In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize

Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0–100 μM) and five auxins (0–5 μM). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 μM) and a higher concentration of picloram (5 μM) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation.     

Effects, distribution and uptake of silicon in banana (Musa spp.) under controlled conditions

Three contrasted genotypes of Musa spp. (M. acuminata cv Grande Naine, M. acuminata spp. Banksii and M. balbisiana spp. Tani) were grown for 6 weeks under optimal conditions in hydroponics and were submitted to a wide range of Si supply (0–1.66 mM Si) to quantify the Si uptake and distribution in banana, as well as the effect of Si on banana growth. The level of Si supply did not affect plant growth, nor the rate of water and nutrient uptake. The rate of Si uptake and the Si concentration in plant tissues increased markedly with the Si supply. At the highest Si concentrations (1.66 mM), silicon absorption was essentially driven by mass flow of water (passive transport). However, at lower Si concentrations (0.02–0.83 mM), it was higher than its uptake by mass flow and caused the depletion of silicon in the nutrient solution, suggesting the existence of active processes in silicon transport. The distribution of silicon among shoot organs (pseudostem < petiole and midrib < young lamina < old leaf) confirmed the major role of transpiration in silicon accumulation and was not dependent on silicon supply. However, other mechanisms of transport might be operating in the roots and in the petiole and midrib of young leaves, whose silicon concentration was unexpectedly high at low Si supply (0.02 mM) compared to higher levels of Si. The three genotypes did not exhibit consistent differences in their responses to silicon supply.     

Long-termin vitro storage ofColocasia esculenta under minimal growth conditions

The storage of a clone ofColocasia esculenta at 28/24°C over a 12-h photoperiod in the absence of mannitol, was not feasible with transfer intervals of more than eight months. Mannitol had a positive effect on survival at this temperature, but caused abnormalities at high concentrations. At 9°C in total darkness,C. esculenta could be stored for more than eight years with transfer intervals of approximately three years. After this period, more than 90% of the cultures showed living shoots, but not all shoots per culture showed regrowth. Cultures which were transferred three times during the experimental period, had a significantly (=0.05) lower number of regrowing shoots than cultures which were transferred twice. This might be due to the fact that the former cultures were transferred at a less favourable developmental stage. The addition of mannitol to the storage medium did not improve survival and regrowth, nor did a more gradual lowering of the temperature to 9°C.     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variability in storage response within a coffee (Coffea spp.) core collection under slow growth conditions

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.Abbreviations BA 6-benzyladenine - CAR Central African Republic - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - FAO Food and Agriculture Organization - IBPGR International Board for Plant Genetic Resources - IDEFOR-DCC Institut Des Fôrets - Département Café Cacao - ORSTOM Institut français de recherche scientifique pour le développement en coopération     

Control of hyperhydricity in Eucalyptus axillary shoot cultures grown in liquid medium

Shoots of four clones of two Eucalyptushybrids were successfully grown in a liquid medium containing modified MS basal salts supplemented with 0.01 mg l ^–1 (0.04 M) BAP and various concentrations of a proprietary anti-hyperhydricity agent (sold commercially as anti-vitrification agent EM2). Shoots of a single clone were also grown using the same medium but with the anti-hyperhydricity agent replaced with various concentrations of a commercially available gelling agent containing pectin (M-Gel) or a polysaccharide extract (iota-carrageenan) from seaweed. A range of supplements applied to the media were effective in reducing the hyperhydricity of shoots. The addition of EM2 and M-Gel led to a significant decrease in hyperhydricty, without showing any significant detrimental effect on the numbers of shoots produced for three of the four genotypes tested. At a concentration of 5 g l^–1, for either compound, the percentage rooting and percentage survival of shoots, both in vitro and ex vitro, either equalled or exceeded those of untreated shoots. Addition of iota-carrageenan, although beneficial in reducing hyperhydricity and improving percentage rooting in vitro, was detrimental to shoot production and acclimatisation ex vitro. Common factors of these agents' contributions to their anti-hyperhydric properties are discussed.     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l^–1 biotin, 100 mg l^–1 glutamine, 100 mg l^–1 malt extract (Sigma), 1.0 mg l^–1 2,4-D and 45 g l^–1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO₂ and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.     

The transmission of banana bunchy-top virus in micropropagated bananas

Plants were established in vitro from banana bunchy0top virus (BBTV) infeeted plants. Explants containing either vegetative shoot apices or terminal floral apices were used to initiate cultures. Plants multiplied in culture were indistinguishable from non-infected (control) plants and lacked characteristic symptoms of BBTV infection. After 16 months in culture plants were established in the glasshouse and after 1 month in pots some plants started to show symptoms of the disease. After a further 5 months, 73% of the plants showed characteristic symptoms of the disease while 27% were symptomless and similar in appearance to control plants. These plants have been grown to maturity in the field without showing recognizable symptoms. This study demonstrates that BBTV can be transmitted in an apparently symptomless condition in culture and has important consequences for the dissemination of banana germplasm within Australia and internationally.     

Low-cost alternatives for the micropropagation of banana

A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m⁻² s⁻¹ and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m⁻² s⁻¹ and photoperiods ranged from 8–16 hours. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hormonal and histological studies related to in vitro banana bud formation

Shoot apices of Musa subgroup AAA `Grande Naine' were used for in vitro culture establishment. The endogenous hormone levels and their effects on bud formation were evaluated during a 75-day period. Cytokinins, IAA and ABA were separated by HPLC and quantified by means of ELISA. Enzymatic degradation of IAA was determined by the colorimetric method. Explants were maintained on establishment medium for 60 days. The endogenous cytokinins were higher in the basal portion of the explant. Subculture to proliferation medium (65 to 75 days) resulted in a substantial increase of cytokinins in the basal portion and in a decline in the apical portion. 2iP was the predominant cytokinin in the tissue. The endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65th day of culture. The level of ABA was reduced from the time of inoculation up to the 75th day of culture. Histological analysis indicated that buds formed at the leaf base at the 65th day of culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mineral nutrition in carnation tissue cultures under different ventilation conditions

Growth and propagation rates, hyperhydricity percentages, macronutrientabsorption and pH evolution were evaluated in Dianthuscaryophyllus CV Nelken cultured in vitro under different ventilationconditions. Culture in well ventilated conditions (HVC) i.e. low relativehumidity, generated lower percentages of hyperhydric explants, with highermicropropagation coefficients and dry weight increments, than in less ventilatedcultures (LVC). Macronutrient absorption was similar in both types of cultures,except for ammonium, nitrate, chloride and phosphate. In LVC, after 15 days ofculture, carnation explants absorbed more nitrate than ammonium and chlorideuptake was 5 times greater than in HVC. Phosphate uptake was more pronounced inLVC after 15 days of culture, reaching similar values in both types of culturevessels at the end of the experiment, and led to growth limiting conditions formore prolonged cultures. Medium pH decreased to acid values after 15 days ofculture and even more at the end of the experiment; however, these acidconditions seem not be an obstacle for nutrient absorption.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

Lenticel hypertrophy in shoot cultures of Ceratonia siliqua L.

Numerous white surface proliferations appeared in cultures of Ceratonia siliqua L. grown three to four weeks on medium containing 0.5 mg l^-1 BA and 0.1 mg l^-1 IBA. It was histologically confirmed that these proliferations were hypertrophied lenticels. Proliferations appeared first at the basal shoot internode and gradually spread acropetally, covering eventually the whole shoot except the uppermost internodes. Increase of BA concentration in the medium increased both the number of hypertrophied lenticels per shoot and the shoot multiplication index.Abbreviations BA 6-benzylamino-purine - IBA indole-3-butyric acid Dubravka Bojovi-Cveti deceased July 8, 1991.     

Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

Genotype and medium affect shoot regeneration of melon

The effects of basal media, growth regulators and gelling agents on the morphogenetic response of eleven Cucumis melo L. var. reticulatus and inodorus genotypes were examined. Regeneration was achieved from cultured cotyledons of all genotypes and the morphogenic response was affected by the genetic background. Among the combination of factors tested, MS basal medium supplemented with 2.8 M 6-benzyladenine (BA) and 1.0 M abscisic acid (ABA) solidified with agar gave the highest frequency of shoot regeneration.Abbreviations ABA abscisic acid - BA 6-benzyladenine - TDZ thidiazuron     

Effect of l-arginine,casein hydrolysate,banana powder and sucrose on growth and solasodine production in shoot cultures of Solanum laciniatum

The addition of l-arginine, casein hydrolysate, banana powder and the reduction of the concentration of sucrose in the media could increase the solasodine content in the shoot cultures of Solanum laciniatum. No linear correlations between growth index, chlorophyll content and solasodine content were observed, however a positive linear correlation between solasodine productivity and chlorophyll content occurred in these shoot cultures.Abbreviations Ch chlorophyll content - DW dry weight - fl flask - FW fresh weight - GI growth index - LOD limit of detection - LOQ limit of quantitation - SDc solasodine content - SDp solasodine productivity - w week