A guide to Agrobacterium binary Ti vectors

Agrobacterium-based plasmid vectors allow the transformation of a wide range of plant species by capitalizing on a natural bacterial system to introduce DNA into the nuclear genome of plants. It is often a complex task to consider fully all the possible plasmid vectors and Agrobacterium strains available, and it can thus be difficult to take full advantage of these research tools. This practical guide is a survey of the many binary Ti plasmid vectors and Agrobacterium strains available, and aims to help researchers to make an informed decision about the system that is best suited to their needs...

Mendel, 150 years on

Mendel's paper 'Versuche über Pflanzen-Hybriden' is the best known in a series of studies published in the late 18th and 19th centuries that built our understanding of the mechanism of inheritance. Mendel investigated the segregation of seven gene characters of pea (Pisum sativum), of which four have been identified. Here, we review what is known about the molecular nature of these genes, which encode enzymes (R and Le), a biochemical regulator (I) and a transcription factor (A). The mutations are: a transposon insertion (r), an amino acid insertion (i), a splice variant (a) and a missense mutation (le-1). The nature of the three remaining uncharacterized characters (green versus yellow pods, inflated versus constricted pods, and axial versus terminal flowers) is discussed.

MYB transcription factors that colour our fruit

Anthocyanin concentration is a primary determinant of plant colour. Fruit anthocyanin biosynthesis is controlled by a distinct clade of R2R3 MYB transcription factors. In apple, three recent papers describe the discovery of MYB genes activating skin, flesh and foliage anthocyanic colour. These findings lead the way to new approaches in the breeding and biotechnological development of fruit with new colour patterns.

Optimising ketocarotenoid production in potato tubers : effect of genetic background, transgene combinations and environment

Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits,as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring.

Triploid plants from endosperm cultures of sandalwood by experimental embryogenesis

Embryogenesis has been induced from endosperm callus cultures of sandalwood (Santalum album L.). Viable plantlets developed from the embryoids on subculture to White's basal medium supplemented with 0.5 mg/l of indole acetic acid. Chromosomal analysis of the root tips showed the triploid number 3n = 30.

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

Discovery of genes associated with fruit ripening in arica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded:chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Progress towards the eradication of mikania vine (Mikania micrantha) and limnocharis (Limnocharis flava) in northern Australia.

To eradicate a weed invasion, its extent must be delimited and each infestation must be extirpated. Measures for both of these criteria are utilized to assess the progress of current eradication programs targeting mikania vine and limnocharis in northern Australia. The known infested area for each species is less than 5 ha and has remained largely static for the last 3 or more years against a backdrop of refined and enhanced detection methods. This suggests that delimitation has been approached, if not achieved. Different methods of detection have their places, relative to the stage of the program and the spatial distribution of infestations. Although all known infestations of both species are effectively monitored and controlled, ongoing emergence from persistent seed banks limits progress towards the extirpation of infestations to a slow, but measurable, rate. Nomenclature: Glyphosate. N-phosphonomethyl)glycine; fluroxypyr, [(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl)oxy]acetic acid; limnocharis, Limnocharis flava (L.) Buchenau LIFL5; mikania vine (mile-a-minute), Mikania micrantha Kunth MIKMI.

Lantana (Lantana camara) Seed Bank Dynamics: Seedling Emergence and Seed Survival.

