Effects of VA-mycorrhizal fungi on growth and nutrient uptake of cuttings ofRosa multiflora in two container media with three levels of fertilizer application

Rooted cuttings ofRosa multiflora ‘Brooks 56’ were grown in a medium of 1 mineral soil: 1 sand (v/v) or bark: 1 sand (v/v) inoculated with the VA-mycorrhizal (VAM) fungiGlomus mosseae (Nicol. and Gerd.) Gerd. and Trappe andG. fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe or left as noninoculated controls. The slow release fertilizer osmocote was applied at rates of either 0, 1.2, or 4.2 kg/m³ (18N-6P-12K) and incorporated into the container medium. After 180 days plants were evaluated for growth, development and chemical leaf analysis. Greatest growth responses occurred after the highest fertilizer application of 4.2 kg/m³, and the poorest one after 0 kg/m³. Combination bark: sand medium was superior to mineral soil: sand medium for growth of mycorrhizal plants. At 0 kg/m³, mycorrhizal plants in bark: sand medium had longer shoots than other treatments. At 1.2 kg/m³, VAM plants compared to nonmycorrhizal plants in park:sand medium had greater effect on growth parameters. At the highest fertilizer application of 4.2 kg/m³, greatest growth responses occurred with VAM plants in bark:sand medium. Mycorrhizal plants compared to nonmycorrhizal plants in bark:sand medium had greater K and Zn uptake at 0 kg/m³, and greater K, Ca, S, Mn and Zn uptake at 1.2 kg/m³. Texas Agr. Expt. Sta. Journal Series No. TA-22264.     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

In vitro bulb production fromAllium spp.

Summary A procedure for bulb formation from onion, garlic, and shallot explants is described. Explants from cut stem bases were cultured in shoot induction medium composed of Murashige and Skoog (MS) medium with or without N⁶-benzyladenine. Shoots produced were then transferred to bulb induction medium composed of MS medium containing 5 g/liter activated charcoal and 120 g/liter sucrose under a long-day photoperiod and 28° C. Bulbs were also produced from onion and garlic directly, without passing through shoot formation, when explants were cultured in the bulb induction medium described above. Bulbs were transferred to soil without acclimatization and produced viable plants.     

In vitro shoot proliferation and production of sets from garlic and shallot

A procedure is described to regenerate shoots and bulbs in vitro with high frequency from shoot tips of garlic and shallot plants using benzyladenine or thidiazuron. Regenerated shoots were induced to form bulbs in Murashige and Skoog medium (1962) containing 5 g l^-1 activated charcoal and 120 g l^-1 sucrose under a long-day photoperiod. Bulbs formed in vitro were transferred to soil without acclimatization and produced viable plants. This method could be useful to produce low-cost bulbs, which are easy to handle and store until needed.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog's (1962) medium - NAA naphthaleneacetic acid - TDZ thidiazuron     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

Efficient micropropagation of Robinia ambigua var. idahoensis (Idaho Locust) and detection of genomic variation by ISSR markers

Robinia ambigua var. idahoensis, presumably originated from interspecific hybridization of R. pseudoacacia L. and R. hispida L., is a multipurpose tree. Several reports have showed that in vitro micropropagation is a feasible method to produce large quantities of ‘clonal’ plants from R. pseudoacacia, however, no information is available on micropropagation of R. ambigua or the other assumed parental species, R. hispida. Here, we report on a tissue culture system for efficient micropropagation of R. ambigua plants by enhanced branching of axillary buds taken from a single branch of a donor tree. The culture system consists of sequential use of three media, namely, the bud-induction medium (MS medium supplemented with 0.8–1.4 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA), elongation medium (MS medium added with 0.35–0.5 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA) and root-induction medium (1/4 MS medium fortified with 1.7–2.5 mg l⁻¹ IAA and 0.1–0.5 mg l⁻¹ IBA). In addition, we investigated the genetic stability (relative to the donor plant) of a sample of 41 morphologically normal plants randomly taken from ca. 13,000 micropropagated plants, by using the inter-simple sequence repeat (ISSR) marker with 32 selected primers. We found that of the 226 reproducible bands scored, 24 were polymorphic (10.62%), thus pointing to the occurrence, though at a relatively low level compared with an earlier study on R. pseudoacacia, of genomic variation in these micropropagated plants. Further sequencing on seven loci underlying the variations showed that two had significant homology to known or predicted plant genes.     

