Selection of the optimal concentration of indole-3-butyric acid (IBA) in the nutrient medium for effective micropropagation of Populus  berolinensis

. The study describes the features of the effective micropropagation of Populus berolinensis using indolyl-3-butyric acid (IBA). Internode sections without axillary buds are the most frequently used type of explant for Agrobacterium-mediated genetic transformation of woody plants. The application of growth regulators, such as indolyl-3-butyric acid (IBA), allows to increase the length of the internodes and thereby solve the problem of the availability of a sufficient amount of material for genetic transformation. Our results showed that 0.15 mg L -1 of IBA in the nutrient medium is the most suitable concentration for the effective micropropagation of Berlin poplar. Shoots rooted or regenerated from axillary buds in case of replanting of the rooted stump at medium supplemented with IBA in mentioned concentration, have expanded internodes in comparison with the control group of plants and may be successfully used as a good source of internode sections in Agrobacterium-mediated plant transformation.


Introduction
Micropropagation of plants is a powerful tool for the rapid increase of planting material and the preservation or multiplication of rare genotypes of woody or hardly breading plants [1][2].Using the exogenous growth regulators makes possible not only to control the speed of plant growth and development but also change the percentage-mass portion of certain organ in the whole plant organism [3].This property is very useful in the genetic engineering of plants, where it is often necessary to quickly obtain a strictly defined type of tissue that is most effectively subjected to Agrobacterium-mediated genetic transformation.In particular, a number of studies have shown that not all tissue types of a plant may be effectively modified by Agrobacterium-mediated genetic transformation, which often occurs in a species-or/and tissue-specific manner [4].
An important parameter for transgenesis is the physical amount of the organ or tissue in plant organism which is suitable for genetic transformation.In particular, some plants in vitro have an extensive root system, others are characterized by a large number of leaves, and others have very long leaf petioles or internodes.Often, the tissue that is maximally suitable for genetic transformation is found in insufficient quantity and it is required either to increase the quantity of plant samples or to regulate plant growth with exogenous phytohormones to increase the amount of the desired tissue.In such a way, applying the effective concentrations of exogenous growth regulators allows to increase roots length and quantity, internodes length, leaves size and other parameters helping to obtain the sufficient quantity of material for genetic transformation.
Another aspect of the preparation for genetic transformation of plants is the maximum simplification and unification of the process of target tissue growth in the necessary amount.Plants grown in the presence of high concentrations of exogenous growth regulators, e.g.auxins and cytokinins, in nutrient medium may have undesirable phenotypic effects as well as their explants with shifted hormonal balance may demonstrate unpredictable directions of regeneration after Agrobacterium-mediated transformation.Therefore, it is important to choose minimal concentrations of growth regulators that would effectively contribute to rapid micropropagation, high output of the target tissue, and have a nonessential effect on hormonal balance during plant regeneration after Agrobacteriummediated genetic transformation.
Among woody plants, representatives of the genus Populus are most often the object of genetic transformation [5][6].Poplars grow well and quickly in vitro, are easily exposed to cuttings and regeneration, which makes them an efficient model system for developing the technique of micropropagation and genetic transformation of woody plants.The most frequently used type of explant for Agrobacterium-mediated genetic transformation of poplars are internode sections without axillary buds.Because of the direct morphogenesis of new plants from the internode cells, the chance of the appearance of chimeric plants decreases, which in turn often occurs if callus using for genetic transformation [7].
In case of a nutrient medium with no growth regulators, in vitro poplars have fairly short internodes (3-4 mm), which complicates internode sections cutting and increases the time spent on preparing the necessary amount of plant material for genetic transformation.The application of growth regulators, such as indolyl-3-butyric acid (IBA), allows to increase the length of the internodes [8] and thereby solve the mentioned problem.
In such way, our study is aimed to select the optimal concentration of indolyl-3-butyric acid (IBA) in the nutrient medium for maximally efficient and rapid micropropagation of poplar with expanded internodes length.

The plant material
Berlin poplar (Populus  berolinensis K. Koch) is one of the convenient objects for woody plant micropropagation and genetic transformation.It has a high percentage of rooting (up to 100%) after the cutting during in vitro cultivation, takes up little space in test tubes due to narrow leaves (in vitro only), and also easily regenerates from roots and internodal segments.Relatively fast growth (25-30 days from rooting to cutting) makes berlin poplar a very useful object for genetic transformation.
For the study, we used Populus  berolinensis from our in vitro collection set up in 2012 using as a donor the tree growing in the arboretum of Siberian Institute of Plant Physiology and Biochemistry.Ten years of poplar in vitro cultivation were done by sequential cutting and rooting of the apical parts of the plant with simultaneous replantation of the rooted stump to the new nutrient medium (Figure 1) without growth regulators.Following this approach, up to 4-5 rooted poplars from one plant per 30 days may be obtained.

