly equal frequency. Formation of embryo-likestructures was infrequent. Zamia pumila, callus differentiation, callus formation, embryo culture, naphthaleneacetic acid, N⁴-benzylaminopurine     

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     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Promotion of Plant Cell Division by an Epidermal Growth Factor

In some specified treatments, an epidermal growth factor (EGF)promoted adventitious root formation in epicotyl cuttings ofVigna angularis. The number of the roots induced in cuttingstreated with 0.1 mg liter^-1 EGF during the first 24 h and with210^-4 M IAA during the second 24 h was 15% greater than thatof the roots in cuttings treated without EGF and with IAA. Analysisof the optimum timing of EGF application was performed by dividingthe first 24 h period into three sequential 8 h periods (0–8h, 8–16 h and 16–24 h). The most effective timeperiods in terms of the root formation were 8–16 h and16–24 h. The 0–8 h period was ineffective with respectto the formation. When carrot suspension cells were culturedfor 15 days at a very low cell density (1,000 cells/3 ml Murashigeand Skoog's medium) with more than 0.1 mg liter^-1 EGF, cellnumbers were 72% higher than those cultured without EGF. Theseresults suggest that EGF promotes cell division of plants. (Received October 5, 1992; Accepted May 24, 1993)     

Differentiation of Shoot Buds in vitro in Tissue 1Sisymbrium irio L.

Shoot bud formation was induced in the stem callus of Sisymbriumirio L., a Cruciferous plant. The callus was established onMurashige and Skoog medium with IAA (1?0 mg l^–1) and kinetin(0?5 mg l^–1). The effect of three purines (kinetin, 6-benzylaminopurine,and 6-methylaminopurine) incorporated singly along with IAAin MS medium was investigated. It was found that kinetin orMAP (3–5 mg l^–1) along with IAA (0?5 mg l^–1)were the most effective in inducing shoot bud formation. Adeninesulphate (10 mg l^–1) with kinetin (1?0 mg l^–1) alsoinduced bud differentiation. The morphogenetic potential of the callus to differentiate shootbuds was seemingly lost in 2 year old callus cultures. However,on successively subculturing on a regeneration medium shootbuds differentiated and the number of buds formed improved onfurther subculture. Two types of meristematic outgrowths were recognized: (i) arisingfrom superficial cells and (ii) arising from deep-seated cellsin the vicinity of tracheidal elements. However, both typesformed meristematic nodules on the surface of which shoot budsdifferentiated. Some embryoids were also recognized arisingsuperficially.     

Effects of Growth Regulators and Glutamine on In Vitro Development of Zygotic Embryos of Taro (Colocasia esculenta var. antiquorum)

Zygotic embryos of taro, Colocasia esculenta var. antiquorumcultured on Linsmaier-Skoog (LS) medium without the additionof hormones develop into mature plants only in the presenceof endosperm tissue. Growth is usually evident within the firstweek of culture when embryos swell and become green. Embryosexcised from endosperm and cultured on LS containing 0-01 mg1^–1 naphthaleneacetic acid (NAA), and 0–01 mg 1^–16-dimethylaminopurine (6-DMAP) grow at a rate comparable withcontrols for the first week of culture. During the second week,growth rates are higher than controls primarily because embryosform elongated hypocotyl regions which often produce swollentissues and/or callus. In the presence of 200 mg 1^–1 glutamineand a range of concentrations of 6-dimethylaminopurine, benzyladenine,or NAA, elongation of the hypocotyl axis is inhibited, and acompact callus may develop. Embryos grown on LS containing 200mg 1^–1 glutamine and 2.0 mg 1^–1 2, 4, 5-trichlorophenoxyaceticacid form friable callus which was used to generate short-livedsuspension cultures. Growth Regulators, Glutamine, tygotic embryos, Colocasia esculenta, endosperm     

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, Culture, and Plant Regeneration of Protoplasts of Two Shrubby Oxalis Species from South America

