Biotechnological approaches to the reproduction of remontant forms of red raspberry

Rubus idaeus L. is one of the oldest and most widespread berry crops, which is cultivated for the excellent taste as well as for medical and dietary properties of berries. Rubus fruits contain a significant amount of vitamins A and C, anthocyanins, polyphenolic substances, which determines their high antioxidant activity. Remontant red raspberry forms are able to berry on annual shoots in the second half of summer, which extends the term of consumption of fresh berries by 1.5-2 months. However, many forms of remontant raspberry have a low potential for vegetative propagation compared to summer varieties, which makes them difficult to reproduce and to use in the breeding process. We investigated the possibility of increasing the efficiency of in vitro micropropagation of a remontant raspberry variety ʻBiryulevskaya’. The effects of 6-benzylaminopurine (6-BAP) at concentrations of 0.5–3 mg l and thidiazuron (TDZ) at concentrations of 0.05–0.2 mg l as well as doubled and tripled iron chelate Fe-EDTA (Ferric ethylenediamine-tetraacetic acid) doses were studied. We found the adding 1.0 mg l 6-BAP to the MS medium containing a triple dose of iron chelate, provided intensive proliferation of high quality adventitious shoots.


Introduction
The raspberry (Rubus idaeus L.) belongs to the Rosaceae family. It is one of the oldest and most widespread berry crops, which is cultivated for the excellent taste as well as for medical and dietary properties of berries. Raspberry enters the fruiting season very early. The next year after spring planting, it already gives the first berries, and after another year, the yield is greatly increased. Berries are consumed fresh. They serve as valuable raw materials for the food and confectionery industry. In addition, they are used for drying and freezing. When frozen, raspberry fruits retain their taste, aroma and all useful properties. Rubus fruits contain a significant amount of vitamins A and C, anthocyanins, polyphenolic substances, which determines their high antioxidant activity (McGhie et al., 2002;Moyer et al., 2002;Çekiç and Özgen, 2010;Lee et al., 2012). Raspberry is an effective medicine against many diseases. Due to the valuable biochemical composition of the berry, it is successfully used for the treatment and prevention of cardiovascular, gastrointestinal, skin and other diseases, as well as vitamin deficiencies. In addition, it is shown that аppreciable amounts of polyphenols and dietary fiber in red raspberry suggest metabolic benefits for people at risk for diabetes mellitus (Xiao et al., 2017). Remontant raspberry forms are unique berry plants, which, unlike ordinary raspberry varieties, are capable of berrying on annual shoots in the second half of summer. Cultivation of remontant raspberry varieties, in addition to varieties of the usual type, allows us to extend the period of consumption of fresh berries by 1.5-2 months. When selecting varieties of different ripening periods, you can create a continuous conveyor of fresh raspberry berries from late June to October (Ivanova-Khanina, 2014). The best of modern varieties of remontant type have high yield, large-fruited, ecological adaptability, suitable for low-cost technologies of cultivation (Evdokimenko, 2009). However, many forms of remontant raspberry have a low potential for vegetative propagation compared to summer varieties, which makes them difficult to reproduce and to use in the breeding process. The using the method of clonal micropropagation allows to solve the problem of accelerated reproduction of valuable breeding material. Compared with traditional methods of raspberry reproduction by layering and bush division as well as root, leafy and green cuttings (Dziedzic and Jagła, 2013;Marchi et al., 2018) this method has a number of undoubted advantages. The main ones are the high reproduction rate and the possibility of improving planting material from a number of harmful microorganisms, including viral infection (Tsao et al., 2000). Over the past decades, numerous studies have been conducted in different countries to improve the method of clonal micropropagation in order to produce high-quality raspberry planting material. It is known that the coefficient of reproduction of plants in culture in vitro depends on the genotype, the composition of the nutrient medium, the physical conditions of cultivation, the stability of the reproduction process when subculturing shoots (Stoevska et al., 1995;Mezzetti et al., 1997;Tsao and Reed, 2002;Gajdosova et al., 2006;Zawadska and Orlikowska, 2006a;Clapa et al., 2008;Wu et al., 2009;Reed, 2014, 2015;Hunková et al., 2016;Borodaeva et al., 2017). However, the biological features of the remontant forms of raspberries, associated with their complex interspecific origin, have led to the low effectiveness of the proposed biotechnological methods for the reproduction of raspberries at some stages of in vitro cultivation. In this regard, it became necessary to optimize the process of clonal micropropagation of remontant forms of raspberries. In vitro cloning involves several stages, the main of which are the introduction of explants into a sterile culture, micropropagation, in vitro rooting and adaptation of regenerants to ex vitro conditions.
To increase the efficiency of the method, it is necessary to improve the technology for all of the above steps (James et al., 1980;Shornikov et

Materials and Methods
The object of the study was the variety of red raspberry (Rubus idaeus L.) ʻBiryulevskaya'. This variety was chosen due to their high yield, large-fruited, as well as the possibility of use in home gardens. Regenerant plants were placed on the Murashige and Skoog medium (MS) (Murashige and Skoog, 1962) with doubled and tripled iron chelate (FeEDTA) concentration supplemented with various growth regulators. The effects of cytokinins 6-benzylaminopurine (6-BAP) at concentrations of 0.5-3 mg l -1 and thidiazuron (TDZ) at concentrations of 0.05-0.2 mg l -1 were studied. The control was a hormone-free medium with a single dose of iron chelate. Cultivation was carried out at 24 ± 1 ºС, in a photoperiod of 16/8 hours. The duration of the passage was 25-30 days. We estimated the number of shoots (pcs./explant); height of the shoot (mm); the number of leaves on the shoot (pcs.); presence of callus + \ -. The experiment was performed in 5 replicates. Five explants per each replicate were used. Mean values were compared according to least significant differences test (LSD) at P < 0.05. Statistical data processing was performed using the software package Microsoft Office Excel 2007. The studies were conducted in the Altai Center for Applied Biotechnology of the Altai State University (Barnaul, Russia).