Genetic transformation of <Emphasis Type="Italic">Torenia fournieri</Emphasis> using the PMI/mannose selection system

Transgenic torenia plants were obtained using the selectable marker gene phosphomannose isomerase (manA), which encodes the enzyme phosphomannose isomerase (PMI) to enable selection of transformed cells on media containing mannose. We found that shoot organogenesis in torenia leaf explants was effectively suppressed on medium supplemented with mannose, which indicated that torenia cells had little or no PMI activity and could not utilize mannose as a carbon source. Leaf pieces from in vitro-germinated plants were inoculated with Agrobacterium tumefaciens EHA105 containing the binary vector pKPJ with both hpt and ManA genes, and subsequently selected on shoot induction (SI) medium (half strength MS basal + 4.4 μM BA + 0.5 μM NAA) supplemented with 20 g l⁻¹ mannose and 5 g l⁻¹ sucrose as carbon sources. Transformed plants were confirmed by PCR and Southern blot. The transgene expression was evaluated using Northern blot and the chlorophenol red assay. The transformation efficiency ranged from 7% to 10%, which is 1–3% higher than that obtained by selection with hygromycin. This system provides an efficient manner for selecting transgenic flower plants without using antibiotics or herbicides.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of light quality and gibberellic acid (GA3) on photosynthesis and respiration rates of pea seedlings

CO₂ exchange were measured on pea seedlings (Pisum sativum L. var. Bördi) cultivated from seeds imbibed either in water (C-plants) or in gibberellic acid (GA₃) at the concentration of 25 g/1 (GA-plants), and then grown under 17 W/m² blue light (B-plants) or 11 W/m² red light (R-plants).When measured under the same light conditions as during growth the net photosynthesis (APS) rate in B-plants was about twice higher than that in R-plants. Dark respiration (DR) rate was 70% higher in B- than in R-plants. Red light retarded the development of photosynthetic activity, but GA₃ suppressed this effect. The hormone enhanced net photosynthesis and dark respiration to the same extent.When measured under saturating white light net photosynthesis rate of C-plants was also two times higher in B-plants than in R-plants. Growth conditions had only a slight effect on the APS of GA-plants under white light. APS rates of GA-plants grown under red light were higher under white light than those of C-plants, but lower than those of plants grown under blue light.We assume that blue light induced formation of plants that were adapted to higher light intensity: red light had an opposite effect, whereas gibberellic acid induced formation of plants that were adapted to medium light intensity.     

Comparison of physical and chemical characters of tomato fruits grown from plants received from seeds or obtained in vitro

Results of experiments concerning comparison of tomato fruit properties which were collected from plants obtained in three manners are described. Control plants were received from seeds. Remaining plants were derived in vitro from leaf and shoot fragments on MS medium supplemented with IAA 0.2 mg·l⁻¹ and BA 2 mg·l⁻¹ (Górecka and Krzyżanowska 1991, Górecka et al. 1994) or with Fari’s et al. (1992) method of obtaining plants by decapitation of sterile seedlings and culture on MS medium without hormones. Evaluation of physical and chemical fruit characters was performed. In the spring experiment the biggest diameter (72 mm), weight (154 g) and volumne (151 ml) were characterized to fruits from plants obtained in vitro on MS medium with IAA and BA. Also fruits from plants received by Fari’s methods were significantly superior in these characters over fruits from the control plants. The fruits from the plants obtained in vitro on MS medium with IAA and BA had highest sugar content (2.95 % f.wt.) and fruits from in vitro plants after Fari’s method contained highest vit.C-13.4 mg·100 g⁻¹ f.wt (significant differences in comparison to control fruits). In other characters fruits from in vitro did not differ as compared to control ones. In the autumn experiments significant differences among fruit groups and characters evaluated were not stated. Generally, yield quality was poorer in the all autumn treatments.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Sucrose and light effects on in vitro cultures of potato,chokecherry and saskatoon berry during low temperature storage

Cultures of potato (Solanum tuberosum) cv. Atlantic, chokecherry (Prunus virginiana L.) cv. Garrington and saskatoon berry (Amelancher alnifolia Nutt.) cv. Northline grown in vitro for 3 weeks at 24/22 °C, 16-h photoperiod, 150 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) mixed fluorescent/incandescent light were stored for 6, 9 and 12 weeks at 4 °C under 0 (darkness) and 3 μmol m⁻² s⁻¹ PPFD (690 nm red light continuous illumination). Growth regulators free MSMO medium either with or without 30 g l⁻¹ sucrose was used to store the cultures. All cultures retained capacity to re-grow after storage. Tested factors, sucrose, light and the length of the storage period had an impact on shoot quality and re-growth capacity of the cultures. For either light treatment sucrose was essential for the low temperature maintenance of vigorous stock plants of potato, if stored for over 6 weeks. Chokecherry and saskatoon cultures stored well without sucrose; although chokecherry benefited from sucrose in the storage medium when the stock cultures were kept at the low temperature for 12 weeks. Low light significantly improved quality of the stored potato cultures, but had very little effect on both chokecherry and saskatoon berry cultures. The woody plant cultures grew during storage, and the longer the stock plants were stored, the more vigorous cultures they generated. The results indicate that growers can successfully use their existing facilities, small refrigerators and coolers with low light intensity, set at 4 °C, for short term storage of potato, chokecherry and saskatoon berry cultures. The potato cultures, which are known to be sensitive to prolonged low temperature storage, should be frequently monitored and subcultured as required. On the other hand, the woody plant stock cultures do not require any special attention when kept at 4 °C and re-grow the most vigorous shoots if stored for at least 12 weeks. This revised version was published online in August 2006 with corrections to the Cover Date.     

