Improvement of the elements of technologyof micropropagation Cornus mas L.

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Improvement of the elements of technologyof micropropagation Cornus mas L.

Filipova L., Matskevych V., Cand. of Agricultural Sciences

Bila Tserkva National Agrarian University

Існує ряд проблем з розмноженням і поширенням кизилу - надзвичайно цінної плодової культури в Україні. Перспективний спосіб розмноження кизилу - застосування мікроклонального розмноження in vitro. Шляхом ряду експериментів нами встановлено технологічні прийоми, які дозволяють удосконалити процес мікроклонального розмноження Cornus mas L. (сорти Ніжний та Екзотичний) на етапі введення в асептичну культуру: 1) відбір експлантів у фазу зелений конус; 2) деконтамінація препаратом Бланідас 300 (7 г/л автоклавованого дистиляту); 3) відбір експлантів з бруньок медіальної частини пагона; 4) вирощування донорних рослин в умовах депозитарію; 5) сумісне застосування як антиоксидантів аскорбінової кислоти (15 мг/л) та полівінілпіролідону (0,5 г/л) способом додавання їх у живильне середовище.

Ключові слова: in vitro, антиоксидант, деконтамінант, експлант, кизил, рослина-донор.

Совершенствование элементов технологии микроклонального размножения Cornus mas L.

Существует ряд проблем с размножением и распространением кизила - ценнейшей плодовой культуры в Украине. Перспективным способом размножения кизила есть применение микроклонального размножения in vitro. Путем ряда экспериментов нами установлены технологические приемы, которые позволяют усовершенствовать процесс микроклонального размножения Cornus mas L. (сорта Нежный и Экзотический) на этапе ввода в асептическую культуру: 1) отбор эксплантов в фазе зеленый конус; 2) деконтаминация препаратом Бланидас 300 (7 г/л автоклавированного дистиллята); 3) отбор эксплантов из почек медиальной части побега; 4) выращивания донорных растений в условиях депозитария; 5) совместное применение как антиоксидантов аскорбиновой кислоты (15 мг/л) и поливинил- пирролидона (0,5 г/л) способом добавления их в питательную среду.

Ключевые слова: in vitro, антиоксидант, деконтаминант, эксплант, кизил, растение-донор.

Improvement of the elements of technology of micropropagation Cornus mas L.

L. Filipova, V. Matskevych

There is a number of problems with the reproduction and spread of cornel - extremely valuable fruit crop in Ukraine. A promising way of cornel reproduction is the use of in vitro microclonal propagation.

Through a number of experiments, we have established a number of technological techniques, that can improve the process of microclonal propagation of Cornus mas L. (varieties Nizhnyi and Exotic) can be improved of at the stage of introduction into an aseptic culture by the following technological methods: 1) the explants selection in the phase of "green cone"; 2) decontamination with Blanidas 300 preparation (7 g/l of autoclave distillate); 3) selection of explants from the buds of the medial part of the shoots; 4) cultivation of donor plants under the conditions of the depositary; 5) combined use of ascorbic acid antioxidants (15 mg/l) and polyvinylpyrrolidone (0.5 g/l) through their adding to the nutrient medium.

Key words: in vitro, antioxidant, decontaminant, explant, cornel, donor plant.

Introduction

Cornel (dogwood), also referred to as cornelian cherry (Cornus mas L.), a European species, is the only edible of many other species of cornel [1, 2]. The plants of this genus are common in the Eastern and Southern Europe, the Caucasus, Asia Minor, China and Japan. In Ukraine, cornel occupies nets to the most important place among the rare crops due to its unique consumer, medical, technical and other values, as well as to its undemanding to growing conditions. Cornel plants have high decorative qualities due to their early and abundant flowering, dense intensively green leaves, bright and beautiful fruits. They can withstand the cutting well and are resistant to dust and gases. They are used as a hedge in sunny and semi-shadowed areas. The wood is solid, very good, highly valued as a material for joinery and lumber. Cornel has wide ecological amplitude, it can grow in a variety of conditions, spread in the mountains to an altitude of 1000-1500 meters above sea level, grow on any soil.

Cornel is grown in Ukraine on private plot mainly s. The largest in Ukraine and the world industrial cornel orchard of 10.5 thousand trees grown on 14 hectares is located on basis of "Tokmatske 2010" farm. An important fact is that the seedlings were purchased in the Crimea, which makes problems with the purchase of planting material nowadays.

