The Role of a New Compound Micronutrient Multifunctional Fertilizer against Verticillium dahliae on Cotton

Verticillium dahliae Kleb., the causal pathogen of vascular wilt, can seriously reduce the yield and quality of many crops, including cotton (Gossypium hirsutum). To control the harm caused by V. dahliae, considering the environmental pollution of chemical fungicides and their residues, the strategy of plant nutrition regulation is becoming increasingly important as an eco-friendly method for disease control. A new compound micronutrient fertilizer (CMF) found in our previous study could reduce the damage of cotton Verticillium wilt and increase yield. However, there is little information about the mode of action of CMF to control this disease. In the present study, we evaluated the role of CMF against V. dahliae and its mechanism of action in vitro and in vivo. In the laboratory tests, we observed that CMF could inhibit hyphal growth, microsclerotia germination, and reduce sporulation of V. dahliae. Further studies revealed that the biomass of V. dahliae in the root and hypocotyl of cotton seedlings treated with CMF were significantly reduced compared with the control, and these results could explain the decline in the disease index of cotton Verticillium wilt. Furthermore, those key genes involved in the phenylpropanoid metabolism pathway, resistance-related genes defense, and nitric oxide signaling pathway were induced in cotton root and hypocotyl tissue when treated with CMF. These results suggest that CMF is a multifaceted micronutrient fertilizer with roles in inhibiting the growth, development, and pathogenicity of V. dahliae and promoting cotton growth.

In Vitro Regeneration of Coral Tree from Three Different Explants Using Thidiazuron

The coral tree (Erythrina variegata) is a multipurpose horticultural plant with a plethora of medicinally important alkaloids. Regeneration via tissue culture can provide an efficient alternative to seed-grown plantlets and reduce the cost of the plant significantly. Thidiazuron (TDZ) is an efficient plant growth regulator and is effective in numerous species. However, the response to it varies with the type and position of the tissue on the plantlet treated. This study was carried out to ascertain the best tissue types for micropropagation of the coral tree using TDZ. Three tissue types (shoot tip, nodal, and hypocotyl), originating from different strata of the plantlet were evaluated. Adventitious shoots were observed in all three explants at the tested concentrations. However the quality and the shoot number varied significantly with the type of explant. Explants with a meristematic zone (shoot tip and nodal) were more responsive to the treatment compared with hypocotyl tissue lacking preexisting meristem. Nodal explants produced the maximum number of shoots (about eight) per explant after 4 weeks of culture, whereas shoot tips produced about only five shoots per explant at an equimolar concentration (1.5 µm). Approximately three shoots were observed in hypocotyl explants. Moreover, growth and rooting of the regenerated shoots was influenced by the origin of the explants. The molecular characterization of the regenerants using intersimple sequence repeat (ISSR) markers revealed genetic homogeneity among regenerants. An efficient micropropagation method for the coral tree is described.

Quantitative proteomic analysis of auxin signaling during seedling development

Auxin induces rapid gene expression changes throughout plant development. How these transcriptional responses relate to changes in protein abundance is not well characterized. We have identified auxin regulated proteins in whole seedlings, roots and hypocotyls and at three different time points (30 min, 120 min and 3 hours) using an iTRAQ (isobaric tags for relative and absolute quantification) based quantitative proteomics approach. These profiling experiments detected 4,701 proteins from seedling tissue, 6,740 proteins from root tissue and 3,925 proteins from hypocotyl tissue. Comparisons between the differentially expressed proteins data sets showed little overlap, suggesting that auxin proteomes exhibit both temporal and spatial specificity. Numerous proteins showed significant changes in abundance following auxin treatment independent of changes in cognate transcript abundance. This includes several well characterized proteins with various roles in auxin pathways, suggesting that complex gene regulation mechanisms follow auxin signaling events. Specifically, regulation of translation may play a role as inferred from MapMan categorization analyses and protein interaction networks comprised of auxin regulated proteins. Additionally, functional categorization of auxin regulated proteins indicates rapid and complex metabolic changes occur in both roots and hypocotyls in response to auxin which are not apparent from transcriptome analyses. Altogether these data describe novel auxin-regulated proteomes and are an excellent resource for identifying new downstream signaling components related to auxin-mediated plant growth and development.

Embedding in Filter-Sterilized Alginate Enhances Brassica Oleracea L. Protoplast Culture

The influence of sodium alginate sterilization on the viability and mitotic activity of embedded protoplasts was studied in protoplasts of Brassica oleracea subsp. alba and rubra isolated from hypocotyl tissue and leaves of seedlings or plants grown in vitro. Both leaf and hypocotyl-derived protoplasts were more viable and divided more frequently when embedded in filtrated alginate. Division frequency was highest in cv. Reball F1 and the mitotic activity of its protoplasts was three times higher when embedded in filtrated alginate (36.1 ± 6.8%) than when cultured in autoclaved alginate (10.9 ± 5.0%). Protoplast-derived calli colonies were transferred to solid regeneration media and plants of all tested accessions were obtained.

