Basilio Carrasco

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.

Genetic structure based on EST–SSR: a putative tool for fruit color selection in Japanese plum (Prunus salicina L.) breeding programs

Prunus salicina is one of the most economically important stone fruits. However, there is scarce genetic information available, which makes it difficult to implement marker-assisted selection (MAS) in genetic improvement programs. Recently, next-generation sequencing has greatly enhanced breeding program strategies, generating information associated with the identification of expressed sequence tag–simple sequence repeats (EST–SSRs) and single-nucleotide polymorphisms (SNPs), two of the most used molecular markers in MAS. Few studies have focused on developing EST–SSR markers considering both gene expression levels of contrasting phenotypes and specific transcription factors of metabolic pathways. This study investigated the transcriptome profile of P. salicina in fruits with contrasting skin colors, obtaining 54,224 unique contigs. From this dataset, 44 EST–SSRs have been generated, considering gene expression levels of contrasting phenotypes and specific transcription factor from three metabolic pathways: citric acid, carbohydrate metabolism and flavonoid pathways. Three EST–SSR markers developed from the putative flavonoid pathway transcription factors PsMYB10, PsMYB1 and PsbHLH35 were selected to determine genetic structure in 29 cultivars. This structure was contrasted with the genetic structure generated using genomic SNPs obtained by genotyping-by-sequencing (GBS). The analysis using SNPs identified two groups, while the use of selected EST–SSRs identified three. In contrast to the structure given by the SNPs, the EST–SSRs grouped all the yellow cultivars in one cluster, which was composed mainly of cultivars of this color. The EST–SSRs developed in this study may be considered as candidate markers to be evaluated in MAS strategies in genetic improvement programs.

Breeding in peach, cherry and plum: from a tissue culture, genetic, transcriptomic and genomic perspective

This review is an overview of traditional and modern breeding methodologies being used to develop new Prunus cultivars (stone fruits) with major emphasis on peach, sweet cherry and Japanese plum. To this end, common breeding tools used to produce seedlings, including in vitro culture tools, are discussed. Additionally, the mechanisms of inheritance of many important agronomical traits are described. Recent advances in stone fruit transcriptomics and genomic resources are providing an understanding of the molecular basis of phenotypic variability as well as the identification of allelic variants and molecular markers. These have potential applications for understanding the genetic diversity of the Prunus species, molecular marker-assisted selection and transgenesis. Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNPs) molecular markers are described as useful tools to describe genetic diversity in peach, sweet cherry and Japanese plum. Additionally, the recently sequenced peach genome and the public release of the sweet cherry genome are discussed in terms of their applicability to breeding programs.

Advances in breeding and biotechnology of legume crops

Legume crops are relevant globally to the feeding and the nutrition of humans and animals because of their relatively high seed content of protein and essential amino acids. Additionally, they are related to sustainable agriculture, considering their ability to associate with atmospheric nitrogen fixing bacteria (Rhizobia). Despite this, several technical constraints of legumes crops have maintained their worldwide production far behind from cereals. This review article focuses in current information about recent advances in breeding and biotechnology of the major leguminous crops. Conventional breeding has mainly focused in improving multiple vegetative and reproductive traits that have associated to distinct heritability values, which reflects how amenable each character is for genetic improvement. Legumes have strongly entered into the genomics era through the complete genome sequencing of several species in the last decade. Moreover, a wealth of tools and techniques of Fabaceae genomics are now available and discussed throughout this article. In addition, there is an increasing amount of quantitative trait loci, candidate genes, and genes associated to abiotic and biotic resistance and to agronomic traits that have been reported, which will potentially allow more rapid progress of legume genetic improvement. Two successful examples of genetically modified legume crops are examined in this paper: glyphosate-resistant transgenic soybean and transgenic common bean resistant to Bean golden mosaic virus. Finally, legumes genomics and breeding programs, using classical breeding methods, marker-assisted selection, and biotechnological tools face a promising momentum for further application of technology and information that could boost their global production.

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.

Genetic variability and structure of Gomortega keule (Molina) Baillon (Gomortegaceae) relict populations: geographical and genetic fragmentation and its implications for conservation

Gomortega keule (Molina) Baillon is a rare Chilean species classified as endangered and currently under conser- vation. Currently, conservation of this species is carried out by private companies and government agencies, but is done so based more on pragmatic criteria than scientifically based strategies. In this context, information concerning the genetic variability of the species among the different populations over its entire distribution range is incomplete, making it difficult to coordinate management and conservation strategies. Here we report a study to evaluate the genetic variability and popu- lation structure of G. keule over its entire distribution in Chile. Twenty-five ISSR primers for 223 individuals from 11 geo- graphical populations were tested to evaluate genetic variability. Results showed a high level of genetic diversity for the species. A high level of genetic differentiation among populations was found, while genetic structure of the geographic populations was also shown by using STRUCTURE 2.1 software. Despite the fact that this species has been highly af- fected by human intervention and habitat destruction, a high level of genetic diversity was present. A likely explanation for this is that it reflects the previous distribution of this long-lived species and the current lack of sexual regeneration. Ge- netic diversity data must be considered in the development of a management strategy for further developing the protected areas, for identifying isolated small populations for conservation, and using larger populations with higher genetic diversity as seed sources for ex situ propagation and replanting.

