UDC 581.1

APPLICATION OF CELLULAR BIOTECHNOLOGY FOR STORAGE OF ASPEN BIODIVERSITY (Populus tremula L.)

Lyutsiya Aubakirova*, Elena Kalashnikova ***

* Eurasian national university named after Lev Nikolaevich Gumilev, 010000, Astana, Republic of Kazakhstan, web: www.enu.kz

*** Russian State Agrarian University –MSAU named after Konstantin Timiryazev, web:www.timacad.ru

The technology of clonal micro propagation of the Populus tremula L. at the expense of activation the meristem existing in a plant and induction of adventives buds’ formation from primary and graft callus tissue is elaborated.

Key words: the Populus tremula L, crop of a tissue, micro sprouts, adventives buds, callus genesis, meristem.

Aspen (Populus tremula L.) - member of the genus Populus (poplar), is a tree specie widely spread in Russia, growing on a wide variety of soils, both in pure plantations and in mixed forests. For the Republic of Kazakhstan Aspen is one of the most important wood forming species, where it presents by itself the tree of the first magnitude: its height is up to 35-40 m, diameter is up to 80-90 cm. In the forests of the Republic of Kazakhstan aspen plantations grow in the area of 238 thousand hectares, which is more than 21% of the area covered by forest vegetation. Despite of the fact that nowadays more than 14 thousand hectares (more than 6% of the breed) took over-mature aspen, its annual average growth in the forests of the republic more than 4.7 m/ha. Current growth plantations of the best forms often exceeds 20 m 3 / ha.

Until recently, the aspen were viewed as "secondary" low-grade species, often rotten from the first years of its life. Due to this in many instructions it is recommended to replace aspen plantations with pine or oak plantations. Currently, however, in most countries the major challenge is to grow the forest fast growing trees to produce wood for pulp and paper, chemical and lumber industries.

Fast-growing species allow preserving a balance between consumption and the reduction of wood pulp for many years. Great significance in the normalization of the balance between consumption and reproduction of forest plantations from becoming fast growing species of wood. At the present time in Russia and another foreign country have developed technologies for cultivation of such plantations.

To the establishment of plantations of energy forests great attention was paid by many European countries - Britain, France, Germany and others. In Kazakhstan while energy plantations are still poorly developed. In the stage of experimental-industrial are located power stations, for which the organized cultivation of energy forests, that is, working for the burning of wood. Widely used waste products of wood recycling and lumbering and also energy peat for the production of properties of heat and power energy (countries of Scandinavia) (Alekseev, Krasovskaya, 1995).

Awareness of the problem of energy security in the world first occurred in connection with the repeated increases in oil prices.

Particularly great importance of aspen tree as of a fast-growing tree species, easily reviving in natural way. Its stands’ age of maturity comes in 2-2,5 times faster in comparison with the coniferous and oak species. This allows using wood efficiently in the quality of heat sources. The cost of cultivation of aspen wood is cheaper than pine, fir and oak, and consequently the demand for aspen wood will grow. However, the main plantations of aspen in the Republic of Kazakhstan is characterized by extremely low marketability, due to the defeat of fungal diseases, causing for rot and reduces in the quality of the wood. At the same time, there are clones of aspen which are resistant to fungal diseases, and characterized by full arborous trunks and high productivity.

With the increase of anthropogenic impact of human activities on the environment leads to the sharp rise of biodiversity of vegetable, as in the level of species and of ecosystem. The burning issue of the day in the republic is the preservation of different types of aspen (clone) and the diversity of stable aspen phytocenosis.

One of the promising methods for the preservation biodiversity of plants is the application of biotechnology, cell and particular method of clonal micro propagation, significantly reduces the time needed to obtain high-quality of the material (Kalashnikov, 2009).

