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     

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.     

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     

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     

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).     

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     

The Establishments of Tissue 4

Callus cultures of 12 temperate grasses were established, somefor the first time, by incubating detached roots or whole seedlingson a Linsmaier and Skoog basal medium which contained 2, 4-dichlorophenoxyaceticacid (2, 4-D) at 1.0 mg 1^–1 as the only growth hormone.The callus, which developed in the pericycle of the roots andon the embryo of the seeds, was subcultured on the same medium;further growth of the callus varied from good in the case ofDactylis glomeraia, Agrostis tennis, Cynosurus cristatus, andPoa trivialis, to poor in several Lolium species and varieties.Most cultures developed root primordia which sometimes grewinto visible roots, but shoot primordia, none of which grewinto shoots, were found only in the callus of Lolium multiflorumvar. westerwoldicum. Cell suspension cultures were also readily established and maintainedusing the same culture medium. Most cultures contained a highproportion of round or oval cells, which ranged from 18 to 77µm in diameter or length, while many also had a significantproportion of larger, more elongated cells which varied in lengthfrom 46 to 182 µm. The cells of Dactylis glomerata werecharacteristically larger and more convoluted than the cellsof other grass species that were examined. The addition of kinetinat 0.1 mg 1^–1 to the 2, 4-D-containing culture mediumincreased the proportion of irregular-shaped cells and reducedthe dispersion of the cells, perhaps by improving cellular contactand adhesion; in some species, such as Agrostis tenuis and Phleumpratense, the presence of kinetin promoted the deposition ofstarch granules in cells.     

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.     

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     

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     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Organogenesis in the Cultured Female Gametophyte of Ephedra foliata

The female gametophyte of Ephedra foliata was used as an explantfor the production of haploids as it is composed of haploidcells, all of the same genotype. The regeneration of roots wasdependent upon the presence of NAA, while BAP had a modifyingeffect. At lower concentrations (0.05 parts 10^–6 and 3.5parts 10^–6) BAP enhanced the root promotion of NAA (0.05–4.0parts 10^–6). At higher concentrations of BAP (1–6parts 10^–6), roots and shoot buds were formed. Kinetinat 4.0 parts 10–6 with 0.5 parts 10–6 2, 4-D wasoptimal for shoot bud production in explants at the archegonialstage and 2, 4-D at 2.0 parts 10–6 with 0.5 parts 10–6kinetin was optimal for root formation. Cells of the callusand root tip had the haploid number of chromosomes, n = 7. Meristemoidswere located on the surface or embedded in the callus tissue.The deep seated meristemoids organized only root primordia,but the peripheral ones gave rise to root as well as shoot budprimordia. Initially, there was no vascular connection betweenthe shoot-bud and the callus. This was established later. Key words: Ephedra, Female gametophyte, Haploid, Tissue 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     

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     

Callus Growth and Plantlet Regeneration in Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae)

Explants of taro cultivars belonging to Colocasia esculentavar. esculenta have been nearly impossible to culture untilrecently. Here, we describe a method which induces callus formationfrom bud explants of Colocasia esculenta var. esculenta cv.Akalomamale, brings about shoot and root production, and leadsto plantlet regeneration. The medium used is half-strength Murashige-Skoog(HMS) solution containing 25 ml taro tuber extract (TE), 2 mg2, 4, 5-trichlorophenoxyacetic acid and 200 mg glutamine 1^–1.TE is an important requirement for bud explants and callus tissues.Root induction on callus-derived shoots (i.e. plantlet formation)occurs on HMS containing only 25 ml TE and 100 ml coconut water1^–1. Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae), coconut water, micropropagation, plantlet regeneration, root formation, taro extract, tissue culture     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Regeneration of Shoots on Root Explants of Flax

Regeneration of shoots from adventitious root explants of flaxwas achieved for two of five cultivars tested. Root explantsof the cultivar Bombay regenerated buds on MS medium with varioussupplements, the best combination being 002 mg I^–1 NAA,1 mg I^– 6-BA, 20 mg I^– adenine and 500 mg I^–cefotaxime. Adenine, in the presence of 6-BA, and cefotaxime,in the presence of both 6-BA and adenine, were found to stimulatebud initiation, but the most important factor influencing budregeneration from flax roots was genotype     

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l⁻¹) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l⁻¹) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l⁻¹ TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.     

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