Peter D.S. Caligari

High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar.

* The provision of sequence-tagged site (STS) anchor points allows meaningful comparisons between mapping studies but can be a time-consuming process for nonmodel species or orphan crops. * Here, the first use of high-resolution melt analysis (HRM) to generate STS markers for use in linkage mapping is described. This strategy is rapid and low-cost, and circumvents the need for labelled primers or amplicon fractionation. * Using white lupin (Lupinus albus, x = 25) as a case study, HRM analysis was applied to identify 91 polymorphic markers from expressed sequence tag (EST)-derived and genomic libraries. Of these, 77 generated STS anchor points in the first fully resolved linkage map of the species. The map also included 230 amplified fragment length polymorphisms (AFLP) loci, spanned 1916 cM (84.2% coverage) and divided into the expected 25 linkage groups. * Quantitative trait loci (QTL) analyses performed on the population revealed genomic regions associated with several traits, including the agronomically important time to flowering (tf), alkaloid synthesis and stem height (Ph). Use of HRM-STS markers also allowed us to make direct comparisons between our map and that of the related crop, Lupinus angustifolius, based on the conversion of RFLP, microsatellite and single nucleotide polymorphism (SNP) markers into HRM markers.

Comparison of transcriptional profiles of flavonoid genes and anthocyanin contents during fruit development of two botanical forms of Fragaria chiloensis ssp. chiloensis

Difference in fruit pigmentation observed between two botanical forms of Fragaria chiloensis ssp. chiloensis (form chiloensis and form patagonica) was studied through transcriptional and chemical approaches. The proportion of different anthocyanins was demonstrated to be characteristic of each botanical form, with pelargonidin 3-glucoside being the most abundant in f. patagonica fruit and cyaniding 3-glucoside as the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and flavonoid biosynthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-specific primers in order to perform transcriptional analyses by qRT-PCR. These genes showed spatial, developmental, and genotypic associated patterns. The red fruit of f. patagonica exhibited higher transcript levels of anthocyanin-related genes and higher levels of anthocyanins compared to the barely pigmented fruit of f. chiloensis. The anthocyanin accumulation in F. chiloensis ssp. chiloensis fruits was concomitant with the particular progress of the transcriptional activity of genes involved in the biosynthesis of flavonoid pigments. The differences in anthocyanin contents, both in terms of type and quantity, between the two botanical forms of F. chiloensis ssp. chiloensis were coincident with the differential transcriptional patterns found in the anthocyanin-related genes.

Plant tissue culture: Current status, opportunities and challenges

R. Garcia-Gonzales, K. Quiroz, B. Carrasco, and P.D.S. Caligari. 2010. Plant tissue culture: Current status, opportunities and challenges. Cien. Inv. Agr. 37(3): 5-30. In the last two decades plant biotechnology applications have been widely developed and incorporated into the agricultural systems of many countries worldwide. Tissue culture tools have been a key factor to support such outcomes. Current results have allowed plant biotechnology and its products –including transgenic plants with several traits- to be the most assimilated technology for farmers and companies, representing several benefits such as: 125 millions ha of transgenic crops in 2008, the reduction of pesticides application by up to 9% in the last ten years, transgenic plants with a better nutritional quality, mass propagation of selected and healthy plants, and the production of proteins for industrial or therapeutic use. The rapid and extensive assimilation for this technology has improved the competences of the agricultural systems both in industrial and in developing countries, based on the proper application of research programs. Several theoretical and practical aspects supporting plant tissue culture applications, as well as the main results and current status of the technology are discussed in this review. The reader will find key elements to evaluate the potential of plant tissue culture tools for the development of agriculture, livestock, human health and nutrition, and human well being in general.

Chlorophyll, anthocyanin, and gas exchange changes assessed by spectroradiometry in Fragaria chiloensis under salt stress

Chlorophyll and anthocyanin contents provide a valuable indicator of the status of a plants physiology, but to be more widely utilized it needs to be assessed easily and non-destructively. This is particularly evident in terms of assessing and exploiting germplasm for plant-breeding programs. We report, for the first time, experiments with Fragaria chiloensis (L.) Duch. and the estimation of the effects of response to salinity stress (0, 30, and 60 mmol NaCl/L) in terms of these pigments content and gas exchange. It is shown that both pigments (which interestingly, themselves show a high correlation) give a good indication of stress response. Both pigments can be accurately predicted using spectral reflectance indices (SRI); however, the accuracy of the predictions was slightly improved using multilinear regression analysis models and genetic algorithm analysis. Specifically for chlorophyll content, unlike other species, the use of published SRI gave better indications of stress response than Normalized Difference Vegetation Index. The effect of salt on gas exchange is only evident at the highest concentration and some SRI gave better prediction performance than the known Photochemical Reflectance Index. This information will therefore be useful for identifying tolerant genotypes to salt stress for incorporation in breeding programs.