Materials and Methods. As an object of research served one-year sprouts of 15-20 cm long, isolated from the crone of an adult tree of the Populus tremula L. Sprouts contained top and axillary sleeping buds. Cut buds were placed in a vessel with water and kept in the room temperature until the stage of a green cone’s and young sprouts’ occurrence, which was further, used for introduction to the culture in vitro [4].

Sprouts were processed with the use 96 % spirit, after what they were divided into segments (1-1,2 cm) and put in the saturated solution of hypochlorite sodium for 30-45 minutes or in 3 % solution chloramines. Further, the sterilized segments were washed out with the use of the sterile distilled water and put it into sterile nutrient medium of Murasiga and Skuga [5] containing BAP(three times 6-benzilaminopurin) in the concentration of 0,5 mg/l and 2,4-D (2,4-dichlor phenolacetic acid) 0,2 - 1,5 mg/l or IAA (0.5 mg/l) and NAA(naftilacetic acid) 0,2 mg/l inducing the process of callusogenesiss or the activation of developed existing meristems. [6, 7].

Explants grew up plants in a light room, where the sustained temperature was about 26°С and 16-hours photoperiod and 3 thousand lux intensity of light were supported. [8].

For the regeneration of sprouts from the callus tissue there applied a nutrient medium, containing mineral salts under the prescription of Murasiga and Skuga or WPM with an addition of various cytokine to regeneration of runaways (BAP, kinetin) - 2,0 mg/l in a combination with 2,4-D, NAA or IAA in the concentration of 0,5 mg/l.

The rootages of micro sprouts in vitro were spent in a nutrient medium WPM containing IMK in the concentration of 3 mg/l.

Results and discussion. Researches have shown that in the process of cultivation explants in vitro within 3-4 weeks in a nutrient medium MC, containing 2,4-D and BAP there was observed the formation unorganized growing callus tissues fabrics of friable type in a basal parts of a sprout. It is experimentally fixed that the process of callusogenesis is in direct dependence on the concentration 2, 4-D in a nutrient medium. However, with the raised concentration of the given auxin there observed the decrease of a considered indicator (Tab. 1) which is revealed by the intensity in growth of callus tissue.

Tab. 1 The influence of various concentrations 2,4-D on callusogenesis in the crop of Populus tremula L tissues

Concentration 2,4-D, mg/l	The formation of a callus, %	Increment of callus tissues, mg
0,2	0	0
0,5	40,1±8,2	39,7±1,4
1,0	66,7±9,8	62,0±2,1
1,5	43,4±7,9	56,0±1,2

Thus, it has been revealed that the basic condition for the callusogenesis in the crop of Populus tremula L tissues is an addition exogenous auxin 2, 4-D in concentration of 1 mg/l.

As the main factor, initiating secondary differentiation of cells is the balance of phytohormones , the influence of auxins- indolylasetic acids (IAA) has been investigated; naftilasetic acids (NAA); 2,4-dichlorfenolasetic acids (2,4-D) and cytokine - 6-furfuralaminopurine (kinetin) and 6-benzilaminopurine (BAP) to the formation morphogenetic structures in the callus tissue of the Karelian birch. As an initial material heterogeneous globular calluses of green color were used. As the basic environment МС environment was used. The account of the received data was drawn in 30 days from the moment of the cultivation of callus tissues in inducing environments (Tab. 2).

Tab. 2 Influence of phytohormones (mg/l) to the morphogenesis of the crop of Populus tremula L tissues.

Considered indicators	Kinetin 2			BAP 2
	IAA 0,5	NAA 0,5	2,4-D 0,5	IAA 0,5	NAA 0,5	2,4-D 0,5
The number of morphogenetic callus , %	42,3±3,7	33,2±2,6	12,3±2,7	73,6±6,4	45,3±4,7	15,7±4,3
Average number of adventives buds for 1 callus, piece	4,6±0,4	3,7±0,3	3,3±0,7	8,1±0,9	4,7±1,3	4,0±1,0

After four-week cultivation from callus tissue there started the formation of morphogenetic structures presented by dense dark- green areas (meristematic centre), consisting of a cone of increase and leaf rudiments. Further plants-regenerators were formed from them. From the table 2 it follows, that the maximum increase in coefficient of reproduction is reached by addition to the nutrient medium IAA in concentration of 0, 5 mg/l and BAP 2 mg/l. The average of the induced buds de novo in this variant makes 8, 1 pieces on one callus. In other variants considered indicators were for 1, 5-2 times lower, and generated adventives buds distinguished with reduced growth.

