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.     

Callus culture and plantlet production in Carica papaya (Var. Honey Dew)

Summary In order to develop techniques for efficient callus production and regeneration in Carica papaya (Var. Honey Dew), lamina, petiole, stem and root explants from in vitro plantlets were cultured in media supplemented with 2.0 mg/1 IBA and 0.5 mg/1 BAP. Use of in vitro-grown plantlets as an explant source helped to avoid contamination common in papaya tissue culture. Callusing was maximum in root explants cultured in a modified MS (half-strength) medium. Shoot reganeration was maxium in root-derived callus grown in full-strength modified MS medium supplemented with 0.5 mg/1 IBA and 1 to 2 mg/1 kinetin. A histological study indicated that shoot buds originated from peripheral cell layers of the callus. Each shoot regenerated from callus was subcultured using a multiplication medium. Root formation was induced in all shoots treated in half-strength of modified MS medium containing 2 mg/1 IBA and rooted shoots were transferred successfully to the field.Abbreviations MS Murashige and Skoog medium, 1962 - LS Linsmaier and Skoog medium, 1965 - BAP 6-Benzylaminopurine - FAA Formalin Acetic acid 30% Ethanol, 1110 - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA Napthaleneacetic acid - SH Schenk and Hilderbrandt medium, 1972     

In vitro clonal propagation of dioecious Carica papaya

A procedure for in vitro propagation of dioecious papaya clones is described. A high rate of success in culture estbalishment was obtained when axillary buds were taken from lateral shoots of hedged rooted cuttings grown in a greenhouse. Seasonal endophytic contamination was suppressed by shaking propagules for 24 h in 300 mgl^-1 rifampicin or by incorporating it at 50 mgl^-1 into the medium. Murashige & Skoog (MS) basal medium supplemented with 0.5 mgl^-1 6-benzyladenine and 0.1 mgl^-1 naphthaleneacetic acid was used for establishment and proliferation. The addition of 160 mgl^-1 adenine sulfate improved multiplication and shoot growth. An elongation stage on MS medium supplemented with 1.0 mgl^-1 kinetin and 0.05 mgl^-1 naphthaleneacetic acid was necessary before rooting. Rooting was obtained at a high rate on half-strength macroelements of MS medium supplemented with 1.0 mgl^-1 indole-3-butyric acid. Commercial plots of papaya plants obtained through this procedure already exist.     

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.     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Pichia cecembensis sp. nov. isolated from a papaya fruit (Carica papaya L., Caricaceae)

The ascogenous yeast YS16T was isolated from a decaying papaya fruit. Phenotypic traits such as multilateral budding, spheroidal or elongate shape, pseudohyphae formation, asci with one or more ascospores, ability to ferment d-glucose, inability to assimilate nitrate and the presence of Q7 ubiquinone suggest its affiliation to the genus Pichia. The nearest phylogenetic neighbor, based on D1/D2 domain sequence of the 26S rRNA gene and ITS region sequence, was identified as Issatchenkia orientalis (NRRL Y-5396T, a synonym of Pichia kudriavzevii) with similarities of 98.2% and 97% respectively. In addition to the difference in the D1/D2 and ITS region sequence, YS16T differs from I. orientalis with respect to a number of phenotypic traits. However, in the phylogenetic analysis, YS16T showed close relatedness to the P. membranifaciens clade. Thus, it is proposed to assign the status of a new species to YS16T, for which the name P. cecembensis sp. nov. is proposed. The type strain of P. cecembensis sp. nov. is YS16T (=NRRL Y-27985T=JCM 13873T=CBS 10445T).     

In vitro propagation of Vicia faba L. by micro-cutting and multiple shoot induction

The influences of nitrogen sources, culture temperature and activated charcoal supplements were studied in relation to the rooting ability of V. faba cuttings. The interaction of these factors led to quantitative and qualitative modifications of the culture responses. Low temperatures (14–18°C) were suitable for in vitro culture, limiting the formation of phenolics in plant material and making activated charcoal supplement unnecessary. Nitrogen supplements contributed in modifying the different plant responses, in accordance with temperature. Multiple shoot formation was obtained from the cotyledonary node and from the stem nodes cultivated in the presence of 6-benzylaminopurine (BAP). BAP at 4 mg l^-1 was the most effective concentration in promoting high rates of shoot development. The original position of stem nodes was found to determine the explant response to plant growth regulator treatments, possibly due to the effect of residual apical dominance.     

In vitro regeneration of shoot buds and plantlets from seedling root segments of Brassica napus L.

Root segments obtained from aseptically germinated seedlings of Brassica napus cv. Westar were used to optimize conditions for high-frequency shoot bud differentiation. The presence of low kinetin (0.5 M) and relatively high indole-butyric acid (1.0 M) levels facilitated optimum shoot bud differentiation. Modified MS medium (MMS) was superior to the other three basal media tested (MS, B5 and White's). Elevated sodium dihydrogen phosphate levels increased the differentiation of shoot buds. Increasing or decreasing the level of sucrose from 3% reduced the frequency of explants forming shoot buds. Addition of glutamine enhanced both the frequency of responding explants, as well as the number of shoots per responding explant. Root segments from 13-day-old seedlings produced the highest response (58%) in the presence of 100 mg l^-1 glutamine. The position of the segment on the main root, size, and the presence or absence of lateral roots altered the morphogenic response. Sealing of the donor seedling cultures with Parafilm^® instead of Stretch' n seal^® resulted in a higher production of shoot buds, although root segment cultures were not affected by the type of sealing. Spontaneous rooting occurred on all developed shoots.Dedicated to Dr. Friedrch Constabel on the occasion of his 60th birthday     

