An improved procedure for the propagation in vitro of grapevine (t Vitis vinifera cv. Pinot noir) using axillary-bud microcuttings

A method has been developed for rapid propagation in vitro of Vitis vinifera cv. 'Pinot noir' from axillary-bud microcuttings harvested from plantlets initially cultured in vitro. The requirement of plant growth regulators for the different stages of the micropropagation was examined. BA at 8.9 μM added to MS basal medium was found to be optimal for culture establishment, while for subcultures, the concentration of BA was reduced to 4.4 μM to prevent hyperhydricity. Among the two auxins (NAA and IBA) tested for rooting, IBA at 2.5 μM was found to induce a good rooting- system in 100% of the shoots. The advantages of this method, using microcuttings from established plants in vitro, is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Populus trichocarpa ‘Nisqually-1’: the genotype deriving the Populus reference genome

Populus serves as a model tree for biotechnology and molecular biology research due to the availability of the reference genome sequence of Populus trichocarpa (Torr. & Gray) genotype ‘Nisqually-1’. However, ‘Nisqually-1’ has been shown to be very recalcitrant to micropropagation, regeneration and transformation. In this study, a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’ was established. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite^? and 5–10 g L⁻¹ of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. This highly effective protocol provides a consistent supply of quality leaf and stem materials throughout the year for transformation experiments and other in vitro manipulations, therefore eliminating inconsistency due to seasonal and greenhouse environmental variations and the need for repetitive tissue sterilization.     

Environmental control for the large-scale production of plants through in vitro techniques

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO₂ concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets. Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation with 3-methyleneoxindole: Its obligatory role in indole-3-acetic acid-mediated auxin action

Summary 3-Methyleneoxindole (MO), a metabolite of the plant auxin indole-3-acetic acid (IAA), was more active than IAA in supporting Stage II and III micropropagation of several plant species. In Stage II micropropagation, characterized by the rapid numerical increase of shoots, the optimal IAA concentration was 0.01 mM compared to 0.1 mM MO for most plants. In Stage III micropropagation where auxin is required for the rhizogenic response, 0.1 μM MO was more effective than 0.01 mM IAA. Inhibition analysis of plant growth with chlorogenic acid (CGA) suggested an obligatory role for MO in IAA-mediated auxin reactions: CGA, which blocks the enzymatic oxidation of IAA to MO, in vivo, completely abolished IAA's ability to support the growth of explants during micropropagation. In contrast, CGA did not inhibit the auxin activity of MO, the product of the blocked reaction. The growth rate and rooting efficiency of tobacco propagules in Stage III medium was improved substantially if these were first exposed to a high concentrations of MO and subsequently transferred to media containing low or no MO.     

Photoautotrophic micropropagation

Summary Sugar in the medium is considered to be an essential cause for the high production costs of plantlets in conventional, heterotrophic micropropagation. Chlorophyllous explants, shoots, and plantlets in vitro have high photosynthetic ability to develop photoautotrophy, but their photosynthetic activity is restricted largely by the low CO₂ concentration in the vessel during the photoperiod and in part by the presence of sugar in the medium. The growth of plantlets in vitro is often greater under photoautotrophic conditions than under heterotrophic conditions, provided that the in vitro environment is properly controlled for promoting photosynthesis. The advantages and disadvantages of photoautotrophic micropropagation are discussed.     

In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.     

Orchid micropropagation: the path from laboratory to commercialization and an account of several unappreciated investigators

A commonly held view is that the ideas and basis for the practice of orchid micropropagation arose de novo in 1960 from the work of Georges Morel in France. In this paper we argue that the crucial developments in micropropagation were made by Gavino Rotor in 1949 in the USA and Hans Thomale in 1957 in Germany, and that Morel's work needs to be seen in the context of a long line of research achievements in the in vitro culture of a wide range of explanted tissues and organs from plants of many species. A critical, historical, analysis of the events as they relate to clonal orchid multiplication is offered here. Two important technical innovations for orchid micropropagation — the use of activated charcoal to darken nutrient media and the adoption of liquid culture environments for part of the process — are examined in detail. In addition, an unusual US patent claiming invention of 'a method for rapidly reproducing orchids', especially cattleyas, is analysed. The origin of the micropropagation process claimed in this patent, said by the nominal inventor to go back as far as 1950, is discussed, but the claim remains unsubstantiated. Finally, consideration is given to the problems of adjudicating unequivocal priority for 'discovery' of a process as complicated and as broad as micropropagation.     

