The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.     

Kalanchoe blossfeldiana plants expressing the Arabidopsis etr1-1 allele show reduced ethylene sensitivity

Transgenic Kalanchoe blossfeldiana Poelln. with reduced ethylene sensitivity in flowers was obtained by Agrobacterium tumefaciens-mediated transformation using the plasmid pBEO210 containing the mutant ethylene receptor gene etr1-1 from Arabidopsis thaliana under the control of the flower-specific fbp1-promoter from Petunia. Three ethylene-resistent T0 lines, 300, 324 and 331, were selected and analyzed for postharvest-performance and morphological characteristics. Line 324 was found to be infertile and only slightly less ethylene-sensitive than control-plants, but lines 300 and 331 had significantly increased ethylene-resistance and were fertile. These two lines were analyzed for copy-number of the etr1-1 gene by Southern blotting and were crossed with the ethylene-sensitive cultivar ‘Celine’ to create T1 progeny. Line 300 contains two T-DNA copies per nucleus, one of which is rearranged, and these are unlinked according to segregation data from the crossing to ‘Celine’ and PCR-analysis of progeny plants. For control plants all flowers were closed after 2 days at 2 μl l⁻¹ ethylene, but for line 300 only 33% were closed after 10 days. Line 331 contains three T-DNA copies per nucleus and is more sensitive to ethylene than line 300. In the line 300 the etr1-1 gene was found by RT-PCR to be expressed in petals and stamens but not in carpels and sepals. Both lines 300 and 331, and their progeny, appear morphologically and physiologically identical to control plants except for the higher ethylene resistance. Line 300 and its progeny with only one T-DNA copy have very low ethylene sensitivity and may be useful in future breeding.     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

Alcohol induced silencing of gibberellin 20-oxidases in Kalanchoe blossfeldiana

Reduction in the amounts of active gibberellic acids (GA) in elongating cuttings from the ornamental crop Kalanchoe blossfeldiana were pursued by genetic manipulation as an alternative to synthetic growth regulators. An alcohol inducible promoter system was used to control silencing of GA activating enzymes. Apart from affecting the stem length, abnormal levels of GA can lead to altered flowering time, lacking seed maturation and changes in morphology. The effects of down regulating a group of GA 20-oxidases were investigated in fast growing cuttings of K. blossfeldiana Poelln. cv. Molly. The transgenic plants were phenotypically indistinguishable from wild type plants until silencing was induced by low concentrations of ethanol. Treated plants were reduced in height but otherwise appeared normal; flowering was delayed but with large variations in time between the transgenic lines. These data indicate that optimisation of the ethanol treatments can enable us to produce more compact growing plants still maintaining normal flowering.     

Hybrid seed production and the challenge of propagating male-sterile plants

The introduction of hybrid crop varieties has enabled spectacular increases in productivity owing to hybrid vigor and increased uniformity. To produce hybrid seeds, a pollination control system is required to prevent unwanted self-pollination. In crop species with hermaphrodite flowers, this can be a major challenge. Over the past decade, new pollination control systems have been developed with the aid of genetic engineering, mainly based on the generation of nuclear-encoded male sterility. The successful application of these systems for large-scale hybrid seed production depends on whether the male-sterile female parent line can be multiplied efficiently and economically. In spite of its relevance, the propagation of the male-sterile line has often been overlooked in the development of pollination control systems.     

从长寿花嫩叶直接诱导体细胞胚和出芽

1　植物名称　长寿花 (Kalanchoeblossfeldiana) ,又名寿星花、矮生伽蓝菜。2　材料类别　盆栽植物的嫩叶片。3　培养条件　基本培养基为MS。诱导培养基 :( 1 )MS +NAA 1 .0mg·L- 1 (单位下同 ) + 6 BA1 .0 ;( 2 )MS +NAA 1 .0 ;( 3)MS + 6 BA 1 .0。生根培养基为 :( 4 ) 1 /2MS不加任何生长调节剂。以上培养基含 2 %蔗糖、0 .6%琼脂 ,pH 5 .8。培养温度( 2 5± 2 )℃。4　生长与分化情况　嫩叶外植体经 70 %酒精消毒 1 5s和 0 .1 %升汞消毒 8min后 ,以无菌水清洗。切成 1cm× 1cm大小的切段 ,接种于诱导培养基上培养。嫩叶外植…     

Effects of Different Nitrogen Sources and Concentrations on CAM Photosynthesis in Kalanchoe blossfeldiana

When Kalanchoë blossfeldiana Poelln. cv. Hikan plants werecultured in solutions containing 0.2, 1.0, 5.0 or 10 mM of nitrateor ammonium under a long-day photoperiod, some criteria of CAM(Crassulacean acid metabolism) photosynthesis (diurnal changesof CO₂ uptake, titratable acidity and malate content in leaves)were examined. The plants absorbed 90 to 100% of CO₂ duringthe light phase regardless of the supplied nitrogen. Nitrate-grownplants absorbed about 10% of CO₂ during the dark phase regardlessof the supplied concentration, whereas in ammonium-grown plantsthe nocturnal CO₂ uptake occurred at 0.2 mM, at which the plantsdepleted nitrogen and no uptake was observed at the higher concentrations.Changes of nocturnal increase in titratable acidity and malatecontent almost corresponded with the changes in the amount ofnocturnal CO₂ uptake. Also K. daigremontiana plants suppliedwith 10 mM of ammonium had a less CAM-like pattern of diurnalCO₂ uptake than the plants supplied with 10 mM of nitrate. Theseresults suggest that a sufficient supply of ammonium depressesCAM photosynthesis.     

