Improvement of micropropagation of Genista monosperma Lam. by abscisic acid treatment

Genista monosperma Lam. (Leguminosae) is an ornamental shrub cultivated for the production of its attractive flowered branches. In order to overcome the low rate of in vivo propagation, micropropagation protocols and somatic embryogenesis procedures were established. In this work, the effect of ABA treatments on somatic embryo development has been taken into consideration with the aim of improving the conversion of G. monosperma somatic embryos into plants. A statistically significant higher number of regulary torpedo-shaped somatic embryos occurred on modified Murashige and Skoog medium supplemented with 1 μM ABA. When subcultured in light, the embryos induced in the presence of ABA, elongated and converted in a significantly higher percentage than those formed in absence of ABA. Sixty percent of the plantlets were successfully acclimatized in the greenhouse. A sample of the somatic embryo-derived plants were grown in the field, flowered during the third year and were phenotipically normal. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

Triacontanol-supported micropropagation of woody plants

 The effectiveness of triacontanol in the micropropagation of two woody, economically important fruit plant species was investigated. Triacontanol was added to the routine multiplication and rooting media of apple (Malus domestica cv. JTE-E4) and sour cherry (Cerasus fruticosa cv. Probocskai) rootstocks at concentrations of 2, 5, 10 and 20 μg/l. It was found to increase the number of shoots and the fresh weight of apple in the multiplication phase and to enhance root number and chlorophyll content in the rooting phase. The addition of indole-3-butyric acid (IBA) to the media further improved the effect of triacontanol. A less pronounced effect could be seen in the multiplication phase of sour cherry, although there was an enhancement of shoot proliferation. In the rooting phase, however, the application of triacontanol caused a significant increase in the number of roots per plant, and this effect was further improved when triacontanol was combined with 0.5 mg indole-3-butyric acid/l. Received: 29 March 2000 / Revision received: 1 September 2000 / Accepted: 4 September 2000     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Plant growth regulators affect biosynthesis and accumulation profile of isoflavone phytoestrogens in high-productive in vitro cultures of Genista tinctoria

The influence of plant growth regulators on biomass growth and the accumulation of medicinally-relevant isoflavone phytoestrogens, derivatives of genistein and daidzein (8 compounds including aglycones, glucosides and glucoside esters) in callus cultures of Genista tinctoria (Fabaceae) was examined. The experiments included 10 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, β-naphthoxyacetic acid, picloram, 2,3,5-triiodobenzoic acid (TIBA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] and 7 cytokinins [6-benzylaminopurine, forchlorfenuron, 1,3-diphenylurea, 2-isopentenyladenine, kinetin (KIN), thidiazuron, zeatin] applied at 0.5 and 5.0 mg l^?1, jointly with 5.0 or 0.5 mg l^?1 KIN or 2,4-D (for auxins and cytokinins, respectively—36 phytohormone combinations in total). Statistical analysis of the relationships between callus growth [expressed as growth index (Gi)] and the accumulation of isoflavones showed positive correlation in the cytokinin group (r_xy values from 0.13 to 0.61) and negative correlation within auxins (r_xy values from ?0.31 to ?0.39). Among the cytokinins tested, the highest isoflavone content (6,436.26 mg/100 g dry weight) and the fastest biomass growth (Gi = 892.46 %) were obtained for 0.5 mg l^?1 KIN used jointly with 5.0 mg l^?1 2,4-D. In the group of auxins, the combination of 0.5 mg l^?1 TIBA and 5.0 mg l^?1 KIN provided the fastest culture growth (Gi = 983.07 %) and the isoflavone concentration of 10,474.23 mg/100 g dry weight, which is so far the highest amount of these metabolites achieved in callus cultures of higher plants.     

A general approach for developing a commercial micropropagation system

Summary Five distinct steps can be recognized in the establishment of a plant in a commercial micropropagation system, especially if the most utilized approach (shoot culture) is the focus. Failure at any one step can make the total system commercially unworkable. When one considers a plant without extensive previous history of microculture, the first step involves an analysis of the potential market (economic reality) as well as the plant’s general growth habit (biological reality). For the latter, the general growth habit of the plant can provide valuable predictive information as to the potential ease of microculture. For example, plants showing indeterminant herbaceous growth (e.g., Chrysanthemum, Solanum, Dieffenbachia) or continuous woody seasonal growth (e.g., Betula, Ulmus, Thuja) are generally much more amenable to microculture than those that are determinant herbaceous (e.g., Panix, Paeonia) or episodic woody organisms (e.g., Quercus, Pinus). At times, an episodic habitat can be overcome in microculture (Syringa, Rhododendron). The next four steps involve the actual manipulation and microculture of the plant and include the initiation, stabilization, optimization, and production phases. The most intensive analytical step is usually the optimization phase in which plant growth regulator response curves, replication, repetition through multiple subcultures, and evaluation of productivity and product quality are involved. The intent of this discussion is to help develop a decision tool to be used as a first approach to designing a potential new micropropagation system for an untested plant genotype.     