Cornel is propagated mostly by vegetative method in amateur horticulture [3, 4], but propagation by seeds is also quite possible. In propagation by seeds, unstratified seeds sprout only in two years, what is more, not all the seeds sprout. The stratified seeds sprout a year after sowing, but the trees fruit only in 7-10 years. In the case of reproduction the two years old seedlings are grafted by occulation and a yearling can be obtained by fall under appropriate care. Thus, a grafted year-old cultivated cornel seedling, which gives the signal yield a year after planting in the garden, can be obtained 5-6 years after the seeds harvesting. Consequently, there are a number of problems with the reproduction and spread of this extremely valuable fruit crop in Ukraine. A promising way of fruit crops reproduction, cornel in particular, is the use of in vitro microclonal reproduction. This technology will reduce the cost of cornel seedling material, reduce the period of seedlings growing from 5-6 to 1-2 years and the seedlings will yield in the second year of cultivation. At the same time, microclonal propagation of trees is complicated as compared to herbaceous plants. Biotechnologists face the problems with endogenous contamination, self-poisoning with phenolic exudate, and hyperhydration of plant tissues at the very first stage [5, 6]. There is no exception to the aseptic culture of cornel [7, 8]. Therefore, it is urgent to develop a protocol for cornel accelerated breeding which allows to obtain up to 1 million seedlings per year from one mother plant of cornel.

The aim of the research is to develop elements of the industrial technology of cornel microclonal propagation.

The objective the research - to develop advanced technological techniques based on experimental studies on Cornus mas L microclonal propagation at the stage of introduction into the aseptic culture.

Materials and methods. The experiment was carried out in the laboratory of MKR NPO Prime- Agro Ltd. Nizhnyi and Exotic varieties of Cornus mas L. (selected by Klymenko S.V., Grishko National Botanic Garden) were used for the study.

Technological process of aseptic cultivation has been investigated at the stage of introduction into aseptic culture.

For cultivation, an agar medium was used according to the MS (Murazig and Skoog) in its own modification. Modification of the medium involved changes in the macroelements amount (NH₄NO₃ 1250 mg/l; KNO₃ 1100 mg/l; MgSO₄x7H₂O 770 mg/l; KH₂PO₄ 970 mg/l; CaCl₂ was replaced by Ca(NO₃)₂ 440 mg/l; Ferum and Chelating agent was replaced with Ferylen Fe-EDDHA (ferum by the Italian firm Valagro) in the amount of 183.4 mg/l; ascorbic acid content made 3 mg/l. Light emitting diodes were used for light with the intensity range of 1.8-2.2 kL.

The explants were placed by 5 pcs in 200 ml jars (volume of nutrient medium - 20-22 ml), covered with transparent, suitable for autoclaving polypropylene lids (manufacturer Selena, Smila).

The following substances were used as decontaminants: sodium hypochlorite (control), mercuric cloride, PPM biocide, Blanidas 300. The study of the influence of the donor plants cultivation conditions on the number of decontaminated explants was carried out in open soil and in the depository.

The effect of ascorbic acid, L-cysteine, polyvinylpyrrolidone and activated carbon on the effectiveness of antioxidants was studied. The findings of the research were based on a continuous sampling (100 plants of each variant in a triple repetition).

Research results

Obtaining aseptic plants is the first stage of microclonal propagation. Its success predetermines whether the plant object (sort, breeding number) will be involved in microclonal reproduction. We have investigated different factors influencing the release of primary explants from contaminating microflora: decontaminating substances, explants types, explants selection terms, conditions for donor plants cultivation.

The output of sterile and live explants was influenced by both different decontaminants and the biological features of the cornel variety. Among the studied variants of decontaminant, the least amount of live and sterile cuttings was obtained for the primary explants processing by mercuric cloride (Table 1). In the Nizhnyi variety, 4 % took roots, of which only 2 % were microorganisms free; 2 % took roots in the Exotic variety with no decontaminated explants.

The best results were obtained on the control variant, which provided the use of a solution of sodium hypochlorite. In the Nizhnyi variety, 8 % took roots with 4 % of decontaminated explants. High rates were obtained for daily processing in a 50 % solution of PPM biocide. However, the use of this option has the following disadvantages: firstly, the high cost of the drug, and, secondly, the staff must be available all day long while the shaker is working. The variant with dipping into the nutrient medium was unacceptable due to the death of most explants with hypoxia.

The most effective variant was the domestic Blanidas 300 preparation (7 g / l autoclave distillate). The explants had no burns. The percentage of live explants output made 81 % in the Nizhnyi variety and 44 % in the Exotic variety; the yield of decontaminated explants made 32 % and 18 % respectively.