Seed persistence is poorly quantified for invasive plants of subtropical and tropical environments and Lantana camara, one of the world's worst weeds, is no exception. We investigated germination, seedling emergence, and seed survival of two lantana biotypes (Pink and pink-edged red [PER]) in southeastern Queensland, Australia. Controlled experiments were undertaken in 2002 and repeated in 2004, with treatments comprising two differing environmental regimes (irrigated and natural rainfall) and sowing depths (0 and 2 cm). Seed survival and seedling emergence were significantly affected by all factors (time, biotype, environment, sowing depth, and cohort) (P < 0.001). Seed dormancy varied with treatment (environment, sowing depth, biotype, and cohort) (P < 0.001), but declined rapidly after 6 mo. Significant differential responses by the two biotypes to sowing depth and environment were detected for both seed survival and seedling emergence (P < 0.001). Seed mass was consistently lower in the PER biotype at the population level (P < 0.001), but this variation did not adequately explain the differential responses. Moreover, under natural rainfall the magnitude of the biotype effect was unlikely to result in ecologically significant differences. Seed survival after 36 mo under natural rainfall ranged from 6.8 to 21.3%. Best fit regression analysis of the decline in seed survival over time yielded a five-parameter exponential decay model with a lower asymptote approaching −0.38 (% seed survival = [( 55 − (−0.38)) • e (k • t)] + −0.38; R2 = 88.5%; 9 df). Environmental conditions and burial affected the slope parameter or k value significantly (P < 0.01). Seed survival projections from the model were greatest for buried seeds under natural rainfall (11 yr) and least under irrigation (3 yr). Experimental data and model projections suggest that lantana has a persistent seed bank and this should be considered in management programs, particularly those aimed at eradication.

Weed eradication - an economic perspective

There has been recent interest in determining the upper limits to the feasibility of weed eradication. Although a number of disparate factors determine the success of an eradication program, ultimately eradication feasibility must be viewed in the context of the amount of investment that can be made. The latter should reflect the hazard posed by an invasion, with greater investment justified by greater threats. In simplest terms, the effort (and hence investment) to achieve weed eradication comprises the detection effort required to delimit an invasion plus the search and control effort required to prevent reproduction until extirpation occurs over the entire infested area. The difficulty of estimating the required investment at the commencement of a weed eradication program (as well as during periodic reviews) is a serious problem. Bioeconomics show promise in determining the optimal approach to managing weed invasions, notwithstanding ongoing difficulties in estimating the costs and benefits of eradication and alternative invasion management strategies. A flexible approach to the management of weed invasions is needed, allowing for the adoption of another strategy when it becomes clear that the probability of eradication is low, owing to resourcing or intractable technical issues. Whether the considerable progress that has been achieved towards eradication of the once massive witchweed invasion can be duplicated for other weeds of agricultural systems will depend to a large extent upon investment (. $250 million over 50 yr in this instance). Weeds of natural ecosystems seem destined to remain more difficult eradication targets for a variety of reasons, including higher impedance to eradication, more difficulty in valuing the benefits arising from eradication, and possibly less willingness to pay from society at large.

Comparative seed and dispersal ecology of three exotic subtropical Asparagus species

The genus Asparagus includes at least six invasive species in Australia. Asparagus aethiopicus and A. africanus are invasive in subtropical Australia, and a third species, A. virgatus is naturalized and demonstrates localized spread in south east Queensland. To better understand how the attributes of these species contribute to their invasiveness, we compared fruit and seed traits, germination, seedling emergence, seed survival, and time-to-maturity. We further investigated dispersal ecology of A. africanus, examining the diet of a local frugivore, the figbird (Sphecotheres viridis) and the effect of gut passage on seedling emergence. Overall, A. aethiopicus was superior in germination and emergence, with the highest mean germination (98.8%) and emergence (94.5%) under optimal conditions and higher emergence (mean of 73.3%) across all treatments. In contrast, A. africanus had the lowest germination under optimal conditions (71.7%) and low mean seedling emergence (49.5%), but had fruits with the highest relative yield (ratio of dry pulp to fruit fresh weight) that were favored by a local frugivore. Figbirds consumed large numbers of A. africanus fruits (~30% of all non-Ficus fruits), and seedling germination was not significantly affected by gut passage compared to unprocessed fruits. Asparagus virgatus germinated poorly under cool, light conditions (1.4%) despite a high optimum mean (95.0%) and had low mean performance across emergence treatments (36.3%). The species also had fruits with a low pulp return for frugivores. For all species, seed survival declined rapidly in the first 12 mo and fell to < 3.2% viability at 36 mo. On the basis of the traits considered, A. virgatus is unlikely to have the invasive potential of its congeners. Uniformly short seed survival times suggest that weed managers do not have to contend with a substantial persistent soil-stored seed bank, but frugivore-mediated dispersal beyond existing infestations will present a considerable management challenge.

