Sherry L. Kitto

Microbe-Associated Molecular Patterns-Triggered Root Responses Mediate Beneficial Rhizobacterial Recruitment in Arabidopsis

This study demonstrated that foliar infection by Pseudomonas syringae pv tomato DC3000 induced malic acid (MA) transporter (ALUMINUM-ACTIVATED MALATE TRANSPORTER1 [ALMT1]) expression leading to increased MA titers in the rhizosphere of Arabidopsis (Arabidopsis thaliana). MA secretion in the rhizosphere increased beneficial rhizobacteria Bacillus subtilis FB17 (hereafter FB17) titers causing an induced systemic resistance response in plants against P. syringae pv tomato DC3000. Having shown that a live pathogen could induce an intraplant signal from shoot-to-root to recruit FB17 belowground, we hypothesized that pathogen-derived microbe-associated molecular patterns (MAMPs) may relay a similar response specific to FB17 recruitment. The involvement of MAMPs in triggering plant innate immune response is well studied in the plant’s response against foliar pathogens. In contrast, MAMPs-elicited plant responses on the roots and the belowground microbial community are not well understood. It is known that pathogen-derived MAMPs suppress the root immune responses, which may facilitate pathogenicity. Plants subjected to known MAMPs such as a flagellar peptide, flagellin22 (flg22), and a pathogen-derived phytotoxin, coronatine (COR), induced a shoot-to-root signal regulating ALMT1 for recruitment of FB17. Micrografts using either a COR-insensitive mutant (coi1) or a flagellin-insensitive mutant (fls2) as the scion and ALMT1pro:β-glucuronidase as the rootstock revealed that both COR and flg22 are required for a graft transmissible signal to recruit FB17 belowground. The data suggest that MAMPs-induced signaling to regulate ALMT1 is salicylic acid and JASMONIC ACID RESISTANT1 (JAR1)/JASMONATE INSENSITIVE1 (JIN1)/MYC2 independent. Interestingly, a cell culture filtrate of FB17 suppressed flg22-induced MAMPs-activated root defense responses, which are similar to suppression of COR-mediated MAMPs-activated root defense, revealing a diffusible bacterial component that may regulate plant immune responses. Further analysis showed that the biofilm formation in B. subtilis negates suppression of MAMPs-activated defense responses in roots. Moreover, B. subtilis suppression of MAMPs-activated root defense does require JAR1/JIN1/MYC2. The ability of FB17 to block the MAMPs-elicited signaling pathways related to antibiosis reflects a strategy adapted by FB17 for efficient root colonization. These experiments demonstrate a remarkable strategy adapted by beneficial rhizobacteria to suppress a host defense response, which may facilitate rhizobacterial colonization and host-mutualistic association.

Regeneration of peppermint and orange mint from leaf disks

Leaf disks from peppermint, spearmint, orange mint, lavender mint and Scotch spearmint were cultured on various Murashige-Skoog-based media in order to regenerate shoots. A significantly larger average number of orange mint leaf disks regenerated shoots on basal medium containing 44.4 μM benzyladenine (BA) and 250 ml l-1 coconut water (CW). Shoots regenerated from peppermint leaf disks cultured on basal medium containing 44.4 μM BA and 250 ml or 450 ml l-1 CW. The most shoots regenerated from orange mint leaf disks cultured on medium containing 10 g l-1 washed Difco Bacto-agar. Disks excised from the bases of the first expanding pair of orange mint leaves cultured under dark conditions regenerated a significantly larger average number of shoots. Histological studies suggested that shoots regenerated from the palisade parenchyma cells associated with vascular tissue.

Mechanical purification of torpedo stage somatic embryos of Daucus carota L.

A filtering unit was designed and constructed that concentrates and purifies torpedo-stage embryos, from carrot (Daucus carota L.) embryo suspensions. The filtering unit was composed of three sections, each containing a screen disk. Purification efficiency of a series of three screen size combinations was evaluated using 10 to 15 ml of 23- to 30-day-old embryo suspensions. Under optimal conditions where the first screen was a 1000 micron in size, >84% of the total number of torpedo embryos were concentrated in the purified fraction. Purified fractions contained more than double the percentage of torpedo embryos when compared to unfiltered suspensions. This filtering unit provided a simple method for concentrating and purifying torpedo embryos from a suspension.

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.

Fluid drilling as a delivery system for somatic embryo-derived plantlets of carrot (Daucus carota L.)☆

Abstract Somatic embryos and/or somatic embryo-derived plantlets (SEPs) of carrot cultivar ‘Orlando Gold’ were subjected to various treatments during either suspension culture or a subsequent incubation period in fluid-drilling gel. Conversion of SEPs in the glasshouse into plants containing primary leaves was greatest when SEPs were incubated for 1–2 weeks in hydroxyethyl cellulose fluid-drilling gel (1.67%, w/v of N-gel™) hydrated with a solution containing Murashige and Skoog salts and vitamins and 2% (w/v) sucrose. Providing 3 days of chilling at 4°C then 3 days at 25°C all under light (60 μmol m−2 s−1) during suspension culture (at the globular to torpedo stage) led to the greatest SEP growth following fluid drilling. Inclusion of either 250 mg TrubanR fungicide or 10 mg chitosan glutamate per litre of gel improved SEP conversion.

Fluid Drilling as a Delivery System for Somatic Embryo-Derived Plantlets

Four systems have been proposed to facilitate the bulk handling and delivery of in vitro-derived somatic embryos: fluid drilling (Drew 1979), encapsulation of somatic embryos in alginate gel (Redenbaugh et al. 1986), desiccation of somatic embryos (Gray et al. 1987), or desiccation of somatic embryos encapsulated in water-soluble resin (Kitto and Janick 1985).