Effects of auxins and cytokinins on epigenetic instability of callus-propagated Kalanchoe blossfeldiana Poelln.

A main problem in the vegetative propagation of ornamental plants in vitro is the epigenetic instability of cells removed from their organized environment. With calluses of leaf explants of Kalanchoe blossfeldiana Poelln., cv. Yucatan, the role of plant growth regulators (PGRs) in the occurrence of fasciation was studied.In various combinations of auxins and cytokinins, the auxin 2,4-D (2,4-dichlorophenoxyacetic acid) gave only deformed, inseparable shoot primordia. The most rapid callus induction with regeneration of well-developed sprouts was obtained with the natural IAA (indoleacetic acid) and Z (zeatin).As a first symptom of fasciation, aberration in decussate phyllotaxis can be observed. At increasing concentrations of IAA + Z, this symptom gradually decreased but fasciation proper increased. The optimum concentration was at 1 M for both PGRs. Reduction of exposure to the PGRs from six to three weeks reduced the epigenetic instability.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens

A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity was observed ranging from 25% to 55% out of the cocultivated FEC cultures, when FEC cultures were infected for 30 min with 50 μM AS, 1:10 dilution of bacteria, and then cocultivated at 24°C in the dark for 7 days with Agrobacterium strain LBA4404 (pTOK233) that carried gus, nptII and hpt genes. Seven independent experiments produced a total of 1300 transformed somatic embryos with shoots from 3.5 g of FEC. Of these germinated embryos, 50% developed into plants in vitro. Thus, on average, 500 mg of FEC infected with A. tumefaciens produced approximately 80–100 transgenic plants within 6–8 months via a selection process with 2.5–20 mg L^?1 hygromycin. Additionally, transformation was also performed with Agrobacterium strain AGL1 (containing the uidA and ppt genes), and this showed that luciferase‐based selection was less detrimental to the transgenic lines than was herbicide‐based selection. The transformation efficiency was 18.6% for the luciferase‐based selection and 7.6% for the PPT‐based selection, although with luciferase‐based selection, more false positives were obtained (about a quarter of the lines were escapes). The nptII and uidA genes were detected by polymerase chain reaction analysis in nine of the 19 tested lines. The results indicate that the system developed here can be used as an alternative to particle bombardment of Alstroemeria.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Enzymatic Properties of Phosphoenolpyruvate Carboxylase of Purified Chloroplasts from CAM-Performing Kalanchoe blossfeldiana Poelln. Plants

A phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.3 [EC] ) activitywas associated with, the Percoll purified chloroplasts fromKalanchoe blossfeldiana leaves performing crassulacean acidmetabolism (CAM) (plants grown under short-day conditions).Very little PEPC activity was detected in the chloroplasts whenthe plants were grown under long days, performing a C₃-typephotosynthetic metabolism. The PEPC activity measured in thechloroplasts from CAM-plants was very sensitive to such effectorsas glucose-6-phosphate (G-6-P) and malate: the initial activityof PEPC in the presence of 1.2 mM PEP was 400% activated by10 mM G-6-P and was 25% inhibited by 1 mM malate. These resultsshow that the PEPC in the chloroplasts has the enzymatic characteristicsdescribed by Brulfert and Queiroz [(1982) Planta 154: 339] forPEPC extracted from CAM-performing K. blossfeldiana leaves. (Received November 1, 1985; Accepted April 25, 1986)     

Flower inhibition in Kalanchoe blossfeldiana. Bioassay of an endogenous long-day inhibitor and inhibition by (±) abscisic acid and xanthoxin

Summary The inhibition of flowering in Kalanchoe by crude sap expressed from leaves held in non-inductive long-day conditions is described, using a bioassay technique of leaf injection, which confirms the existence of a transferable inhibitor.This technique has also revealed that ± abscisic acid and Xanthoxin are inhibitory to flowering at 50 and 100 ppm respectively. The previously known inhibitory effects of gibberellic acid on flowering have also been confirmed.     

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

 The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10^–3–10^–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M₂ doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M₂ doubled-haploid lines carrying mutations per line analysed was 3.8% when 10^–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10^–5 and 10^–4 M sodium azide were applied to freshly isolated microspores from model cultivars. Received: 18 April 2000 / Revision received: 28 September 2000 / Accepted: 28 September 2000     

Pre-illumination as a Factor in the Dark Fixation of Carbon Dioxide by Leaf Disks of Kalanchoe blossfeldiana var. Feuer Blute

Dark fixation of CO₂ by leaf disks or whole leaves taken fromplants of variety ‘Feuer Blute’ was measured using¹⁴CO₂. Results indicate that dark fixation by leaf disks isindependent of photoperiodic induction of the plant, but isquantitatively related to the amount of light, over a fairlywide range, to which the leaf is exposed in the single precedinglight period.