Development and application of photoautotrophic micropropagation plant system

Research has revealed that most chlorophyllous explants/plants in vitro have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is not due to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Moreover, numerous studies have been conducted on improving the in vitro environment and investigating its effects on growth and development of cultures/plantlets on nearly 50 species since the concept of photoautotrophic micropropagation was developed more than two decades ago. These studies indicate that the photoautotrophic growth in vitro of many plant species can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. This paper reviews the development and characteristics of photoautotrophic micropropagation systems and the effects of environmental conditions on the growth and development of the plantlets. The commercial applications and the perspective of photoautotrophic micropropagation systems are discussed.     

The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L

Summary Comparative studies of carnation micropropagation under four different ventilation rates showed that using gas-permeable filters, with gelled or liquid media and modifying the volume of culture medium, it was possible to establish a suitable hydric state to obtain good proliferation rates with gelled and liquid medium, as well as optimal acclimatization of microcuttings. The following parameters were measured: ventilation rate, gas exchange coefficients, relative water loss, increase of agar concentration, micropropagation rates, percentage of hyperhydricity, and acclimatization rates. Our results confirm that it is possible to avoid hyperhydric plants cultured in liquid medium with the use of ventilated culture vessels through the control of the water relations during the multiplication phase and, at the same time, keeping the micropropagation rate.     

A protocol for Ulmus minor Mill. micropropagation and acclimatization

Here we report the establishment of a simple protocol for the micropropagation and acclimatization of U. minor. Branches with dormant buds were collected from mature elms and sprouted in a greenhouse. Tip and node segments were used as starting material for in vitro proliferation in a medium (designated here as DKW1) already used for the micropropagation of a clone of the English Elm (U. procera SR4). In the first assay, in which explants from nine different trees were used, 88.5% of the tip segments produced new axillary shoots thus proving to be the best explant type. Afterwards, material from four different trees (F4, F7, F13, F14), that had the highest sprouting rate in the greenhouse, was used to test for genotype influence. F14 proved to be the best genotype in culture and it was used for all the subsequent experiments. Shoots from F14 were used to assay in vitro rooting using five DKW based media. Rooting percentages were high for all media and varied between 80% and 100%. For acclimatization two approaches were assayed: the use of previously rooted in vitro plants and the direct acclimatization of shoots from cultures in DKW1. After 6 weeks, 86.4% of the in vitro rooted plants were successfully acclimatized and a slightly higher value, 88.6%, was attained by direct acclimatization of shoots with thick stems and hard leaves. These results proved that there is no need for a previous in vitro rooting step and that direct acclimatization can effectively reduce time and costs. Thus U. minor micropropagation and acclimatization can be divided into only two steps: proliferation of shoots in DKW1 and direct acclimatization of these shoots in a sterile soil mixture.     

Factors affecting the efficiency of micropropagation from lateral buds and shoot tips of <Emphasis Type="Italic">Rubus</Emphasis>

Factors affecting micropropagation efficiency of 32 selections of Rubus, including the pre-treatment and initiation culture stages, were investigated. Chilling at 4°C for 6 weeks as a pre-treatment significantly promoted in vitro initiation culture; up to 65% of initiation cultures post chilling were successful. The cytokinin 6-benzyladenine (BA) was the most effective of the three tested for promotion of multiple shoot development in culture, with an average of three to seven shoots or plantlets developed from each single node. Alternating the concentration of BA between 4.44 and 13.31 μM from sub-culture to sub-culture improved recalcitrant Rubus micropropagation, and reduced the problems associated with long-term culture in the presence of high concentrations of BA. Reduction of the strength of macro- and micro-elements in the basal medium from half to one-third, with addition of 0.49 μM indole-3-butyric acid and 0.05% activated charcoal, and placing the cultures under reduced light intensity (17 μmol m⁻² s⁻¹), was found to alleviate chlorosis and improve micropropagation with high quality Rubus plantlets. Response to in vitro culture differed greatly and consequently different methods of micropropagation were required by different genotypes. From these selections, three types of micropropagation including micro-cutting, micro-shoots and multi-shoots were observed, and their efficiency was characterized.     

