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     

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 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     

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     

Effects of Sucrose Concentration on the Photosynthetic Ability of Rose Shoots In Vitro

Reducing the concentration of sucrose in the culture mediumover successive subcultures has been tested as a method forincreasing the ability of rose shoots grown in vitro (Rosa cvsIceberg and Peace) to take up CO₂. Shoots maintained on ‘constant’10, 20 and 40 g I^–1 sucrose showed decreased levels ofCO² uptake at higher sucrose concentrations, although cv. Peacegrew least at 10 g l^–1 and showed correspondingly lowamounts of CO₂ uptake compared with 20 and 40 g l^–1. Bothcultivars died when sucrose was omitted from the medium. Assucrose concentration was reduced in the medium, so CO₂ uptakeof shoots initially cultured on 20 and 40 g l^–1 sucrosewas found to increase, although a concentration of 10 gl ^–1sucrose seemed to be limiting, below which the growth and chlorophylllevels of shoots declined. Rosa hybrid, rose, shoot culture in vitro, photosynthetic ability, sucrose, infra-red gas analysis     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

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     

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     

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     

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.     

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).     

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     

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     

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     

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.     

Soya Bean Seed Growth and Maturation In vitro without Pods

Immature Glycine max (L.) Merrill seeds, initially between 50and 450 mg f. wt, were grown and matured successfully in vitro.Excised seeds were floated in a liquid medium containing 5 percent sucrose, minerals and glutamine in flasks incubated at25 °C under 300 to 350 µE m^–2 s^–1 fluorescentlight. During 16 to 21 d in culture, seeds grew to a matured. wt of 100 to 600 mg per seed at an average rate of 5 to 25mg d. wt per seed d^–1 depending on initial size. Growthrates were maximal during the first 8 to 10 d in vitro but declinedwith loss of green colour in the cotyledons. Seed coats rupturedwith rapid cotyledon expansion during the first 2 d in culture.Embryos were tolerant to desiccation and 80 to 90 per cent germinatedif removed from culture before complete loss of green colour.The growth of excised seeds in vitro exceeded the growth ofseeds in detached pods, but when windows were cut in pods topermit direct exposure of seeds to the medium, seed growth wascomparable. Glycine max (L.) Merrill, soya bean, seed culture, seed growth, seed maturation, germination     

The Detection of the Accumulation of Silicon in Phalaenopsis (Orchidaceae)

Small particles along the veins of leaves in Phalaenopsis containsilicon. The silica bodies are spherical in shape and 5-20 µmin diameter. In the in vitro cultured plantlets, they grow differentlyin size, depending on the developmental stage of the plantletsand the concentration of silicon added to the medium. The growthof the silica body was increased by increasing the concentrationof CaSiO₃ from 0·01 to 0·5 mg l^-1 and was maximizedfrom 0·5 to 1·0 mg l^-1. In the medium with 1·0mg l^-1 CaSiO₃, they grew to a size larger than that of the greenhouseplants after 6 months in culture. The sensitivity of the growthof the silica bodies to the environmental concentration of siliconwas then suggested to be a useful indicator for studying theuptake of silicon in plants.Copyright 1995, 1999 Academic Press Phalaenopsis, Orchidaceae, silica body, stegmata     

The Uptake and Distribution of Copper in Some Forage Grasses as Affected by Nitrate-nitrogen Supply in Flowing Solution Culture

The effect of changes in nitrate-nitrogen supply on the absorptionand distribution of copper was examined in grasses grown inflowing solution culture with a maintained concentration ofcopper. Absorption by roots (µg Cu g^–1 dry root)decreased markedly when nitrogen had been depleted or was maintainedat 0.1 mg l^–1 N, but there was an immediate increase whennitrogen was maintained at 1.0 or 10.0 mg l^–1. There werealso large increases in the concentration of copper in the shootsof plants grown with 1.0 and 10.0 mg 1^–1 N. The rootsof plants grown with 0.1 or 1.0 mg 1^–1 N retained similarproportions of uptake, but a lower proportion was retained whenthe plants were grown with 10.0 mg 1^–1. Although a lowerproportion of the copper was associated with cell walls in theplants grown at 10.0 mg 1^–1 N this was the result of alower content of cell walls rather than an effect on copperitself. In a longer-term experiment in conventional solutionculture with a range of nitrogen concentration, the concentrationof copper in shoots was largely determined by shoot growth. Dactylis glomerata, Festuca arundinacea, Lolium perenne, cell walls, copper absorption, copper distribution, flowing solution culture, nitrate-nitrogen     

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