Repeated cultivation of callus tissue on investigated variants of nutrient mediums again led to the formation of adventives on the average of 6-8 pieces for one callus, from which, in consequence, micro sprouts were formed.

Generated sprouts propagated with the use of the method of activation of development in a plant meristem (cutting) which is based on removal of apical dominance. Thus divided into the segments, containing two buds and cultivated on non hormonal to a nutrient medium with half maintenance in its structure of macro salts under the prescription WPM or Murasige and Skuga. In these conditions an active growth of buds and formation of the micro sprouts characterized by normal morphology are observed. FiG.1

A B

FiG. 1 - Development of the aspen micro shoots of axillary buds: A - WPM, B - Murasige and Skuga

The multiplied micro sprouts implanted on nutrient medium WPM containing IAA in concentration of 3 mg/l. The percent of the root plants has made 85, 3 %.

Thus micro cuttings received in this way were transferred to rooting medium containing IBA at a concentration of 2 mg / liter. Cultivation of microshoots during 1-1,5 months in these conditions contribute to gross-perpetuating of microshoots, which later were ready to transfer to the conditions in vivo (FiG. 2). The entire cycle of micro propagation realized in a light room where the temperature was 23, 24-hour photoperiod and the light intensity of 1250 lux.

Formed aspen plants under in vitro conditions differ on biometric indicators, despite the same period of their growth in vitro. Plants regenerated the triploid forms were characterized by more rapid growth of (2,0-5,5 cm height) and 95% had well-developed root system.

FiG. 2. - Tube plants of aspen

An adaptation vitro plant was carried out in greenhouses, which were equipped with the installation of artificial fog. Relative humidity ranged from 90% to 65% as the acclimation of plants. Our experience has shown, the term transplant plants from the conditions of the in vitro conditions in vivo, has a significant impact on the survival rate of test-tube plants. The best time to transplant test-tube plants is spring or early summer. Plants with 2-3 leaves and well developed root system capable of adapting to the conditions in vivo. Regenerated plants were grown in pricked out packets or transferred directly into the soil, consisting of accessible forms of nitrogen (NH4 + NO3) - not less than 130mg/kg, phosphorus (P2O5) - not less than 250 mg / kg of potassium (K2O) - not less than 400 mg / kg, the mass fraction of moisture - not more than 65%, the pH of saline suspension - 6,6. Fertilizing of plants was conducted daily with mineral fertilizers "Kemira lux” by adding a chalk or lime.

Equally important, in our view, in vitro adaptation of plants to soil conditions has the height and physiological state (e.g., degree of rooting) of regenerated plants. The received data testifies about the existence of genotypic dependence and the ability of plants to adapt to the conditions in vivo. It was established that the triploid aspen were characterized by high adaptative capacity (65%) compared with diploid forms (30%) and had a high shoot 2 times larger than the diploid form.

Adapted plants reached a height of 25-30 cm were carried to the area of plantations. An inventory of uterine plantation in the end of September showed that the growth of seedlings in height of open ground for triploid and diploid forms of aspen was 25.8 and 15.2 cm respectively.

Thus, in vitro methods to allow receiving successfully sufficient quantity of planting material of highly productive aspen, and resilience to the core rot. Potential method is far from exhausted, and some of its stages can still be improved. Genetic identity of planting material with maternal copy allows carrying forestry practically on the basis of grain - to grow plants with known properties (Shabunin, Podolskaya, 2005). The most promising in terms of the Republic of Kazakhstan

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