Chemodifferentiation of diosgenin in Dioscorea composita

Diosgenin was isolated from different parts of a three-year-old plant of Dioscorea composite. The amounts (% on a dry wt basis) present were: tubers, 3.6; vine internodes and nodes with their leaves from first 20 nodes from the tubers, 1.6; similarly from intermediate 20 nodes, 0.039 and from upper 20 nodes, 0.03. The amounts (% on a dry wt basis) from tissue culture of nodal explants were: 30-day-old callus, 0.89; 90-day-old callus, 1.61; emergent shoots, 2.5; regenerated roots, 0.08.     

Callus induction and plant regeneration from excised zygotic embryos of the seed-propagated yams Dioscorea composita Hemsl. and D. cayenensis Lam.

A tissue culture method for regeneration of plantlets from calluses of Dioscorea composita Hemsl. and Dioscorea cayenensis L. is described. Zygotic embryos were used as initial explants. Calluses were obtained on Murashige & Skoog basal medium supplemented with 18 M 2,4-D and plantlets were regenerated on media containing 0.1 M zeatin and 3.3 mM glutamine according to previously described protocols [3]. Inclusion of 0.3% (w/v) activated charcoal in media did not increase callusing. Regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (2.25 M) contained in the media tested. The results indicated that there were genotype-dependent differences between the yam species in their ability to regenerate plantlets in vitro.     

水杨酸对菊叶薯蓣中薯蓣皂素生物合成的影响

薯蓣皂素为甾体激素药物合成起始原料,主要来源于菊叶薯蓣等薯蓣属植物的块茎或根状茎,因而关于提高菊叶薯蓣中薯蓣皂素含量的研究有重要意义。利用水杨酸处理菊叶薯蓣的离体植株,研究其对薯蓣皂素生物合成的影响及作用机制。100μmol·L－1的水杨酸处理使薯蓣皂素积累量最大,且提高了叶绿素含量和可溶性糖含量,降低可溶性蛋白含量。半定量 RT-PCR 检测基因表达发现,除了法尼基二磷酸（FPP）基因,水杨酸增强菊叶薯蓣角鲨烯合酶（SQS）基因、甲基戊二酰辅酶 A 还原酶（HMGR）基因、环阿屯醇合成酶（CAS）基因的表达。研究结果为提高菊叶薯蓣中薯蓣皂苷的含量、揭示水杨酸促进薯蓣皂素生物合成的机制等研究提供了基础。     

In vitro shoot culture of wild Oryzae and other grass species

A method was developed for the in vitro clonal propagation of shoots from a range of wild rice and other grass species that have important genetic traits such as drought resistance and salinity tolerance. The axenic multiple shoot cultures, which were suitable for DNA and protein extraction or direct protoplast isolation, could be maintained without subculture for between 2 and 3 months or rapidly multiplied for the subsequent production of mature plants and seeds. Basal sections of the micropropagated shoots also provided novel explants for the production of highly embryogenic callus, from some species, that could be regenerated into green plants. It is envisaged that this clonal propagation technique could aid the genetic manipulation of cultivated rice by providing a means to vegetatively conserve valuable genetic resources, a technique to rapidly multiply novel hybrid material and a source of embryogenic callus that will allow the application of biotechnological techniques, such as somatic hybridization and genetic transformation, to previously unexploited species.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - PAR photosynthetically-active radiation     

In vitro culture ofXanthium strumarium (Cocklebur)

Xanthium strumarium L. was micropropagated by rooting shoots proliferated from shoot-tip explants. The best shoot proliferation was obtained from explants growing on Murashige and Skoog medium supplemented with 4.4 to 8.9 M benzyladenine (BA) and 1.1 to 2.1 M naphthaleneacetic acid (NAA). The micropropagated plants were transferred to potting media and maintained under high humidity conditions in the greenhouse. The media that produced best shoot proliferation from shoot-tip explants also produced the most callus from hypotocotyl, cotyledon and shoot-tip explants, whereas more callus was produced on leaf explants with a lower BA concentration (1.1 M) and 1.1 M NAA.Abbreviations BA benzyladenine, 2 4-d-2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog Technical contribution No. 3319 of the South Carolina Agricultural Experiment Station, Clemson University.     

In vitro tuberization in white yam (Dioscorea rotundata Poir)

Nodal cuttings of white yam were induced to produce microtubers on a MS-revised medium supplemented with various concentrations of sucrose, 20 mgl^–1 L-cysteine, 0.5 mgl^–1 kinetin and 0.7% agar. The frequency of tuberization was affected by the daylength, which is optimal at 12 and 16 h of light depending on the sucrose concentration. The microtubers were planted in a seed bed and grown to maturity. The importance of in vitro tuberization of yam as a means of international germplasm distribution or exchange as well as for the propagation of planting material is discussed.     

In vitro plant regeneration of Aristolochia indica through axillary shoot multiplication and organogenesis

Protocols for in vitro plant regeneration via axillary and adventitious shoot regeneration were established in an important medicinal plant, Aristolochia indica L. (Aristolochiaceae). Basal Murashige and Skoog's (MS) medium supplemented with 0.54 μM α-naphthaleneacetic acid (NAA) and 13.31 μM benzyladenine (BA) induced the maximum number of shoots (45-50) from shoot tip and nodal segment cultures. Phenolic accumulation in leaf and internodal stem derived callus cultured in MS medium containing NAA or 2,4-dichlorophenoxyacetic acid and BA or kinetin was controlled by the addition of 1.0 mg l^-1 phloroglucinol (PG) to the callus induction medium. Basal medium supplemented with 2.69 μM NAA, 13.31 μM BA and 1.0 mg l^-1 PG induced the best results in terms of shoot bud regeneration from leaf derived callus. Direct de novo development of shoots from leaf segments was achieved using 13.31 μM BA along with 50 mg l^-1 activated charcoal. The microshoots were rooted in White's medium supplemented with 2.46 μM indolebutyric acid. More than 85% of rooted plants survived in the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

