Genetic Stability of the Endangered Species Salix lapponum L. Regenerated In Vitro during the Reintroduction Process.

Abstract

Simple SummarySalix lapponum, a downy willow, is a boreal relict species, threatened with extinction in Poland. In order to save populations in their refugia on the southern limit of the specie’s range, some activities were undertaken to rebuild their resources. The in vitro propagation was chosen to produce new plants, as it allows obtaining many individuals in a relatively short time with no harm to natural populations. The collected shoot pieces were multiplicated on a special growing media, containing all the necessary macro- and micronutrients, with addition of plant growth regulators to make them form shoots and roots. The obtained plants were then planted into soil and acclimated to natural habitat conditions. On the basis of the conducted genetic analysis and flow cytometry, it was stated that the new plants were genetically unchanged in comparison to the mother plants. The research results confirmed that the tissue culture may be applied in the propagation of the endangered willow species and the obtained plants may be used to build new populations or to strengthen the present ones. Salix lapponum L. is a boreal relict species, threatened with extinction in Poland. An 80% decrease in the number of its stands was confirmed in the last half-century, so that to prevent the loss of downy willow, attempts were made to reintroduce this species in natural habitats. Micropropagation was chosen as a first stage of its active conservation. S. lapponum shoots were collected and disinfected with NaOCl, AgNO3, or HgCl2 or with a two-step disinfection with NaOCl and then placed on MS medium with BA 1 mg·dm−3 and IBA 0.1 mg·dm−3. Regenerated shoots were cultivated with addition of BA, KIN, or 2iP, alone or in combination with auxins, to find the highest multiplication rate. Inter-simple sequence repeat (ISSR) analysis and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. Disinfection was quite difficult and the use of HgCl2 was the most efficient. The highest multiplication rate was obtained in presence of KIN at 0.5 mg·dm−3 + IAA at 0.5 mg·dm−3. The analysis confirmed the genome size stability, which is in agreement with the results obtained by ISSR, revealing no somaclonal variation in plantlets and therefore allowing the use of the obtained plants for reintroduction.