Plant regeneration from Bulgarian rose callus

Plant regeneration capacity of Bulgarian rose callus tissue was examined. Adventitious bud formation could be successfully attained, depending on the kinds of mineral salts used in the medium, auxin and cytokinin used. When callus tissues were cultured on the medium without ammonium nitrate and contained indoleacetic acid and benzyladenine, buds were formed in the callus. The number of buds were significantly increased by the simultaneous addition of calcium ionophore. When the cultures were transferred to the medium without cytokinin, roots were formed in the basal part of the buds.Abbreviations BA benzyladenine - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250 M BA for 3 h and then culture them on medium containing 5 M each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.     

Morphogenesis in Sitka spruce (<Emphasis Type="Italic">Picea sitchensis</Emphasis> (Bong.) Carr.) bud cultures – tree maturation and explants from epicormic shoots

The adventitious bud forming ability of Sitka spruce (Picea sitchensis (Bong.) Carr.) buds in vitro was found to be dependent upon the age of tree from which the explants were taken. Bud formation declined exponentially with increasing tree age when 1.0 and 10 μM 6-benzylaminopurine (BA) were used to induce adventitious buds. When less BA was used (0.1 μM) bud production was much lower with all ages of tree and no mathematical relationship between declining bud production and tree age was found. By a tree age of 6 years bud-forming ability had declined severely. Even the few buds that developed on older tree tissues failed to elongate into shoots, became necrotic and eventually died, indicating that adventitious bud induction in this species is not a rejuvenative process. Callusing of bud explants also declined with increasing tree age when 0.1 μM BA was used whilst very little callusing occurred at the higher cytokinin concentrations (1.0 and 10 μM BA). Tissue necrosis in vitro increased with tree age, whilst the ability of BA to retard necrosis declined with increasing tree age. Buds from epicormic shoots, formed on the lower trunks of 20-year-old trees when these were exposed to light, were not significantly better at forming adventitious buds in vitro than buds taken from the lower branches of the crown.     

Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro

Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained from 22.0 M cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.Abbreviations (BA) 6-benzyladenine - (NAA) 1-naphthaleneacetic acid     

Role of hormones on flower bud formation in thin-layer expiants of tobacco

Flower bud formation was studied in thin-layer tissue expiants of epidermis plus subepidermal cortex from the inflorescence ramifications ofNicotiana tabacum cv. Samsun. With appropriate hormone concentrations of BA and NAA expiants from flowerv and fruitbearing stalks regenerate flower buds only, while those from the internodes of the inflorescence ramifications produce generative as well as vegetative buds. In both types of expiants the number of buds formed depend mainly on the hormone concentrations but, in addition, the age of stalks and internodes from which expiants are taken also affects bud formation. Both ABA and JA inhibit flower bud formation in expiants of flower stalks. JA was shown to particularly inhibit bud initiation.     

Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tsatsai</Emphasis>)

Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.     

Hormonal and histological studies related to in vitro banana bud formation

Shoot apices of Musa subgroup AAA `Grande Naine' were used for in vitro culture establishment. The endogenous hormone levels and their effects on bud formation were evaluated during a 75-day period. Cytokinins, IAA and ABA were separated by HPLC and quantified by means of ELISA. Enzymatic degradation of IAA was determined by the colorimetric method. Explants were maintained on establishment medium for 60 days. The endogenous cytokinins were higher in the basal portion of the explant. Subculture to proliferation medium (65 to 75 days) resulted in a substantial increase of cytokinins in the basal portion and in a decline in the apical portion. 2iP was the predominant cytokinin in the tissue. The endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65th day of culture. The level of ABA was reduced from the time of inoculation up to the 75th day of culture. Histological analysis indicated that buds formed at the leaf base at the 65th day of culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

大岩桐花瓣切块离体培养高频率花芽再生

该文报道了大岩桐花瓣切块离体培养再生花现象,花瓣切块再生花有两种方式：一种是仅再生花芽（命名为BF）;另一种是既再生花芽也再生营养芽（命名为BF＋V）。花芽再生的能力与光照、花芽大小及培养基中赤霉素和细胞分裂素浓度紧密相关。当培养基中含有1.0 mg/L GA3时,BA的添加会显著增加总花芽（BF＋BF＋V）的形成率,添加0.5 mg/L BA时,总花芽形成率达100%。在暗中培养时,BF达93.4%。不同大小花芽的花瓣再生花的能力不同,7 mm直径花芽的BF最高,达86.7%。同时,对花芽再生过程中花瓣切块的组织结构形态变化也进行了观察。     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Adventitious shoot bud formation and plant regeneration from <Emphasis Type="Italic">In vitro</Emphasis>-cultured stem segments of reed (<Emphasis Type="Italic">Phragmites communis</Emphasis> Trin.)

Summary A protocol for large-scale propagation of Phragmites communis Trin. by adventitious bud formation and plant regeneration was established. Adventitious buds were induced through either the indirect pathway or the direct pathway from stem explants of Phragmites communis. In the indirect pathway, it was essential to decrease the level of 2,4-dichlorophenoxyacetic acid from 9.1 to 0.5 μM to induce adventitious buds and achieve plant regeneration. In the direct pathway, the effects of different benzylaminopurine (BA) concentrations in the medium, and different positions of the explants, on adventitious bud formation were determined. Murashige and Skoog (MS) medium supplemented with 5.4μM α-naphthaleneacetic acid (NAA) and 53.4 μM BA, and the bottom part of stem explants were most responsive for the differentiation of adventitious shoot buds. The highest differentiation frequency was 20–30 adventitious shoot buds per stem node tissue. Elongation and proliferation of adventitious buds were achieved on MS medium supplemented with 13.3 μM BA and 5.4 μM NAA. Shoots were rooted in liquid half-strength MS medium with 5.4 μM NAA+4.9 μM indole-3-butyric acid. Rooted plants survived (87.5%) and grew well after transfer into soil for 4 wk. More than 20 000 regenerated plants of a salt-tolerant variant line of Phragmites communis have been produced. This protocol is useful for clonal micropropagation and possibly for Agrobacterium- mediated gene transfer in P. communis.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

In vitro morphogenesis from excised leaf explants of Digitalis obscura L.

The morphogenic capacity of Digitalis obscura leaf explants cultured in vitro has been studied, noting factors promoting the differentiation of roots, buds and shoots as well as those promoting callus proliferation. Complete plant regeneration was obtained only by first culturing the leaf explants in a medium with NAA and BA to induce formation of buds, and subsequently transferring them to a medium without growth regulators to achieve the further development of shoots.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid