Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.     

Micropropagation of a Local Olive Cultivar for Germplasm Preservation

In vitro shoot culture was applied to an Italian local cultivar Nebbiara of olive (Olea europaea L.) to preserve its endangered germplasm. This cultivar showed a notable difficulty for the in vitro establishment due to heavy pathogen contamination. Mercury chloride and sodium hypochloride in the sterilisation step and antibiotics in culture media allowed to overcome the problem. Proliferation of shoot apical bud on olive culture medium with 36 g dm^–3 mannitol and 4.56 M zeatin appeared very satisfactory. All the explants tested rooted during a subculture (1 month) preceeded by a 5-d long dark pre-treatment.     

Micropropagation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) and application of mycorrhiza to improve plantlet establishment

Micropropagation methods were developed for the three French varieties of olive (Olea europaea L.), Aglandau and Tanche, that have the Appelation d’Origine Contrôlée (AOC) status and one ecotype (05300, Laragne, France). Explants consisting of partially lignified nodal segments were collected from rejuvenated glasshouse-grown plant material. Nodal explants with axillary buds were cultured on different media. For AOC varieties, olive medium modified (OM mod) to contain half the concentration of macroelements was the most efficient in inducing bud break and growth when supplemented with 30 g l^?1sucrose and 4 mg l^?1 zeatin. The resulting shoot buds were further multiplied and maintained on OM mod medium. Rooting was best achieved on OM supplemented with 4 mg l^?1 indole-3-butyric acid (IBA). For the Laragne ecotype, maximal shoot proliferation occurred when explants were cultured on woody plant medium supplemented with 15 g l^?1 sucrose and 0.1 mg l^?1 zeatin. Efficient rooting was achieved with 1 mg l^?1 IBA combined with 0.75 mg l^?1 naphthaleneacetic acid. After acclimatization in the glasshouse, survival rates ranged from 57 to 92%, depending on the genotype. Inoculation of Laragne ecotype microplantlets with the arbuscular mycorrhizal fungus Glomus mosseae significantly improved plant survival and subsequent plant development and growth.     

Relationship between reproductive behavior and new shoot development in 5-year-old branches of olive trees (Olea europaea L.)

The development of new shoots plays a central role in the complex interactions determining vegetative and reproductive growth in woody plants. To explore this role we evaluated the new shoots in the olive tree, Olea europaea L., and the effect of fruiting on new shoot growth and subsequent flowering. Five-year-old branches served as canopy subunits in order to obtain a global, whole-tree view of new shoot number, size and morphological origin. The non-bearing trees had many more shoots than the fruit-bearing trees, and a greater number of longer shoots. In both bearing conditions, however, the majority of shoots were less than 4 cm long, with shoots of progressively longer lengths present in successively decreasing frequencies. Six major shoot types were defined on the basis of apical or lateral bud origin and of parent shoot age. On fruit-bearing trees, the new shoots originated predominantly from the shoot apex, while on non-fruiting trees, they formed mainly from axillary buds, but in both cases, they tended to develop on younger parent shoots. The previous bearing condition of the tree was the main determinant for subsequent inflorescence development, which was independent of both shoot type and length. Thus, reproductive behavior strongly affected both the amount and type of new branching, but subsequent flowering level was more influenced by previous bearing than by the potential flowering sites on new shoots.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

Trehalose 6‐phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.)

Trehalose 6‐phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2‐oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration‐dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.     

A rooting procedure for lentil (Lens culinaris Medik.) and other hypogeous legumes (pea,chickpea and Lathyrus) based on explant polarity

The present study assessed the rooting response of lentil nodal segments in relation to explant polarity, hormone, salt and carbohydrate concentrations of the medium. Nodal segments of lentil with an axillary bud cultured in an inverted orientation (apical end in medium) showed higher rooting frequencies than explants cultured in a normal orientation (basal end in medium). The highest rooting percentage (95.35%) and average number of shoots regenerated per explant (2.4) were obtained from explants placed in an inverted orientation on Murashige and Skoog (MS) medium salts with 3% sucrose, supplemented with 5 microM indole acetic acid (IAA) and 1 microM kinetin (KN). Reducing or increasing phytohormone concentration did not alter significantly root regeneration of inverted explants. Sucrose at 3% allowed higher root regeneration frequencies compared to 1.5% sucrose. MS full concentration permitted regeneration of longer shoots with more nodes per regenerated shoot, compared to MS half-strength, which regenerated more shoots of shorter length and with less nodes. Inverted nodal segments of other hypogeous legumes (pea, chickpea and Lathyrus) also exhibited higher rooting frequencies than explants cultured in a normal orientation on MS medium with 3% sucrose and supplemented with 5 microM IAA and 1 microM KN. The most novel application of this study is the culture of nodal segments of hypogeous legumes in an inverted orientation. This procedure is a considerable improvement over other published procedures concerning in vitro rooting of lentil, pea, chickpea and Lathyrus.     