Protoplasts were successfully isolated from internodal callustissues of both Oxalis glaucifolia and O. rhombeo-ovata whenthey were digested in a solution containing 0.1% (w/v) MacerozymeR-10, 0.5% (w/v) cellulase Onozuka R-10 and 0.3 mmol m^–3sucrose. Protoplasts proliferated to give cell colonies on Gamborget al.'s B5 medium supplemented with 0.3 mmol m^–3 mannitol,0.5 mg dm^–32, 4-D, and 2.0 mg dm^–3 kinetin. Calluswas produced upon transfer of cell colonies to Murashige andSkoog medium containing 2.0 mg dm^–3 l-naphthaleneaceticacid (NAA) and 0.1 mg dm^–3 kinetin for O. glaucifolia,or with 5.0 mg dm^–3 NAA and 0.5 mg dm^–3 6-benzylaminopurine,for O. rhombeo-ovata. Plants were regenerated from O. glaucifoliaprotoplasts on a medium containing 0.1 mg dm–3 NAA, 1.0mg dm^–3 kinetin and 1.0 mg dm^–3 gibberellic acid,but only vascular nodules were differentiated by O. rhombeo-ovataprotoplast-derived calli. Key words: Tissue culture, protoplasts, plant regeneration, Oxalis spp     

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     

Epidermal Growth Factor Interacts with Indole-3-Acetic Acid and Promotes Coleoptile Growth

We used coleoptile sections of Avena sativa, Sorghum bicolor,and Zea mays seedlings to examine interactions between epidermalgrowth factor (EGF) and indole-3-acetic acid (IAA) that mayaffect plant growth and development. Our 24-h bioassays employedthree controls ranging in dilution from 10^–4 to 10^–8g ml^–1: (1) 50 mM potassium-phosphate buffer solution(pH=6.0), (2) bovine serum albumin, a nonspecific protein; and(3) IAA; plus two treatments: (1) mouse epidermal growth factor(EGF) ranging from 10^–6 to 10^–10gml^–1, and(2) EGF + IAA. In all three species growth in IAA, EGF, andEGF + IAA treatments showed significant increases over controls;EGF+IAA showed significant increases in growth over IAA alone.As the concentrations of IAA decreased, the EGF and IAA interactionbecame more pronounced. At the highest IAA concentrations, EGF+ IAA increased growth rates ca. 2% to 39%, whereas at lowerIAA concentrations EGF + IAA promoted growth as much as 121%,thereby lowering the normal IAA physiological set point up tothree or four orders of magnitude. Our data suggest that aninteraction between EGF and IAA may allow plants to recognizeand respond to animal biochemical messengers, resulting in changesin plant cell elongation that ultimately may alter plant growthpatterns. (Received April 27, 1994; Accepted September 5, 1994)     

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     

Morphogenetic Responses of Browallia Leaf Sections and Callus

Leaf sections of Browallia viscosa and B. speciosa were placedon Murashige and Skoog (1962) salts and vitamins medium (MS)containing auxins and cytokinins, singly or in combination,to elicit morphogenetic responses. B. viscosa developed extensiveroots in 4 weeks on media supplemented with indolebutyric acid(IBA), indol-3-yl acetic acid (IAA) or naphthalene acetic acid(NAA) (0·01, 0·1, 1·0, 5·0 and 10·0mg^–1), but with 2, 4-D (0·1 mg^–1) only lightyellow friable callus was obtained. Shoot initiation and elongationoccurred consistently in 4–6 weeks on leaf sections inthe presence of 6---dimethylallyl amino purine (2iP). Similarly,shoot regeneration from leaf-derived callus, initiated and sub-culturedon MS + benzyladenine (BA) + NAA only induced callus on leafexplants of both species. B. speciosa did not respond exceptfor moderate and prolific callus formation on MS + BA + NAAand Uchimiya and Murashige (1974) media respectively. Browallia viscosa, Browallia speciosa, tissue culture, regeneration, morphogenetic potential