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     

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 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     

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     

In Vitro Micropropagation of Delphinium malabaricum (Huth) Munz.--a Rare Species

A micropropagation technique was developed for Delphinium malabaricumusing nodes from inflorescence stalks Maximum shoot proliferationwas obtained on Murashige and Skoog's medium supplemented with2-1P (10 mg l^–1) and inositol (100 mg l^–1) Fromthe sixth passage onwards, shoots could be multiplied by omissionof inositol and reduction of 2-1P (0.5 mg l^–1) concentrationBest rooting response was obtained with a 24-h pulse treatmentof shoots with 0.5 mg I^–1 IBA in the dark, transfer oftreated shoots to hormone-free half-strength MS medium and incubationunder 24-h light. Regenerated plants were established successfullyin the field Cytological examination of root tips of in vitroand control plants showed identical chromosome number (2n =16) Delphinium malabaricum (Huth) Munz, micropropagation, tissue culture, rare plant     

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     

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.     

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     

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.     

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 in Cassava: The Anatomy and Morphology of the Regeneration Process

Anatomical and morphological studies demonstrated that somaticembryos developed similarly on mature seed and clonal leaf explantsof cassava (Manihot esculenta Crantz) cultured for 20–24d on Murashige and Skoog (MS2) basal medium supplemented with4.0 mg l^–1 2,4-D (Stage 1) before transfer to MS2 basalmedium supplemented with 0–01 mg l^–1 2,4-D and 0–1mg l^–1 6-benzylaminopurine (Stage II medium). Within 7d of inoculation onto Stage I medium, cell divisions occurredin the adaxial tissues of cotyledon-piece and leaf-lobe explants,and associated with this was the development of embryogeneticprotusions and ridges on the adaxial surface. Foliose structuresand somatic embryo initials developed from these tissues oncotyledon, embryonic axis and leaf-lobe explants and, when cultureswere transferred to Stage II medium, further somatic embryodevelopment occurred. Somatic embryos apparently originatedfrom groups of cells and were identified by the presence ofa closed root axis, a shoot axis and cotyledons of similar shapeand venation to those of zygotic embryos. Somatic embryos hadno vascular connection with parental cultures. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, anatomy, morphology, morphogenesis     

Shoot and Plantlet Formation in Organ and Callus Cultures of Albizzia lebbek Benth

Plantlets were produced in vitro from root and hypocotyl explantstaken from seedlings of the tree legume, Albizzia lebbek. Theseexplants formed shoots when cultured with 5.0 mg l^–1 kinetinand 1.0 mg l^–1 IAA in MS medium. Shoots were also inducedin large numbers from callus treated with benzylaminopurine.About 20 per cent of the shoots rooted and were grown into plants. Albizzia lebbek Benth, tree legume, hypocotyl, root, in vitro cultures, shoot-plantlet induction     

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     

In Vitro Multiple Shoot Regeneration in Syzygium aromaticum

Multiple shoots were induced from nodal segments of seedlingsof Syzygium aromaticum, on Murashige and Skoog's (MS) basalmedium at half strength salts and Gamborg's medium (B5), supplementedwith BAP and NAA. Six to eight shoots were obtained when 3 mgl^–1 BAP and 0.5 mg l^–1 NAA were used in the medium.Both MS medium and B5 medium showed more or less similar resultsregarding the proliferation of the explants. Syzygium aromaticum (L) Merr and Perry (clove), multiple shoots, regeneration     

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     

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     

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).     

In vitro Propagation of Narcissus

Adventitious shoots were induced on leaf, scale and stem explantstaken from the basal plate region of flowering-size bulbs onmedia containing 2–16 mg l^–1 6-benzylaminopurineand 0·25–4·0 mg 1^–1 1-naphthal-eneacetic acid (NAA). Only the levels of NAA had a significanteffect on the numbers of shoots produced. When trimmed and splitin half, 6 mm or more diameter in vitro shoots regenerated furtheradventitious shoots which in turn grew in size suitable forsplitting within 16 weeks. The vigour of the first generation of shoots was proportionalto the hormone levels used for their initiation. All shootseventually declined in vigour, senesced and formed dormant bulbils.Split senescent shoots regenerated only a few secondary shootswhich quickly became senescent. A total of 500–2000 bulbilscould be obtained from each initial bulb within 18 months. Bulbilsrequired 10 weeks at low temperature before planting to breakdormancy. Histological observations showed that in twin scales and splitshoots, adventitious shoots were regenerated from at least twosuperficial layers of menstematic cells near to the basal plate.This multicellular mode of origin suggests that plants multipliedfrom in vitro adventitious shoots could be as genetically uniformas those from natural vegetative increase. Narcissus, tissue culture, propagation, adventitious shoots, histology     

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     

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