Two-stage designs for experiments with a large number of hypotheses

MOTIVATION: When a large number of hypotheses are investigated the false discovery rate (FDR) is commonly applied in gene expression analysis or gene association studies. Conventional single-stage designs may lack power due to low sample sizes for the individual hypotheses. We propose two-stage designs where the first stage is used to screen the 'promising' hypotheses which are further investigated at the second stage with an increased sample size. A multiple test procedure based on sequential individual P-values is proposed to control the FDR for the case of independent normal distributions with known variance. RESULTS: The power of optimal two-stage designs is impressively larger than the power of the corresponding single-stage design with equal costs. Extensions to the case of unknown variances and correlated test statistics are investigated by simulations. Moreover, it is shown that the simple multiple test procedure using first stage data for screening purposes and deriving the test decisions only from second stage data is a very powerful option.     

In vitro cloning and micropropagation of Lavandula Stoechas from field-grown plants

In vitro clonal propagation of native Mediterranean Lavandula stoechas has been achieved from mature field-grown plants. Procedures have been developed for reducing shoot hyperhydricity during in vitro culture establishment and shoot multiplication stages. Shoot multiplication was obtained, in 4–5 weeks, from single node explants cultured on a basal medium containing Margara N30K macrosalts and supplemented with 217.2 M adenine hemisulphate (AdS) and 0.05 M NAA. In vitro rooting (100%) of the shoots was observed on basal medium containing 5.4 M NAA.Abbreviations BA N⁶-benzyladenine - AdS adenine hemi-sulphate - GA₃ gibberellic acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Heterosis as an explanation for large amounts of genic polymorphism

By using both numerical and analytical approaches, we have shown that heterosis alone is not a mechanism for maintaining many alleles segregating at a locus. Even when all heterozygous are more fit than all homozygotes, the proportion of fitness arrays that will lead to a stable, feasible equilibrium of more than 6 or 7 alleles is vanishingly small. More alleles can be maintained if, in addition to heterosis, it is assumed that there is very little variation in fitness from heterozygote to heterozygote, with the ratio of mean heterosis to standard deviation of fitness among heterozygotes in the neighborhood of 10. When such conditions hold, the allelic frequency distribution and equilibrium will be very uniform, with all alleles very close to equal frequency (see PDF). It is much more likely that stable equilibria for multiple alleles will be best explained by multiple niche selection.     

Proliferation of meristematic clusters in disposable presterilized plastic bioreactors for the large-scale micropropagation of plants

Summary Proliferation of meristematic clusters of several plants in an inexpensive airlift bioreactor system, consisting of a disposable presterilized light transmittable plastic film vessel is described. The optimal shape, size, and structural function of the disposable plastic bioreactor are based on the bubble column and airlift glass bioreactors. The disposable bioreactors are designed in a conical configuration with a single inoculation and harvest port and multiple use dispensing and mixing accessories. Shearing damage and foaming problems known to exist in bioreactors due to the plant's rigid cell wall and size were greatly reduced in the disposable plastic bioreactors. The disposable bioreactors were used for propagule proliferation and growth, using meristem and bud clusters of potato, fern, banana, and gladiolus. The clusters' biomass increased five-to eightfold over a period of 26–30 d, depending on the species. The clusters were separated mechanically by a chopper made of a grid of knives. The chopped propagules were inoculated to agar medium for further growth and developed into transplantable plants. In the case of gladiolus and potato, corms and tubers developed in a sucrose-elevated storage organ induction medium, respectively, after the initial formation of small shoots. The plantlets and storage organs were transplanted to an acclimation greenhouse and continued to grow with a 95–100% survival, depending on the species. Plant development was followed for a period of 16 wk in fern and 12–14 wk in potato, banana, and gladiolus and normal shoot and leaf growth was observed. The feasibility of large-scale liquid cultures for plant micropropagation is discussed.     

