David J. Wolyn

Activation of defense responses to Fusarium infection in Asparagus densiflorus

Defense responses to Fusarium oxysporum f. sp. asparagi and F. proliferatum were compared after root inoculation of the asparagus fern, Asparagus densiflorus vars. Myersii and Sprengeri, and cultivated asparagus, A. officinalis cv. Guelph Millennium. Both varieties of A. densiflorus exhibited a hypersensitive response with rapid death of epidermal cells within 8–24 h and restricted the fungal growth. In A. officinalis roots, rapid cell death was not found, and necrotic lesions were observed 8–14 d after fungal inoculation. Peroxidase and phenylalanine ammonia-lyase activities increased significantly in inoculated A. densiflorus but not A. officinalis plants. Local and systemic induction of peroxidase activity was detected after pathogen inoculation in root and spear tissues, respectively, of A. densiflorus. POX activity decreased in roots of inoculated A. officinalis by 8 d post-inoculation. Germination and germ tube growth were inhibited when spores of F. oxysporum f. sp. asparagi were incubated in root exudates and on root segment surfaces of inoculated A. densiflorus plants exhibiting hypersensitive cell death. Spore germination of F. proliferatum and three fungi non-pathogenic to cultivated asparagus was inhibited as well. Rapid induction of hypersensitive cell death in A. densiflorus was associated with restriction of fungal growth, and activation of peroxidase and phenylalanine ammonia-lyase, two defense enzymes thought to be important for plant disease resistance.

High frequency production of haploid embryos in asparagus anther culture.

SummaryA method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l−1 casein hydrolysate, 800 mg l−1 glutamine, 2 mg l−1 NAA, 1 mg l−1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l−1 NAA, 0.1 mg l−1 kinetin and 0.65 mg l−1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l−1 GA3. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.

A mitochondrial plasmid and plasmid-like RNA and DNA polymerases encoded within the mitochondrial genome of carrot (Daucus carota L.)

The mitochondrial genome of mitochondrial type (mitotype) SW3 of carrot (Daucus carota L.) encodes intact reading frames for a RNA polymerase (Rpo) and a DNA polymerase (Dpo) similar to those encoded by linear mitochondrial plasmids from plants. A BLAST search of translated nucleotide sequences in GenBank revealed previously unreported plasmid-like Rpo or Dpo sequence fragments in many plant mitochondrial DNAs. Phylogenetic analyses of the relationships between mitochondrial (mt)DNA-encoded and plasmid-encoded Rpos and Dpos from plants suggest that the mitochondrial sequences were derived from integrated plant plasmid sequences. A linear mitochondrial plasmid was detected in a different mitotype (FG21) of carrot by Southern hybridization of the Rpo and Dpo to undigested mtDNAs. Transcripts of the mtDNA-encoded Rpo and Dpo in mitotype SW3 were detected by RT-PCR.

The effects of ancymidol, abscisic acid, uniconazole and paclobutrazol on somatic embryogenesis of asparagus

SummaryThe effects of ancymidol, abscisic acid (ABA), uniconazole, and paclobutrazol on asparagus somatic embryogenesis were evaluated. Calli induced from seedlings of genotype G447 were transferred to embryo induction medium (MS plus 3% sucrose, 0.1 mg L−1 NAA, 0.5 mg L−1 kinetin and 3% gelrite), with different concentrations of these compounds. After 8 weeks, the recovered bipolar or globular embryos were placed on germination medium (MS plus 6% sucrose, 0.1 mg L−1 NAA, 0.1 mg L−1 kinetin, 0.75 mg L−1 ancymidol, 40 mg L−1 adenine sulphate dihydrate, 0.17 mg L−1 sodium phosphate monobasic and 3% gelrite) for conversion to plantlets. Inclusion of ancymidol, ABA, uniconazole and paclobutrazol in the embryo induction medium did not affect the total number of somatic embryos produced relative to the control without these compounds. However, ancymidol, ABA and uniconazole significantly improved embryo development by increasing the production of bipolar embryos 250–750% and decreasing that of globular embryos 8–35% relative to the control. The bipolar embryos produced with any of the four compounds in the embryo induction medium converted to plantlets at rates 700–1100% greater than the control. None of the globular embryos converted to plantlets. Ancymidol (0.75 mg L−1) and ABA (0.05 mg L−1) were the most effective treatments; 61 and 46 bipolar embryos g−1 callus were produced, and 38% and 37% of the bipolar embryos converted to plantlets, respectively. These results indicated that ancymidol, ABA, uniconazole and paclobutrazol significantly enhanced the production of asparagus somatic embryos and their conversion to plantlets, and ancymidol and ABA were more effective than uniconazole and paclobutrazol.

