Optimization of marker techniques to estimate somaclonal variation in “in vitro” propagated sugarcane

SEPÚLVEDA TUSEK, M.; PERERA, M. F.; GARCÍA, M. G.; NOGUERA, A. S.; FILIPPONE, M. P. AND CASTAGNARO, A. P.

Cali, Colombia

Workshop; ISSCT IX Plant Pathology and VI Molecular Biology Workshop.; 2008

By means of “Vitroplantas” project, created by Estación Experimental Agroindustrial Obispo Colombres (Argentina), 30000 sugarcane seedlings are produced annually through “in vitro” meristem culture. These later undergo a rustification process and three more stages of conventional propagation (in Basic, Registered and Certified Nurseries) before being distributed among sugarcane growers. This project is supposed to guarantee seedling phytosanitary quality and genetic purity. However, it has been proved that “in vitro” culture generates genetic and epigenetic changes known as somaclonal variation; due to DNA sequence alterations, methylation, chromosome structure and number alterations, etc. This work aimed at optimizing a molecular methodology to quantify and detect somaclonal variation in “Vitroplantas” project propagation scheme. Thus, two molecular marker techniques with different sensibility levels, RAPDs and AFLPs, were compared, as well as two DNA extraction methods; one based on the use of meristemic tissue (method A), and the other involving the use of tender leaves (Method B). Seedlings propagated through “Vitroplantas” project after 6 successive multiplication cycles (M6) of 5 sugarcane genotypes (TUC87-3, TUC95-37, TUC97-7, TUC97-8 and CP85-384) were studied in comparison with conventionally propagated controls (of the same genotypes). In the assessed genotypes, no profile differences were found with respect to the controls for the primer combinations selected, with either of the marker techniques used. Moreover, the use of different DNA extraction methods did not produce different results, so the faster and more practical method based on tender leaves (B) was used. These results show that “Vitroplantas” project propagation protocol leads to scarce or no somaclonal variation at all, at least as far as the assessed genotypes are concerned. In cases where this variation does occur, it would be possible to detect it before releasing the material thus propagated.Estación Experimental Agroindustrial Obispo Colombres (Argentina), 30000 sugarcane seedlings are produced annually through “in vitro” meristem culture. These later undergo a rustification process and three more stages of conventional propagation (in Basic, Registered and Certified Nurseries) before being distributed among sugarcane growers. This project is supposed to guarantee seedling phytosanitary quality and genetic purity. However, it has been proved that “in vitro” culture generates genetic and epigenetic changes known as somaclonal variation; due to DNA sequence alterations, methylation, chromosome structure and number alterations, etc. This work aimed at optimizing a molecular methodology to quantify and detect somaclonal variation in “Vitroplantas” project propagation scheme. Thus, two molecular marker techniques with different sensibility levels, RAPDs and AFLPs, were compared, as well as two DNA extraction methods; one based on the use of meristemic tissue (method A), and the other involving the use of tender leaves (Method B). Seedlings propagated through “Vitroplantas” project after 6 successive multiplication cycles (M6) of 5 sugarcane genotypes (TUC87-3, TUC95-37, TUC97-7, TUC97-8 and CP85-384) were studied in comparison with conventionally propagated controls (of the same genotypes). In the assessed genotypes, no profile differences were found with respect to the controls for the primer combinations selected, with either of the marker techniques used. Moreover, the use of different DNA extraction methods did not produce different results, so the faster and more practical method based on tender leaves (B) was used. These results show that “Vitroplantas” project propagation protocol leads to scarce or no somaclonal variation at all, at least as far as the assessed genotypes are concerned. In cases where this variation does occur, it would be possible to detect it before releasing the material thus propagated.(Argentina), 30000 sugarcane seedlings are produced annually through “in vitro” meristem culture. These later undergo a rustification process and three more stages of conventional propagation (in Basic, Registered and Certified Nurseries) before being distributed among sugarcane growers. This project is supposed to guarantee seedling phytosanitary quality and genetic purity. However, it has been proved that “in vitro” culture generates genetic and epigenetic changes known as somaclonal variation; due to DNA sequence alterations, methylation, chromosome structure and number alterations, etc. This work aimed at optimizing a molecular methodology to quantify and detect somaclonal variation in “Vitroplantas” project propagation scheme. Thus, two molecular marker techniques with different sensibility levels, RAPDs and AFLPs, were compared, as well as two DNA extraction methods; one based on the use of meristemic tissue (method A), and the other involving the use of tender leaves (Method B). Seedlings propagated through “Vitroplantas” project after 6 successive multiplication cycles (M6) of 5 sugarcane genotypes (TUC87-3, TUC95-37, TUC97-7, TUC97-8 and CP85-384) were studied in comparison with conventionally propagated controls (of the same genotypes). In the assessed genotypes, no profile differences were found with respect to the controls for the primer combinations selected, with either of the marker techniques used. Moreover, the use of different DNA extraction methods did not produce different results, so the faster and more practical method based on tender leaves (B) was used. These results show that “Vitroplantas” project propagation protocol leads to scarce or no somaclonal variation at all, at least as far as the assessed genotypes are concerned. In cases where this variation does occur, it would be possible to detect it before releasing the material thus propagated.