番木瓜的离体繁殖

建立番木瓜离体繁殖体系.用0.1%HgCl2溶液对番木瓜的新生嫩茎进行消毒,适宜的消毒时间为12min,1mm茎尖的成苗率达到87.6%.随蔗糖浓度的提高,番木瓜试管苗的株高显著降低,增殖系数显著增加.在附加IBA0.3mg/L的1/2MS培养基上新梢的生根率达到89.3%,试管苗大规模移栽的成活率达90%以上.基因型显著地影响番木瓜离体增殖的效率.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

In vitro propagation of Paphiopedilum orchids

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.     

In vitro propagation of Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae)

Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae), a West African liana producing naphthylisoquinoline alkaloids, was successfully raised from seeds in vitro. Clonal propagation was best achieved growing nodal stem segments on 1/5 Linsmaier and Skoog medium with full strength organics and supplemented with 0.02 μM thidiazuron, 4.44 μM 6-benzylaminopurine and 0.05 μM 1-naphthaleneacetic acid. Detached axillary shoots were grown on Anderson's Rhododendron medium devoid of phytohormones and rooted within one month when dipped in 4.92 μM indole-3-butyric acid. Rooted plants became acclimatized to nonsterile greenhouse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid     

Enhancement of in vitro growth of papaya multishoots by aeration

Efficient micropropagation of papaya (Carica papaya L.) has become crucial for multiplication of specific sex types of papaya or transgenic lines resistant to virus infection. In this study, aeration at different intervals with a 0.02 μm filter disc in the closure of culture flasks ensured exchange of gas components. The effect of aeration on development of multibuds to multishoots was investigated. Multibuds grown in culture flasks after one-week without aeration followed by a two-week aeration treatment caused a 41% increase in the number of shoots ≥0.5 cm, 42% increase in leaf expansion, and 17% increase in leaf numbers in comparison with unaerated materials. Ethylene and oxygen concentrations in the culture flasks were measured by gas chromatography and oxygen electrode at weekly intervals during the culture period. Oxygen concentrations were slightly different between aerated and unaerated culture flasks. Ethylene in the unaerated flask reached the highest level (0.11 ppm) 2 weeks after the treatment, while accumulation of ethylene in the aerated flasks was not detected. The multishoots grown for 3 weeks without aeration showed growth retardation on leaves and epinasty on petioles. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of Amaryllis belladonna

Amaryllis belladonna L. plants were multiplied successfully by means of tissue culture techniques. Different plant parts were tested as explant material, but plantlets could only be generated from the twin-scales and immature scapes. These in vitro-formed plantlets were divided into four parts and used for further multiplication. The twin-scale explants had the highest multiplication rate when a medium with 22.2 M benzyladenine and 0.54 M naphthaleneacetic acid was used. The sucrose concentration played an important role in the initiation of new plantlets, and the best results were obtained when a sucrose concentration of 2–3% was used. Anatomical observations were made during the initiation of the new plantlets.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - Benomyl (methyl [1-[(butylamino) carbonyl]-1H-benzimidazol-2-yl] carbamate) - Folpet (2-[(trichloromethyl)thio]-H-isoindole-1,3(2H)-dione phthalimide(I))     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

In vitro propagation of mature Liquidambar styraciflua

Mature specimens of liquidambar styraciflua were propagated in vitro. Components of the nutrient medium and culture conditions were first determined for one-year-old seedling material. Mature material responded similarly to seedling material in culture, but alterations in frequency of early transfers and components of the medium were required. Explants responded best to Woody Plant Medium of Lloyd and McCown supplemented with 0.2 mg l^-1 BA and 0.05 mg l^-1 NAA. Root formation occurred on shoots placed on media containing 0.5–1.0 mg l^-1 IBA. Growth in culture and percentage of rooting of mature explants were markedly affected by the individual selection, with rooting percentages varying from 33–100% among selections.     

