Growth and organogenesis in moth bean callus cultures as influenced by triazole growth regulators and gibberellic acid

The triazole plant growth regulators, paclobutrazol and uniconazole, reduced in vitro growth of moth bean callus by 20–25% when added to the culture medium at 1 mg/L (3.4 μM). The addition of 10 mg/L (29 μM) gibberellic acid (GA₃) to the culture medium in combination with the triazoles restored callus growth to a level equivalent to that of the untreated control. GA₃ alone had little effect on callus growth. When added to a regeneration medium at 1 mg/L both paclobutrazol and uniconazole reduced the percentage of cultures that formed roots, as well as the mean number of roots per culture. In contrast, GA₃ increased root formation and counteracted the inhibitory effects of the triazoles on rooting. The addition of triazoles or GA₃ to the regeneration medium reduced the formation of green meristematic nodules, which are precursors of shoots in moth bean callus. When callus was grown in the presence of either paclobutrazol or uniconazole, subsequent root and green meristematic nodule formation were reduced upon transfer to a growth regulator-free regeneration medium. The results of this study indicate that exposure of moth bean callus tissue to micromolar concentrations of triazoles or GA₃ can significantly alter in vitro growth and differentiation.     

Growth and organogenesis in moth bean callus cultures as influenced by triazole growth regulators and gibberellic acid

The triazole plant growth regulators, paclobutrazol and uniconazole, reduced in vitro growth of moth bean callus by 20–25% when added to the culture medium at 1 mg/L (3.4 M). The addition of 10 mg/L (29 M) gibberellic acid (GA₃) to the culture medium in combination with the triazoles restored callus growth to a level equivalent to that of the untreated control. GA₃ alone had little effect on callus growth. When added to a regeneration medium at 1 mg/L both paclobutrazol and uniconazole reduced the percentage of cultures that formed roots, as well as the mean number of roots per culture. In contrast, GA₃ increased root formation and counteracted the inhibitory effects of the triazoles on rooting. The addition of triazoles or GA₃ to the regeneration medium reduced the formation of green meristematic nodules, which are precursors of shoots in moth bean callus. When callus was grown in the presence of either paclobutrazol or uniconazole, subsequent root and green meristematic nodule formation were reduced upon transfer to a growth regulator-free regeneration medium. The results of this study indicate that exposure of moth bean callus tissue to micromolar concentrations of triazoles or GA₃ can significantly alter in vitro growth and differentiation.     

Hormonal Control of Morphogenesis in Leaf Explants of Perilla frutescens Britton var. crispa Decaisne f. viridi-crispa Makino

Leaf discs of Perilla frutescens var. crispa f. viridi-crispawere cultured on a defined medium to investigate factors influencingbud and root formation, callus induction, somatic embryogenesis,and floral bud formation. Addition of naphthalene-acetic acid(NAA) to the culture medium caused compact callus whereas 2,4-dichlorophenoxyacetic acid (2,4-D) promoted soft and friable callus formationon the surface of the explants. Benzyladenine, when appliedwith auxin, suppressed callus and root formation. Somatic embryogenesisoccurred, when the explants were first grown on nutrient mediumcontaining 2,4-D and organic elements, and then transferredto the 2,4-D free medium. Treatments with cytokinins, N-phenyl-N'-(4-pyridyl)urea and its derivatives induced bud formation. A low concentrationof NAA and naphthoxy-acetic acid promoted bud development. Occasionalfloral bud formation was observed depending on the originalleaf positions on mother plants from which the leaf discs wereexcised. A gradient of floral bud forming capacity along thestem was noted. Perilla frutescens, tissue culture, embryogenesis, morphogenesis, benzyl adenine, kinetin, naphthalene-acetic acid, naphthoxy-acetic acid, 2,4-dichlorophenoxy acetic acid, indol-3yl-acetic acid, cytokinins, auxins     

An Anatomical Study of Rhizome Bud Formation Induced by Paclobutrazol and Adventitious Root Formation in In Vitro Cultures of Lapageria rosea (Ruiz et Pav.)

An anatomical study was made of bud dimorphism in in vitro shootcultures of Lapageria rosea cv. Nashcourt, utilizing the presenceand absence of the gibberellin-biosynthesis inhibitor paclobutrazol(10 µM) in the medium to control the development of axillarybuds. Patterns of axillary bud development differed betweenthe aerial pattern of shoot extension (in the absence of paclobutrazol)and rhizome bud formation (in the presence of paclobutrazol),with respect to planes of cell division, cell expansion andthe formation of adventitious root primordia. These differencesare examined and discussed. Lapageria rosea cv. Nashcourt, Chilean Bellflower, rhizome bud, paclobutrazol, gibberellin biosynthesis inhibitor, micropropagation     

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.     

