Charles A. Leslie

Salicylic acid: A new inhibitor of ethylene biosynthesis.

Salicylic acid and acetylsalicylic acid at concentrations of 10−6M to 10−4M effectively inhibit ethylene production by pear cell suspension cultures. Results suggest these acids act by blocking the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene.

Improved efficiency of the walnut somatic embryo gene transfer system.

SummaryAnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5–6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.

Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut

The polyphenol oxidase enzyme functions in the metabolism of the amino acid tyrosine in walnut. The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

Silencing crown gall disease in walnut (Juglans regia L.)

Crown gall disease can severely limit the growth and productivity of many tree crops and ornamental plants. We have developed a novel crown gall disease resistance strategy that is based upon the silencing of two highly conserved Agrobacterium tumefaciens oncogenes which are required for crown gall development. The tryptophan monooxygenase (iaaM) and isopentenyl transferase (ipt) genes are horizontally transferred from A. tumefaciens to the plant cell, mediating the de novo auxin and cytokinin production that initiates tumorigenesis. By expressing inducers of post-transcriptional gene silencing homologous to iaaM and ipt in plant cells, the corresponding oncogene mRNA transcripts are degraded in planta, providing functional resistance to crown gall disease. This strategy is highly efficacious in English (Persian) walnut (Juglans regia L.), as several transformed lines expressing self-complementary iaaM and ipt transgenes display a highly specific accumulation of small-interfering RNAs and a broad-spectrum suppression of tumorigenesis. Potential applications and limitations of this technology in the future development of crown gall-resistant fruit and nut crop are also discussed.

High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos

Abstract A full-length synthetic version of the cry IA(c) gene, expressing a Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP), was transferred into walnut somatic embryos. Sixty one transgenic embryo lines or clones were obtained and bioassayed with first instar codling moth larvae. In 34% of these lines, designated as ‘class A’, expression was high enough to obtain 80–100% mortality. A total of 20% were designated ‘class B’, which produced a mortality between 25 and 70% and also caused a decreased rate of larval development. Insect mortality and development from the remaining 46% of the lines were indistinguishable from that of the control. Expression of a chimeric gene encoding β -glucuronidase (GUS) was evaluated to serve as a linked but unselected marker gene. About 62% of the class A embryo lines showed correspondingly high activity for the GUS gene. However, 38% of the class A would have been missed if the level of GUS activity was used as the sole indicator. Detectable levels of protein corresponding to cry IA(c) could be found only in class A, but not in class B or C clones. Southern analysis of border regions revealed single inserts for class A clones and multiple inserts for classes B and C.

Susceptibility of Walnut and Hickory Species to Geosmithia morbida

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Low levels of expression of wild type Bacillus thuringiensis var. kurstaki cryIA (c) sequences in transgenic walnut somatic embryos

Abstract Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis encoded by cry IA (c) and cry IA (b) were previously shown to be lethal to key target walnut insect pests (codling moth, navel orangeworm and Indianmeal moth). One of these genes, cry IA (c), was used to transform walnut somatic embryos using a binary vector (pWB139) in which this gene was expressed as a protein fusion with the kanamycin resistance gene from bacteria. Transgenic embryos representing individual transformation events were germinated to produce shoots that were maintained as micropropagated shoot lines in the laboratory. Transgenic shoots were then grafted onto seedling rootstocks and transplanted into the field. DNA analysis (Southern blotting) demonstrated that these shoots were transformed by pWB139 or by the control construct pWB149 (which does not contain the cry IA (c) gene). Insect feeding trials of walnut embryos grown in vitro and infested with neonatal Indianmeal moth, codling moth or navel orangeworm larvae showed non-significant mortality; confirming insufficient or incomplete expression of ICPs in the transgenic embryos. Therefore, transformation of somatic walnut embryos with the vector pWB139 was ineffective in protecting the embryos from damage by lepidopteran insect larvae.

Transformation of pecan and regeneration of transgenic plants

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of ‘Elliott’, ‘Wichita’, and ‘Schley’ were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line ‘Elliott’6, 3 from ‘Schley’5/3, and 3 from ‘Wichita’9. Transgenic embryos of ‘Wichita’9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Salicylic acid inhibition of ethylene production by apple discs and other plant tissues

Ethylene production by apple discs is effectively inhibited by salicylic acid. Inhibition is pH dependent, being greatest from pH 3.5–4.5 and minimal at pH 6.5 and above. With 100 μM salicylic acid maximal inhibition, approximately 90%, is achieved in 3 h with an apparent Ki of 40 μM. At somewhat higher concentrations salicylic acid also inhibits the conversion of 1-aminocyclo-propane-1-carboxylic acid to ethylene by pear discs and mung bean hypocotyls. Salicylic acid interferes with action of the putative ethylene-forming enzyme and in this respect is somewhat more effective than cobalt ion. The inhibitory effects of salicylic acid and cobalt ion are not additive. Implications for the limits and locus of salicylic acid inhibition are discussed.