Лаборатория In vitro

Not all poplars can be easily propagated in vivo. Some only through microclonal propagation in the laboratory. How did you manage to grow entire collections of tubes?

There have been many attempts to propagate turanga before. But they did not learn by success. And now, 2016-2017 at the Institute of Plant Biology and Biotechnology of the Ministry of Education and Science approached the issue from a scientific point of view. At this time, the scientific work was carried out: "Development of a biotechnological regulation for the cloning of commercially valuable difficult to propagate poplar species and hybrids." The main goal of the project was to accelerate the production of elite seedlings of commercially valuable difficult to propagate species and poplar hybrids. Before laboratory experiments, natural populations of Turanga variegated (Populus diversifolia Schrenk), and hybrid poplar plantations in JSC Forest Nursery and RSE Republican Forest Breeding Center were examined. In them, plus trees were selected to create clone collections, the best by breeding characteristics - fast-growing, highly decorative, resistant to salinity and drought, with an even trunk and a beautiful, voluminous crown (turanga - 4 clones, hybrid poplar forms 2/67, 1/86 No. 39 and the Hybrid Excellent).

Already in the laboratory, the protocols of isolation of poplar explants into aseptic culture, clonal micropropagation and rhizogenesis of microplants in vitro, as well as transplantation into non-sterile conditions in vivo were optimized. The optimal isolation period of shoots in vitro was established.

It was experimentally found that the best initiation of shoot growth occurs from the sleeping buds of cuttings after the dormancy period in January-March, and during the period of active growth in June-August. Kidney germination is good in a solution of ½ macro- and micronutrients according to Murashige and Skoog (MS) with the addition of 1.0 mg / l of gibberellic acid, pH 5.6. The best sterilizing agent for saprophytic microflora was 1% HgCl2 in an exposure of 5 minutes (95% efficiency).

It has been established that the regeneration and propagation of poplar in vitro culture is influenced by the mineral and hormonal composition of culture media. Of the recommended, standard environments for woody crops - Woody Plant Medium (WPM), Murashige and Skoog (MS), the best was MS (reproduction ratio 1: 9 per passage), and the best growth regulators: 6-benzylaminopurine (BAP) at a concentration of 0, 3 mg / L and β-indolyl-3-butyric acid (IMA) - 0.01 mg / L.

The composition of the nutrient medium for the clonal micropropagation of poplar was modified, which differs from the MS by a 2-fold increase in the content of trace elements and a 2-fold decrease in the content of CaCl2 and MgCl2. In such an environment, the multiplication factor increases significantly (1:15 per passage) and the quality of the shoots improves.

        

The best medium for rhizogenesis is an agarized MS medium containing ½ concentration of macro- and micronutrients with pre-soaking microprobe in 25 mg / l IMC for 16 hours. On which the root system is formed in 80-85% of mini-shoots. A favorable substrate for transplanting aseptic shoots into non-sterile conditions is horse peat, low peat, sand, limestone flour, expanded clay drainage, complex mineral fertilizer at a pH of 5.5–6.5 (survival rate 65–70%). The survival rate of seedlings adapted in the greenhouse from containers to the nursery is 85-90%.

Based on the results obtained, a biotechnological regulation of clonal micropropagation of poplar was developed, consisting of 5 stages: isolation of samples in vitro, cloning under aseptic conditions, rhizogenesis, adaptation in non-sterile conditions in vivo, and transplantation of plants in open ground. In vitro clonal collections have been created, including 4 clones of Turangi multifolia and 4 clones of hybrid poplars (2/67, 1/86, No. 39 and the Prevoshodnyi hybrid).