Table 1 - Decontaminants effect on sterile primary explants output (average of three repetitions)

"N" corresponds to the name of the Nizhnyi variety of cornel

** "E" corresponds to the name of the Exotic variety of cornel

Regarding the different periods of primary explants selection for their sterility (Table 2), the highest selection efficiency in the phase of the "green cone" is found out. Thus, in the Nizhnyi variety, there were 81 % of live explants, of which 44 % were sterile; in the Exotic variety the figures made 32 % and 18 % respectively. The selection in tranquility state - both deep and forced turned to be ineffective.

Table 2 - Effectiveness of different periods of primary explants selection for the output of sterile ones (decontamination Blanidas 300, average of three repetitions)

* "N" corresponds to the name of the Nizhnyi variety of cornel

** "E" corresponds to the name of the Exotic variety of cornel

The results of the study of the influence of origin of buds from the donor plant shoots reveal a low efficiency of meristem explants decontamination (Table 3). In both varieties, the number of live explants made 6-7 %, of which only 6 % were infection free. The highest yield of live and sterile plants was obtained under selection of explants from the medial buds - 80 % and 35 % in the Nizhnyi variety, and 46 % and 16 %, respectively, in the Exotic variety.

Table 3 - Influence of cornel explant type on decontamination efficiency

* "N" corresponds to the name of the Nizhnyi variety of cornel

** "E" corresponds to the name of the Exotic variety of cornel

The second stage of the research was to study the influence of conditions for the donor plants cultivation on the number of decontaminated explants among the live ones (Fig. 1). The best variants of the previous experiments were used for this experiment, namely: the selection of explants into the phase of the "green cone", the treatment with Blanidas 300 preparation, the selection of explants from the buds of the medial part of the shoot. High decontamination effect under conditions of growing in the depository was established for both types as compared to growing in the open soil . Conditions of donor explants cultivation.

Fig.1. Influence of conditions of donor explants cultivation on the efficiency of decontamination, %.

Cornel plants, like most tree crops, are characterized by phenol-like substances release in the wound in order to protect the plant against pathogens that can get into injured areas. These substances are oxidized to tannins in the air (for example, gallic acid) and become toxic to microorganisms. In an open environment, tannins are not toxic to the plant. However, for in vitro cultivation in small closed volumes (e.g. in a testtube), plants are self-destructed by their own phenol-like exudate [5, 9]. Therefore, it is relevant to find the conditions that would minimize the problem during the stabilization period. For this purpose, we investigated the effectiveness of antioxidant use (in pre-selected optimal concentrations: ascorbic acid - 15 mg/l; L-cysteine - 5 mg/l; polyvinylpyrrolidone - 0.5 g/l; activated carbon - 1.0 g/l).

Having compared the effect of adding antioxidants, we established their different activity, which was displayed in reducing the percentage of explants with phenolic exudate (Fig. 2). Cysteine adding proved to by inefficient as an antioxidant. A significant reduction in the percentage of explants with phenolic exudate was observed in variants where polyvinylpyrrolidone and co-administration of ascorbic acid with polyvinylpyrrolidone were added.

Fig. 2. Effect of antioxidants adding to the nutrient medium on the number of primary explants with phenolic exudate, %.

*«asc. acid+pvp» corresponds co-administration of ascorbic acid with polyvinylpyrrolidone

Conclusions

The results of the research reveal that the process of microclonal propagation of Cornus mas L. (varieties Nizhnyi and Exotic) can be improved of at the stage of introduction into an aseptic culture by the following technological methods: 1) the explants selection in the phase of "green cone", 2) decontamination with Blanidas 300 preparation (7 g/l of autoclave distillate), 3) selection of explants from the buds of the medial part of the shoots, 4) cultivation of donor plants under the conditions of the depositary, 5) combined use of ascorbic acid antioxidants (15 mg/l) and polyvinylpyrrolidone (0.5 g/l) through their adding to the nutrient medium.

List of references

1. Morphological Characteristics of Best Cornelian Cherry (Cornus mas L.) Genotypes Selected in Serbia / S. Bijelic, B. Golosin, J. NiniC-Todorovic, S. Cerovic // Genet. Resour. Crop Ev., 2011.- 58 (5). - P. 689-695.

2. Antioxidant, Physical and Chemical Characteristics of Cornelian Cherry Fruits (Cornus mas L.) at Different Stages of Ripeness / K. Gunduz, O. Saracoglu, M. Ozgen, S. Serce // Acta Sci. Pol. Hortorum Cultus, 2013. - 12 (4). - P. 59-66.

