Cryopreservation of Plant Tissues in Poland: Research Contributions, Current Status, and Applications

Cryopreservation of vegetatively propagated plant material is an increasingly widely used method for the efficient and safe storage of germplasm resources around the world. In Poland, there are currently four cryobanks in use for long-term plant protection programs. However, plant tissues propagated in vitro constitute only a small portion of the accessions stored in them. To date, cryogenic storage techniques have been developed and adopted in this country for ornamental plants (roses, chrysanthemums, and geophytes), crop species (potato and garlic), forest tree species (the genera Quercus and Fraxinus), and some ferns. Polish researchers have used suspension cultures of Gentiana spp. and shoot tips of Lamprocapnos spectabilis to improve cryopreservation knowledge. A better understanding of the benefits of cryopreservation and its widespread implementation in plant biodiversity conservation programs is required. The objective of this review is to provide a concise synthesis of the scientific contributions, current status, and applications of cryogenic techniques for the conservation of in vitro culture-derived plant tissues in Poland. First, the results contributing to research that has been achieved using cell suspensions and advances related to the use of nanoparticles and plant extracts to improve cryopreservation efficiency are discussed. Then, the applications and advances in cryopreservation of ornamental plants (roses, radiomutants, plant chimeras, Lamprocapnos spp., and geophytes), crop species (potato and garlic), forest trees, and ferns are summarized

Somatic Embryogenesis of Norway Spruce and Scots Pine: Possibility of Application in Modern Forestry

Somatic embryogenesis (SE) is an important method for the vegetative propagation of trees. SE is the developmental in vitro process in which embryos are produced from somatic cells. This method can be integrated with other biotechnological techniques, genomic breeding and cryopreservation, which enables commercial-scale sapling production of selected high-yielding genotypes in wood production combined with fast breeding cycles. The SE is potential tool to improve plant stock in comparison with seed orchards. It can be useful for ecologically and economically important species, such as Norway spruce (Picea abies L. Karst.) and Scots pine (Pinus sylvestris L.), ensuring stable production in the era of climate change and biodiversity crisis. In this review, we summarize the current state of research on problems associated with somatic embryogenesis in P. abies and P. sylvestris

Propagation of Juniper Species by Plant Tissue Culture: A Mini-Review

The genus Juniperus (of the Cupressaceae family) is the second most prevalent group of conifers on Earth. Juniper species are widely dispersed in the Northern Hemisphere, in Europe and Asia, and in Africa and Central America. Juniper species are resistant to dry climates and can adapt to difficult environmental conditions. Most juniper species are important in both ecological and economic terms. However, today, many forests in which junipers occur are being reduced in size due to both natural causes (fires, for example) and human activity (uncontrolled exploitation of forests, etc.). Also, climate changes may have adversely affected the range of populations of different juniper species. For this reason, some juniper species are now categorized as rare or endangered, and require immediate protective action. Therefore, there is an urgent need to develop effective strategies for ex situ conservation, including reliable procedures for Juniperus sp. reproduction for future reintroduction and restoration programs. The conservation strategies used until now with traditional forestry techniques (seed propagation, rooted cuttings, grafting) have not been satisfactory in many cases. Thus, increasing attention is being paid to the possibilities offered by in vitro culture technology, which enables the conservation and mass clonal propagation of different coniferous tree species. In this mini-review, we summarize the current state of knowledge regarding the use of various methods of the propagation of selected Juniperus species, with a particular emphasis on in vitro culture techniques

Somaclonal variation during Picea abies and P. omorika somatic embryogenesis and cryopreservation

Embryogenic cultures of plants are exposed to various stress factors both in vitro and during cryostorage. In order to safely include the plant material obtained by somatic embryogenesis in combination with cryopreservation for breeding programs, it is necessary to monitor its genetic stability. The aim of the present study was the assessment of somaclonal variation in plant material obtained from embryogenic cultures of Picea abies (L.) Karst. and P. omorika (Pančić) Purk. maintained in vitro or stored in liquid nitrogen by the pregrowth-dehydration method. The analysis of genetic conformity with using microsatellite markers was performed on cotyledonary somatic embryos (CSE), germinating somatic embryos (GSE) and somatic seedlings (SS), obtained from tissues maintained in vitro or from recovered embryogenic tissues (ETc) and CSE obtained after cryopreservation. The analysis revealed changes in the DNA of somatic embryogenesis-derived plant material of both Picea spp. They were found in plant material from 8 out of 10 tested embryogenic lines of P. abies and in 10 out of 19 embryogenic lines of P. omorika after in vitro culture. Changes were also detected in plant material obtained after cryopreservation. Somaclonal variation was observed in ETc and CSE of P. omorika and at ETv stage of P. abies. However, most of the changes were induced at the stage of somatic embryogenesis initiation. These results confirm the need for monitoring the genetic stability of plants obtained by somatic embryogenesis and after cryopreservation for both spruce species

Stimulation of somatic embryo growth and development in Picea spp. by polyethylene glycol

The objective of the presented study was to compare the effect of osmotic stress, induced by various concentrations of polyethylene glycol (PEG) MW 4000, on the development, maturation, and conversion of somatic embryos of Picea abies (model species) and P. omorika (endemic species) into somatic seedlings. Embryogenic tissue (ET) of P. abies (line A and B) and P. omorika were incubated on a maturation medium with 20 μM abscisic acid (ABA), 1 μM indole-3-butyric acid (IBA) and PEG 4000 at 0, 5, and 7.5%. After 5 weeks, the number of somatic embryos produced, and the number of cotyledonary somatic embryos per 1 gram of ET, was assessed. Cotyledonary somatic embryos were selected and transferred to a Margara germination medium for two months. Their growth, as measured by hypocotyl and radicle length, was evaluated after one month, and the somatic embryos were transferred to fresh medium. After the second month of incubation, the conversion frequency of germinated embryos into the seedlings was assessed. In general, ETs of both of the tested spruce species cultured on a maturation medium amended with 5% PEG 4000 exhibited a tendency to produce a higher total number of embryos, as well as a higher number of cotyledonary embryos. The B line of P. abies produced a significantly greater number of cotyledonary embryos, relative to the control (no PEG). PEG only stimulated hypocotyl growth in embryos of both lines (A and B) of P. abies during the germination stage. Somatic embryos of Picea omorika were characterized by poor growth of both hypocotyls and radicles, irrespective of PEG treatment. The addition of PEG 4000 to the maturation medium did not improve the conversion frequency of germinated somatic embryos into somatic seedlings in either spruce species after a month of incubation on Margara medium. The effect of PEG 4000 on somatic embryogenesis efficiency was more evident in the ET of the two P. abies lines, relative to the P. omorika ET line, during both the maturation and the germination stage. Results indicated that the P. omorika ET line was almost completely insensitive to all of the PEG concentrations tested

Somatic embryogenesis of selected coniferous tree species of the genera Picea, Abies and Larix

Experiments on somatic embryogenesis in selected spruce, fir and larch species were performed to determine if this method of micropropagation enables production of quality seedlings for forest nurseries. High frequencies of embryogenic callus in spruce (23-31%) and fir (29%) were achieved when mature zygotic embryos were used as explants, while in larches (36%) only when megagametophytes (endosperms with immature embryos) were used. The possibility of somatic embryogenesis initiation also from somatic embryos (resulting in secondary and third generation of embryogenic callus) indicate high efficiency of this method of micropropagation. The best results at all stages of somatic embryogenesis (good proliferation of embryogenic callus, high rate of embryo regeneration and their survival) were obtained for Norway spruce, European larch and some hybrid larches