In vitro propagation of pummelo (Citrus grandis L. osbeck)

Summary Several experiments were carried out to develop protocols for the in vitro propagation of pummelo (Citrus grandis L. Osbeck) using shoot-tip explants from seedlings. Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and thidiazuron (TDZ), singly or in combination with α-naphthaleneacetic acid (NAA), was used to determine the rate of shoot proliferation. The response of explants to all concentrations of TDZ was very poor. After 6 wk culture, the most adventitious shoots per explant (average 5.2) were obtained on medium supplemented with 1.8 μM BA. NAA with cytokinin in the medium did not improve the rate of shoot multiplication significantly. Addition of 5.8 μM gibberellic acid in shoot-proliferation medium during the second subculture improved shoot elongation significantly. Shoot multiplication increased 3.5-fold in each successive subculture. NAA was superior to indolebutyric acid for in vitro root induction. Over 75% of the shoots developed roots when transferred to half-strength MS medium with 1.3, 2.7, or 5.4 μM NAA.     

Propagation of Morus indica L. (Mulberry) by encapsulated shoot buds

Axillary buds of mulberry (Morus indica L) were encapsulated in alginate and agar to produce individual beads. The beads could be stored at 4°C for 45 days without loss of viability. Amongst the encapsulating agents tested, sodium alginate was found to be a better matrix. Encapsulated buds regenerated complete plantlets on an appropriate medium. This technique would provide an easy and novel propagation system for the elite as well as difficult-to-root species of mulberry.Abbreviations BAP benzylaminopurine - MS Murashige and Skoog (1962).     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.     

In vitro shoot propagation of Dendrocalamus strictus nees

Multiple shoots were induced from seedling and axillary buds of mature plants of Dendrocalamus strictus on Murashige and Shoog's medium supplemented with BA and kinetin. About 35–45 shoots were obtained within 20–25 days from a nodal explant of seedling and 3–8 shoots were obtained from a nodal explant of mature plants in the primary culture. The seedling derived cultures were separated into groups of 5–7 and transferred to fresh subculture medium. Rooting of the shoots was achieved under in vitro and ex vitro conditions. 85–90% of rooting was achieved by the ex vitro method using IBA. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro shoot proliferation and propagation of guava (Psidium guajava L.) from germinated seedlings

Guava seeds were germinated on Murashige and Skoog (MS) medium with or without 8.8 μM benzyladenine (BA). BA increased the rate of germination and the number of lateral shoots (3.4 vs 1.2 per seedling). Stem nodes from these lateral shoots were cultured on proliferation media with 4.4 μM BA, and multiple shoots (3.5) were formed within 4 weeks of culture. Increasing the concentration of BA or the addition of naphthaleneacetic acid (NAA) did not affect shoot formation. Shoots produced from explants and lateral shoots from germinated seedlings were rooted in media containing activated charcoal (AC) or 9.8 μM indolebutyric acid (IBA). Shoots rooted with IBA had a higher rooting percentage (100% vs 75%) and a greater number of roots (5.5 vs 3.2) but the shoots were shorter (2.6 vs 3.4 cm) than when rooted in AC, and they required an additional 4 weeks of culture in media with AC to achieve shoot elongation. About 80% of the shoots with roots survived in the glasshouse and produced normal phenotypic plants.     

In nature dormant buds and in vitro dormant-like buds ofFraxinus excelsior L.

Summary Microcuttings ofFraxinus excelsior, sampled from adult trees during the period of cell cycle blockage of bud in G_0–1, developed long rejuvenated sprouts on the Woody Plant Medium (WPM) with benzylaminopurine (BAP) (4.0 mg/l) and indolebutyric acid (IBA) (0.03 mg/l). These sprouts had the ability to enter a resting period, building dormant-like buds when maintained on the original WPM. Sprouts developed from subcultures also entered a resting period without any transfer. Comparison of in nature buds in active growth and dormancy with buds of growing sprouts and in vitro dormant-like buds revealed similarity in behaviour at the shoot apical level. In particular, in dormant-like buds in a constant environment, shoot apical functioning was suppressed while the cell cycle of the shoot apex was blocked at the G_0–1 phase, like in nature dormant buds.Abbreviations a.u. arbitrary unit - BAP benzylaminopurine - CF cumulative frequency - d₁, d₂ diameters of the shoot apex - IBA indolebutyric acid - P_n last opposite primordia of range n - WPM woody plant medium     

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun')

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and β-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 μM brassin, benzylaminopurine and β-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'. Received: 9 April 1997 / Revision received: 27 August 1997 / Accepted: 20 September 1997     

In vitro shoot proliferation and plant regeneration from kurrat (Allium ampeloprasum var. kurrat) seedlings

Shoots were produced from kurrat seedling and mature plant explants cultured in Murashige and Skoog medium (MS) alone or supplemented with 4.4 M benzyladenine (BA). Shoots were also produced from explants through a two-step procedure. Regenerated shoots were induced to form roots on MS medium with 5 g I^-1 activated charcoal. Plants were successfully established in soil.Abbreviations AC activated charcoal - BA benzyladenine - MS Murashige & Skoog's (1962) medium - 2,4-d 2,4-dichlorophenoxyacetic acid