Paul Chavarriaga

Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene

Cassava is a very important staple crop, especially in the arid tropics where it is a chief source of carbohydrates, but the provitamin A content in the storage root is insufficient to sustain a healthy life. The work presented shows that a single amino acid exchange in a conserved region of the enzyme phytoene synthase leads to substantive accumulation of β-carotene (provitamin A) in the roots. Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

Development and application of transgenic technologies in cassava.

The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails.

Quantitative analysis of transgenes in cassava plants using real-time PCR technology

To speed up the molecular analysis of cassava transgenic plants, we developed real-time polymerase chain reaction (PCR)-based methods that could be implemented as a tool in the primary scrutiny of putative transgenic plants. We tested for the presence of transgenes, estimated copy number, and quantified messenger RNA (mRNA) levels of genes introduced through Agrobacterium. Copy numbers for the genes ß-glucuronidase and hygromycin phosphortransferase were estimated in 15 transgenic lines. Most lines contained one or two copies of each gene; in some, the copy number was different for the two genes, suggesting rearrangements of the transferred DNA. Six of the 15 lines were analyzed by Southern blot. The copy number so estimated was concordant in most cases. Although real-time PCR was efficient for classifying transgenic lines with one or more transgenes inserted, for conclusive analysis of gene copy number, i.e., in a potential breeding line, the Southern blot may still be required. The transcript levels from both genes were determined in eight lines. High, medium, and low levels of mRNA expression were detected. No direct relationship between copy number and expression level of transgenes was obvious, suggesting that factors like position effects or DNA rearrangements led to differential expression. Quantitative mRNA expression data for the ß-glucuronidase gene agreed with results from histochemical staining. With real-time PCR we could detect high levels of transgene expression in 3-y-old cassava plants maintained and propagated as clones in the greenhouse. This is the first time that real-time PCR is reported to be used for transgene analysis in cassava.

Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz)

Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding.

A simple hydroponic hardening system and the effect of nitrogen source on the acclimation of in vitro cassava (Manihot esculenta Crantz)

Plant tissue culture technology is being widely used for large-scale, rapid, clonal multiplication and genetic transformation in cassava. The main limitation of this technology is the period of acclimation of the fragile in vitro plants after they have been multiplied or regenerated. Most losses of in vitro plants occur when the plantlets are moved directly from the test tubes to the ex vitro conditions. Our aim was to design a simple, rapid, low-maintenance hydroponic system to improve survival rate of transplanting to the ex vitro conditions through the rapid acclimation process of in vitro plants. In this paper, we have developed a simple hydroponic system to accelerate the cassava acclimation and multiplication process. This system considerably increased the survival percentage of in vitro and/or transgenic lines and reduces the time requirement for multiplication by hydroponic acclimation. In order to assess the effectiveness of the acclimation of seedlings on their establishment, we analyzed plant growth and field survival rate with response to different nitrogen (N) sources using different cassava accessions. Nitrogen sources of NO3− and NH4NO3 increased plant growth and root length compared to NH4+ alone, or water treatments. The greenhouse and field survivability of N-hardened plants, including transgenic lines, were significantly different in growth and development. We present a simple NO3− hydroponic acclimation system that can be quickly and cheaply constructed and used by the cassava community around the world. The efficiency of our proposed N hydroponic acclimation system is validated in the transgenic development pipeline which will enhance the cassava molecular breeding.

Evidence of Epigenetic Mechanisms Affecting Carotenoids

Epigenetic mechanisms are able to regulate plant development by generating non-Mendelian allelic interactions. An example of these are the responses to environmenal stimuli that result in phenotypic variability and transgression amongst important crop traits. The need to predict phenotypes from genotypes to understand the molecular basis of the genotype-by-environment interaction is a research priority. Today, with the recent discoveries in the field of epigenetics, this challenge goes beyond analyzing how DNA sequences change. Here we review examples of epigenetic regulation of genes involved in carotenoid synthesis and degradation, cases in which histone- and/or DNA-methylation, and RNA silencing at the posttranscriptional level affect carotenoids in plants.