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UDC 634.1:581.143.6
EFFECT OF GENOTYPE OF THE BLACK CURRANT ON THE EFFICIENCY OF PROPAGATION IN CULTURE IN VITRO
Skovorodnikov N.A., Sazonov F.F., Associate Professors Lebedev A.A., Student Bryansk State Agricultural Academy, Bryansk, Russia Phone: +7 (920) 605-26-37, E-mail: skovorodnikov [email protected]
ABSTRACT
Clonal micropropagation of varieties and elite forms of black currants is used in a breeding program of the Kokinskiy branch of the RBTIHN. The influence of a genotype on the frequency of the regeneration of the original explants at the introduction in culture in vitro and propagation factor of the microplants at the stage of proliferation are determined in this paper. To a lesser extent varietal peculiarities of currant influenced the rooting indices and the adaptation indices of plants to non-sterile conditions. The low regeneration ability in vitro is stated in varieties which are difficult to be bred by woody cuttings (Istok, Debryansk).
KEY WORDS
Black currant; Micropropagation; in vitro culture.
The method of clonal micropropagation is an important component in the technology of getting improved planting material of the highest quality fruit crops. This method of reproduction can greatly increase the plants propagation factor, eliminate the possibility of reinfection, provide an opportunity to work throughout the year, reducing the area under the uterine plantings, as well as store the material for a long time in culture in vitro [1, 3, 4, 6].
The need to use biotechnological techniques in accelerated breeding of elite forms and new varieties of the currants appeared at Kokinskiy branch of the VSTISP , that is conducting the selection work with the culture very actively. The aim of the reserch was to study the features of in vitro propagation of valuable genotypes of black currants, which were obtained at the Kokinskiy branch of the VSTISP.
RESEARCH METHODOLOGY
The work has been done in the scientific-educational center of biotechnology of the Bryansk State Agricultural Academy. The object of the study were 4 varieties and 9 elite forms of black currant (the authors I.V. Kazakov and F.F. Sazonov). The introduction into the culture was carried out in September 2012. The axillary buds (3-5 mm) without several covering scales were used as the explants sources. The plant material had been sterilized in
0.1% solution of thimerosal and 0.3% SDS for 3 minutes followed by five-times washing in sterile distilled water.
The cultivation of the explants was carried out on the nutrient medium Murashige-Skoog (1962) in Florinskiy test tubes with 0.2 mg/l CPPU as a source of cytokinin: N-(2-chloro-4-pyridyl)-N-phenylurea (C12H10ClN3O).
At the stage of proliferation the nutrient medium MS was enriched with vitamin-mineral complex "Complivit" in a concentration of 2 g / l. The 6-BAP in a concentration of 1 mg / l was used as a source of cytokinin. The test tubes for maintaining sterility and reducing evaporation from the nutrient medium were covered with two layers of the food wrap.
At the third stage the elongation of the microcuttings was combined with their rooting. The shoots were cultured on the hormoneless and twice diluted MS medium with the multicomplex "Complivit" (2 g/l).
Rooted plants were planted in minigreenhouses filled with prepared nutrient substrate based on "Nesterov" peat mixed with river sand in a ratio of 3:1. Unrooted microcuttings were treated with preparation "Kornevin" and planted in the substrate.
RESULTS AND DISCUSSIONS
The use of 0.1% thimerosal as an anticeptic agent has allowed to get a sterile culture of all 13 samples of the currants studied. The frequency of contamination ranged from 4% in variety "Bryanskiy Agat" to 47.4% in variety "Istok" and composed 23.9% on the average (Table 1). We believe that the genotype of the currant does not significantly affect the rate of microbial contamination of crops in vitro, and is random and depends on the mechanical damage to the buds and growing conditions of the parent plants. The variation in the frequency of explants contamination of the currant of the same types when introduced in the culture in vitro in different years proves the facts mentioned above. So, in 2011,the index of the variety "Istok" was only 13.2%.
The infected cultures must be taken away to prevent the equipment and space contamination. The plant tissues which are damaged by the fungus, usually die quickly. If a microflora is of bacterial nature and has a weak growth rate, then the contaminated currant explants can have shoot regeneration. Such plants can be planted in non-sterile soil with pretreatment with auxins to stimulate root formation. This technique can be used when there is a limited number of the variety of special value. In our study, we have got 76 blackberry plants, contaminated by the bacterial microflora. In January these plants were 10-20 cm high.
As the explants isolation was conducted in the autumn the buds of the currants were differentiated by flower type. In the successive removal of the covering scales the flowers primordiums of different sizes located in the central part of the bud can be detected. After the first few weeks of cultivation there was an increase in the flower buds, the buds emergence and their blossoming. Flowers inflorescences were of green color. Regeneration of the plant took place on the periphery of buds, at the base of the covering scales.
After a month of cultivation the maximum height of the regenerants (10.9 mm) was observed in elite form 55-41-5. The smallest shoots (3.5 mm) were formed in the variety «Istok2 (Table 1). The size of plants at the first stage of clonal micropropagation depends primarily on genotype of the currants. Recently we have got varieties which have both the significant growth force in the cultivation of isolated tissues in vitro (varieties: Vera,Gamayun, Bryanskiy Agat), and the "problem" varieties, which form very small shoots - «Istok» Debryansk.