Nutrient utilization in liquid/membrane system for watermelon micropropagation

Watermelon [Citrullus lanatus (Thunberg) Matsumura and Nakai] proliferating shoot meristems from established shoot cultures were inoculated on modified Murashige and Skoog salts medium supplemented with 10 μM 6-benzyladenine (BA) for shoot proliferation and on similar medium supplemented with 1 μM BA and 10 μM gibberellic acid (GA₃) for shoot elongation. Agar-solidified medium and microporous polypropylene membrane rafts in liquid medium were used to support the tissues. Growth over culture time of proliferating and elongating tissues in liquid and agar-solidified media were compared. Nutrient depletion in liquid medium was monitored and quantified using ion selective electrodes. Tissue fresh weights in both proliferation and shoot elongation media were greater in liquid than in agar-solidified medium. Relative dry matter content, however, was greater in agar-solidified than in liquid medium. More shoots elongated in agar-solidified than in liquid medium. The numbers of buds or unelongated shoot meristems, however, were comparable for both the liquid and agar-solidified medium. Proliferating and elongating tissues in liquid medium used Ca⁺⁺ and K⁺ minimally. NO ₃ ⁻ was utilized but not depleted by proliferating tissues. NH ₄ ⁺ , however, was depleted. Most of the NH ₄ ⁺ was utilized by the proliferating tissues within 21 days of culture when growth rate was greatest. At 35 days, residual Ca⁺⁺, K⁺, NO ₃ ⁻ , and NH ₄ ⁺ in proliferation medium were 81.0%, 67.8%, 55.7%, and 1.2% of initial levels, respectively. NO ₃ ⁻ and NH ₄ ⁺ in shoot elongation medium were depleted. The greatest NO ₃ ⁻ and NH ₄ ⁺ utilization was observed during the first 14 days of culture when the largest growth rate was obtained. The residual Ca⁺⁺, K⁺, NO ₃ ⁻ , and NH ₄ ⁺ in shoot elongation medium at 38 days were 63.5%, 37.9%, 21.2%, and 24.3% of initial concentrations, respectively. At the end of experiment, 72.3% and 42.8% of initial sugars were still remaining in the shoot proliferation and shoot elongation medium, respectively. Technical Contribution No. 3236 of the South Carolina Agricultural Experiment Station.     

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.     

Safe and acceptable strategies for producing foreign molecules in plants

The ability to express foreign genes using transgenic technologies has opened up options for producing large quantities of commercially important industrial or pharmaceutical products in plants. These technologies have made it possible to use well-developed systems of commercial agriculture that were developed principally to produce raw material for large-scale food, feed or processing applications for the production of foreign molecules. The possibility of the novel industrial or pharmaceutical molecules produced in such plants, or components derived from them, contaminating the environment and food chains has become especially controversial. This potential contamination has prompted detailed consideration of how such crops and the molecules that they produce can be effectively isolated and contained. First, the crop can be completely isolated physically from its food or feed counterpart during every aspect of its development and commercialization. Second, genetic isolation systems or genetic barriers that prevent normal reproduction can be used to reduce the likelihood of the industrial or pharmaceutical crop entering the food chain.     

Implementation of functional genomics for gene discovery in alkaloid producing plants

Two centuries after the discovery of the first alkaloids, many enzymes involved in plant alkaloid biosynthesis have been identified. Nevertheless, the biosynthetic pathways for most of the plant alkaloids still remain incompletely characterised and understanding the regulatory mechanisms controlling the onset and flux of alkaloid biosynthesis is virtually inexistent. This information is however crucial to allow modelling of metabolic networks and predictive metabolic engineering. In the postgenomics era, new functional genomics tools, enabling comprehensive investigations of biological systems, are continuously emerging and are now gradually being implemented in the field of plant secondary metabolism as well. Here we discuss the advances these promising new technologies have already brought and may still bring with regard to the dissection of plant alkaloid biosynthesis. Encouraging results were obtained in alkaloid producing species such as Papaver somniferum, Catharanthus roseus and Nicotiana tabacum. Therefore we anticipate that functional genomics and the knowledge it brings along, will eventually allow a better exploitation of the plant biosynthetic machinery.     

Sucrose uptake and metabolism in a double layer system for micropropagation ofRosa multiflora

Uptake and metabolism of sucrose in micropropagatedRosa multiflora using the double layer technique was investigated. In the multiplication as well as the root induction stage, hydrolysis of sucrose in the culture medium was observed. A mathematical model was developed to quantify sucrose hydrolysis and the uptake of sucrose, glucose and fructose, based on the time series for the different sugars in the culture medium. These data were linked to a study of the sugar metabolism in the microshoots. After 48 h of incubation on¹⁴C-[U]-glucose containing medium, the incorporated label was mainly detected in the ethanol soluble fraction; within this fraction sucrose was the most important compound. This indicates a significant re-synthesis of sucrose in the plant material after the uptake of hexose. To assess the extent that different enzymes of sucrose metabolism (invertases, sucrose synthase and sucrose-P-synthase) were involved, their activity in different plant parts (of final stage III microshoots) were assayed. A decreasing gradient for sucrose metabolising enzymes from the roots toward the leaves gave a good indication of how the different tissues depend on sucrose absorbed from the medium.     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.