Project: CONEMDE - Development of zygotic and somatic embryos in conifers – from basic
studies to practical application
Person in Charge: prof.RNDr.Ladislav Havel, CSc.
Country of Origin: Slovakia
Country of scientific activity: Italy
Project duration:36 months
Scientific panel: Environment and Biotechnology
The economically relevant clonal plantation forestry as well as new forest-based biotechnology presumes effective mass-propagation systems to produce high quality static embryo clones with specific traits. The CONEMDE project is aiming at studying zygotic embryogenesis in conifers to develop protocols for mass-scale production of Abies alba and Picea abies, for either conventional tree breeding purposes or for cultivation in specific areas. The general outcomes of the project will be increased knowledge of the zygotic embryogenesis in conifers and efficient protocols for somatic embryogenesis. The aims will be reached by the comparison of zygotic and somatic embryogenesis on morphological, biochemical, and molecular levels followed by comprehensive statistical analyses and modelling to recognize the specific properties and features needed for effective somatic embryogenesis. The toolbox includes testing the effects of heavy metal ions and investigations of polar auxin transport on development of zygotic and somatic embryos with various analytical and molecular methods including e.g. analyses on energetic status. Thus, it presents a novel way to study plants which do not have mutant genotypes available. The research fellow Assoc. Prof. J. Krajnakova and her future career plans, as an independent researcher (or group leader) at universities or as a R&D sector expert, will greatly benefit for the updated skills in molecular biology as well as from the lectures / practical training in management and pedagogic skills provided by the MENDELU Brno. The research area of the CONEMDE project is important for the Europe and, therefore, also her career plans are quite feasible. The research group will be headed by Prof. Havel from MENDELU Brno (Czech Republic), in collaboration with Dr. Reinöhl (polar auxin transport), Assoc. prof. Kizek (heavy metals), Prof. Häggman, Finland (zygotic and somatic embryogenesis of conifers) and Prof. Vianello, Italy (bioenergetics).
The ongoing project summary
The CONEMDE project is aimed on studying zygotic embryogenesis in conifers to develop protocols
for mass-scale production of Abies alba and Picea abies, for either conventional tree breeding
purposes or for cultivation in specific areas. The general outcomes of the project will be increased
knowledge of the zygotic embryogenesis in conifers and efficient protocols for somatic
Five elite mother trees of Abies alba were selected in the Forest District Habrůvka, Training
Forest Enterprise Masaryk Forest Křtiny (Fig. 1 A). During the summer 2010, immature (Fig. 1 B) and
mature cones were collected for the establishment of 2 independent experiments.
The first set of experiments focused on the determination of bioenergetical parameters (cellular
levels of ATP and glucose-6P) considering the effect of mother tree, sampling day and seed part
(developing zygotic embryo and megagametophyte). The second set of experiments was related to
the induction of embryogenic cell masses on immature zygotic embryos. The experiments were
focused on the effects of the mother tree, sampling period and the preferable position on the
zygotic embryo where the induction of embryogenic cell mass has taken place. The induction
frequencies were determined. Altogether 1 573 immature zygotic embryos were isolated and yielded
191 Abies alba embryogenic cell lines. Mucilaginous embryogenic cell masses (ECMs) induced on
induction medium were visible at 3 – 8 weeks. ECMs with a clear, glassy appearance were
subsequently transferred on proliferation medium (Fig. 1 D) and sub-cultured regularly. The
intensive proliferation of more than 170 cell lines was stabilized over a period of 5 months. These
embryogenic cell lines are going to be used in experiments with heavy metals.
Twenty five A. alba embryogenic cell lines representing 5 mother trees and 5 different
genotypes within each mother tree were randomly chosen for maturation experiment (Fig. 1 F). The
objectives of maturation experiment were to study the effect of mother tree, different cell lines on
the capability of embryogenic cell masses to form somatic embryos, average number of
cotyledonary somatic embryos per 1 g of fresh weight of the ECMs. The maturation has taken place
within 10 weeks. The embryogenic cell lines were monitored regularly in two-week intervals.
Samples for determination of bioenergetical parameters were prepared in each sampling week.
The suitability of anti-AtPIN1 antibodies for PIN1 immuno-localization in somatic embryos of
Abies alba was repeatedly tested with a so far negative result. If the final test with the new anti-
AtPIN1 antibody will not be successful, commercial preparation of the anti-PaPIN1 antibody will be
The Mendel University has gained embryogenic cell lines originating from the zygotic embryos
from the Training Forest Enterprise Masaryk Forest Křtiny which could become an active part of gene
pool storage and in a future perspective, after cryopreservation, also a part of a gene bank.