Results and Discussion
The essential point of ensuring active proliferation of microshoots in vitro is the correct choice of cytokinin and its concentration.
Previously, we found that 6-BAP and TDZ were the most effective growth regulators of the cytokinin series. In this experiment, we revealed that the MS medium with 1.0 and 1.5 mg l -1 of 6-BAP provided the highest reproduction rate of the ʻBiryulyovskaya' variety (2.6 ± 0.3 and 2.7 ± 0.4 pcs./exp. respectively) ( Table 1). The height of the shoot was 22.6 ± 2.4 and 21.7 ± 2.3 mm; the number of leaves on the shoot was 13.2 ± 0.9 and 12.9 ± 1.2 pcs. which was also the maximum result. Adding 6-BAP to the nutrient medium, regardless of the concentration, increased the number of shoots in comparison with the control variant. The use of higher doses of this cytokinin (2-3 mg l -1 ) resulted in a decrease in the reproduction rate of remontant raspberry. Note: Data are in the form of mean ± SEM, and means followed by the same letter within the columns are not significantly different at P < 0.05.
In addition, when higher concentrations of 6-BAP were added to the nutrient medium (within 2.5-3.0 mg l -1 ), we observed anomalous development of microshoots. Morphological disorders included the formation of twisted leaves, vitreous organs with signs of hyperwatering, shortened deformed stems. It is known that shoots with signs of vitrification during in vitro reproduction produce the same plants. They rarely take root and, as a rule, can only live in vitro. Even with a slight manifestation of the degree of vitrification, it is very problematic to transfer such plants to non-sterile conditions. When used TDZ, almost the same multiplication rate was observed as with 6-BAP. The medium containing 0.1 mg l -1 of the hormone was the most productive. However, it was noted callus formation at the base of the shoot, which is undesirable during micropropagation, as it is difficult for nutrients to enter the plant. This is confirmed by a decrease in the height of the shoots and the number of leaves. Similar results were obtained by other authors (Kulkhanova et al., 2012). Zawadzka and Orlikowska (2006a) recommended TDZ at a concentration of 0.1 mg l -1 as the most effective cytokinin for direct regeneration of adventitious shoots from leaf explants. The authors found that at higher TDZ concentrations, the number of regenerated shoots did not increase, while excessive hyperhydricity and dying were observed. One of the most important components of nutrient media is iron, because it participates in regulatory processes, in redox transformations and is a part of the coenzymes. Its role is extremely important for the chlorophyll biosynthesis. Therefore, iron deficiency strongly limits plant growth and provokes the development of chlorosis. It also changes the structure of chloroplasts and affects the efficiency of photosynthesis. Iron is most often used in the chelated form of Fe-EDTA (Ferric ethylenediamine-tetraacetic acid). Some early papers reported addition of a double amount of Fe-EDTA to MS or to Anderson medium (Anderson, 1980) to reduce chlorosis (Sobczykiewicz, 1984). Zawadzka and Orlikowska (2006b) used MS salts with MgSO 4 increased by 200% and Fe-EDTA increased by 50% as the basic medium for propagating various red raspberry varieties. In some varieties, these additives prevented chlorosis for up to 3-4 weeks. They also investigated the effect of Fe-EDDHA (Ethylenediamine di-2-hydroxy-phenyl acetate ferric) as an alternative to Fe-EDTA on the shoot multiplication and adventitious regeneration from leaf explants as well as on the rooting and acclimatization of 5 varieties of R. ideaus. The change in Fe-EDTA to Fe-EDDHA in the medium increased the availability of iron, which led to a decrease in tissue hyperhydration and an increase in the level of chlorophyll in the leaves, earlier and more abundant rooting, higher fresh and dry weights. As a result, they managed to significantly improve the quality of microshootings Orlikowska, 2006b, 2009). We studied the effect of different doses of iron chelate on the effectiveness of clonal micropropagation of remontant red raspberry ʻBiryulyovskaya'. Single, double and triple concentrations of this substance were added to the nutrient medium MS containing 1.0 and 1.5 mg l -1 of 6-BAP. The maximum multiplication rate was achieved with a combination of 1.0 mg l -1 cytokinin with a triple dose of iron chelate (Figure 1). The number of microshoots per explant averaged 4.2, varying from 3 to 5. Reproduction of remontant raspberry ʻBiryulyovskaya' on MS medium with a triple dose of iron chelate and 1.5 mg l -1 6-BAP was also successful, but somewhat inferior to the previous version. We managed to get an average of 3.6 microshoots per explant. It should be noted that with the increased iron content, well-developed shoots with large leaves of dark green color were formed (Figure 2). Shornikov et al. (2010) found that for raspberries and raspberry-blackberry hybrids a similar effect can be achieved by doubling the content of iron chelate in MS medium, while iron deficiency provoked the development of chlorotic shoots.

Conclusion
So we found the adding 1.0 mg l -1 6-BAP to the MS medium containing a triple dose of iron chelate Fe-EDTA, provided intensive proliferation of high quality adventitious shoots of remontant red raspberry ʻBiryulyovskaya'. The use of higher doses of this cytokinin (2-3 mg l -1 ) resulted in a decrease in the reproduction rate of the variety. The medium containing 0.1-0.2 mg l -1 of TDZ induced callus at the base of the shoots decreasing their height and the number of leaves.