Influence of growth regulators on somatic embryogenesis,plantlet regeneration,and post-transplant survival of Echinochloa frumentacea

After placement on Murashige and Skoog's basal medium supplemented with 3–5 mg/l 2,4-D, immature inflorescence expiants of Echinochloa frumentacea gave rise to three distinct types of callus: a) loosely arranged and soft; b) compact and translucent; c) compact, sticky and mucilaginous. Somatic embryo formation occurred in type b callus in about 18–24 d. Callus types a and c did not produce somatic embryos. The highest percentage of cultures exhibiting somatic embryogenesis occurred on the medium containing 5 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryos also formed directly on the inflorescence (without intervening callus formation) in about 15% of the expiants placed on this medium. The addition of paclobutrazol or uniconazole (0.25 or 1 mg/l) to the medium had no influence on the percentage of cultures exhibiting direct somatic embryogenesis, but paclobutrazol slightly increased the mean number of somatic embryos per culture. Many of the callus-derived somatic embryos germinated when subcultured on basal MS medium supplemented with kinetin. Addition of paclobutrazol or uniconazole to the culture medium at 0.25 or 1 mg/l decreased somatic embryo germination and shoot elongation but increased root length and leaf width. Both paclobutrazol and uniconazole increased survival of the plantlets following transplanting to soil. Increased post-transplant survival was accompanied by reduced water loss from plantlets produced on culture media containing triazoles.     

Effect of carbohydrates and inhibitors of GA3 biosynthesis on embryogenenic potential of salt tolerant and non-tolerant callus lines of orange (Citrus sinensis osbeck)

Embryogenesis in callus lines of Citrus sinensis (orange) selected for tolerance to NaCl stress was compared to an unselected line. Addition of inhibitors of gibberellic acid (GA₃) synthesis, 2.6 × 10⁻⁶M paclobutrazol or 6.2 × 10⁻⁶M daminozide (butanedioic acid mono-(2-2-dimethylhydrazide) to the medium enhanced embryogenesis in the unselected line and two of the three salt tolerant lines, and also overcame embryo browning in the salt tolerant line R13. Sucrose as a carbon source was generally ineffective for embryogenesis. Two percent (w/v) glycerol was more effective than 2% (w/v) galactose, especially in older cultures. Formation of embryos was quicker in the non-selected line. Only a slight improvement in embryogenic potential was noted upon removal of salt tolerant lines for three passages from NaCl containing medium to basal medium without salt.     

In vitro germination and induction of direct somatic embryogenesis in 'Bottle Palm' [Hyophorbe lagenicaulis (L. Bailey) H.E. Moore], a critically endangered Mauritian palm

Hyophorbe lagenicaulis is a critically endangered palm of Mauritius. Zygotic embryos were isolated from seeds and germinated in vitro on MS salts and vitamins containing activated charcoal. When seedlings were pre-treated in vitro for 2 weeks in liquid medium containing 0.05 mg l^-1 paclobutrazol, 80% survived the transfer to soil. Three-week-old seedlings were sectioned longitudinally and partially embedded in medium that contained MS salts and vitamins, 30 g l^-1sucrose and 3 mg l^-1 2,4-dichlorophenoxyacetic acid. Somatic embryos were formed, in profusion, directly (without callus) from the haustorium, plumule and radicle. Direct regeneration is important for the conservation of endangered species, as fewer somaclonal variants are likely to arise than from indirect regeneration. When the haustorium, plumule and radicle of the longitudinally sectioned seedlings were separated, they formed more callus but fewer embryos. Plantlets derived from somatic embryos have not yet been successfully established in soil.     

Growth Behavior of a Liverwort, Jungermannia subulata Evans, in a Cell Suspension Culture. The Role of Organic Acids Required for Cell Growth

A green callus of a liverwort, Jungermannia subulata Evans,was induced from gametophytes, and a cell suspension culturewas obtained from the callus. Both callus and suspension-culturedcells grew in a modified Murashige and Skoog medium if organicacids of the TCA cycle were supplied. In the cell suspensionculture of J. subulata, ammonium was taken up preferentiallyby the cells particularly at the earliest stage of growth, whileonly a negligible amount of nitrate was utilized as long asammonium was present in the medium, and this unbalanced utilizationof the two nitrogen sources caused an abrupt drop in the pHof the medium. The organic acids of the TCA cycle supportedthe growth of this cell line by preventing the abrupt drop inthe pH of the medium. (Received June 13, 1981; Accepted October 8, 1981)     

The Meristem and Quiescent Centre in Cultured Root Apices of the gib-l Mutant of Tomato (Lycopersicon esculentum Mill.)