Nutrient media preparation and experiment design
Nutrient media for rooting, cultivation and micropropagation was prepared according to the composition of Murashige and Skoog [9] basal salt mixture manufactured by Sigma-Aldrich (MS5524).The full base medium was supplemented by thiamine (1 mg L -1 ), pyridoxine (0.5 mg L -1 ), nicotinic acid (0.5 mg L -1 ), and meso-inositol (50 mg L -1 ).Sucrose (2%) was used as a source of carbohydrates.To obtain a solid medium, agar (A7002, Sigma-Aldrich) was used at a concentration of 7 g L -1 .The acidity of the medium was adjusted to pH 5.7.Tested rooting media contained IBA in the final concentrations of 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5 mg L -1 .Freshly prepared medium was autoclaved (15 min at 121 °C) and dispensed by 10 ml into anticipatorily autoclaved and dried borosilicate glass tissue-culture tubes (Z681784, Sigma-Aldrich) covered by semi-transparent lids (118158C, Globe Scientific Inc.).Using the approach illustrated in Figure 1 required amount of poplar plants were obtained on the nutrient medium without any growth regulators.Then, the obtained plants were divided into one control and five experimental groups.Each group contained six plants (three for rooting and three for shoots growth from axillary buds) replanted to the experimental media supplemented by IBA in different concentrations.The general experimental design is illustrated in Figure 2. The experiment proceeded for 3 months and included 3 sequential passages per each 30 days on the same medium for each group of plants.
During the experiment, in vitro plants were grown at 24 °C in an air-conditioned room with 16/8 hours of the day/night photoperiod under T5 fluorescent lamps at 5 000 Lux [10].
E3S Web of Conferences 463, 01032 (2023) EESTE2023 https://doi.org/10.1051/e3sconf/202346301032Fig. 2. Experiment design.Plants grown and rooted in the nutrient medium with no IBA were divided into two parts in manner showed in Figure 1 and replanted to the fresh media supplemented with different concentrations of IBA.

Results and discussion
Our results showed, that all tested concentrations of IBA in the nutrient medium led to 100% rooting of the cut apical parts of the Berlin poplar (Figure 3: 1A, 2A, 3A, 4A, 5A, 6A).Using IBA concentrations of 0.4 and 0.5 mg L -1 led to the formation of the highest plants from all experimental groups (Figure 3: 5A, 6A).At the same time, applying the aforementioned concentrations led to the serious necrotic processes in apical meristems and suppression of new shoots growth from the axillary buds located on rooted stump transferred into fresh nutrient media (Figure 3: 5B, 6B).Moreover, the death of the apical buds was also observed for plants obtained on nutrient media with IBA in concentrations of 0.2 mg L -1 (Figure 3: 3B) and 0.3 mg L -1 (Figure 3: 4A).
We found that the best IBA concentration, which causes optimal rooting and the growth of new shoots from the axillary buds, was 0.1 mg L -1 .The rooted plants were higher than the control plants obtained on a hormone-free medium, and also differed from the latter with more elongated internodes (Figure 3: 2A).The rooted stems that remained after cutting the apical part of the plant, transferred to a fresh nutrient medium supplemented with IBA (0.1 mg L -1 ) also showed a good ability to sprout axillary buds and to form new shoots without necrosis of the apical meristems (Figure 3 -2B).The obtained results allowed us to hypothesize that there might be another concentration of IBA, lying in the range between 0.1 and 0.2 mg L -1 , which would be more effective for the micropropagation of the Berlin poplar.To confirm this assumption, an additional experiment was carried out to study the influence of IBA at a concentration of 0.15 mg L -1 on the effectiveness of micropropagation of the Berlin poplar.The results showed that IBA at concentration of 0.15 mg L -1 in the nutrient medium did not lead to the necrosis in plants regenerated from axillary buds located on rooted stump transferred to fresh nutrient media (Figure 4B).In addition, the cut apical parts of the Berlin poplar moved to this nutrient media rooted well (up to 100%) and demonstrated good growth and the presence of elongated internodes in comparison with the control group on a hormone-free nutrient medium (Figure 4A).In the next step of the experiment, we determined the differences in the average stem length between plants rooted in a nutrient medium containing IBA (0.15 mg L -1 ) and the control group on a hormone-free nutrient medium.The results showed that poplars, rooted and grown for 30 days on a nutrient medium containing IBA (0.15 mg L -1 ) were on average almost 2 cm higher than control plants grown on a hormone-free medium (Table 1).At the same time, from a single plant grown on a medium with the addition of IBA (0.15 mg L -1 ) it is possible to obtain from 8 to 12 pieces (5 mm) of internode sections without axillary buds.Berlin poplar plants, grown on hormone-free medium, have shorter internodes, which complicates the process of cutting the stem into segments.From such plants it is possible to obtain 4-6 cuttings of internode sections (3-4 mm) without axillary buds, which is two times less than the number of internode sections obtained from plants rooted in the presence of IBA (mg L -1 ).

Conclusion
It has been shown that 0.15 mg L -1 of IBA in the nutrient medium is the most suitable concentration for the effective micropropagation of Berlin poplar.Shoots rooted or regenerated from axillary buds in case of replanting of rooted stump at medium supplemented with IBA in mentioned concentration, have expanded internodes in comparison with the control group of plants and may be successfully used as a good source of internode sections in Agrobacterium-mediated plant transformation.The other studied concentrations of IBA are suitable only for effective rooting of cut apical parts of the Berlin poplar but not for propagation from axillary buds located on the rooted stumps.

Fig. 1 .
Fig. 1.Scheme of Populus  berolinensis micropropagation combining two ways.The upper way illustrates a sequential cutting and rooting of the apical parts of the plant.The bottom path illustrates a replantation of the rooted stump containing axillary buds.

Fig. 4 .
Fig. 4. Populus  berolinensis after 3 passages of plants on the medium supplemented with IBA in final concentration 0.15 mg L -1 .Arooting of apical part of the plant after cutting, B -shoots grown from axillary buds on rooted stump.

Table 1 .
Stem length of poplar plants obtained on the two rooting media.