INVESTIGADORES
CASTAGNARO Atilio Pedro
congresos y reuniones científicas
Título:
Optimization of marker techniques to estimate somaclonal variation in in vitro propagated sugarcane
Autor/es:
SEPÚLVEDA TUSEK, M.; PERERA, M. F.; GARCÍA, M. G.; NOGUERA, A. S.; FILIPPONE, M. P. AND CASTAGNARO, A. P.
Lugar:
Cali, Colombia
Reunión:
Workshop; ISSCT IX Plant Pathology and VI Molecular Biology Workshop.; 2008
Resumen:
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.