Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium

The development of a more efficient in vitro regeneration system for somatic embryos (SEs) of avocado (Persea americana) would facilitate the development of new superior cultivars for this valuable horticultural crop. In this study, we report a new and efficient method for maintenance and regeneration of avocado SEs. Avocado SEs of four cultivars remained healthy and viable in vitro for 11 months on a medium used for mango somatic embryogenesis, compared with 3-4 months on Murashige and Skoog medium. Various supplements and media modifications were investigated to improve the low conversion rate of regenerated plants from avocado SEs reported previously. The one-step system for regeneration of white-opaque somatic embryos (WOSEs) used solid medium only over a period of 12-14 weeks (sub-culturing every 6 weeks). Addition of praline and glutamine improved the total regeneration from 0 to 17.5% and 10.5%, and plant/shoot recovery from 0 to 12.5% and 5%, respectively. A two-step culture system involving the transfer of WOSEs of cultivar 'Reed' after 6 weeks on solid to liquid medium for 12-15 days as an intermediate step, followed by subculturing again onto solid medium for 6 weeks improved total regeneration to 29% and plant/shoot recovery to 18.3 from 0% when regenerated by subculturing on solid medium only. Supplementation with proline in the solid as well as liquid medium in the two-step culture system at 0.4 g/L increased total regeneration to 35% and plant/shoot recovery to 20%. We were able to achieve highest regeneration using glutamine at 1 g/L in the two-step culture system in terms of both total regeneration (58.3%, including 43.3% bipolar regeneration) and plant/shoot recovery (36.7%) rates, which were significantly higher than in any other treatment investigated. (C) 2013 Elsevier B.V. All rights reserved.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

A new culture method for lesser mealworm, Alphitobius diaperinus

A new culture method for lesser mealworm, Alphitobius diaperinus (Panzer), was developed to provide large numbers of adult lesser mealworms of approximately the same age for insecticide resistance testing. Culturing entailed allowing 100 adults to reproduce for 4 days in a wheat-based culture medium contained inside a plastic culture box, removing the adults from the medium, and then rearing their progeny to adulthood therein, in approximately 56 days at 32 degrees C and 55% RH. During their development, progeny were supplied water via apple slices at 0, 21 and 35 days, and a foam substrate in which to pupate, also at 35 days. During 2004-2005, adult lesser mealworms were collected from six broiler-house populations and then cultured with this method. Each population produced 4500 adults required to complete resistance testing with one insecticide within ten culture boxes, at an average of 798 adults per culture box.

In Vitro Conditions for a Successful Biolistics transformation System of Pineapples (ananas comosus merr.), in 'Contributing to a Sustainable Future'

In order to develop an efficient and reliable biolistics transformation system for pineapples parameters need to be optimised for growth, survival and development of explants pre- and post transformation. We have optimised in vitro conditions for culture media for the various stages of plant and callus initiation and development, and for effective selection of putative transgenic material. Shoot multiplication and proliferation is best on medium containing MS basic nutrients and vitamins with the addition of 0.1 mg/L myo-inositol, 20 g/L sucrose, 2.5 mg/L BAP and 3 g/L Phytagel, followed by transfer to basic MS medium for further development. Callus production on leaf base explants is best on MS nutrients and vitamins, to which 10 mg/L of BAP and NAA each was added. Optimum explant age for bombardment is 17-35 week old callus, while a pre-bombardment osmoticum treatment in the medium is not required. By comparing several antibiotics as selective agent, it has been established that a two-step selection of 2 fortnightly sub-cultures on 50 μg/mL of geneticin in the culture medium, followed by monthly sub-cultures on 100 μg/mL geneticin is optimal for survival of transgenic callus. Shoot regeneration from callus cultures is optimal on medium containing MS nutrients and vitamins, 5% coconut water and 400 mg/L casein hydrolysate. Plants can be readily regenerated and multiplied from transgenic callus through organogenesis. Rooting of shoots does not require any additional plant hormones to the medium. A transformation efficiency of 1 – 3.5% can be achieved, depending on the gene construct applied.

Optimising indoor phosphine fumigation of paddy rice bag-stacks under sheeting for control of resistant insects

Three indoor, sheeted bag-stack fumigations of paddy rice using aluminium phosphide were undertaken in Guangdong Province, southern China. We measured the effect of two types of sheeting (polyvinylchloride [PVC] or polyethylene [PE]) and two types of floor sealing (clips or fixing into a slot with a rubber pipe) on phosphine concentration and retention. The aim was to test the feasibility of retaining fumigant at a sufficient concentration for long enough to control known resistant insect pests. Each stack was pressure tested and phosphine concentrations measured daily during the fumigation. Cages of test insects in culture medium, including resistant and susceptible strains, were placed inside each stack and could be observed through the clear sheeting. Highest concentrations for the longest period were obtained in a PVC-covered stack that included a ground sheet and wall sheets sealed to the floor with rubber pipes. A similar PVC-covered stack sealed to the floor with clips instead of pipe did not retain gas as efficiently and required re-dosing. A PE-covered stack, with no ground sheet but also with wall sheets sealed to the floor with pipe, produced an acceptable fumigation. Susceptible Rhyzopertha dominica were controlled in 2 days and the most resistant strain in 15 days. Resistant Cryptolestes ferrugineus survived until day 21. The paddy was still free of insect infestation 7 months later when the bag-stack was opened to mill the rice. Pressure half-lives correlated with gas concentration and retention. Sorption appeared to be a major limiting factor, reducing potential fumigant dosage by about 50%. The trials demonstrated the feasibility of sealing bag-stacks to a standard high enough to control all known resistant strains.