Rapid in vitro propagation of Aloe barbadensis Mill

Axillary bud development and adventitious bud formation was obtained with decapitated shoot explants of Aloe barbadensis Mill. Maximal bud growth and rooting of shoots was obtained on a modified medium of Murashige and Skoog supplemented with 5 M IBA. More adventitious and axillary buds developed on nutrient media supplemented with IBA than with NAA. Axillary buds but not adventitious buds developed with IAA in the medium. Morphogenesis was inhibited by 2,4-D. Kinetin, benzyladenine and thidiazuron were toxic to the explants and did not stimulate the development of axillary of adventitious buds. The optimal temperature for bud growth and development was 25°C. Axillary bud growth and the formation of adventitious buds was slowed down at 10°C and totally inhibited by 30°C. The optimal sucrose concentration was 3% with the inhibition of bud growth and development by higher sucrose levels.     

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [<Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.     

Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

Lateral Bud Growth in Phaseolus vulgaris L. and the Levels of Ethylene in the Bud and Adjacent Tissue

Ethephon and the ethylene inhibitors Ag⁺ and aminoethoxyvinylglycine (AVG) inhibited outgrowth of the axillary bud of thefirst trifoliate leaf in decapitated plants of Phaseolus vulgaris.Endogenous ethylene levels decreased in the stem upon decapitationalthough it is not conclusive that a causal relationship existsbetween this decrease and the release of axillary buds frominhibition. The proposition that auxin-induced ethylene is responsiblefor the suppression of axillary bud growth in the decapitatedplant when the apical shoot is replaced by auxin is not borneout in this study. Application of IAA directly to the axillarybud of intact plants gave rise to a transient increase in budgrowth. This growth increment was annulled when AVG was suppliedwith IAA to the bud despite the fact that the dosage of AVGused did not affect the normal slow growth rate of the bud ofthe intact plant or bud outgrowth resulting from shoot decapitation.     

Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

In vitro response of apical bud explants from mature trees of jackfruit (Artocarpus heterophyllus)

A tissue culture technique for rapid vegetative propagation of mature jackfruit trees using apical bud cultures has been developed. Shoot-tip cultures were established on MS medium with 5–10 mm explants dissected from terminal buds of new growth from trunk. After initial culture of bud explants, one- to two-node pieces were taken from the microshoots formed and used to proliferate further axillary shoots for multiplying and maintaining shoot cultures. Benzyladenine and kinetin (4.5–9.0 µM), either separately or together, supported shoot proliferation; higher concentrations of the cytokinins inhibited bud breaking and favoured callus formation at the explant bases. Bud explants taken from emerging trunk sprouts invariably produced clumps of multiple shoots, whereas buds obtained from actively growing top branches generally elongated to form a solitary shoot. November to January was the best season for initiation of cultures from field-grown trees. Shoots proliferated at the initial subcultures had mature morphology and were difficult-to-root. Shoots assumed to be juvenile-like developed at the later passages and could be rooted with 60–80% success using 1/2-MS salts and 10 µM of indolebutyric acid or naphthaleneacetic acid. Regenerated plantlets were transferred to the soil and about 50% survived.     

In vitro multiplication of cashew (Anacardium occidentale L.) using shoot node explants of glasshouse-raised plants

Using glasshouse-raised plants (1 month, 1 year and 5 years old), factors affecting shoot development from shoot nodes of two Brazilian and one Tanzanian elite selections of cashew (Anacardium occidentale L.) were assessed. Sprouting of buds decreased strongly with increasing age of mother plants. Solidified media, mainly when purified agar was used, gave better results than liquid medium. Murashige and Skoog salts containing 1/2-strength macroelements were the most suitable for bud sprouting and shoot elongation. Vitamins and sucrose concentration did not have a significant effect but by replacing 20 g/l sucrose with glucose or maltose all estimated parameters were improved. Gibberellins supported bud sprouting and shoot elongation but blocked rooting. Shoots developed in the presence of cytokinins were short and produced axillary branches. Activated charcoal, cultivation of explants in darkness for the first 7 days and superoptimal temperature (35 °C) decreased bud sprouting and supported shoot elongation. Microshoots rooted in vitro at a frequency of 42% when cultured for 5 days with 100 μμ indole-3-butyric acid. Over 40% of rooted microshoots survived weaning. Received: 20 October 1996 / Revision received: 24 January 1997 / Accepted: 1 June 1997     

