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.     

In vitro propagation and shoot encapsulation as tools for ex situ conservation of the aquatic plant Ludwigia palustris (L.) Ell.

Ludwigia palustris (L.) Ell. is an aquatic perennial herb present in several regions of Italy, which is one of its native countries. In this research, micropropagation and encapsulation protocols were established from axillary buds of L. palustris. Shoots proliferated on half-strength Murashige and Skoog medium without growth regulators. Different culture vessels were tested. Shoots in GROWTEK bioreactor showed the highest fresh and dry weight and total length while the plantlets grown in the RITA bioreactor showed the highest shoot number per explant. Encapsulation of L. palustris microcuttings with sodium alginate formed small and whitish beads which were stored for 14 or 28 days at 7° or 25°C. Storage for 14 days at both temperatures gave the best results but prolonged storage at 25°C decreased the shoot viability to 73%. After 4 weeks of recovery, all the plantlets showed the typical features of the species. Even though the latest Italian IUCN Red List does not mention L. palustris, conservation measures are proposed at local level because this species locally remains vulnerable mainly due to the loss of adequate habitats.Our protocol could be one of the methods for ex situ conservation of L. palustris particularly because its seed storage behavior is uncertain.     

Influence of explant,temperature and different culture vessels on <Emphasis Type="Italic">in vitro </Emphasis>culture for germplasm maintenance of four mint accessions

Two experiments were conducted with four accessions of mint (Mentha spp.) on MS medium for their in vitro performance. In the first experiment apical and nodal explants were cultured at both 20 °C and 25 °C. Data was recorded at second and at fifth week. Both apical and nodal explants of mint showed better leaf production at 25 °C than 20 °C. Nodal explants of mint cultured at 25 °C in both cultivation periods exhibited the highest number of leaves. In the second experiment apical explants were cultured in four different culture vessels viz., industrial glass jar (IG), magenta vessel (MV), Erlenmeyer flask (EF) and culture tube with 1(CT1) and 2(CT2) explants at 25 °C for 6 weeks. The highest weight loss from the media, evapo-transpiration and fresh weight gain were recorded in IG and next in MV. The lowest weight loss from the media and fresh weight gain both were found in CT2. However the lowest evapo-transpiration was noted in EF. The highest numbers of leaves were recorded from MV. Without explants, depletion of medium and increase of headspace were both higher in IG than in the other vessels. Overall, Magenta vessel GA 7 showed the best in vitro performance.     

In vitro shoot culture and microtuber induction in the steroid yam Dioscorea composita Hemsl

The individual effects of sucrose, plant growth regulators and basal salt media formulations were investigated on microtuber induction and development in shoot cultures of the steroid yam Dioscorea composita. Sucrose at 8% (w/v) was the single most significant medium constituent for microtuber induction. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5 μM showed strong inhibitory effects on microtuber induction. By contrast, the auxins α-naphthaleneacetic acid and indole-3-butyric acid at 5.0 μM showed striking promotive effects on microtuber induction and growth. In the presence of either one of these auxins at 5.0 μM shoot cultures produced microtubers weighing 300–400 mg fresh weight whilst kinetin, 6-(γ,γ-dimethylallylamino)-purine, 6-benzyladenine and abscisic acid failed to promote microtuber growth (microtubers weighed generally <200 mg). Media formulations Lloyd and MacCown and White supported the lowest frequencies of microtuber induction when kinetin was present at 2.5 μM. Anderson Rhododendron was as effective as Murashige and Skoog overall in promoting both microtuber induction and growth. When removed from cultures and planted in sterilized moist sand, microtubers sprouted readily (60–87% within 2 weeks) and produced vigorous shoot growth and after 5–7 months minitubers of sizes (30–80 g) suitable for direct field planting. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro Growth and Shoot Multiplication of Achras zapota in a Controlled Carbon Dioxide Environment

