UDC 630. 161:4:581.143.6

IN VITRO PROPAGATION OF WOODY PLANTS USING TISSUE CULTURE AND ITS APPLICATION TO FOREST IMPROVEMENT IN CENTRAL KAZAKHSTAN

L.S.Aubakirova¹, PhD doctoral candidate, E.A.Kalashnikova², a Doctor of biological sciences.

¹ Eurasian national university named after L.N.Gumilev, 010000, Astana, Republic of Kazakhstan

² Russian State Agrarian University- MAU named after K.A.Timiriazev, 127550, Moscow

The technology of clonal micro reproduction of the Karelian birch at the expense of activation the meristem existing in a plant and induction of adventive buds’ formation from primary and graft callus tissue is elaborated.

Keywords: the Karelian birch, crop of a tissue, micro sprouts, adventives buds, callus genesis, meristem.

In connection with an increasing interest and demand for new plants, and also with an internal and external arrangement of green spaces in cities the problem of mass reproduction of decorative-arborous cultures becomes actual in Kazakhstan. It is connected with practical absence of arboretums for their manufacturing in Kazakhstan and with the decrease of a share for an import planting material which does not always correspond to the standard

Now the most perspective way of plants’ reproduction is the method clonal micro reproduction in vitro, which is widely applied in agriculture and is rarer in forestry. For the majority of tree species there are no technologies of mass reproduction in the conditions of in vitro. Therefore the elaboration of new and improvement of existing technologies of clonal micro reproduction is an actual problem.

A special interest for selectors, experts and silviculturists is represented by the Karelian birch, distinguishing from other kinds of birches with a high decorative veiny structure of wood. Therefore, the given breed is widely used in the furniture and cottage craft industries. The basic way of the Karelian birch’s’ reproduction is seeding. However, such way of reproduction in posterity bears the splitting of a sign into figured and disfigured forms, which leads to the loss of economically valuable forms of the given kind of a plant in natural populations. The replacement of a seeding way of reproduction by the vegetative one eliminates lacks of the first [1-3]. However the Karelian birch enters to the list of hardly propagating breeds by the use of vegetative way, for which it is expedient to conduct researches on the elaboration of new technologies of reproduction with an application of cellular biotechnological ways.

Methodology. As an object of research served one-year sprouts of 15-20 cm long, isolated from the crone of an adult tree of the Karelian birch. 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 Betula pendula Roth, var, carelica Mercl 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 Betula pendula Roth, var, carelica Mercl 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 colour 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 Betula pendula Roth, var, carelica Mercl 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.

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 %.

Adaptation of micro sprouts is realized in the conditions of 90-95 % of humidity of air at temperature of 23-25°С. As a substratum used a peat and sand mix in the ratio 1:1. After 2 weeks of adaptation a plant is transferred to a hothouse. After 6 months entirely generated plants had the height of 24-32 cm.

Thus, as a result of multi planned experiments the conditions of cultivation of Karelian birch have been optimized (Betula pendula Roth, var, carelica Mercl) in vitro and the technology clonal micro reproduction of the given culture both through callus tissue , and at the expense of development activation sinus buds is elaborated.

References:

1. Kalashnikova E.A., A.R.Rodin Reception of a landing material of wood, flower and grassy plants with use of methods of cellular and gene engineering: the Manual. The edition 2, М-2001

2. Ualikhanova Zh, Biotechnology of a plant. Almaty, ЖСШ «Дәуір», 2009.

3. Taliansky M.E. Biotechnology and plant growing / in book. Biology of our days. М, Knowledge, 1987. - s.77-95.

4. Butenko R.G.Biology of cells of tall plants in vitro and of biotechnology on their basis. М, the FBK-PRESS, 1999.

5. Kalinin Ф.Л., Sarnatskaya Century В, Polishchuk V. E. Methods of crops of tissue and cells in physiology and biochemistry of plants. Kiev, Naukova Dumka, 1980.

6. Murashige Т, Skoog F. A revised medium for rapid growth and bioassays with tobacco cultures//Physiol. Plant, 1962. Vol.15, № 3. P. 473-497.

7. Kataeva N.V., Butenko R.G. Clonal micro reproduction of plants. М: the Science, 1983.

8. Laboratory-practical lesson of an agricultural biotechnology: Methodical instructions. Under the editorship of V.S.Shevelukh M MACA, 1991.