Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet Cultured In Vitro

Detrez, C., Tetu, T., Sangwan, R. S. and Sangwan-Norreel, B.S., 1988. Direct organogenesis from petiole and thin cell layerexplants in sugar beet cultured in vitro.—J. exp. Bot.39: 917–926. Plant regeneration was obtained by direct bud formation frompetiole as well as from thin cell layer explants taken fromsugar beet (Beta vulgaris L.) plants grown in vitro. The budswere mainly induced in the blade-petiole transition zone ofthe explants. High frequency bud regeneration was observed inpetiole and thin layer explants of 10 different breeding linesof sugar beet tested. Organogenesis resulted when petiole explantsexcised from 8-d-old seedlings grown on half-strength Murashigeand Skoog medium (MS) containing 3.0 mg dm^–3 naphthaleneacetic acid (NAA), 3.0 mg dm^–3 6-benzylaminopurine (BAP)and 1.0 mg dm^–3 2, 3, 5, triiodobenzoic acid (TIBA) werecultured on MS with 3.0 mg dm^–3 NAA and 3.0 mg dm^–3BAP. Thin cell layer strips isolated from shoot apices culturedon MS medium supplemented with 0–9 mg dm^–3 BAP or1.0 mg dm^–3 indolebutyric acid (IBA) formed adventitiousbuds on MS medium containing 0–5 mg dm^–3 NAA + 5.0mg dm^–3 BAP. Histological studies confirmed the sub-epidermalorigin of shoots. Key words: Beta vulgaris, direct organogenesis, in vitro culture, petiole, regeneration, thin cell layer     

Production of Plantlets of Shorea roxburghii G. Don. from Embryonic Axes Cultured In Vitro

Development of axillary shoots was induced in embryonic axesof the dipterocarp Shorea roxburghii G. Don. cultured on a modifiedMS medium containing 6-benzyl-aminopurine (BAP) at an optimumconcentration of 5 mg I^–1. Excised axillary shoots wereused in multiplication and rooting experiments. Vigorous rootdevelopment occurred in shoots supported on filter paper bridgesin liquid medium containing naphthaleneacetic acid (NAA) andindolebutyric acid (IBA) (0.1 mg I^–1 each). Shorea roxburghii, Dipterocarpaceae, tissue culture, plantlet formation     

Morphogenesis in Cultured Floral Parts of African Violet

Callus tissue was induced from floral parts of African violetcultured on MS medium containing NAA (2 mg I^–1) and BAP(0.2 mg I^–1). When maintained on this medium in the presenceof light, the callus produced many shoots and roots. Large numbersof adventitious shoot buds were formed apparently in the absenceof callusing when ovary, sepal, and petal tissue was culturedon MS medium supplemented with BAP (1 mg I^–1) and NAA(1 mg I^–1). In contrast, culturing the same floral partson MS medium augmented with kinetin (1 mg I^–1) and NAA(0.5 mg I^–1) and NAA (0.5 mg 1-¹) led to the profuse developmentof roots. Organs seemed to be initiated from the epidermis ofcultured floral parts and did not appear to be related to particularcells or loci. Transfer of shoots to MS medium deviod of growthsubstances resulted in the formation of plantlets, which ata height of 3 cm could be transferred to soil and grown to maturitywithout variation in morophology or cytology.     

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     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP     

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 Embryo Culture and Induction of Multiple Shoots in Lychee (Litchi chinensis Sonn.)

Immature embryos of different sizes and ages from commercialvarieties of lychee (Litchi chinensis Sonn.) were cultured ina range of different media. Embryos as small as 3 mm could becultured using in vitro techniques and subsequently grown intoplants. MS solid medium with 2% sucrose supplemented with 150ml l^–1 coconut water was most effective in stimulatingthe germination of immature lychee embryos. Embryos of lycheewere treated to induce adventitious buds from embryonic shootsas a means of achieving multiplication. The different varietiesexhibited differences in response, with Bengal embryonic shootsproducing 15 adventitious buds after pretreatment with 100 mgl^–1 BAP for 3 h. Root formation was achieved in 65% ofadventitious shoots using MS medium supplemented with 0.5 mgl^–1 NAA and activated charcoal. These plants were successfullydeflasked and grown on in the glasshouse. This technique providesof means of producing some multiple shoots from lychee embryosand has value for multiplication in a breeding program wherea method of micropropagation is unavailable. Litchi chinensis Sonn., lychee, embryo culture, multiple shoots, in vitro     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

Multiplication In Vitro of Limonium estevei Fdez. Casas

Plantlets of Limonium estevei Fdez. Casas, an endangered Spanishspecies, were successfully regenerated from nodal segments excisedfrom young seedlings. Initiation of multiple adventitious budswere obtained in MS modified medium plus 1 mg l^–1 IBAand 0·1 mg l^–1 BAP. Rooting was achieved by transferof the isolated shoots to fresh MS medium without plant growthregulators. Fully grown plants were established in a pottingmix and are growing well in a greenhouse. Limonium estevei, in vitro multiplication, adventitious regeneration     

In vitro Regeneration from Excised Leaf Discs of Three Brassica Species

Excised leaf discs of three Brassica species, B. oleracea, B.napus, and B. campestris were induced to produce adventitiousbuds and subsequently entire plants by culture on media withspecific combinations of 6-benzylaminopurine (BAP) and -naphthylaceticacid (NAA). Each species required a particular hormone concentrationfor optimum growth and differentiation: B. oleracea, BAP 10mg^–1 and NAA 1 mg 1^–1; B. napus, BAP 10 mg 1^–1and NAA 10 mg 1^–1; B. campestris, BAP 1 mg 1^–1 andNAA 10 mg 1^–1. In a more detailed study on one of these species, namely B.oleracea, the relative influence of other media components suchas amino acids and other organic additives was examined. Itwas also found that the source and size of the explant greatlyaffected the growth response, as did the size of the culturevessel. The regenerated plants dislayed a range of ploidy as well asphenotypic abnormalities. Findings are discussed in relation to results from other tissueculture systems.     

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     

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 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     

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     

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.     

Micropropagation of Indian Rosewood by Tissue Culture

Multiple shoots were induced on excised hypocotyl segments andshoot tips of in vitro germinated seedlings of Indian rosewood(Dalbergia latifolia) on Murashige and Skoog's medium supplementedwith cytokinins and auxins. Roots were induced when individualshoots were treated first with half strength MS medium supplementedwith NAA, IAA and IBA (1 mg 1^–1 each) and subsequentlytransferred to hormone-free half-strength MS medium. The plantletswere then transferred to pots and grown in the greenhouse. Dalbergia latifolia, micropropagation, tissue culture, clonal propagation     

Micropropagation of Betula celtiberica

Plantlets were successfully regenerated from shoot segmentsof Betula celtiberica excised from young seedlings. Initiationand elongation of multiple shoot-buds were obtained after 20d culture in MS-modified medium plus BAP 0.6 mg l^–1 followedby 20 d culture in the same medium in the presence of a reducedBAP concentration (0.1 mg l^–1). Rooting was achieved 7d after having transplanted the isolated shoots to fresh medium,supplemented with IBA (0.2 mg l^–1). Betula celtiberica, birch, micropropagation, organogenesis     

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     

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