Media derived from brown seaweeds <Emphasis Type="Italic">Cystoseira myriophylloides</Emphasis> and <Emphasis Type="Italic">Fucus spiralis</Emphasis> for in vitro plant tissue culture

In the present study, the effect of seaweed extract (SE) from Fucus spiralis (Fs), Cystoseira myriophylloides (Cm) and Laminaria digitata (Ld) on in vitro plant tissue culture was examined. Combination of 25?% of SE from Cm with 25?% of MS medium increased adventitious shoot regeneration from Nicotiana benthamiana leaf discs explants by 620?%, when compared to the conventional regeneration medium. Similarly SE from Fs and Ld enhanced regeneration by about 500?%. However, when increasing SE to 50?%, only Cm significantly enhanced shoot regeneration. The effect of SE was also evaluated on in vitro micropropagation of N. benthamiana, grape, plum and apricot by assessing shoot length, number of leaves and internodes. When used alone but at lower concentrations (2.5 and 12.5?%), SE from Fs and Cm resulted in at least the same efficacy as MS alone for micropropagation of N. benthamiana shoots. However, for micropropagation of grapevine, plum and apricot woody plants, a combination of 50?% of SE from Cm or Fs with 50?% of their conventional micropropagation media was necessary. Rooting was also enhanced in N. benthamiana and grapevine, and was correlated with their higher concentrations of indole acetic acid when compared to SE from Ld. This finding, in addition to mineral analysis data, suggests that SE of Fs and Cm contain necessary nutrients and growth regulators to allow their use as medium for in vitro plant culture.     

Luteolin content and antioxidant activity in micropropagated plants of <Emphasis Type="Italic">Poliomintha glabrescens</Emphasis> (Gray)

Mexican oregano is composed by a wide range of endemic species which support important economic activities, but with serious limitations. In this work a micropropagation protocol was established to regenerate oregano plants (Poliomintha glabrescens Gray) from apical buds. At the same time we evaluated the impact of this process in the phytochemical profile of the new plants. The optimal induction of apical buds was observed when shoot-tip explants were incubated on solidified Murashige and Skoog (MS) medium supplemented with 0.5 mg ml⁻¹ α-naphthalenacetic acid (NAA) and 0.02 mg ml⁻¹ 6-benzyladenine. Also, the optimal response to root induction was with MS solid medium added with 0.5 mg ml⁻¹ of NAA. The micropropagation protocol took 17 weeks with an efficiency of 40%, and plants achieved were successfully acclimatized under greenhouse conditions. The total phenolics (TP) concentration, antioxidant capacity (oxygen radical absorbance capacity, ORAC), and luteolin content from methanol extracts of wild type (WT) and in vitro (IN) and ex vitro (EX) plants were determined. The results show that TP content and ORAC were similar for WT and EX plants, but IN plants had the highest TP content and ORAC activity (25 and 28% respectively). Luteolin content showed significant differences between WT, IN and EX, with EX plants having the highest luteolin content (10% more compared to WT). In summary, we successfully microprogated oregano plants from WT and they contained similar amount of their TP content and ORAC activity and an increase in luteolin content.     

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.     

Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology

Members of the Chrysanthemum-complex include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of culinary, medicinal and (ethno)pharmacological interest. The last 35 years have seen a tremendous emphasis on their in vitro tissue culture and micropropagation, while the latter 10–15 years has seen a surge in transformation experiments, all aimed at ameliorating aesthetic and growth characteristics of the plants. This review highlights all available literature that exists on ornamental Chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

Towards ex situ conservation of globally rare Turkish endemic Tripleurospermum fissurale (Asteraceae)

In Vitro Cellular & Developmental Biology - Plant - A rapid and efficient in vitro micropropagation system was developed to conserve Tripleurospermum fissurale (Sosn.) E.Hossain (Asteraceae), a...     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

Correction to: Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Plant Cell, Tissue and Organ Culture (PCTOC) - In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect...     

In vitro biotechnological approaches on <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews: advancements and opportunities

In vitro biotechnological advancement of Vanilla plays a major role in germplasm conservation, genetic engineering, accelerated clonal multiplication and production of disease-free plants with enviable aromatic properties. Several attempts have been taken place for the establishment of efficient in vitro protocol for Vanilla in the past few decades. Optimization of various conditions during different phases of micropropagation, for instance development of in vitro aseptic cultures, multiple shoot regeneration, rooting and acclimatization of the plantlets are discussed in this review. In addition to basic micropropagation techniques, various other in vitro biotechnological applications such as clonal fidelity assessment, genetic transformation, synthetic seed technology and cryopreservation are also highlighted. Apart from the existing data, applied aspects like embryo rescue, mutation breeding, genetic engineering, protoplast fusion, somaclonal variation, in vitro enhancement of vanillin production through cell suspension culture, hairy root culture or bioreactors and cryopreservation need to be investigated further. Overall, the current review gives a synopsis on progress and prospect of in vitro culture of Vanilla.