Physiologic responses and gene diversity indicate olive alternative oxidase as a potential source for markers involved in efficient adventitious root induction

Olive (Olea europaea L.) trees are mainly propagated by adventitious rooting of semi-hardwood cuttings.However, efficient commercial propagation of valuable olive tree cultivars or landraces by semi-hardwood cuttings can often be restricted by a low rooting capacity. We hypothesize that root induction is a plant cell reaction linked to oxidative stress and that activity of stress-induced alternative oxidase (AOX) is importantly involved in adventitious rooting. To identify AOX as a source for potential functional marker sequences that may assist tree breeding, genetic variability has to be demonstrated that can affect gene regulation. The paper presents an applied, multidisciplinary research approach demonstrating first indications of an important relationship between AOX activity and differential adventitious rooting in semi-hardwood cuttings. Root induction in the easy-to-root Portuguese cultivar 'Cobrançosa' could be significantly reduced by treatment with salicyl-hydroxamic acid, an inhibitor of AOX activity. On the contrary, treatment with H2O2 or pyruvate, both known to induce AOX activity, increased the degree of rooting. Recently, identification of several O. europaea (Oe) AOX gene sequences has been reported from our group. Here we present for the first time partial sequences of OeAOX2. To search for polymorphisms inside of OeAOX genes, partial OeAOX2 sequences from the cultivars 'Galega vulgar', 'Cobrançosa' and 'Picual' were cloned from genomic DNA and cDNA, including exon, intron and 3'-untranslated regions (3'-UTRs) sequences. The data revealed polymorphic sites in several regions of OeAOX2. The 3'-UTR was the most important source for polymorphisms showing 5.7% of variability. Variability in the exon region accounted 3.4 and 2% in the intron. Further, analysis performed at the cDNA from microshoots of 'Galega vulgar' revealed transcript length variation for the 3'-UTR of OeAOX2 ranging between 76 and 301 bp. The identified polymorphisms and 3'-UTR length variation can be explored in future studies for effects on gene regulation and a potential linkage to olive rooting phenotypes in view of marker-assisted plant selection

Isolation and functional characterization of a lycopene β-cyclase gene that controls fruit colour of papaya (Carica papaya L.)

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to β-carotene (yellow) is catalysed by lycopene β-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene β-cyclases (lcy-β1 and lcy-β2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-β2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-β1 and lcy-β2 genes is similar and low in leaves, but lcy-β2 expression increases markedly in ripe fruit. Isolation of the lcy-β2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties

Functional marker development from AOX genes requires deep phenotyping and individualized diagnosis

The development of new ‘deep phenotyping’ techniques for functional markers (FM) development on alternative oxidase (AOX) gene sequences are expected to greatly increase the efficiency of association studies between the candidate FM sequences and the desired phenotype. However, it is critical to perform these studies in the appropriate target tissue/cell at the correct time point. AOX genes, due to their diversity and with differential methylation marks, are likely also subjected to such interplay between sequence and regulatory mechanisms. Polymorphisms in coding sequences may directly affect protein function, but expression regulatory switches are more abundant in non-coding regions. Transgenic technology continues to contribute to crop improvement programme, if efforts are directed more towards FM-assisted plant breeding. Hence, AOX can be put into best use if a dual approach involving genetic transformation and conventional plant breeding go hand in hand

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect

Biological Resource Management-Connecting Science and Policy TRANSGENIC CROPS: RECENT DEVELOPMENTS AND PROSPECTS

ABSTRACT It is now more than fifteen years since the first transgenic plants were generated experimentally. In that period there have been dramatic advances in our understanding on both basic and applied aspects of plant biology. Transgenic plant research depends on the availability of procedures for plant transformation. Two types of method for plant transformation exist, the use of Agrobacterium as a biological vector for foreign gene transfer, and direct gene transfer techniques, in which DNA is introduced into cells by the use of physical, electrical or chemical means. Agrobacterium can be used to transform a wide range of plants, but there are α number of species which are of interest for basic or applied research in which of Agrobacterium -mediated transformation is not reproducible or efficient. Using this procedures thousands of transgenic crops have been developed experimentally or field tested, while few of them are currently cultivated world wide, predominately on temperate zone crops and on conditions prevailing in industrial countries offering the potential increasing and improving food production capacity while limiting the use of agrochemicals and protect the environment. The " first generation " of transgenic crops were aimed at improving traits involving single genes. Now we are on the verge of a new step in crop modification, fueled by the rate at which new genes ( important for plant growth and development metabolism and stress tolerance ) characterised. Transgenic technology has been pivotal in the full spectrum of these new developments, from gene identification to an improved understanding of their regulation, as well as genetic transformation involving more complex transfers of many genes simultaneously. This will further help in managing natural resources like water, soil, e.t.c. in a better way. Our view of the nature of crop products can also be expected to change in the short to medium term, as plants are exploited for the production of novel compounds such as biodegradable plastics and new pharmaceuticals. However, it is the case that the extent to which the potential of transgenic research is realised will depend on public acceptance. To a significant extent this will require that the biology of transgenics is fully understood, and that a maximum degree of predictability of transgene effect, both phenotypic and genotypic, can be ensured. There is a need for diffusing this technology to tropical plants and adapt it to benefice the small farmer in the developing world were food demands will increase. To achieve this it requires: to find ways integrating biotechnology research into their national agricultural research activities in one hand horizontal and vertical networking cooperation on the other. Finally the implication of advancement in this relatively new technology especially in the area of biosafety, production patterns, biodiversity, inertecnal property rights and other critical factors should be sufficiently dismissed and understood

Characterization of Lupin Cultivars Based on Phenotypical, Molecular and Metabolomic Analyses

Lupins are an important source of protein that could replace soybeans in the diet of ruminants and monogastrics, without reducing their performance. Lupinus albus (L. albus) is the main species of the genus Lupinus that is cultivated in the Mediterranean region. The aim of the present research was to study commercial cultivars and advanced breeding lines of L. albus by using phenotypical, molecular and biochemical data, in order to be used in breeding projects. Seven commercial cultivars (Estoril, Fas Sweet, Multitalia, Magnus, Orus, Ulysse Sulimo and Figaro) and three advanced lines from the company AGROLAND (LKML, LKAP and LKAU) were used. Eleven morphological traits were described using UPOV Guidelines (International Union for the Protection of New Varieties of Plants). Additionally, agronomical traits and yield components were measured. Regarding the nutritional value, grain samples were analyzed for N and the crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), total alkaloids (TA), total phenolic content (TP), total tannins content (TT) and condensed tannins (CT) were calculated. Genetic diversity among genetic materials was assessed by SSRs molecular markers. The metabolomic analysis for four selected cultivars (Figaro, Magnus, Multitalia and Sulimo) was performed on the seeds with the GC/EI/MS technique. According to the results, the advanced lines were most productive but also with higher content of total alkaloids than the commercial cultivars. The only exception was the cultivar Multitalia that was characterized by a high content of alkaloids. Based on the SSRs, the cultivars Magnus, Orus and Estoril were grouped together while the breeding lines LKAP, LICML and LKAU were grouped with Multitalia. Regarding the metabolomic profile, the cultivars Multitalia and Magnus were together, while Sulimo was grouped with Figaro. Finally, the content of several beneficial metabolites for human and animal nutrition was significantly increased in Sulimo and Figaro, compared to Magnus and Multitalia. Both commercial varieties and lines have characteristics that can be exploited and used in breeding programs