Currently, research on plant tissues cultured in vitro on artificial growth media
presents a field of biology that contributes to the successful solution of theoretical and
practical problems, such as non-traditional ways of multiplication of tissues, meristems or
various plant organs (Cachiţă et al., 2004; Cachiţă, Sand, 2000). Tissue culture is used to
obtain perfectly healthy initial plants. Over the years, this method has become a modern
technique of rapid propagation of species of interest. Plants have proven to be suitable for
multiplication by tissue culture methods, and achievements in this direction have progressed
rapidly. The goal of this research has been the rapid multiplication and propagation of plants,
obtaining virus-free propagating material, creating genotypes with desired characters, highly
resistant to diseases, pests, physical and chemical stressors.
The conservation of plants by tissue culture methods is of great interest and will
probably become even more common in the future. The advantages of this method are
numerous, but we find it most important that the initiation of cultivation requires only a single
plant, a seed or a single explant (apex, meristem, bud, piece of leaf, stem etc.), thus, the
available plant specimens (which can be only a few) will not be affected by harvesting them
from their habitat (Clapa Doina, Fira Alexandru, 2018).
Despite the risks associated with tissue culture, such as somaclonal variability caused
by the regeneration methods used, but also of medium and long-term maintenance of crops,
this propagation method plays an extremely important role in conserving endangered plant
species, helping prevent the extinction of species or populations in the wild and is a
continuous source of plants used in environmental restoration programs. The success of a
method of ex situ conservation of genetic variability of plant species depends strictly on the
collection of samples in situ (Clapa Doina, Fira Alexandru, 2018).
In vitro conservation, as a modern technique of ex situ conservation, involves cell
culture techniques used to maintain and preserve plant resources cultured and grown in vitro
through processes involving successive subcultures.
In the Laboratory of Embryology and Biotechnology of the «Alexandru Ciubotaru»
National Botanical Garden (Institute), the biomorphological features of in vitro microcloning
and micropropagation processes and the adaptation of valuable species to the soil and climatic
conditions of the Republic of Moldova have been studied for several decades. Tissue culture
methods have been applied to medicinal, ornamental, horticultural and rare plants that are
widely known and used as a remedies and raw material for the manufacture of medicines –
the medicinal ones, or for decorative purposes, indoors and outdoors – the ornamental ones.
Rare plants are multiplied and reintroduced in their natural habitats.
The biological material subjected to in vitro micropropagation in the laboratory is
diverse, but the most important species that have been propagated are Withania somnifera
Dunal, Stevia rebaudiana Bertoni, Polymnia sounchifolia Poepp. end Endl., Lavandula
angustifolia Mill., Stachys sp., Actinidia arguta (Siebold & Zucc.) Planch. ex Miq., Actinidia
kolomikta (Maxim. & Rupr.) Maxim., Lycium barbarum L., Rubus sp., Syringa sp., Lilium
martagon L., Fritillaria montana Hoppe ex W. D. J. Koch, Bellevalia sarmatica (Georgi)
Woronow.
In order to obtain high inoculation efficiency, prior sterilization of the propagating
material is required, which consists in removing the contaminating microorganisms from the
surface of the explant. Apical meristems (most often), pieces of bulbs (for rare plants) and
pieces of leaves were taken as explants for micropropagation. As a result of the research, the
optimal concentration, time required for sterilization and exposure to certain sterilizing
reagents were determined.
For the researched species, several reagents (ethyl alcohol, diacid (of different
concentration and exposure time)) were tested according to the methods developed and
optimized in the laboratory.
The explants were disinfected with chemical sterilizers, after which, it was possible to
inoculate them on nutrient media. The most effective inocula for inducing morphogenesis
have been meristems and pieces of leaf blade, for callus induction. For the rare plants studied,
the most effective inocula were the fragments of bulbs.
In order to accomplish the morphogenic processes in plants by tissue culture, the
selection of the media was first performed. It is known that culture media supplemented with
different growth regulators used in microclonal development are not always useful for
callogenesis and organogenesis. It is necessary to select those media that induce
morphogenesis. Morphogenesis induction was obtained on MS 100 % or Standarti media
supplemented with growth regulators. Both the quality and quantity of growth regulators and
of the culture medium have profound effects on the morphogenic processes.
It is necessary to choose correctly the growth regulators and it is important to mention
that the regenerative processes are conditioned by a certain balance of auxin-cytokinin,
cytokinin-cytokinin characteristic for each plant species. The media used contained different
amounts of agar-agar. In the MS 100 % medium, such growth regulators as BAP, α NAA,
KIN, IAA were added.
For the induction of organogenesis in all species, the composition of the medium was
unchanged, MS 100% was always used, but the growth regulators and their amount differed.
For a more efficient development, the explants were cultured in a room with a temperature of
20 ± 2 °C, the relative air humidity of 75 %, the light intensity of 2500-3000 lux and the
photoperiod of 16 light hours. Apical and lateral meristems, fragments of shoots and callus
from previous cultures were taken as explants for organogenesis induction.
Micropropagation consists of several stages – the selection of the explants, their initial
growth, which is performed in vitro, followed by the transfer of the obtained plantlets to a
greenhouse or field. During the in vitro culture, the plants grow under special, controlled
conditions. Thus, after the transfer to a more natural environment, they need several weeks for
acclimatization, so that a sufficient number of plants can survive under the new conditions.
The research was carried out within the project 20.80009.7007.19 «The introduction
and development of technologies for propagation and cultivation of new species of woody
plants by conventional techniques and tissue culture».
References
Cachiţă C. D., Deliu C., Rákosy-Tican E., Ardelean A. Tratat de biotehnologie
vegetală, Ed. «Dacia», Cluj-Napoca, 2004. Vol 1. 433 p.
Cachiţă C.D., Sand C. Biotehnologie vegetală. vol I: Baze teoretice şi practice, Ed.
Mira Design, Sibiu, 2000 . 288 p.
Clapa Doina, Fira Alexandru. Înmulțirea plantelor prin culturi in vitro. Ed. Risoprint,
Cluj-Napoca. 2018. 205 p. ISBN 978-973-53-2239-7.