Genetic improvement of pyrethrum (Tanacetum cinerariifolium Sch. Bip.) through gamma radiation and selection of high yield stable mutants through seven post-radiation generations.

PURPOSE: To increase the size of the flowers for easy plucking, flower yield, pyrethrins content (%), and elite mutant selection in pyrethrum. MATERIALS AND METHODS: To increase pyrethrum production and acclimatize in north Indian plain condition, a genetic improvement program was undertaken to widen the range of variations for size and yield of flowers and pyrethrins content (%) in pyrethrum crop. Pyrethrum seeds of the variety Avadh were irradiated with gamma rays at 20 to 300 Gy doses in Gamma chamber 5000 (cobalt-60 research irradiator). RESULTS: Observations gathered visually in M1 based on vigor, synchronization of flowering, and flower's size. Out of 90 M2 families, 20 mutants were raised in M3 along with the check-in preliminary evaluation trial. The four promising mutants, 1 (20 Gy-3), 7 (40 Gy-5), 10 (40 Gy-8), 14 (60 Gy19-10) was grown for four years in a bench-scale trial (randomized block design, replicated thrice) to test the yield performance and selection of high yielding elite mutant (s). It has been found that pyrethrum is sensitive to gamma rays irradiation and produced a high range of qualitative and quantitative variations. After massive screening over four years, two promising mutants for high dry flower yield and pyrethrins content, namely 7 (40 Gy-5), and 10 (40GY-8) were isolated. CONCLUSIONS: The mutagenesis changed traits mean in positive or negative directions. Pyrethrum plant is highly sensitive to gamma irradiation and produced a high range of variability in the qualitative and quantitative traits. The mutagenesis changed the mean of traits in both positive and negative directions. Due to mutagenic efficacy, two mutants 7 (40 Gy-5), and 10 (40GY-8) were expressed high performance for pyrethrin percent i.e., 87.23 and 59.78% improvement over the check variety 'Avadh', with synchronous flowering. These two mutants are in the pipeline for release as a variety for cultivation in the North Indian plains.

Coexpression Analysis Identifies Two Oxidoreductases Involved in the Biosynthesis of the Monoterpene Acid Moiety of Natural Pyrethrin Insecticides in Tanacetum cinerariifolium.

Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.

Progress and future of pyrethroids.

After the chemical structure of "natural pyrethrins," the insecticidal ingredient of pyrethrum flowers, was elucidated, useful synthetic pyrethroids provided with various characteristics have been developed by organic chemists throughout the world, leading to the advancement of pyrethroid chemistry. Even in pyrethroids with high selective toxicity, a chemical design placing too much importance on efficacy improvements may invite loss of the safety margin. It is strongly hoped that the development of household pyrethroids and their preparations for use in living environments around humans and pets will be achieved in the future by retaining the characteristics of natural pyrethrins.

Development of a cryopreservation procedure using aluminium cryo-plates.

A cryopreservation procedure using an aluminium cryo-plate was successfully developed using in vitro-grown Dalmatian chrysanthemum (Tanacetum cinerariifolium) shoot tips. Shoot cultures were cold-hardened at 5 degree C on MS medium containing 0.5 M sucrose over a period of 20 to 40 days. Shoot tips with basal plate (1.0-1.5 x 1.0 mm) were dissected from shoot cultures and precultured at 5 degree C for 2 days on MS medium containing 0.5 M sucrose. Precultured shoot tips were placed on aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 or 60 min in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 1.4 M sucrose). For dehydration, the loading solution was replaced with PVS 7M vitrification solution (30 percent glycerol, 19.5 percent ethylene glycol and 0.6 M sucrose in liquid MS basal medium), which was applied for 40 min. After rapid immersion in liquid nitrogen, shoot tips attached to the cryo-plates were rewarmed by immersion in cryotubes containing 2 ml 1 M sucrose solution. Using this procedure, regrowth of cryopreserved shoot tips of line 28v-75 reached 77 degree. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65 to 90 percent. By contrast, the modified vitrification protocol tested as a reference produced only moderate regrowth percentages. This new method displays many advantages and will facilitate large scale cryostorage in genebank.

Effect of delivery method and population size of Trichoderma harzianum on growth response of unrooted chrysanthemum cuttings.

In a previous study, addition of Trichoderma harzianum Rifai isolate T-12 to a propagative medium resulted in improved performance of chrysanthemum cuttings. However, root and shoot growth of one cultivar, 'Dark Bronze Charm', were more responsive to a lower (5 g T-12/kg medium) than higher (25 g T-12/kg medium) rate of fungal propagules, suggesting potential phytotoxicity at higher concentrations. The objectives of this study were to investigate higher rates of T-12 medium amendment for phytotoxicity, and to examine an alternative method of delivering the fungus to the propagative medium in order to obtain a more uniform response from cuttings. Isolate T-12 was added to the propagative medium as either a powdered peat-bran amendment (0, 5, or 50 g T-12/kg medium) or as alginate prills (80 or 800 g T-12/kg medium). There were no differences among treatments on day seven, but by day 21, shoot fresh weight and heights were significantly greater for plants treated with prills at 800 g T-12/kg medium. Both prill treatments resulted in greater shoot height on day 14 and 21 than all other treatments, which were similar to controls. Amendment with T-12 powder at 50 g/kg increased root length, but 80 g/kg medium added as prills decreased root dry weight compared to the control. The highest rate of T-12 (800 g prills/kg medium) had no effect on root growth. This suggests that moderate, rather than high rates of T-12 are more effective in promoting rooting of unrooted chrysanthemum, and that there is a potential for phytotoxic effects on root growth with higher rates.

Enhancement of plant stem growth by flocculation of the antibiotic-producing bacterium, Pseudomonas fluorescens S272, on the roots.

The antibiotic-producing bacterium, Pseudomonas fluorescens, is assumed to be important in protecting plants from soilborne diseases. S. fluorescens S272, a hyper-producing strain of pyoluteorin (PT) and 2,4-diacetylphloroglucinol (DG), had previously been isolated from soil. The present paper reported that the growth of water-cultivated Kaiware radish was promoted to 120-140% of its normal level by the coaddition of an S272 culture broth (0.01-1% v/v) and a polysaccharide flocculant (1-100 ppm) from Klebsiella pneumoniae H12. Tight adhesion of S272 cells to the root tissue was microscopically observed. The growth promotion is assumed to have been caused by antibiotic effects for the following two reasons: 1) PT (4 mg/l) and DG (24 mg/l) addition to a radish culture enhanced stem growth to 130% of the normal level; 2) a culture solution containing the S272 culture broth (0.01-1% v/v) markedly inhibited the decomposition of hypersensitive chrysanthemum leaves. A soil-cultivation experiment with Gomphrena globosa under natural conditions also exhibited enhanced stem length (160%) by coaddition of the S272 culture broth and H12 polysaccharide. These results suggest that polysaccharide-enhanced adhesion of P. fluorescens S272 cells might be useful for promoting plant growth through the increased antibiotic effect.

[A preliminary study on benthyloid wasp control for chrysanthemum stem borer].

The Chrysanthemum stem borer was parasitized by Benthyloid wasp. In the lab, the parasitic rate is 83.9%, and in the fields, 57.2%. The best ratio for the released Bethyloid wasp and the host is two to one.