Journal Highlights

Detection of Epigenetic Variation in Tissue-culture-derived Plants of Doritaenopsis by Methylation-sensitive Amplification Polymorphism(MSAP) Analysis

For centuries the orchid in all its sizes, shapes, colors and fragrances has symbolized all that is exotic and mysterious. With an estimated 25,000-30,000 species worldwide, it is probably the largest flowering plant family. Also the market potential for both orchid cut flowers and potted orchids is very favorable. This has also paved the way for the development of tissue culture techniques for mass clonal propagation. However, there are also many problems associated with the commercial production including slow growth, low multiplication rate, somaclonal variation, poor rooting and low survival rate during acclimatization. Among others, poor quality of planting materials due to somaclonal variation constrains the full expansion of the orchid industry.  This article focuses on the recent findings in understanding the physiology of clonal variation of Doritaenopsis during micropropagation. Orchids regenerated from tissue culture techniques show a wide range of variability, ranging from temporary changes in the phenotype to sexually heritable mutation. Changes in DNA methylation has been hypothesized as an underlying mechanism of tissue-culture–induced mutagenesis which includes a high frequency of quantitative phenotypic variation, activation of transposable elements, heterochromatin-induced chromosome breakage events, and sequence changes due to deamination of 5-methylcystosine to thyamine.  Somaclones exhibiting variations with flower characteristics were recovered from tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation sensitive amplification polymorphism (MSAP) analyses were used to characterize the somaclones. RAPD analysis using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6-10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal V1, V2, V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 336 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cystosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants. Park, S.Y., Murthy and Paek, K.Y. Rapid propagation of Phalaenopsis from floral stalk derived leaves.S. Park, H. Murthy, D. Chakrabarthy, and Kee Paek.  Detection of Epigenetic Variation in Tissue-culture-derived Plants of Doritaenopsis by Methylation-sensitive Amplification Polymorphism (MSAP) Analysis, In Vitro Cellular & Developmental Biology-Plant, 45:104-108, 2009

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Quantitative Analysis of Transgenes in Cassava Plants Using Real-time PCR Technology

The research developed in cassava (Manihot esculenta Crantz) is justified by the importance of this crop as a source of carbohydrate in the tropics, which makes it a staple food for millions in Africa, Asia and South- and Central America. Our laboratory participates in the Harvest Plus initiative (www.harvestplus.org) to improve the nutritional status of the roots through conventional breeding and genetic modification. Several major projects have been carried out lately in functional genomics of cassava, and its genome is being decoded, which partially explains the recent interest in developing and applying transgenic technologies in the hope of accelerating cassava development to benefit producers and consumers. Initially we focused on improving the efficiency of genetic modification, and on adjusting methods based on quantitative real-time PCR to estimate copy number and quantify mRNA levels of transgenes introduced by Agrobacterium-mediated transformation. Real time technology turned out to be far more efficient than conventional methods to detect and quantify transgenes, and provided an alternative for rapid screening of multiple transgenic events. Our study showed that most of the events were low copy number, which we consider relevant when bio-safety issues come into play. We were able to confirm, within a varied range, high levels of expression in transgenic cassava plants maintained and propagated as clones in the greenhouse for more than three years. In collaboration with leading research groups we currently study the role of root-specific promoters and their potential to direct expression of transgenes of agronomic interest, such as genes involved in the accumulation of β-carotene to improve the content of pro-vitamin A in the roots of plants grown in the field. Our results are of vital importance for the characterization of expression in organs and tissues of transgenic cassava plants.  Jesus Beltrán, H. Jaimes, M. Echeverry, Yaneth Ladino, Danilo Lopéz, M. Duque, Paul Chavarriaga, and Joe Tohme.  Quantitative analysis of transgenes in cassava plants using real-time PCR technology, In Vitro Cellular & Developmental Biology-Plant 45:48-56, 2009.