ASSESSMENT OF GENETIC STABILITY IN CRYOPRESERVED ROMANIAN ELITE TOMATO


(LYCOPERSICON ESCULENTUM Mill.) CULTIVARS

This work is supported by a grant of the Romanian National Authority for Scientific Research CNCS-UEFISCDI

        Project codePCE_2011-3-0435

        Project nr. PN-II-ID-PCE-2011 - 3 - 280   


HOST INSTITUTION

Institute of Biological Research Cluj-Napoca branch of NIRDBS Bucharest

Republicii str. 48

RO - 400015 Cluj-Napoca

Tel/Fax: +40 264591238

www.icbcluj.ro


   

Aim of the project


Analysis of genetic stability of five Romanian tomato cultivars of economic importance regenerated from cryopreserved shoot tips vs. donor plants.


Summary


In the last years there is an increasing need to conserve plant genetic resources, not just to maintain biodiversityy, but also to support biotechnology and plant breeding programmes. The recent progress in cryogenic technology is attracting attention due to its significance as a safe approach for preservation of plant genetic resources. Cryopreservation is an attractive alternative for the long-term storage of plant germplasm, consisting in the conservation of plant material at ultra-low temperatures, in general the temperature of liquid nitrogen (-196°C). The main objective is the preservation of five tomato (Lycopersicon esculentum Mill.) cultivars of economic importance, using cryopreservation and assessment of genetic stability of preserved genotypes. Cryostorage imposes a series of stress factors to the plant material, which are susceptible of inducing modifications in cryopreserved and regenerated plants. A sine qua non condition for a successful cryopreservation is to maintain the genetic integrity of plant material after freezing. It is desirable to assess the ‘true-to-typness’ of plants regenerated after cryopreservation vs. donor plants. For tomato genotypes this will be analyzed at molecular level by detection of putative genetic modifications in structure of AFLP markers and by DNA sequencing of chloroplast lycopene beta cyclase gene after cryopreservation of tested cultivars. These analyses will establish whether and how influent are possible mutations in the structure of involved gene or if they could affect ß-cyclase synthesis involved in lycopene cyclisation to ß-caroten.


Objectives


2011


O1:
Assessment of studied resources in conformity with intern law requirement related to tomato varieties.

O2: Elaboration of a model based on the analysis methods used for identification of genetic variability. Assignment of the experimental methods to be used for cryopreservation of tomato cultivars, AFLP analysis and gene sequencing.

2012


O3:
Development and optimization of a cryopreservation protocol for tomato genotypes. This protocol will be based on previous results obtained (vitrification based protocols) for other plant species. Parameters like shoot tip size, preculture and dehydration duration, biochemical changes which showed a positive correlation with post-thaw regeneration will be considered for further improvement of the existing protocols for tomato cultivars.

O4: AFLP analysis of tomato cultivars and DNA sequencing of chloroplast lycopene beta cyclase gene before cryopreservation of plant material. After cryopreservation tomato plant material will be used to analyze genetic stability or polymorphisms appeared due to ultra-low temperature treatment applied.  

2013


O5:
Validation of the improved cryopreservation protocols. It is expected a better understanding of the mechanisms involved in tolerance towards ultra-low temperatures (-196°C).

O6: AFLP polymorphism assessment of tomato cultivars after cryopreservation of plant material and detection of putative genetic modifications in structure of AFLP markers.  

2014

   O7: Assessment of genetic stability and true-to-typeness of plants regenerated after cryopreservation vs. donor plants.  

2015


 O8: Identification of parameters influencing survival and plant regrowth following storage in liquid nitrogen (-196°C).


        2016


O9: Validation of the optimized cryopreservation protocols for the tomato genotypes based on investigated parameters.



       Team members

Dr. Adela HALMAGYI (project manager)

Dr. Sergiu VALIMAREANU

Dr. Constantin DELIU

Dr. Ana COSTE

Dr. Dana SUTEU

Dr. Ioan BACILA

Dr. Ioana ROMAN

Dr. Gabriela SOVAREL

Teh. Emilia KIBEDI