The culture vessels with multiplying shoots of Achras zapota L. on Schenk and Hildebrandt (SH) medium containing 8.88 M 6-benzylaminopurine (BAP) with or without sucrose were kept under varied CO₂ concentrations ranging from 0.6 to 40.0 g m^–3 using different concentrations of sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), potassium bicarbonate (KHCO₃), and potassium carbonate (K₂CO₃) in small acrylic chambers. Complete absence of carbon source caused death of shoots within 20 d. Under elevated concentrations of CO₂ (10.0 and 40.0 g m^–3) the shoots grew photoautotrophically on sucrose-free medium. The growth of cultures was better at 40.0 g (CO₂) m^–3 than on 3.0 % sucrose under ambient air of growth room. However, the best response was obtained at 10.0 g (CO₂) m^–3 and 3.0 % sucrose where maximum number of shoots, shoot length, fresh and dry mass, total number of leaves and leaf area was observed.     

Effect of carbon dioxide and ethylene on tuberization of isolated potato stolons cultured in vitro

Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C₂H₄ which is by itself also inhibitory of tuberization. Furthermore, C₂H₄ inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C₂H₄. The antagonistic actions of CO₂ and C₂H₄ on tuberization are discussed.     

A survey of the response of Rhododendron to in vitro culture

The responses of 7 genotypes of Rhodendron to culture conditions and their establishment as shoot cultures are described. The genotypes represent a broad genetic diversity in the genus. After sterilization and an acclimation period of 3 to 12 months, all the selections were established as shoot cultures on Woody Plant Medium (WPM) supplemented with N⁶(-²-isopenteny) adenine (2iP). Plants with strong episodic growth cycles required the longer acclimation periods. Utilizing shoots from these cultures, the response to a cytokinin series of 0 to 32 M 2iP or BAP (6-Benzylaminopurine) was analyzed. BAP proved toxic to all but the elipidote and lepidote rhododendrons (R. mucronulatum, R. x Boule de Neige, and R. x PJM); however, even with these selections, 2iP stimulated greater shoot multiplication rates. The optimum 2iP level for shoot multiplication varied little with the genotype and levels of 4 to 16 M generally proved optimal, depending on the specific selection. Adventitious shoot production was observed in 3 selections (R. canadense, R. x Boule de Neige and R. x PJM), but only at 2iP levels above 8 M. Shoot multiplication rates of 7 to 21 times were observed, depending on the selection. Using an average utilizable shoot production rate of 40 shoots per culture per 6 week subculture period, some 75,000 shoots can be generated per square meter of culture space per year. The harvested shoots (microcuttings) rooted readily out-of-culture and the resultant plants grew like seedlings.     

Factors controlling adventitious bud induction and plant regeneration in matureJuniperus oxycedrus leaves cultured in vitro

Summary The morphogenetic capacity of matureJuniperus oxycedrus L. leaves cultured in vitro has been studied, noting nutritive, hormonal, and environmental factors inducing differentiation and development of adventitious shoots. Bud primordia formed directly from the leaves. Highest bud differentiation rates were obtained when the explants were cultured for at least 21 days on a modified Schenk and Hildebrandt solidified medium containing 0.5 μM benzyladenine under a 16-h photoperiod. Maximum bud development and elongation was achieved on cytokinin-free medium containing 4% (wt/vol) sucrose and 0.05% (wt/vol) activated charcoal. Regenerated shoots were excised and induced to root on media with auxin. Rooting percentages up to 100% were obtained in the presence of 2.5 μM naphthaleneacetic acid and 4% (wt/vol) sucrose. The inclusion of activated charcoal in the root induction medium drastically reduced the number of rooted shoots. Following conventional procedures, plantlets were ultimately established in soil.     

Studies on the carbon dioxide promotion and ethylene inhibition of tuberization in potato explants cultured in vitro

Ethylene inhibited the tuberization of etiolated potato (Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro. Carbon dioxide did not overcome the C₂H₄ inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C₂H₄ prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin-increased tuberization of sprout sections.     