In vitro axillary shoot proliferation and somatic embryogenesis of yellow pitaya Mediocactus coccineus (Salm-Dyck)

Yellow pitaya (Mediocactus coccineus) seeds were sown on Murashige and Skoog (1962) mineral salt medium. After germination, epicotyls were placed on media enriched with a combination of naphthaleneacetic acid (NAA) (0.05, 0.27 or 0.54 M) and benzyladenine (BA) (2.2 or 4.4 M). The apical tip was excised from half of the shoots and the other half were kept intact. Different values for proliferation rate, shoot length and thickness were observed on each medium. The cotyledons and roots were placed on MS medium supplemented with NAA (2.7 or 5.4 M) and embryogenic calluses were formed. Somatic embryos were induced on these media and then they normally developed on a growth regulator-free medium.Abbreviations BA benzyladenine - MS Murashige and Skoog - NAA -naphthalenacetic acid     

In vitro culture of Ginkgo

Summary Ginkgo biloba L. is an important landscape tree, is resistant to insect, fungi and other pests, and produces a number of chemicals that have pharmaceutical properties (termed ginkgolides). Studies were initiated to establish an in vitro culture protocol for Ginkgo. Explants (intact embryos, embryos with cotyledons removed, and cotyledon tissue) were removed from disinfested seeds and cultured on Murashige and Skoog minimal organics medium with various combinations of either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and either kinetin or benzyladenine (BA). Cultures were incubated in the light and morphological development was recorded. Both embryo and cotyledon explants produced callus (cotyledon tissue produced the most callus). Ginkgolides A and B were detected in callus tissue extracts. Intact embryo cultures initiated on media with 2,4-D plus NAA for 5 wk produced shoots and roots when transferred to media with 4.5 μM 2,4-D alone for an additional 5 wk. Plants were transferred from the 2,4-D media to pots and maintained in the greenhouse.     

Optimization of callus culture and shoot multiplication ofAsparagus densiflorus

The effects of different growth regulators on induction and growth of callus ofAsparagus densiflorus cv. Sprengeri were studied. Calluses grew more rapidly on Murashige and Skoog basal medium supplemented with 5.4 μM p-chlorophenoxyacetic acid (pCPA) and 4.4 μM 6-benzylaminopurine (BA) (medium 1) as compared to the same medium with 11.3 μM 2,4-dichlorophenoxyacetic acid (2,4-d) and 4.6 μM kinetin (medium 2). Calluses on medium 1 were soft and friable, whereas, compact, hard calluses originated on medium 2. Different concentrations and combinations of BA and/or kinetin were also used to study their effects on shoot regeneration. Kinetin was found to be less effective than BA in the initiation of shoots (1.8 shoots/callus). High numbers of shoots were produced in the presence of 0.4 μM BA alone (3.3 shoots/callus). The addition of ancymidol (5 μM) in MS with 0.4 μM BA enhanced multiplication of shoots (9.8 shoots/explant) and also produced well-developed crowns.     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

In vitro multiplication in liquid culture of Syngonium contaminated with Bacillus spp. and Rathayibacter tritici

Contaminated Syngonium clusters were multiplied in an air lift bioreactor in liquid medium containing sucrose with the medium being circulated through a sterilizing filter. After 30 days, the culture in filtered medium produced 19.5 shoot initials per gram fresh weight of inoculum compared to 8.7 shoot initials produced in unfiltered medium. Transfer to an elongation medium with 30 mg l^-1 Rifampicin produced shoots on 67% of the clusters, while transfer to elongation medium without Rifampicin poduced shoots on 40% of the clusters. Clusters grown for three subcultures in a reactor without medium filtration had lost their multiplication ability. Clusters grown for three subcultures in a reactor with filtration, however, continued to show a two-three fold increase in fresh weight and shoot production.Abbreviations MS Murashige and Skoog     

In vitro plant regeneration in Holarrhena antidysenterica wall., through high-frequency axillary shoot prolieferation

Summary An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N⁶-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.     

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 axillary shoot proliferation of apple rootstocks under different ethylene conditions

Summary Ethylene effect on in vitro shoot proliferation of two apple rootstocks, MM111 and M9, was studied. Ethylene biosynthesis was proportionally stimulated by increasing concentrations of the precursor 1-aminocyclopropane-1-carboxylic acid (ACC). When 25 μM or more ACC was applied without any control of the headspace of culture vessels, shoot proliferation of both rootstocks was negatively affected. However, when shoot cultures were transferred to ACC-supplemented medium after the second week of culture, ACC had no effect. Supplementing the medium with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, together with the application of gas traps inside the flasks, significantly enhanced axillary shoot formation and elongation. Steady and high exogenous concentrations of ethylene in the culture flasks had negative effects on shoot proliferation. MM111 appeared to be more sensitive to ethylene than M9. For AVG a threshold dose was noticed, beyond which phytotoxic effects were induced.     

In vitro culture of two threatened South African medicinal trees – Ocotea bullata and Warburgia salutaris

The aim of the project was to develop micropropagation procedures for theheavily exploited and endangered South African trees black stinkwood (Ocotea bullata) and pepperbark (Warburgia salutaris) to facilitateconservation and reforestation. Both species are difficult to establish andgrow in tissue culture because of their high phenolic content. A protocol forthe establishment of explants in vitro was developed comprisingdecontamination, the application of antioxidants and cold treatment.     

In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.