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 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 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     

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     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

In Vitro Somatic Embryogenesis and Plant Regeneration in Clitoria ternatea

A sustainable plant regeneration system in vitro through somaticembryos from mature sexual embryos has been reported in Clitoriaternatea. Somatic embryos developed through callus from seedlingroots on hormone-free MS medium (MS₁). Addition of growth hormones,KN 0.5 mg dm^–3 (MS₂) or KN+1AA 0·5 mg dm^–3of each (MS₃) induced direct somatic embryos, in high frequency,on split root and hypocotyl systems. The embryogenic potentialvaried with the organ, roots or hypocotyls, and also with themedium. The morphogenetic capacity of the somatic embryos isretained for more than 2 years by subculturing at intervalsof 4 weeks on MS₃ in complete darkness. Somatic embryos, underthe appropriate subculture conditions (16 h light/8 h dark photoperiodat 24± 1 °C on media MS₃, MS₄ and MS₅), resultedin recurrent-somatic embryogenesis and was profuse at the shootand root apices of the somatic embryos. Mature somatic embryoswere transplanted to MS₁ to stimulate germination and plantletregeneration. Plantlets, developed from primary and secondaryembryos on MS₁ were successfully hardened and grown in naturaloutdoor conditions. The morphology and histology of the somaticembryo and plantlet and the culture conditions for continuousproduction of plantlets through direct somatic embryogeny arediscussed Key words: Clitoria ternatea, somatic embryos, plant regeneration     

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.     

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     

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     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Wheat Callus Culture: the Initiation, Growth and Organogenesis of Callus Derived from Various Explant Sources

Callus was obtained from mature excised embryos of wheat, fromnodal and internodal stem segments and from rachis segmentsusing the medium of Murashige and Skoog(1962)(M medium), containing1-0mg l^–1 2,4-D, and from immature embryos using the mediumof Green and Phillips (1975) containing 2 mg l^–1 2,4-D.Callus yield from mature embryos depended upon the cultivarused. No callus could be obtained from leaf segments. Callusderived from mature embryos and nodal stem segments was successfullymaintained by serial sub-culture on the M medium containing2,4-D for up to 3 years although its growth rate declined toa lower level as culture proceeded. Such cultures consistently produced roots when transferred toa medium containing a low level of 2,4-D or no 2,4-D. The presenceof the auxin was essential for continued proliferation of thecallus tissue. Shoot initiation was infrequent, did not occurafter the first few sub-cultures and could not be enhanced byvarious auxin and cytokinin additions to the medium. Callusderived from immature embryos did not have an enhanced potentialfor shoot initiation. Triticum aestivum, wheat, callus culture, organogenesis     

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     

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     

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

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 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     

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.     

In Vitro Morphogenesis in Nardostachys jatamansi DC.: Shoot Regeneration from Callus Derived Roots

Callus cultures of Nardostachys jatamansi DC. maintained onMurashige and Skoog's medium containing 3.0 mg 1^–1 of-naphthaleneacetic acid and 0.25 mg 1^–1 of kinetin whenshifted to medium containing 0.25–1.0 mg 1^–1 ofindole-3-acetic acid or indole-3-butync acid showed profuserhizogenesis. The callus-regenerated roots when transferredto medium containing 2.0–6.0 mg 1^–1 of kinetin producedshoot buds. The de novo shoot bud regeneration took place eitherdirectly from cortical cells or from the inner stelar region.In addition, direct, concomitant root-shoot development wasalso observed. Nardostachys jatamansi, organogenesis, root-buds     

Cyclic Nucleotides and In Vitro Plant Cultures: I. INDUCTION OF ORGANOGENESIS IN TOBACCO (NICOTIANA TABACUM CALLUS CULTURES

Mangat, B. S. and Janjua, S. 1987. Cyclic nucleotides and invitro plant cultures. I. Induction of organogenesis in tobacco(Nicotiana tabacum) callus cultures.—J. exp. Bot. 38:2059–2067. The possibility that cyclic nucleotides have a mediatory rolesimilar to cytokinins in plant tissue cultures was examined.Calli obtained from tobacco pith tissue were incubated on growthmedia supplemented with either cyclic AMP, cyclic GMP, adenosineor guanosine, in concentrations ranging from (mg dm^–3)0 to 2·0 together with 2·0 mg dm^–3 of IAA.Results were compared with identical calli grown on media containingcomparable amounts of kinetin and IAA. Increase in callus growthwas observed on all media containing cyclic AMP, cyclic GMP,adenosine, guanosine or kinetin. Adenosine or guanosine didnot promote organogenesis. Low concentrations (0·02 and0·05 mg dm^–3) of kinetin stimulated extensive rootdevelopment. Some root formation was also elicited with higheramounts of cyclic AMP (0·1 and 0·2 mg dm^–3)or cyclic GMP (0·2 and 0·5 mg dm^–3). Bothkinetin and cyclic GMP promoted shoot differentiation. However,in contrast to kinetin, cyclic GMP induced organogenesis atlower concentrations (0·02 and 0·1 mg dm^–3).The addition of 2·0 mg dm ^–3 of cyclic AM P toIAA-free growth media elicited shoot differentiation. This wasalso the case with a similar concentration of kinetin or cyclicGMP. Results suggest cytokinin activity for the two cyclic nucleotides. Key words: Tobacco, Nicotiana tabacum, tissue culture, cyclic nucleotides, cyclic AMP, cyclic GMP organogenesis     

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