3. A Comparison of Grafting Methods for the Production of Quality Planting Material of Promising Cornelian Cherry Selections (Cornus mas L.) in Serbia / S. M. Bijelic, B. R. Golosin, S. B. Cerovic, B. V. Bogdanovic // J. Agr. Sci. Tech., 2016. - Vol. 18. - P. 223-231.

4. Hassanpour H. Propagation of Iranian Cornelian Cherry (Cornus mas L.) by Rooted Stem Cuttings / H. Hassanpour, M. Ali Shiri // Not Sci Biol, 2014. - 6(2). - P.192-198.

5. Кушнір Г. П. Мікроклональне розмноження рослин: теорія і практика: моногр. / Г. П. Кушнір, В. В. Сарнаць- ка; Ін-т фізіології рослин і генетики НАН України. - К.: Наук. думка, 2005. - С. 242-270.

6. Kumar K. Morphophysiologicals problems in acclimatization of micropropagated plants in - ex vitro conditions- A Reviews / K. Kumar, I.U. Rao // J. Ornamental and Horticultural Plants. - 2012. -№ 2. - Р. 271-283.

7. Cabe P.R. Dinucleotide microsatellite lociisolated from flowering dogwood (Cornusflorida L.) / P.R.Cabe, J.S. Liles // Mol. Ecol. Notes, 2002.- №2. - P. 150-152.

8. Durkovic J. Adventitious rooting performance in micropropagated Cornus mas / J. Durkovic, J. Bukovska // Biologia plantarum. - 2009. - 53 (4). - P.715-718.

9. Micropropagation of Buddleja cordata and the content of verbascoside and total phenols with antioxidant activity of the regenerated plantlets / M.E. Estrada-Zuniga, R.C. Aarland , F. Rivera-Cabrera et. al) // Revista Mexicana de Ingenier'ia Qu'imica, 2016. - Vol.15, No. 2. - P. 333-346.

REFERENCES

1. Bijelic, S., Golosin, B., Ninic-Todorovic, J., Cerovic, S. Morphological Characteristics of Best Cornelian Cherry (Cornus masL.). Genotypes Selected in Serbia. Genet. Resour. Crop Ev., 2011,58 (5), pp. 689-695.

2. Gunduz, K., Saracoglu, O., Ozgen, M., Serce, S. Antioxidant, Physical and Chemical Characteristics of Cornelian Cherry Fruits (Cornus masL.) at Different Stages of Ripeness. Acta Sci. Pol. Hortorum Cultus, 2013, 12 (4), pp. 59-66.

3. Bijelic, S. M., Golosin, B.R., Cerovic, S.B., Bogdanovic, B.V. A Comparison of Grafting Methods for the Production of Quality Planting Material of Promising Cornelian Cherry Selections (Cornus mas L.) in Serbia. J. Agr. Sci. Tech, 2016, Vol. 18, pp. 223-231.

4. Hassanpour H., Ali Shiri, M. Propagation of Iranian Cornelian Cherry (Cornus mas L.) by Rooted Stem Cuttings. Not Sci Biol, 2014, 6(2), pp. 192-198.

5. Kushnir, H.P., Sarnatska, V.V. (2005). Mikroklonalne rozmnozhennia roslyn: teoriia i praktyka [Microclonal plant propagation: theory and practice]. In-t fiziolohii roslyn i henetyky NAN Ukrainy [Institute of Plant Physiology and Genetics National Academy of Sciences of Ukraine]. Kyiv, Nauk. dumka, pp. 242-270.

6. Kumar, K., Rao, I.U. Morphophysiologicals problems in acclimatization of micropropagated plants in - ex vitro conditions- A Reviews. Ornamental and Horticultural Plants, 2012, no. 2, pp. 271-283.

7. Cabe, P.R., Liles, J.S. Dinucleotide microsatellite lociisolated from flowering dogwood (Cornusflorida L.). Mol. Ecol. Notes, 2002, no. 2, pp. 150-152.

8. Durkovic J., Bukovska, J. Adventitious rooting performance in micropropagated Cornus mas. Biologia plantarum, 2009, 53 (4 ), pp. 715-718.

9. Estrada-Zu'niga, M.E., Aarland, R.C., Rivera-Cabrera, F., Bernabe-Antonio, A., Buendia Gonzalez, L., Cruz-Sosa, F. Micropropagation of Buddleja cordata and the content of verbascoside and total phenols with antioxidant activity of the regenerated plantlets. Revista Mexicana de Ingenier'ia Qu'imica, 2016, Vol.15, no. 2, pp. 333-346.

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