Flower Morphogenesis in Rosa hybrida 'Mercedes' as Studied by Cryo-scanning Electron and Light Microscopy. Effects on Light and Shoot Position on a Branch

Flower bud development in Rosa hybrida cv. 'Mercedes' was studied in shoots grown at different irradiances and sprouting from axillary buds at different branch positions. Cryo-scanning electron microscopy and light microscopy were used to visualize, characterize and determine flower morphogenesis during early shoot development. Up to the moment of visible flower bud appearance on the plant, flower morphogenesis was divided into nine stages. This classification was based on external and internal characteristics of the primordium. All shoots of the rose 'Mercedes' whether positioned uppermost or second on a branch and whether grown at 300 or 150 μmol m^-2 s^-1 PAR (12 h d^-1) developed equally up to flower stage 7, i.e. the stage just before visible initiation of stamen and pistils. Signs of flower bud abortion were the compactness of the flower bud at developmental stage 7 (height/width < 1·5) and the sprouting of axillary buds positioned just below the flower bud primordium. It was concluded that once a flower bud has reached a height to width ratio larger than 1·5, and once stamen and pistil developmental has started, it has passed the critical developmental stage in which abortion may occur. Flower developmental stage was closely related to shoot length. This relationship was not affected by irradiance level nor by shoot position on a branch. Therefore, cultivation treatments aimed to improve flower production by reducing flower abortion, such as supplementary lighting, will be most effective when applied during the first 2 weeks of shoot growth in which the flower develops up to stage 7.     

Seasonal Changes of the Metabolites in the Leaves, Bark and Xylem Tissues of Olive Tree (Olea europaea. L) II. Carbohydrates

Seasonal changes of starch and soluble carbohydrates in leaves,bark and xylem tissues of olive tree were examined during acomplete annual cycle. Leaf starch and soluble carbohydrateswere detected at high levels during the spring and autumn metabolicallyactive periods. The low level of leaf starch in combinationwith the drastic reduction of soluble carbohydrates and mannitol,defined the summer period of the low metabolic state of thetree. The low leaf starch level in conjunction with the risensoluble carbohydrate levels in leaves in winter were associatedwith cold acclimation processes. The bark and xylem tissueswere performing as starch deposition sites, and differencesin the extent of starch accumulation in these tissues were detectedduring the seasons. The starch fluctuations in bark and xylemwere discussed in relation to the translocation of metabolitesand other physiological processes. Mannitol, the most abundantleaf carbohydrate, was examined in relation to the reducingsugars exported to the bark. The bark mannitol was examinedin conjunction with the sucrose, glucose and starch levels inthe maturing bark tissues and was correlated to the low wintertemperatures. During the winter there was a drastic reductionof mannitol circulation from the bark to xylem. Olive tree, Olea europaea, L, carbohydrates     

Micropropagation of <Emphasis Type="Italic">Sapindus trifoliatus</Emphasis> L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis

An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength MS medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid MS medium with 1.0 mg l⁻¹ IBA for 24 h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

Micropropagation of Eucalyptus tereticornis Smith.

Axillary shoot bud multiplication has been achieved in Eucalyptus tereticornis Smith. using explants from different regions of 8–10 years old elite trees, growing in the field. Results showed that addition of NAA at 0.1 mgl^-1 and BAP at 1.0 mgl^-1 to modified MS medium induced maximum number of shoot buds. For inducing axial growth in regenerated bud promordia, the hormone concentration of the medium was lowered. The addition of charcoal and gibberellic acid to the medium were beneficial. Rooting was best in Knop's medium containing 1.0 mgl^-1 IBA. The key factor in root induction was primarily a dark incubation for a short period. The percentage of both rooting of shoots and survival of the rooted shoots was 60–80.Continuous trials using explants from the elite trees throughout the year showed that the period between July–September was the best season for the explant source for rapid and increased multiplication of axillary buds. Phenolic exudation was also minimum at this period. The experiments were repeated using 50 populations from different plantations. It was observed that during culture, genotypically different populations responded differently in spite of optimal growth conditions.     

Micropropagation of Dendrocalamus asper {Schult. & Schult. F.} Backer ex k. Heyne): an exotic edible bamboo

Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February?CApril) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS?+?15???M BAP. For shoot multiplication, MS medium supplemented with 10???M BAP and 75???M Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ? strength MS medium supplemented with 5???M each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6?months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.