In vitro propagation of Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann (cactaceae)

Summary Development of efficient in vitro propagation, systems for Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann, two endangered Mexican species of cacti, are described. Multiple shoot formation from areoles of in vitro-germinated plantlets was achived in two types of explants (apical and transversal) cultured in Murashige and Skoog (MS) basal media supplemented with 30 or 50 gl⁻¹ sucrose, 10 gl⁻¹ agar and various treatments with cytokins. Shoot production in these proliferation media was evaluated after one (60 d) and three (180 d) culture cycles. In P. asellifornis 13.7 shoots per explant were produced after the first cycle using apical explants in medium with 8.8 μM 6-benzylaminopurine (BA) and 30 gl⁻¹ sucrose. In P. strobiliformis the highest proliferation rate (12.4 shoots per explant) was reached using 8.8 μM Ba and 50 gl⁻¹ sucrose with shoot transverse segments as explants. After the third proliferation cycle, 128.1 and 136.3 shoots per explant were obtained in P. aselliformis and P. strobiliformis, respectively. The shoots were clongated in MS basal medium with 3 gl⁻¹ activated charcoal and rooted in MS basal media indoleacetic acid (2.85 or 5.71 μM) or indolebutyric acid (2.46 or 4.90 μM). On averge, rooting efficiency was 89% for P. aselliformis and 87% for P. strobiliformis. The survival frequency, of the plants once transferred, to soil was on average 88%.     

A protocol for efficient transformation and regeneration of Carica papaya L.

Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya. Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997.     

In vitro propagation of Dipterocarpus alatus and Dipterocarpus intricatus

Seedlings were grown in vitro from embryos of Dipterocarpus alatus and D. intricatus. The problem of explant browning could be overcome by growing embryos initially on a filter paper bridge in liquid medium with activated charcoal. The best basal medium was Woody Plant Medium without the ammonium nitrate. Cytokinin appeared to stimulate seedling growth, 5×10^-5 M 2-isopentenyladenine and 10^-4 M 6-benzyladenine (BA) being the optimum concentrations for D. alatus and D. intricatus respectively. Cotyledonary nodes, excised from the seedlings, were induced to form axillary shoots and in the case of D. intricatus these could be multiplied rapidly. D. intricatus shoots elongated by reducing the BA level from 10^-5 M to 5×10^-7 M. Roots developed when shoots were dipped in 10^-3 M indolebutyric acid for two minutes and subsequently grown in liquid medium supported by a filter paper bridge.Abbreviations AC activated charcoal - BA 6-benzyladenine - 2iP 2-isopentenyladenine - IBA indolebutyric acid - MS Murashige & Skoog medium - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - WPM Woody Plant Medium - 1/2 WPM Woody Plant Medium with half-strength macro salts - WPM (-NH₄NO₃) Woody Plant Medium without ammonium nitrate     

ACC oxidase from Carica papaya: Isolation and characterization

Most of the studies done on 1‐aminocyclopropane 1‐carboxylic acid (ACC) oxidase were done in vivo. It is only recently that in vitro studies have been carried out successfully on the enzyme. Here we report on in vitro studies of the enzyme that was isolated from Carica papaya . The enzyme had a K_m of 37 µ M and was inhibited by n ‐propyl gallate (0.240 m M ), sodium dithionite (0.022 m M ), sodium metabisulphite (0.021 m M ) and cobalt sulphate (0.100 m M ). The activity of the enzyme increased with ripening, the enzyme was somewhat labile and activity was lost after 4 days at 14°C; activity was prolonged when the crude homogenate was kept at −15°C. Isolation and purification were achieved with ammonium sulphate precipitation followed by gel‐filtration (Sephadex G 100‐120) and ion‐exchange chromatography (DEAE‐Sephadex). Gel electrophoresis of the purified enzyme gave a single band which corresponded to a molecular mass of 27.5 kDa. The amino acid content of the enzyme showed a relatively high percentage of valine (10.4%). Enzyme activity was enhanced when dithiothreitol (3 m M ) and bicarbonate ion (30 m M ) were added to the assay medium. The production of ethylene from Carica papaya did not require pretreatment of the fruit with ethylene.     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Clonal propagation of papaya in vitro

