In vitro propagation of three commercial cut flower cultivars of Anthurium andraeanum Hort

In vitro propagation of Anthurium andraeanum Hort. cut flower cultivars viz. Lima White, Tropical White and Tropical Red through organogenesis using mature plant derived leaf explants was established on Murashige and Skoog (MS) medium fortified with different growth regulators. Cultivar, stage and different regions of the source leaf, and type of growth regulators significantly influenced callus induction. Explants from folded brown leaves were superior in induction of callus. Half strength MS medium fortified with 0.88 microM of benzyiadenine (BA), 0.9 microM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.46 microM of kinetin (Kn) at pH 5.5 was most effective for callus induction. Transfer of callus to medium with 0.54 microM of NAA in place of 2,4-D induced higher number of shoots. Subsequent cultures displayed enhanced rate of shoot initiation and multiplication. Transfer of shoots onto half strength MS medium supplemented with 0.54 microM of NAA favoured rooting of shoots. Cultivar Tropical White was superior in callus, shoot and root induction compared to Lima White and Tropical Red. Plantlets after acclimation in greenhouse were transferred to net-house, that exhibited ninety seven per cent survival. Plants flowered normally between 12 and 15 months and were morphologically similar to that of the mother plants.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

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     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Factors affecting shoot regeneration from zygotic embryo and seedling explants of Cycas revoluta thunb

Summary Adventitious shoot induction from zygotic embryo and seedling explants of Cycas revoluta was studied, with reference to both nitrogen formulation of the medium and light. The presence of nitrate as a sole source of nitrogen in Klimaszewska and Keller (KK) medium did not promote shoot induction; on the other hand, shoot induction was promoted by the use of ammonium-containing media, i.e., Schenk and Hildebrandt (SH) and Murashige and Skoog (MS). In the case of zygotic embryos, the percentage of responding explants and the number of regenerated shoots were significantly higher on SH medium than on MS medium. With seedling explants, shoot induction was not affected by the use of SH medium versus MS medium. Photoperiod also influenced bud formation and development. The absence of light during shoot induction stimulated callus formation and very few differentiated shoots. Shoot induction from zygotic embryos was positively affected by the application of 4- and 8-wk photoperiod exposures, the combination SH medium/8-wk photoperiod exposure giving the best results in terms of shoot induction and development. Rooting of shoots was improved by their culture in medium containing NAA.     

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.     

Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars

Protocols were developed to optimize adventitious shoot regeneration from four southern highbush blueberry cultivars. Leaf explants from 6 week-old shoots of the four cultivars were excised and cultured on woody plant medium each containing thidiazuron (4.54 or 9.08 μM), zeatin (18.2 μM), or zeatin riboside (5.7 or 11.4 μM) either separately or in combination with α-naphthaleneacetic acid at 2.69 μM. Optimum medium for shoot regeneration was genotype-dependent. Efficient regeneration was obtained at frequencies of 88.9% for ‘Jewel’, 87.8% for ‘Emerald’, 53.3% for ‘Jubilee’ and 87.8% for ‘Biloxi’. Leaf explants of newly developed shoots from the cultures having undergone five subcultures had higher regeneration frequencies than those having undergone two subcultures. Regenerated shoots, 80–100% for each cultivar, rooted in 8 weeks after transplantation to soil. The regeneration systems described have potential use in genetic transformation of southern highbush blueberry cultivars.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

Factors effecting adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga)

A procedure for adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga Mill.) using thidiazuron (TDZ) was developed. Excised leaves of cultures grown on Murashige and Skoog (MS) medium containing 5 M benzyladenine (BA) and 0.9% Gibco Phytagar were used. Several experiments were conducted to determine optimum concentrations of thidiazuron, -naphthaleneacetic acid (NAA) and sucrose. When the medium contained 1.5 M TDZ and 2.5 M NAA, 85% of the discs regenerated shoots with an average of eight shoots per leaf disc. An incubation period of three weeks in the dark was necessary for optimum shoot regeneration. Leaves excised from four to six-week-old cultures gave a higher percent shoot regeneration than leaves from cultures older than six weeks. Regeneration percentages were significantly reduced when sucrose concentration in the medium was less than 3%. A significantly higher percentage of shoots regenerated when leaf discs were placed on the regeneration medium abaxial side down as compared to the adaxial side.Regenerated shoots were cultured on MS medium containing 5 M BA and rooted on half-strength MS medium containing 10 M NAA. Rooted plantlets were acclimatized to greenhouse conditions for evaluation of any somaclonal variation. The importance of these findings are discussed in relation to in vitro improvement of plants.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962) salt mixture - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N'-1,2,3,-thiadiazol-5-ylurea) Approved for publication by the Director, West Virginia Agric. and For. Expt. Sta. as Scientific Article No.2346     