Carbon dioxide and ethylene interactions in tulip bulbs

The effect of CO₂ on ethylene-induced gummosis (secretion of polysaccharides), weight loss and respiration in tulip bulbs ( Tulipa gesneriana L.) was investigated. A pretreatment with 1-MCP prevented these ethylene-induced effects, indicating that ethylene action must have been directed via the ethylene receptor. Treatment with 0.3 Pa ethylene for 2 days caused gummosis on 50% of the total number of bulbs of cultivar Apeldoorn, known to be sensitive for gummosis. Addition of CO₂ (10 kPa) reduced the ethylene-induced gummosis to 18%. In a second experiment the influence of ethylene and CO₂ on respiration and FW loss of bulbs of the cultivar Leen van der Mark was studied. A range of ethylene partial pressures (0.003–0.3 Pa) was applied continuously for 29 days. Ethylene caused a transient peak in O₂ consumption rate during the first days after the start of application. The relation between O₂ consumption rate and ethylene partial pressure could be described by Michaelis-Menten kinetics. Respiratory peaks were reduced by CO₂. This inhibition by CO₂ could not totally be due to competition with ethylene at the receptor binding-site, as was indicated by the use of an O₂ consumption model. Pre-treatment of bulbs with 1-MCP and subsequent exposure to CO₂ showed that CO₂ could influence respiration irrespective of any interaction with ethylene. Ethylene and CO₂ both stimulated weight loss. The effect of combined treatments of ethylene and CO₂ on weight loss was at least as strong as the sum of the separate effects, which implies that competition between ethylene and CO₂ at the receptor binding-site was unlikely.     

Influence of nutrient salts,auxins and cytokinins on the in vitro growth of Salvia greggii

Experiments were designed to determine the optimal MS salt concentration and the best auxin and cytokinin to use for shoot growth of Salvia greggii A. Gray. Full or 1/2 MS salts were superior to 1/4 MS salts based on number of shoots produced. There were no differences in the various auxins tested (IAA, NAA or IBA) as to their abilities to stimulate shoot production or increased fresh weight. BA, and BA + Kin stimulated the greatest shoot number among the cytokinins tested. A final experiment was designed to determine optimal BA and NAA concentrations for shoot growth. A medium containing 11.1M BA and no NAA produced the best growth of Salvia greggii in vitro. Shoots produced in vitro rooted and acclimatized readily in the green-house.Abbreviations MS Murashige and Skoog salts - IAA indoleacetic acid - IBA indolebutyric acid - NAA napthaleneacetic acid - BA benzyladenine - Kin kinetin - 2iP isopentenyl adenine     

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     

Propagation of Ficus carica L. clones by in vitro culture

This experiment is designed to determine the most suitable conditions and media for propagating three selected fig (Ficus carica L.) clones through tissue culture. The clone 37 displayed a higher performance than clones 50 and 82. As the multiplication medium, the Murashige and Skoog (MS) medium containing 1 mg dm⁻³ α-indole-3-butyric acid (IBA), 1 mg dm⁻³ gibberellic acid and 5 mg dm⁻³ 6-benzyladenine were the best, whereas, MS medium complemented with 1.2 and 2.5 μM IBA or 1-naphthalene acetic acid (NAA) were better in respect to rooting. Peat followed by volcanic tuff gave the best performance for acclimatization to outdoor conditions.     