Cultured root apices of tomato bearing the gib-I mutation, whichreduces the levels of endogenous gibberellins, grew slower andwere thicker than wild-type contols. This was the result ofshorter and broader cells in the menstem of the mutant. Cellsof both cortex and stele were affected, but this did not causeany alteration to the volume fraction occupied by these twotissues in the root meristem. Root caps were longer in the mutantand there were also more layers of rhizodermis. All these effectscould be reproduced in wild-type roots by addition of 0.1µM2S, 3S paclobutrazol (an inhibitor of gibberellin biosynthesis)to the culture medium and could be normalized in mutant rootsby 0.1 µM GA₃. Cell doubling times in the proximal regionof the meristem were similar in mutant and wild-type roots,but were faster in both the quiescent centre (QC) and the capmeristem of the mutant. This latter feature of the mutant rootsis likely to be the cause of their longer caps, while the fasterrate of division in the QC accounts for the additional tiersof cells that were found to build up in the cortical portionof this zone These additional tiers failed to form in mutantroots grown in GA₃, but they could be induced in wild-type rootsby 2S, 3S paclobutrazol. These results suggest that endogenousgibberellins may be partly responsible for the slow rate ofcell growth and proliferation in the QC. Gibberellins, gib-I mutation, Lycopersicon esculentum, meristem, roots, 2S, 3S paclobutrazol, quiescent centre, tomato     

The Effect of Paclobutrazol on Physiological and Biochemical Changes in the Primary Roots of Pea

Primary roots of pea (Pisum sativum L. cv. Taichung No. 11)were treated with 0, 10 and 50 mg dm^–3 paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4, 4-dimethyl-2-(l,2,4-triazol-l-yl)pentan-3-ol]for 1 h at 48 h after germination. Paclobutrazol treatment inhibitedroot extension, promoted swelling (cell expansion was radialrather than longitudinal), and increased cell volume and theactivity of catalase and peroxidase enzymes. Paclobutrazol alsodecreased root respiration and ethylene production. However,under non-stressed conditions, paclobutrazol treatment did notaffect soluble carbohydrate content, water potential, osmoticpotential or water loss. Under osmotic stress with polyethyleneglycol (PEG), paclobutrazol diminished the increase of waterpotential and decreased the rate of water loss caused by theimposed stress, but had no effect on osmotic potential. Catalaseand peroxidase activity were increased in osmotically-stressedroots of treated plants. Key words: Root growth, paclobutrazol, pea, Pisum sativum, water shortage     

Culture of Mesophyll Protoplasts and Stem Segments of Antirrhinum majus (Snapdragon): Growth and Organization of Embryoids

Protoplasts isolated enzymatically from the leaves of Antirrhinummajus L. were cultured on a synthetic medium and their growthand development were followed in vitro. Cultured protoplastsincreased in size, regenerated cell walls, divided and formedsmall colonies and embryoids. Cell wall formation and cell divisionrequired both auxin and cytokinin in the medium. Isolated stemsegments of the same species, when cu1tured on a synthetic mediumwith an added auxin, yielded rapidly growing callus tissuesin which differentiation of embryoids occurred.     

Callus Induction and Plant Regeneration from Barley Mature Embryos

Callus cultures were induced starting from excised mature embryosin spring barley, Hordeum vulgare cv Maxima On a medium containinga high level of auxin, a first primary callus was induced whichwas friable, unorganized and capable of direct plant regenerationin the tested conditions This callus type was characterizedby fast growth and high variability in chromosome number Subsequently,a secondary callus type arose from the primary calli subculturedon the same medium in the light This callus type was white andcompact and consisted predominantly of diploid cells When transferredto hormone-free medium it gave rise to green shoots Completerooting of the shoots was achieved on half-strength basal mediumfollowed by exposure to higher light intensity Regenerated plantletscould then be transferred directly into soil without sufferingany loss in vitality Although showing different degrees in morphologicalvariability, they all maintained the diploid chromosome number Hordeum vulgare L, spring barley, morphogenic calli, organogenesis     

The Production of Plantlets from Tissue Cultures of Brussels Sprout (Brassica oleracea L. var gemmifera D.C.)

Shoots were induced on callus derived from sprout sections andpetiole slices of an inbred parent line of Brussels sprout (Brassicaoleracea L. var gemmifera D.C.). The shoots, when excised andtransferred to fresh medium, enlarged and formed roots. Theseplantlets could be transferred to soil or their number increasedby a multiplication process involving the production of newshoots from the dormant lateral buds. Some of the plantletsderived from sprout callus were grown to maturity in the fieldand their morphology and chromosome number compared to seedgrown plants. There were no significant differences in sproutsize and stem diameter but there were significant differencesin plant shape. None of the plants in the field experiment showedpolyploidy. Plants derived from callus possessed an enhanced ability toform callus and redifferentiate when sections from these plantswere placed back on to nutrient medium.     