Control techniques and management strategies for the problematic Navua sedge (Cyperus aromaticus)

Navua sedge, a member of the Cyperaceae family, is an aggressive weed of pastures in Fiji, Sri Lanka, Malay Peninsula, Vanuatu, Samoa, Solomons, and Tahiti and is now a weed of pastures and roadsides in north Queensland, Australia. Primarily restricted to areas with an annual rainfall exceeding 2500 mm, Navua sedge is capable of forming dense stands smothering many tropical pasture species. Seventeen herbicides were field tested at three sites in north Queensland, with glyphosate, halosulfuron, hexazinone, imazapic, imazapyr, or MSMA the most effective for Navua sedge control. Environmental problems such as persistence in soil, lack of selectivity and movement off-site may occur using some herbicides at the predicted LC90 control level rates. A seasonality trial using halosulfuron (97.5 g ai/ha) gave better Navua sedge control (84%) spraying March to September than spraying at other times (50%). In a frequency trial, sequential glyphosate applications (2,160 g ae/ha) every two months was more effective for continued Navua sedge control (67%) than a single application of glyphosate (36%), though loss of ground cover would occur. In a management trial, single applications of glyphosate (2,160 to 3,570 g ae/ha) using either a rope wick, ground foliar spraying or a rotary rope wick gave 59 to 73% control, while other treatments (rotary hoe (3%), slashing (-13%) or crushing (-30%)) were less effective. In a second management trial, four monthly rotary wick applications were much more effective (98%) than four monthly crushing applications (42%). An effective management plan must include the application of regular herbicide treatments to eliminate Navua sedge seed being added to the soil seed bank. Treatments that result in seed burial, for example, discing are likely to prolong seed persistence and should be avoided. The sprouting activity of vegetative propagules and root fragmentation needs to also be considered when selecting control options.

Indole oxygenase from the leaves of Tecoma stans

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and at 30°C. Oxygen (2 mol) is consumed and anthranilic acid (1 mol) is formed for every mole of indole oxidized. Neither sulfhydryl reagents nor sulfhydryl compounds inhibited the enzyme activity. The oxygenase also attacks, apart from indole, 5-hydroxyindole, 5-bromoindole and 5-methylindole. It is not inhibited by copper specific chelators or non-heme iron specific chelators. Atebrin did not inhibit the enzyme activity suggesting that it is not a flavoprotein, unlike other indole oxygenases and indole oxidases. Dialysis resulted in complete loss of enzyme activity. The inactive enzyme could not be reactivated by addition of various cofactors.

Management of the invasive hill raspberry (Rubus niveus) on Santiago Island, Galapagos: Eradication or indefinite control?

The eradication of an invasive plant species can provide substantial ecological and economic benefits by eliminating completely the negative effects of the weed and reducing the high cost of continuing control. A 5-yr program toward the eradication of hill raspberry (Rubus niveus Thunb.) in Santiago Island is evaluated using delimitation and extirpation criteria, as well as assessment of the ecological community response to management techniques. Currently, hill raspberry is located in the humid zone of Santiago island. It is distributed over three main infestations, small patches, and many scattered individuals within an area of approximately 1,000 ha. New infestations are constantly being found; every year, new detections add an area of approximately 175 ha. Adult and juvenile individuals are still found, both beyond and within known infestations. Both plant and seed bank density of hill raspberry decreased over time where infestations were controlled. Species composition in the seed bank and existing vegetation were significantly different between areas under intensive control and adjacent uninvaded forest. After 5 yr of intensive management, delimitation of hill raspberry has not been achieved; new populations are found every year, increasing the infested area that requires management. Off-target effects on native species resulting from control efforts seem to be substantial. Although a vast increase in economic investment would allow intensive searching that might enable all individuals to be found and controlled, the resultant disturbance and off-targets effects could outweigh the conservation benefits of eradication.