Factors affecting direct organogenesis from flower explants of Allium giganteum

A novel method is described for the propagation of Allium giganteum Regel using direct organogenesis resulting in multiple shoot structures formed on mature flower buds or ovaries. A two step induction and differentiation procedure, similar to that described earlier in onion, was tested. Flowers were inoculated on the induction medium for 6 days and extracted ovaries were placed on the differentiation medium. Optimal formation of multiple shoot structures was obtained using modified BDS medium containing 50 g l^–1 sucrose solidified by a mixture of agar/gellan-gum, with 8.88 M benzylaminopurine (BA) and 9.05 M 2,4 dichlorophenoxyacetic acid (2,4-D) in induction medium and 9.08 M tidiazuron (TDZ) in the differentiation medium. Five plant sources obtained from different European retailers of ornamental bulbs were tested separately. All tested genotypes produced multiple organogenic structures, although induction percentages clustered in two distinctive groups. Shoots formed tended to become dormant, and attempts to improve their growth and rooting included treatment with fluridone. Dormancy was partly broken when shoots were briefly dipped in 1 M fluridone. Genetic analysis of plant sources using random amplified polymorphic DNA method showed that 5 retailers actually distribute only two different clones, one of them more and the other less responsive to shoot organogensis.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Genotype-specific regeneration from a number of Rubus cultivars

Eight Rubus genotypes were examined for their ability to regenerate on 55 media differing in their plant growth substance content. Regeneration was obtained from all cultivars examined, though efficiency varied greatly depending on genotype. A minimum threshold level of thidiazuron was found to be required for regeneration from leaf discs, and the optimum level for efficient regeneration varied among cultivars. Zeatin was not found to be as effective. Thidiazuron supplemented with a range of individual auxins was investigated, and it was found that NAA had a complementary effect, applicable across a wide range of cultivars at relatively low levels. Using five random primers, 28 polymorphic PCR amplification products (RAPD markers) were stably generated and used to discriminate all eight genotypes investigated. A similarity matrix and dendrogram was generated from the markers and this was related to their regenerative ability. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenic regeneration of soybean from hypocotyl explants

Summary Thirteen soybean genotypes representing maturity groups IV−VI were compared for organogenic responses on three media cultured under two lighting conditions with hypocotyl sections excised from 7-d-old seedlings. All soybean lines responsed by producing adventitious shoots on the acropetal end of the hypocotyl explants, confirming genotype-independence of shoot initiation. Media containing 6-benzyladenine (BA; 5.0–10 μM) induced the greatest numbers of shoots. Histological studies confirmed the adventitious nature of arising shoots by indicative formation of meristematic zones and shoot primordia from parenchymatous tissues of central pith and plumular trace regions of the hypocotyl. Incompletely excised cotyledonary buds also contributed to shoot initiation. Degrees of responses were media-dependent and varied with regard to genotype. Centennial, Epps, and Lyon gave the greatest individual responses. Between cultivars (across all treatments), the regeneration potential (percentage of explants producing meristem-like structures or shoot primordia) 4 wk after initiation ranged from 47 to 75%. Four wk later, regenerative ability (number of shoots produced per responding explant) and regeneration efficiency (number of shoots produced per explant plated) yielded 1.4–7.1 and 1.0–5.0 shoots, respectively. The optimized protocol included initiation on a medium containing 5.0 μM BA for 4 wk, then transfer onto a shoot elongation medium (0.36 μM BA) for 4 wk. For 11 genotypes tested, 66–100% of excised shoots produced roots after 4 wk on media containing 12.5–29.2 μM indole-3-butyric acid. Of 109 regenerants transplanted to soil, 94% survived and no sterility has been observed on those mature enough to flower.     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

Direct shoot formation and plant regeneration from cotyledon explants of rapid-cycling Brassica rapa

Summary An in vitro culture system for direct shoot regeneration from cotyledon explants of rapid-cycling Brassica rapa was developed. Cotyledons from 3-d-old seedlings, when cultured on Murashige and Skoog (MS) medium supplemented with 20 μM N⁶-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA), regenerated shoots directly at a frequency of 20%. The addition of 2 μM aminoethoxyvinylglycine (AVG) to this medium increased shoot regeneration to 33%, but silver nitrate drastically inhibited shoot regeneration. Shoot regeneration occurred directly, at the petiolar cut ends of cotyledonary explants, between 10 to 17 d in culture. The highest percentage of regeneration (33%) was obtained from 3-d-old seedlings. NAA was the most effective auxin for root induction and development, with 49% of shoots producing roots after 2 wk on medium containing 1.0 μM NAA. Regenerated plantlets were grown to maturity in pots containing peat moss and vermiculite (1:1). These plants were morphologically normal and fertile. With this protocol, over 100 independently derived, flowering R₀ plants were obtained from 40 regenerating cotyledonary explants within 40 d after culture initiation.     

三叶半夏叶片一步成苗离体培养技术

以药用植物三叶半夏叶片为材料,通过比较直接和间接器官发生两种途径,建立了半夏一步成苗的快速繁殖技术体系。结果表明,经过愈伤组织阶段的一步成苗培养基为MS+0.5mg/L2,4-D+1.0mg/LKT,90d左右方可得到再生植株,植株分化率为74%,每个外植体上分化的块茎数为5.61±1.04。附加NAA与BA两种激素对一步成苗培养基进行优化,筛选出一步成苗最佳培养基MS+0.5mg/LNAA+0.5mg/LBA,60d后就可直接发育成完整植株,植株分化率为76%,每个外植体上分化的块茎数高达9.97±0·81,对这种培养基上的再生小植株进行移栽,1个月后,移栽成活率达100%。     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.