ISSN 1021-4437, Russian Journal of Plant Physiology, 2016, Vol. 63, No. 6, pp. 800–810. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © I.N. Tretyakova, M.E. Park, A.S. Ivanitskaya, N.V. Oreshkova, 2016, published in Fiziologiya Rastenii, 2016, Vol. 63, No. 6, pp. 812–822.
Peculiarities of Somatic Embryogenesis of Long-Term Proliferating
Embryogenic Cell Lines of Larix sibirica in vitro
I. N. Tretyakova, M. E. Park, A. S. Ivanitskaya, and N. V. Oreshkova
Sukachev Institute of Forest, Russian Academy of Science, Siberian Branch, Krasnoyarsk, 660036 Russia
Received October 21, 2015
Abstract—Morphogenesis and maturation of somatic embryos, ploidy, and genotyping of cell lines (CL) of
embryogenic cultures of Larix sibirica Ledeb. in vitro were investigated during 2–6 years. It was revealed that
from 2000 to 11103 globular somatic embryos were formed in proliferating CL. However, the ability of
somatic embryos to the maturation and germination decreased. Cytogenetic study of embryonal-suspensor
masses (ESM) of Larix sibirica demonstrated that cells of long-term cultivated cultures remained diploid.
According to microsatellite analysis, proliferating CL of Siberian larch were characterized by weak allelic
variability, and cell line 6 and cloned seedlings of this line were genetically stable and corresponded to the
donor tree. Embryogenic cell lines composed the collection bank, which will be successfully used for planta-
tion forest growing.
Keywords: Larix sibirica, somatic embryogenesis, somaclonal variation, embryogenic cell lines
Over the past 30 years, somatic embryogenesis is
the most promising direction for the micropropaga-
tion of coniferous species. This method allows not
only the mass reproduction of coniferous species with
the selection of valuable traits but also the perfor-
mance of fundamental developmental biology studies:
morphogenesis and embryology, genetics, and hor-
monal regulation [1–3].
For the successful breeding of conifers through
somatic embryogenesis, it is necessary to know the
genetic stability of the obtained embryogenic cultures
and their compliance with parental genotypes. The
appearance of polyploid cells was observed as a result
of the somatic embryogenesis of Picea abies, Рinus
nigra , Larix marschlinsii , Pinus radiata , and
Larix decidua . Heterogeneous cells with genotypes
completely different from the parent tree were
observed in 2–5-month cell lines (CL) obtained from
Larix sibirica megagametophytes . The analysis of
the genetic stability of nuclear microsatellites in
embryogenic cell cultures of Pinus pinaster , Рinus
sylvestris , Quercus suber and Quercus robur [11,
12], Populus tremuloides , and many other plants
showed a higher frequency of mutations and genetic
instability, which intensified during the long-term cul-
tivation of embryogenic callus.
However, several authors pointed out that, during
the somatic embryogenesis, somaclonal variation was
not detected in some species of spruce (Picea abies,
P. glauca, P. mariana × P. glauca) [14–17] and Pinus
pinaster , and regenerated explants were identical
to the original explant.
In this article, we present the results of the study of
the embryogenic productivity (the number of somatic
embryos) and somaclonal variation (morphogenesis,
ploidy and genotyping) of long-term cultured cell lines
of L. sibirica.
MATERIALS AND METHODS
The objects of study were Siberian larch (Larix sibi-
rica Ledeb.) trees grown in the arboretum of Sukachev
Institute of Forest, in the Pogorelsky Bor research forest
and larch forests of the Republic of Khakassia (Chernoe
Ozero village). The age of trees was 50–70 years. Out of
200 larch tree explants (zygotic embryos), only three
donor trees formed embryogenic callus obtained at dif-
Prior to tissue culture in vitro, the seeds of experi-
mental trees were sterilized with a 10% solution of
hydrogen peroxide for 10 minutes. After sterilization,
under conditions of laminar hood, embryos were
removed from megagametophytes and placed on the
culture medium. The total number of explants from
one tree was 40–100 pcs.
Abbreviations: CL—cell lines; ESM—embryonal-suspensor
masses; IBA—indolebutyric acid.