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.

De novo transcriptome assembly of ‘Angeleno’ and ‘Lamoon’ Japanese plum cultivars (Prunus salicina)

Japanese plum (Prunus salicina L.) is a fruit tree of the Rosaceae family, which is an economically important stone fruit around the world. Currently, Japanese plum breeding programs combine traditional breeding and plant physiology strategies with genetic and genomic analysis. In order to understand the flavonoid pathway regulation and to develop molecular markers associated to the fuit skin color (EST-SSRs), we performed a next generation sequencing based on Illumina Hiseq2000 platform. A total of 22.4 GB and 21 GB raw data were obtained from ‘Lamoon’ and ‘Angeleno’ respectively, corresponding to 85,404,726 raw reads to ‘Lamoon’ and 79,781,666 to ‘Angeleno’. A total of 139,775,975 reads were filtered after removing low-quality reads and trimming the adapter sequences. De novo transcriptome assembly was performed using CLC Genome Workbench software and a total of 54,584 unique contigs were generated, with an N50 of 1343 base pair (bp) and a mean length of 829 bp. This work contributed with a specific Japanese plum skin transcriptome, providing two libraries of contrasting fruit skin color phenotype (yellow and red) and increasing substantially the GB of raw data available until now for this specie.

Molecular Tools for Rapid and Accurate Detection of Black Truffle (Tuber melanosporum Vitt.) in Inoculated Nursery Plants and Commercial Plantations in Chile

Truffle (Tuber melanosporum Vitt.) culture is an agroforestry sector in Chile of increasing interest due to the high prices that truffles fetch in the national market and the recent evidence that its commercial production is possible in Chilean climatic and soil conditions. In this study, the efficiency of three methods of DNA extraction from a mix of 5 g of soil and roots from both nursery and field plants of Quercus ilex L. mycorrhized with T. melanosporum were evaluated, and a simple and reproducible protocol was established. Detection of T. melanosporum was performed by the technique of cleaved amplified polymorphic sequence (CAPS) from amplicons generated with the primers ADL1 (5´-GTAACGATAAAGGCCATCTATAGG-3´) and ADL3 (5´-CGTTTTTCCTGAACTCTTCATCAC-3`), where a restriction fragment of 160 bp specific for T. melanosporum was generated, which allows the discrimination of this species from the rest of the species belonging to the Tuber sp. genus. Direct detection of T. melanosporum in one step was also obtained by polymerase chain reaction (PCR) from total DNA isolated from mycorrhized roots and with the primers ITSML (5´-TGGCCATGTGTCAGATTTAGTA-3´) and ITSLNG (5´-TGATATGCTTAAGTTCAGCGGG-3´), generating a single amplicon of 440 bp. The molecular detection of T. melanosporum by the methods presented here will allow the rapid and accurate detection of mycorrhization of trees, both under nursery and field conditions. This technology will also provide more security to farmers by controlling the quality of the mycorrhized trees they will plant and also by following the mycorrhization status of established orchards.

Combining Niche Modelling, Land-Use Change, and Genetic Information to Assess the Conservation Status of Pouteria splendens Populations in Central Chile

To assess the conservation status of a species with little ecological information is usually a challenging process. Pouteria splendens is an endemic shrub of the coastal range of Central Chile currently classified as lower risk (LR) by IUCN (version 2.3). Knowledge about this species is extremely limited. Currently P. splendens is only found in two small and isolated populations, which are thought to be remaining populations of an originally large metapopulation. However, there is no evidence to support this hypothesis, limiting our ability to gauge the real current conservation status of this species. In this study we combine niche modelling, land-use information, and genetic techniques to test the metapopulation hypothesis and reassess the conservation status of P. splendens using the IUCN criteria. We also evaluated the potential effects of climate change in the species distribution. Our results support the hypothesis of a large metapopulation that was recently fragmented. Future climate could increase the range of P. splendens; however the high level of fragmentation would preclude colonization processes. We recommend reclassifying P. splendens as Endangered (EN) and developing strategies to protect the remaining populations. Similar approaches like the presented here could be used to reclassify other species with limited ecological knowledge.