Table 1 - The results of the introduction to the culture in vitro of the new varieties
and elite forms of black currant
№ Genotype Explants got (number) Contamination frequency, % Height of regenerants in a month, mm Regenerants per explant (number)
1 Gamayun 40 37,5 6,1 ± 0,3 1,6 ± 0,2
2 Istok 57 47,4 3,5 ± 0,3 1,7 ± 0,2
3 Bryanskiy Agat 50 4,0 7,4 ± 0,3 1,5 ± 0,1
4 Etyud 32 15,6 8,8 ± 0,4 1,1 ± 0,1
5 5-4-3/08 40 10,0 6,6 ± 0,3 1,6 ± 0,2
6 5-4-2/08 50 30,2 6,2 ± 0,3 1,3 ± 0,2
7 63-35-1 95 34,7 4,8 ± 0,3 1,2 ± 0,1
8 33-27-1 50 10,0 7,2 ± 0,3 1,2 ± 0,1
9 55-41-5 39 12,8 10,9 ± 0,7 1,5 ± 0,1
10 9-36-1/02 50 16,0 4,3 ± 0,2 2,2 ± 0,1
11 28-03-2 50 24,0 7,1 ± 0,5 1,2 ± 0,1
12 62-03-6 50 42,0 4,5 ± 0,2 1,1 ± 0,1
13 62-03-7 50 26,0 6,4 ± 0,2 1,2 ± 0,1
The number of mature buds / shoots per explant varied in a narrow range from 1.1 to 2.2 of the regenerants (Table 2). This index, as well as the size of plants, was primarily determined by the biological characteristics of the variety.
When growing plants in vitro the differences in the regenerative ability of each genotype is very evident. Sometimes this feature is a limiting factor in reproduction of the
variety desired. Among the the elite forms of currants introduced in the study the genotypes with low propagation coefficient were determined, for example, variety Istok.
Currant plants, cultivated in vitro, have distinctive characteristics in comparison with other berry crops. Such difference consists in the formation of the short shoots at the stage of plants propagation when cultivated in a nutrient medium with cytokinins. The internodes of the shoots formed were very short, and sometimes plants were of rosette structure.
The small size of currant plants causes the technical difficulties when cutting shoots. Besides, the viability of such plants is always less than of the larger ones. The size increase of currants in culture in vitro has been achieved by using vitamin and mineral complex "Complivit"as an additional component in the nutrient medium MS. This preparation in combination with 6-BAP at a concentration of 1 mg/l caused the increase of the the
propagation factor and of the height of the genotypes tested [5]. The plants formed the
elongated shoots, which made it possible to apply the cuttings in the internodes, the leaves increased in size and became intensely green.
The height of the plants at the propagation stage in vitro ranged from 5.9 to 18.3 mm,
and the propagation factor ranged from 1.3 to 2.8.
Table 2 - The propagation coefficient and plant height of the black currant in culture in vitro
(1 -St passage)
№ Genotype Propagation coefficient Height of plants, mm
1 Gamayun 2,5±0,4 18,3±1,5
2 Istok 2,2±0,3 6,5±0,5
3 Bryanskiy Agat 2,2±0,1 11,0±1,4
4 Etyud 1,3±0,1 11,0±2,0
5 5-4-3/08 2,8±0,3 7,2±0,3
6 5-4-2/08 2,0±0,1 6,9±0,5
7 63-35-1 1,4±0,1 7,8±0,7
8 33-27-1 1,3±0,1 10,0±0,3
9 55-41-5 2,3±0,2 12,0±0,7
10 9-36-1/02 2,5±0,1 5,9±0,3
11 28-03-2 1,6±0,1 8,4±0,6
12 62-03-06 2,4±0,1 8,8±1,1
13 62-03-7 2,3±0,3 8,5±0,6
To stimulate the currant plants rooting cultivated in vitro the growth regulators of auxin nature (IAA, IBA) are added in the nutrient medium and, besides, the mineral components content is reduced twice.
We have noted that the black currants also can form roots easily in the hormoneless medium, especially when working with large plants. Therefore microcuttings of the currant cultivars propagated at the final stage of the in vitro cultivation were placed on a twice diluted medium without auxin, with addition of vitamin and mineral complex «Complivit2 at concentration of 2 g/l to enhance the growth processes of the variety.
After rooting the currant plants start to grow rapidly, forming large leaf laminae with characteristic morphology for currants. Therefore, on the basis of the results obtained on the thirteen currants genotypes it can be stated that the index of plant height is more important than the propagation factor, because even at the low propagation rate in currant varieties more than one hundred plants can be obtained from a parent plant by using short-durable subcultivations.
CONCLUSIONS
1. The height of the regenerating shoots and their number in black currant depends on the genotype at the introduction of the culture in vitro and proliferation stages.
2. The genotype affects the index of the rooting and establishment of plants to a lesser extent at rooting and adaptation stages.
REFERENCES
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