Production of haploids and doubled haploids in oil palm

BackgroundOil palm is the worlds most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation.ResultsHere we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences.ConclusionsThis study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

Transcript profiling suggests transcriptional repression of the flavonoid pathway in the white-fruited Chilean strawberry, Fragaria chiloensis (L.) Mill.

Beyond their participation in fruit pigmentation and because of their high antioxidant activity, flavonoids are considered important constituents of fruits and vegetables. We have previously reported that in the ripe receptacles of Fragaria chiloensis only traces of flavonoids can be found, while cinnamic acid derivatives are highly accumulated. In order to characterize the molecular background of this uncommon phenotype we analyzed the transcriptional profile of different biosynthetic genes, with special regard to the gene encoding Cinnamate 4-Hydroxylase (C4H), the enzyme transforming cinnamic acid into the next intermediary of the phenylpropanoid pathway. Northern blot and quantitative RT-PCR showed low transcript abundance for the gene encoding C4H and also for a series of structural genes responsible for flavonoid biosynthesis. Together with this, high transcript levels were found for a repressive transcription factor, suggesting that the pathway would be inhibited at the transcriptional level, thus correlating to our previous findings on the chemical phenotype. Our results contribute to the comprehension of the pigmentation phenotype in strawberries, allowing the utilization of Fragaria chiloensis as a model system for the study of antioxidant pigment biosynthesis.

Management of Auxin-Cytokinin Interactions to Improve Micropropagation Protocol of Henequen (Agave fourcroydes Lem.)

Henequen (Agave fourcroydes Lem.) is a high value plant species both for agricultural and ecological uses. The species is very well known worldwide by the quality of its fiber that is widely used for industrial purposes. This species is propagated at a large scale by asexual methods but current propagation technologies are not satisfying the grower’s demands. A. fourcroydes has been cultivated in vitro but it has shown some recalcitrant behaviour during the multiplication and rooting stages. The combination of 6-benzylamynopurine (BAP) (0.75 mg L -1 ) and indolebutyric acid (IBA) (1.0 mg L -1 ) instead of 2,4-dichlorophenoxyacetic acid (2,4-D) significantly improved explant survival and shooting during the establishment of in vitro young shoots. Combining thidiazuron (TDZ) (0.5 or 0.75 mg L -1 ) with BAP (1.0 mg L -1 ) and IBA (1.0 mg L -1 ) in the basal medium increased the multiplication rate of henequen and significantly speeded out bud dormancy breaking. To improve rooting of the micropropagated shoots, the addition of IBA and naphthylacetic acid (NAA) was tested. The best rooting efficiency was obtained when the basal medium was supplemented with 0.5 or 0.75 mg L -1 of NAA, giving 100% of rooted explants and an average of 9.40 and 11.55 roots per explants, respectively. Over 94% of micropropagated plants survived the ex vitro weaning step and no morphological disorders were observed in any of the plants. Modification of plant growth regulators composition in the medium was a key factor to improve the efficiency of the micropropagation technology of henequen.

In vitro Propagation of Cedar (Cedrela odorata L.) from Juvenile Shoots

Cedrela odorata L. is one of the most important timber species currently traded in the Caribbean and Central America; however, it has been intensively exploited. In vitro techniques and clonal propagation can help to develop new plantations and assist in establishing improvement programs for this species. The aim of this study was to develop a protocol to establish in vitro conditions and to micropropagate this species from nodal explants from juvenile cuttings taken from field trees. Disinfection of node explants with 5% propiconazole CE 25 during 3 min resulted in 100% explant disinfection and 60% morphogenic response on those established explants. Shoot development was optimized by cultivating in vitro node explants in Murashige and Skoog basal medium supplemented with 2 mg L-1 6-bencilaminopurine and 3 mg L-1 naphthaleneacetic acid. This medium resulted in 100% shoot development from the in vitro node explants with a 3.93 cm mean height. Rooting was also stimulated 6 wk after individualization of the regenerated plants on the same micropropagation medium with a mean of 3.9 roots per plant. In vitro plants did not show morphologic differences when compared to ex vitro seeds.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis

BackgroundThe high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR.ResultsHere, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker.ConclusionsA pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.