A procedure for the rapid tissue culture propagation of papaya is being developed. Tissue culture methods using apices of nursery and orchard trees of Carica papaya cv. Sunrise Solo were evaluated. The explants were established in a modified Murashige and Tucker (1969) basal medium with half-strength inorganic salts, 0.5mgl^-1 6-benzylaminopurine (BA) and 0.2mgl^-1 naphthaleneacetic acid (NAA). Established explants were transferred to a proliferation medium consisting of Murashige and Tucker (1969) basal medium, 0.5mgl^-1 BA and 0.1mgl^-1 NAA, which caused extensive multiplication of shoots. Rooting was induced at a higher frequency by subculturing plantlets onto media with indole-3-butyric acid (IBA) than with NAA.     

In vitro propagation of Halesia carolina L. and the influence of explantation timing on initial shoot proliferation

Halesia carolina L., a small, ornamentally valuable tree, is difficult to propagate due to the complexity of seed propagation and the unavailability of propagules for conventional vegetative propagation. A micropropagation system was developed to facilitate easy propagation of this species. Actively growing shoot tips achieved optimum shoot proliferation from axillary buds when placed on Woody Plant Medium supplemented with 1.0 to 2.5 mg/l benzyladenine. The addition of 0.1 mg/l naphthaleneacetic acid had little effect on culture performance. Murashige and Skoog medium was incapable of supporting vigorous shoot proliferation. Non-sterile rooting conditions provided better rooting and subsequent plantlet growth, when compared to an in vitro rooting method. The seasonal fluctuations in the stock plant dramatically affected the shoot proliferating potential of the explants in vitro. Rapidly elongating shoots formed shoot proliferating cultures more slowly than explants taken either before or after the rapid elongation phase.     

In vitro clonal multiplication of 4-year-old plants of the bamboo,Dendrocalamus longispathus kurz

Summary A complete protocol for micropropagation of 4-yr-old plants of the bambooDendrocalamus longispathus is described. Culture initiation was strongly influenced by the nature of the explant and the season. In vitro multiplication was achieved through forced axillary branching. Single node segments from the young lateral branches produced multiple shoots on agar-solidified Murashige and Skoog (MS) medium supplemented with 12µM benzylaminopurine (BAP) and 3µM kinetin. The shoots have been multiplied for 15 passages in liquid and thereafter for over 5 passages on semisolid MS+15µM BAP+1µM indolebutyric acid (IBA)+10% coconut water at a rate of 3.2- and 2.8-fold, every 4 wk, respectively. The nature of the propagule was a critical factor for shoot multiplication and rooting. Seventy-three percent of the shoots rooted on a modified MS medium (major salts reduced to half strength) containing 1µM indoleacetic acid, 1µM IBA, and 68µM coumarin. Through a simple in vitro hardening step, more than 85% of the tissue culture-raised plants were successfully transferred to soil.     

In vitro propagation ofPhebalium equestre andPhebalium hillebrandii (rutaceae)

Summary The endangeredPhebalium equestre D. A. Cooke and the rarePhebalium hillebrandii J. H. Willis were propagated in vitro using shoot tips and nodal segments as explants. For each species, shoot proliferation was initiated on de Fossard MZZM (Medium levels of minerals, Zero auxins, Zero cytokinins and Medium levels of sucrose, growth factors, and amino acids) medium supplemented with 1 μM benzyladenine. ExcisedP. equestre shoots initiated roots when cultured on MZZM medium containing 60 μM 2,4-dichlorophenoxyacetic acid, whileP. hillebrandii shoots required LZZL (Low levels of minerals, Zero auxins, Zero cytokinins and Low levels of sucrose, growth factors, and amino acids) medium containing 10 μM 2,4-D for maximal root initiation. Both species required transfer to MZZM medium without growth regulators after 2 wk to allow root initials to develop and grow. Plantlets were successfully transferred to soil with 80% survival after 2 mo.