Synthesis and turnover of the light-harvesting chlorophylla/b-protein inLemna gibba grown with intermittent red light: possible translational control

Lemna gibba L. G-3 plants grown heterotrophically in the dark with intermittent red light (2 min every 8 h) contain a substantial amount of translatable mRNA encoding the light-harvesting chlorophyll (Chl)a/b-protein. However, very little [³⁵S]methionine is incorporated into the apoproteins during a 1-h labeling period in the dark in these plants compared to plants grown in continuous white light. The Chla/b-protein mRNA is found to be associated with functioning polysomes in plants grown in the dark with intermittent red illumination (R plants). The small amounts of the apoproteins which are synthesized by these plants are found in the membrane fraction; neither the mature apoproteins nor their precursor(s) can be detected immunologically in the soluble fraction. The protein does not accumulate in these plants. Pulse-chase experiments with the R plants demonstrate that the newly synthesized apoproteins have a half-life of about 10 h in the dark. This turnover is not sufficient to explain the observed 20-fold difference in [³⁵S]methionine incorporation into the apoprotein between white-light-grown and R plants. We therefore suggest that the synthesis of the Chla/b-apoproteins can be regulated by a light-dependent step at the level of translation, and that this regulation occurs after the initiation of translation.Abbreviations Chl chlorophyll - W Lemna plants grown in continuous white light - R plants grown heterotrophically in the dark with intermittent red light (2 min/8 h)     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Cultivation and carrageenan yield and quality ofKappaphycus alvarezii in the waters of Vietnam

The carrageenan-producing red algaKappaphycus alvarezii (Doty) Doty was brought to Vietnam from Japan in 1993. Branch fragments of this species were cultivated in a pond, lagoon, inlet and offshore in Vietnam for the first time. The best daily growth rate (DGR) of plants grown in the lagoon area attained 9–11 % day^–1 in May to June (cold season). The water temperature and salinity in this area ranged from 27.2–32.4 °C and 31.4–33.7 °C, respectively. DGR of plants grown in the inlet ranged from 7 to 9% day^–1 in June. Grazing by fish has been observed to occur in this area. The DGR of plants grown in the pond ranged from 5–6% in January–July, but decreased to less than 4% day^–1 in August (hot season). K. alvarezii in Vietnam showed a carrageenan yield of 18.8–24.6% and gel strength of 1566–1712 g cm^–2. These values are similar ones obtained fromK. alvarezii cultivated in the Philippines and Indonesia.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a β-glucuronidase (GUS) gene was transferred into apple cv. ‘Holsteiner Cox’ via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1–2 g l⁻¹ mannose in combination with 30 g l⁻¹ sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l⁻¹ and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.     

Gnotobiotic cultures of rice plants up to ear stage in the absence of combined nitrogen source but in the presence of free living nitrogen fixing bacteriaAzotobacter vinelandii andRhodopseudomonas capsulata

Summary An all glass tight growth chamber, entirely sterilizable, has been constructed to carry out axenic and gnotobiotic cultures of rice plants (Oryza sativa L.). When grown in liquid medium and in the absence of combined nitrogen but in the presence of the diazotrophsAzotobacter vinelandii andRhodopseudomonas capsulata, rice plants exhibited a complete biological cycle from germination up to ear stage, during a period of time similar to the one encountered in french paddy soil of Camargue. In one experiment, mannitol was given to rice culture medium together withAzotobacter vinelandii andRhodopseudomonas capsulata. In another experiment, mannitol was not given together with Rhodopseudomonas, and still positive nitrogen gain was obtained, although it was less than culture with mannitol. When¹⁵N labeled cells of Rhodopseudomonas were added in rice culture medium,¹⁵N was partly transferred to rice plant. Among the nitrogen substances excreted from the bacteria in the rhizosphere medium, large organic molecules were shown to be the most abundant in our experimental conditions. Moreover, the concentration of free ammonia or aminoacids present in the rice rhizosphere were always compatible with a bacterial nitrogenase activity.