Toxicity of ethanol during proliferation and adventitious root formation in apple microcuttings in vitro

We have examined the toxicity of ethanol in tissue culture of the apple rootstock ‘Jork 9’. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro culture of shoots of Pinus caribaea on a liquid medium

Shoot apices of a clone of Pinus caribaea Morelet were cultured and multiplied in vitro by supporting them with their basal cut ends immersed in a liquid nutrient medium.The initial heights of explants and their initial numbers of leaves were positively correlated with the numbers of buds and shoots produced by the explants after a bud induction phase and after a shoot elongation phase. The final numbers of buds and shoots were positively correlated with reductions in the quantities of phosphorus detected in the media and negatively correlated with the numbers of brown leaves produced on the explants.In a comparison between the growth of shoot explants on liquid and solid media, shoots incubated on the liquid medium showed significantly greater increases in length in a four-week period than those cultured on solid medium.This technique, using liquid media, provides a system in which both the nutrient utilization and the growth rates of isolated pine tissues can be readily assessed. Furthermore, the multiplication rate of the tissue can be predicted following the observation of correlated characters early in the micropropagation cycle.     

Effect of low doses of gamma irradiation on in vitro growth of grapevine

Shoot tips and single node explants of two rootstocks (R.99 and 3309) and two varieties (‘Helwani’ and ‘Cabernet Franc’) of grapevine cultured on DSD1 media for a period of 60 days, were irradiated with 0, 2, 5 and 7 Gy doses of gamma irradiation. Shoot length of ‘Helwani’ and ‘Cabernet Franc’ was increased by 7 Gy irradiation. The 5 Gy dose increased the number of roots in plants of the two rootstocks and ‘Helwani’. Root length of ‘Helwani’ and ‘Cabernet Franc’ at the 2 and 7 Gy doses were significantly higher than those of the non-irradiated control. A similar effect was noticed on R.99 rootstock subjected to 5 Gy. Five Gy also increased the dry weight of the R.99 rootstock, whereas 2 and 7 Gy had a similar effect on ‘Helwani’ and ‘Cabernet Franc’. Number of leaves of plants exposed to 5 and 7 Gy was increased when compared with the non-irradiated control. This revised version was published online in June 2006 with corrections to the Cover Date.     

蝴蝶兰的组织培养和快速繁殖

通过诱导残败花梗上的休眠芽萌发,以萌发的幼叶和去茎尖的茎段为外植体进行组织培养,建立了蝴蝶兰(Phalaenopsis amabilis Bl.)的无菌繁殖体系,并筛选出最佳培养基组成.诱导休眠芽萌发的最佳培养基为不加任何激素的MS0培养基;原球茎诱导的适宜培养基为MS 3.0 mg·L-1 6-BA 0.5 mg·L-1 ZT 30 mg·L-1柠檬酸和MS 5.0 mg·L-1 6-BA 30 mg·L-1柠檬酸 30%椰乳(CM),其中茎段的诱导效果明显优于叶片,诱导率达95%;诱导无菌苗生根的最适培养基为1/4 MS 1.0 mg·L-1 6-BA 0.1 mg·L-1 NAA,生根率可达79%.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

Natural light as an alternative light source for the in vitro culture of banana (Musa acuminata cv. ‘Grande Naine’)

The concept of using sunlight for micropropagation systems is proposed as a way of reducing tissue culture costs. Shoot tips of Musa acuminata cultivar ‘Grande Naine’ were cultured in a non-controlled natural light environment at the IAEA Laboratories, Austria during summertime. Significantly more shoots were produced by plantlets cultivated in a sunlit room with photosynthetic photon flux densities (PPFD) fluctuating up to 570 μmol m-2 s-1, temperatures between 23 and 30 °C and photoperiods of 12 to 16-h, than by plantlets under artificial light in a growth chamber providing controlled conditions of a constant PPFD of 65 μmol m-2 s-1, temperatures ranging from 23 to 29 °C and a 16-h photoperiod. Highest multiplication rates were achieved in a greenhouse with PPFD reaching 860 μmol m-2 s-1 and temperatures of 18 – 43 °C, but browning of leaves and loss of turgor occurred. Nevertheless, rooted plantlets showed 100% survival during acclimatisation and normal development. Photoperiods of 12 – 16 h did not affect the multiplication rates. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.