INTER-SIMPLE SEQUENCE REPEAT ANALYSIS OF GENETIC FIDELITY IN IN VITRO MICROPROPAGATED PLANTSOF THE ENDANGERED PLANT SPECIES CENTAUREA DAVIDOVII URUM. (ASTERACEAE)
GALYA PETROVA1*, MARINA STANILOVA2, SVETLANA BANCHEVA2, STEFAN PETROV3
1 - Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria
2 - Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria
3 - Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, Sofia, Bulgaria
*Corresponding author: galiaty@abv.bg
Keywords: Centaurea, Еx situ conservation, Balkan Mountains, ISSR, Genetic fidelity
Abstract: The genus Centaurea s. l. (Asteraceae) is one of the richest in endemics in the Bulgarian flora, represented by more than 70 species. One of them, the endemic Centaurea davidovii Urum., occurs in several very fragmented populations in the floristic region of Balkan Mountains. Habitat destruction and low reproductive capacity of this species, mainly due to weak seed germination and damage of seeds by insects, necessitate its conservation both in situ and ex situ. An ex situ collection of Centaurea davidovii individuals has been established recently at the experimental field of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences. Plants had been obtained from a single seed using in vitro multiplication techniques. They were successfully acclimatized to the open-air field plot, bloomed and formed fertile seeds. True to type clonal fidelity is one of the most important prerequisites in the micropropagation of any plant species. A major problem encountered with the in vitro culture is the potential presence of somaclonal variation. Various factors, such as the in vitro process and its duration, nutritional conditions, in vitro stress, etc. occasionally appear to induce somaclonal variation. Hence, it becomes imperative to regularly check the genetic integrity of the in vitro regenerated plants in order to produce clonally uniform progeny. In the present study, the genetic stability of in vitro propagated Centaurea davidovii plants was tested for the first time using Inter Simple Sequence Repeat (ISSR) markers. The amplification products were monomorphic and no polymorphism was detected. Furthermore, ISSR analysis indicated the genetic integrity of in vitro propagated plants, as well as an identical banding pattern after more than ten passages of subculture. Our results showed that the regenerated plants are similar and therefore the developed micropropagation protocol for in vitro multiplication of C. davidovii is reliable and represents a safe mode for production of true-to-type plants. Besides the establishment of the ex situ collection, planting of in vitro obtained plants in the wild populations has been designed aiming at their strengthening. This in situ measure should be conducted with strict respect to the genetic diversity conservation, which requires improvement of the seed germination rate and regeneration of plants of numerous normal genotypes.