047 Response to Selection for Anthocyanin Pigment Concentration in Wisconsin Fast Plant Populations by University of Wisconsin Students

Wisconsin Fast Plants are rapid-cycling versions of various Brassica species amenable to a variety of genetic studies due to their short life cycle and ease of handling. I have recently developed a model system using Brassica rapa L. Fast Plants for teaching the cyclical selection process known as recurrent selection in the context of a course on plant breeding. The system allows for up to three cycles of recurrent selection during a 15-week semester and enables students to gain experience in planting, selection, pollination, and seed harvest during each cycle. Fourteen cycles of replicated, recurrent mass selection for high (H) and low (L) levels of anthocyanin pigment expression in hypocotyl tissue were practiced by students in Horticulture 502 during a period of four semesters. In addition to bi-directional selection; replicated unselected (D) control populations were maintained forcomparative purposes. Over 14 cycles, highly significant gains and losses in hypocotyl pigment production were realized for H and L populations, respectively. Plants in D populations showed no directional response to random selection and therefore did not exhibit genetic drift. Plants in H populations exhibited production of anthocyanin pigment in organs other than hypocotyls, suggesting selection goals could be modified to include pigmentation of specific organs or whole plants. Results from this selection program suggest significant gains from recurrent selection can be visualized through student-based selection activities in the classroom.

Mechanical stimulation, 4-aminobutyric acid (GABA) synthesis, and growth inhibition in soybean hypocotyl tissue

4-Aminobutyric acid (GABA), a ubiquitous four-carbon nonprotein, amino acid, is synthesized via glutamate decarboxylation. It accumulates in response to mechanical stimulation through the Ca2+/calmodulin activation of glutamate decarboxylase. In this study, dark- and light-grown soybean hypocotyl tissues were used to investigate the relationship between mechanical stimulation, GABA synthesis, and growth inhibition. Mechanical stimulation through stroking resulted in a 65% growth inhibition of dark- or light-grown tissue within 1 min. The 24-h growth increment was reduced by 50% or more. Growth inhibition was accompanied by rapid 4- and 10-fold increases in GABA levels in dark- and light-grown tissue, respectively. Treatment of tissue with exogenous GABA raised GABA levels approximately 6-fold, but did not inhibit growth. In addition, lanthanum or blue light treatment of dark-grown tissue reduced growth by over 60% with no increase in GABA levels. The data demonstrate for the first time, that GABA accumulation accompanies growth inhibition occurring in response to mechanical stimulation. However, GABA accumulation alone appears insufficient to cause growth inhibition.Key words: 4-aminobutyric acid, GABA, L-Glu decarboxylase, GAD, thigmomorphogenesis, mechanical stimulation.

Optimizing the Calcium Content of a Copolymer Acrylamide Gel Matrix for Dark-grown Seedlings

A copolymer acrylamide acrylate gel was investigated as the sole root matrix for dark-grown seedlings of soybean (Glycine max Merr. `Century 84'). Increasing Ca2+ in the hydrating solution of the hydrogel from 1 to 10 mm decreased its water-holding capacity from 97 to 46 mL·g-1, yet water potential of the medium remained high, sufficient for normal plant growth at all Ca2+ concentrations tested. Elongation rate of dark-grown soybean seedlings over a 54-hour period was 0.9, 1.5, and 1.8 mm·h-1 with 1.0, 2.5, or 5.0 mm Ca2+, respectively, but did not increase with further increases in Ca2+ concentration. Further study revealed that Na+ was released from the hydrogel medium and was taken up by the seedlings as Ca2+ increased in the medium. In dry hypocotyl tissue, sodium content correlated negatively with calcium content. Despite the presence of Na+ in the hydrogel, seedling growth was normal when adequate Ca2+ was added in the hydrating solution. Acrylamide hydrogels hold good potential as a sole growth matrix for short-term experiments with dark-grown seedlings without irrigation.

Transformation of Carrot (Daucus carota L.) with Genes Involved in Carbohydrate Metabolism and Partitioning via Hypocotyl Tissue Cultures

A study was initiated to characterize key enzymes that influence sweetness in carrot (Daucus carota L.) roots. Sucrose synthase (SS), sucrose phosphate synthase (SPS), and UDP-glucose pyrophosphorylase (UDPL) genes were isolated from potato (Solanum tuberosum L.) and cloned in an anti-sense orientation into Agrobacterium tumefaciens Bin19, which has a CaMV 35S promoter. Seedling hypocotyl sections of selected carrot lines were pre-incubated on B5 medium for 2 days, co-cultivated with A. tumefaciens Bin 19 for additional 3 days, and then transferred to a modified B5 medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin. In 4 weeks, 18.6%, 33.3%, and 26.7% of the cultures from a breeding line (W204-C) were found to be transformed, respectively, with SS, SPS, and UDPL as determined by kanamycin resistance. In contrast, no kanamycin-resistant calli were obtained from a commercial cultivar (Navajo) in these transformation studies. The transformed calli proliferated in the medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin, whereas non-transformed calli died in the same medium. These transformed calli are currently being used to regenerate plants via asexual embryogenesis using a suspension culture. The influence of these additional genes on sugar metabolism and accumulation in root tissues of transformed carrots will be characterized in the future.