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     

Induction of Callus from the Marine Brown Alga Dictyosiphon foeniculaceus

A method for callus induction from an alga was established forthe first time. Callus tissues from the microthallus of themarine brown alga Dictyosiphon foeniculaceus were induced onASP 12-NTA medium of Provasoli (1963) supplemented with 3% mannitol,0.1% yeast extract and 1.5% agar. The addition of auxins andkinetin to the medium did not show any effect on the formationand growth of the callus. (Received November 24, 1981; Accepted March 30, 1982)     

Gibberellins and the Growth of Excised Tomato Roots: Comparison of gib-1 Mutant and Wild Type and Responses to Applied GA3 and 2S, 3S Paclobutrazol

Clones of excised roots of wild type tomato (Lycopersicon esculentum,Mill., cv. Moneymaker) and a near-isogenic GA-deficient mutant(gib-1/gib-1) were cultured in modified White's medium containing1.5% w/v sucrose. The linear elongation rate of the main axisof the gib-1 mutant was 40% less than that of the wild type.In addition, the main axis of the gib-1 mutant was thicker thanthat of the wild type but main axis volume growth was the samein both genotypes, indicating that the gib-1 allele was affectingthe orientation of root expansion. There was no evidence tosuggest that the gib-1 allele affected either the pattern ofemergence or the density of lateral roots. Elongation rate andthickness of gib-1 mutant roots were restored to those of thewild type by the addition of low concentrations (0.1–1.0µM) of gibberellic acid (GA3). These concentrations ofGA3 caused a slight reduction in extension growth of wild typeroots, indicating that endogenous GAs were not limiting elongationof normal roots in culture. The GA biosynthesis inhibitor, 2S,3S paclobutrazol, at 0.1 µM, significantly reduced elongationof wild type roots and this inhibition was counteracted by 0.1µM GA₃. It is concluded that the difference in growthbetween the gib-1 mutant and the wild type represented GA-dependentgrowth. Low concentrations of 2S, 3S paclobutrazol caused onlya small (5%) reduction in growth of the gib-1 mutant and thisgrowth inhibition was not reversed by GA₃. This observation,and the fact that gib-1 mutant roots grow in the absence ofadded GA₃, suggested that part of root growth was GA-independent.However, the possibilities that the gib-1 mutant is ‘leaky’and that paclobutrazol does not inhibit GA biosynthesis completelycannot be excluded. Key words: gib-1 mutant, gibberellic acid, Lycopersicon esculentum, 2S, 3S paclobutrazol, root growth     

Enhancement of the Division of Equisetum arvense Protoplasts in Culture by Activated Charcoal and Their Further Development

Protoplasts were isolated from subcultured gametophytes of Equisetumarvense by treatment with Driselase and then cultured in vitro.Addition of activated charcoal (AC) to the culture medium enhancedthe rate of cell division, as well as the survival of both protoplastsand regenerated protoplasts. However, subsequent division ofcells was not observed after one or two cycles of replicationin cultures supplemented with AC. When regenerated protoplastswere transferred to fresh medium without AC 3 to 5 weeks afterthe first plating, the transferred cells formed rhizoids anddeveloped into small, young gametophytes without the prior formationof cell clusters or calluses. Furthermore, sprophytic shootsdifferentiated from the protoplast-derived gametophytes whenthey were cultured on medium supplemented with 6-benzylaminopurine(BA). (Received April 5, 1990; Accepted July 30, 1990)     

Generation of Seakale (Crambe maritima L.) Plantlets by Tissue Culture

When placed in shaken liquid culture in MS medium, callus derivedfrom petioles of seakale (Crambe maritima L.) failed to fragmentto produce a cell suspension culture. In the absence of 2,4-D,growth was very good, and hollow, dark green, trumpet-shapedbodies were produced, but in the presence of 2,4-D, solid, palergreen pear-shaped bodies were formed. When grown on filter paperbridges with White's medium, a small proportion of both typesof bodies underwent slow, partly-controlled development culminating,after several months, in the formation of plantlets. Seakale, Crambe maritima L., in vitro propagation, callus culture     

Embryogenesis and Plantlet Formation in Tissue Cultures of Celery

Callus cultures were initiated from sections of petioles ofcelery (Apium graveolens) var. Lathom Blanching on a modificationof the Murashige and Skoog medium. The callus, when isolatedfrom the parent explant, could be sub-cultured at regular intervalsforming both new callus and embryoids during each subculture.The embryoids appeared to be initiated on the callus surfacewhere early embryonic forms were found, but their continueddevelopment into plants only occurred when embryoids were transferredto a hormonefree medium. Embryoids were also formed in suspensionculture following inoculation of the liquid medium by differentiatingcallus, but again development of these structures was limitedto the early embryonic forms. When transferred to a solid hormone-freemedium, the embryoids, from either callus or suspension culture,developed into plantlets which could be transferred to soiland grown to maturity with only a few losses. The relevanceof this system as an aid in the breeding programme of self-blanchingcelery is discussed.