Antisense Expression Of Mitochondrial ATP Synthase Subunits OSCP (ATP5) And γ (ATP3) Alters Leaf Morphology, Metabolism And Gene Expression In Arabidopsis.

Determination of the role of mitochondrial (mt) ATP synthesis in plant metabolism is complicated by chloroplastic ATP synthesis. To differentiate ATP synthesis from these two organelles, we created transgenic Arabidopsis plants in which two different subunits of the mt ATP synthase, the oligomycin sensitivity-conferring protein (OSCP) (=delta) (ATP5) and the gamma (ATP3) subunit, were expressed individually in antisense orientation under the control of a dexamethasone-inducible promoter. The phenotypic effects of antisense expression were identical for both atp5 and atp3. Seedling lethality resulted from induction during germination in the light, demonstrating the essentiality of both gene products. Reduced expression of either gene resulted in stunting of dark-grown (etiolated) seedlings, downward curling or wavy-edged leaf margins of light-grown plants and ball-shaped unexpanded flowers. Antisense induction reduced total ATP levels in dark-grown (etiolated) seedlings germinated on media lacking sucrose, but increased total ATP levels in induced light-grown plants and in induced dark-grown seedlings germinated on media containing sucrose. Induction reduced transcript levels for two transcription factors (TCP3 and TCP4) whose decreased expression is associated with a similar wavy-edged leaf phenotype in Arabidopsis, and increased transcript levels for dynamin-related proteins whose increased expression is associated with increased mt division. Reduced expression of these subunits of the mt ATP synthase is proposed to disturb cellular redox states, which ultimately manifest downstream as diverse and seemingly unrelated phenotypes.

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

Abstract A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line.

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

Abstract To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l–1α-naphthaleneacetic acid and 1 mg l–1 N6-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid.

Interactions of ancymidol with sucrose and α-naphthaleneacetic acid in promoting asparagus (Asparagus officinalis L.) somatic embryogenesis

Abstract Interactions of varying ancymidol concentrations with those of α-naphthaleneacetic acid (NAA) or sucrose in embryo induction medium were related to the production and development of asparagus (Asparagus officinalis L.) somatic embryos, and to the ability of these embryos to germinate. A significant sucrose×ancymidol interaction was observed only for the production of bipolar embryos; 4% sucrose with 0.75 mg l–1 ancymidol gave the best result, 78 g–1 callus. The frequency of globular embryos decreased as sucrose or ancymidol concentrations increased. Sucrose concentration affected embryo germination; 3% and 4% sucrose were optimal with approximately 60% and 40% of bipolar and globular embryos germinating, respectively. Significant ancymidol×NAA interactions were observed for the production of bipolar and globular embryos and their germination. Varying ancymidol concentrations affected embryo production and germination in combination with 0.1 mg l–1 NAA, but not with 1.0 mg l–1 NAA. The treatment combination of 0.1 mg l–1 NAA with 0.75 mg l–1 ancymidol produced the most bipolar embryos, 64 g–1 callus, and the greatest percentages of bipolar and globular embryos germinated, 63% and 42%, respectively.

Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhanced by ancymidol

SummaryThe effect of ancymidol concentration on the development of haploid asparagus embryos was determined. Liquid cultures from anther-derived calli were grown for three weeks in MS medium plus 1.0 mg l−1 2,4-D, 0.1 mg l−1 NAA, 0.2 mg l−1 kinetin, 800 mg l−1 glutamine, 500 mg l−1 casein hydrolysate, 2% sucrose and 0.0–1.0 mg l−1 ancymidol. Cell clumps (224–500 μm) were plated on solid embryo maturation medium (MS medium plus 3% sucrose, 0.1 mg l−1 NAA, 0.5 mg l−1 kinetin and 0.0–1.0 mg l−1 ancymidol) and grown for eight weeks. Ancymidol enhanced embryo maturation and germination and was more critical in the solid than liquid medium. Total embryo number did not vary among most treatments. The best response was observed when ancymidol concentrations were 0.1 and 0.5 mg l−1 in the liquid and solid media, respectively; two-thirds of the embryos produced were bipolar and 35% of bipolar embryos germinated. Seven to 82% of plants recovered from different ancymidol treatments were haploid; the others were diploid, triploid or chimeric for ploidy level.

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

SummaryThe potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.