Alkaloid production by isolates of the sorghum ergot pathogen (Claviceps africana) from Australia and other countries

Isolates of Claviceps africana from Australia, Africa, Asia, and America were tested for the production of dihydroergosine (DHES), and its biogenic precursors dihydroelymoclavine (DHEL) and festuclavine (FEST), in culture. Several growth media were evaluated to optimise alkaloid production with little success. The best of these involved 2-stage culturing on high-sucrose substrate. Australian C. africana isolates varied widely and inconsistently in alkaloid production, with DHES concentrations in mycelium ranging from: <0.1 to 9 mg DHES/kg; <0.1 to 1.6 mg DHEL/kg; and <0.1 to 0.4 mg FEST/kg. In a separate experiment using similar culturing techniques, DHES was produced by 2 of 3 Australian isolates, 1 of 3 USA isolates, 1 of 4 Indian isolates, the sole Puerto Rican isolate, the sole Japanese isolate, but not the sole South African isolate. In this experiment, DHES concentrations detected in mycelium of Australian isolates (0.1-1.0 mg DHES/kg) were of similar magnitude to isolates from other countries (0.2-1.8 mg DHES/kg). Three C. africana isolates, including one that produced only traces of alkaloid in culture after 8 weeks, were inoculated onto panicles of sterile male sorghum plants. After 8 weeks, all 3 isolates produced 10-19 mg DHES/kg in the panicles, demonstrating that the growing plant favoured more consistent alkaloid production than culture medium.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Confirmed case of encephalitis caused by Murray Valley encephalitis virus infection in a horse

A 5-year-old Australian stock horse in Monto, Queensland, Australia, developed neurological signs and was euthanized after a 6-day course of illness. Histological examination of the brain and spinal cord revealed moderate to severe subacute, nonsuppurative encephalomyelitis. Sections of spinal cord stained positively in immunohistochemistry with a flavivirus-specific monoclonal antibody. Reverse transcription polymerase chain reaction assay targeting the envelope gene of flavivirus yielded positive results from brain, spinal cord, cerebrospinal fluid, and facial nerve. A flavivirus was isolated from the cerebrum and spinal cord. Nucleotide sequences obtained from amplicons from both tissues and virus isolated in cell culture were compared with those in GenBank and had 96-98% identity with Murray Valley encephalitis virus. The partial envelope gene sequence of the viral isolate clustered into genotype 1 and was most closely related to a previous Queensland isolate.

An improved simulation model to predict pre-harvest aflatoxin risk in maize

Aflatoxin is a potent carcinogen produced by Aspergillus flavus, which frequently contaminates maize (Zea mays L.) in the field between 40° north and 40° south latitudes. A mechanistic model to predict risk of pre-harvest contamination could assist in management of this very harmful mycotoxin. In this study we describe an aflatoxin risk prediction model which is integrated with the Agricultural Production Systems Simulator (APSIM) modelling framework. The model computes a temperature function for A. flavus growth and aflatoxin production using a set of three cardinal temperatures determined in the laboratory using culture medium and intact grains. These cardinal temperatures were 11.5 °C as base, 32.5 °C as optimum and 42.5 °C as maximum. The model used a low (≤0.2) crop water supply to demand ratio—an index of drought during the grain filling stage to simulate maize crop's susceptibility to A. flavus growth and aflatoxin production. When this low threshold of the index was reached the model converted the temperature function into an aflatoxin risk index (ARI) to represent the risk of aflatoxin contamination. The model was applied to simulate ARI for two commercial maize hybrids, H513 and H614D, grown in five multi-location field trials in Kenya using site specific agronomy, weather and soil parameters. The observed mean aflatoxin contamination in these trials varied from <1 to 7143 ppb. ARI simulated by the model explained 99% of the variation (p ≤ 0.001) in a linear relationship with the mean observed aflatoxin contamination. The strong relationship between ARI and aflatoxin contamination suggests that the model could be applied to map risk prone areas and to monitor in-season risk for genotypes and soils parameterized for APSIM.

An improved simulation model to predict pre-harvest aflatoxin risk in maize

Aflatoxin is a potent carcinogen produced by Aspergillus flavus, which frequently contaminates maize (Zea mays L.) in the field between 40° north and 40° south latitudes. A mechanistic model to predict risk of pre-harvest contamination could assist in management of this very harmful mycotoxin. In this study we describe an aflatoxin risk prediction model which is integrated with the Agricultural Production Systems Simulator (APSIM) modelling framework. The model computes a temperature function for A. flavus growth and aflatoxin production using a set of three cardinal temperatures determined in the laboratory using culture medium and intact grains. These cardinal temperatures were 11.5 °C as base, 32.5 °C as optimum and 42.5 °C as maximum. The model used a low (≤0.2) crop water supply to demand ratio—an index of drought during the grain filling stage to simulate maize crop's susceptibility to A. flavus growth and aflatoxin production. When this low threshold of the index was reached the model converted the temperature function into an aflatoxin risk index (ARI) to represent the risk of aflatoxin contamination. The model was applied to simulate ARI for two commercial maize hybrids, H513 and H614D, grown in five multi-location field trials in Kenya using site specific agronomy, weather and soil parameters. The observed mean aflatoxin contamination in these trials varied from <1 to 7143 ppb. ARI simulated by the model explained 99% of the variation (p ≤ 0.001) in a linear relationship with the mean observed aflatoxin contamination. The strong relationship between ARI and aflatoxin contamination suggests that the model could be applied to map risk prone areas and to monitor in-season risk for genotypes and soils parameterized for APSIM.