In vitro propagation and medium-term conservation of autochthonous plum cultivar 'Crvena Ranka'

Vujović et al. 142 (Glišić, 2015), ranging from autochthonous to newly bred ones. Indigenous cultivars are part of the Serbian tradition, customs, legacy, and cultural identity. In addition to providing the genetic basis for clonal selection, they are also used in different breeding programs aimed at developing new plum cultivars as well as new plum, apricot and peach rootstocks (Milošević et al., 2010). However, the majority of these cultivars are being seriously threatened and are slowly disappearing from orchards. Among autochthonous cultivars, ‘Crvena Ranka’ stands out as a sharka-tolerant cultivar mostly used for producing supreme quality plum brandies (Popović et al., 2015). Fruits of some local genotypes are suitable for fresh use (Milošević and Milošević, 2012). In recent years, there has been an increased interest in establishing new commercial orchards of this valuable cultivar. Therefore, it is of vital importance to develop protocols for the clonal propagation of selected genotypes to obtain a large number of true-to-type plants from a few initial plants, in the shortest period of time. The rapid production of highquality, disease-free and uniform planting stock is only possible through micropropagation. However, long-term successive subculture of in vitro plants on a fresh medium and their maintenance under normal growth conditions can be followed by a decrease in or loss of the cultures’ morphogenetic potential as well as by an increase in the possibility of genetic alterations or propagating material loss due to human errors or microbial contamination (Chauhan et al., 2019). On the other hand, tissue culture technology also enables the conservation of plant genetic resources for either short, medium or long term, depending on the requirement as well as on the technique applied (Engelmann, 1997). In vitro conservation of vegetatively propagated species such as fruit tree species is complementary to field gene banks, which, along with in situ conservation measures, provide an integrated conservation strategy (Rajasekharan and Sahijram, 2015). The aim of this paper was to establish an efficient protocol for the in vitro propagation of autochthonous plum cultivar ‘Crvena Ranka’ by optimizing multiplication and rooting stages, and to examine the possibility of mid-term conservation of this genotype using the slow growth storage method, which involved temperature reduction pooled with the maintenance of cultures under dark conditions. 2. Material and methods Plant material and establishment of aseptic culture A selected clone of autochthonous plum cultivar ‘Crvena Ranka’ (Prunus domestica L.) originating from Gledić Mountains was used as the source of initial explants for in vitro culture. Aseptic culture was established using actively growing axillary leaf buds taken from branches during the spring. The surface sterilization procedure involved washing explants under running water for 2 h, sterilization in 70% ethanol (1 min), and 15 minute-soaking in 10% (v/v) commercial bleach solution (0.4%, w/v, final concentration of sodium hypochlorite), followed by triple rinsing (5 min each) with sterile distilled water. Buds were isolated under a stereomicroscope and placed onto the Murashige and Skoog (1962) medium (MS) containing 2 mg l-1 N6-benzyladenine (BA), 0.5 mg l-1 indole-3-butyric acid (IBA) and 0.1 mg l-1 gibberellic acid (GA3). After four weeks, rates of contaminated and necrotic buds and of those which initiated sterile leaf rosettes were noted. Shoot multiplication and rooting Upon establishment of aseptic culture, single uniform shoots were multiplied on the MS medium of constant plant growth regulator (PGR) composition: 1 mg l-1 BA, 0.1 mg l-1 IBA and 0.1 mg l-1 GA3. The multiplication medium contained 30 g l-1 sucrose and 7 g l-1 agar. The pH value was adjusted to 5.7 before autoclaving at 121oC, 150 kPa for 20 min. Shoots were repeatedly subcultured five times at a constant fourweek subculture interval. Multiplication parameters, i.e. the multiplication index and lengths of axial and lateral shoots were determined upon each subculture. The multiplication index was defined as the number of newly formed axillary shoots (>0.5 cm) per initial shoot tip recorded after the stated subculture interval. To optimize multiplication, the influence of BA concentration and type of auxins [IBA or 1naphthaleneacetic acid (NAA)] on the multiplication capacity and shoot quality were examined in the sixth subculture. The PGR combinations used in this stage of micropropagation are given in Tables 2 and 3. Shoots were subcultured twice at a 28 day-interval on the medium of the same PGR composition, and therefore all parameters were determined in the second subculture. The following multiplication parameters were monitored: multiplication index, length of axial shoots, length of lateral shoots, number of leaves on axial shoots and number of leaves on lateral shoots. After removal from the medium, shoots were washed in distilled water and dried with filter paper, and their fresh weight (FW) was determined. For dry weight (DW), shoots were dried in an oven at 65–70°C for 48 h. Rooting was performed on the MS medium with mineral salts reduced to 1⁄2-strength and organic complex unchanged. Rooting treatments included two PGR combinations, as indicated in Table 4. The percentage of rooted plants was determined after 28 days along with the number and length of roots, and height of rooted plants. Each treatment in multiplication and rooting stages included 45 uniform shoots (three replicates of three culture vessels with five shoots). Shoot cultures were grown in 100 ml culture vessels containing 50 ml of multiplication or rooting medium, at 23 ± 1oC and 16-hphotoperiod (light intensity, 41 μmol m-2 s-1). Slow growth storage and repropagation The slow growth experiment was performed with shoots taken from proliferated cultures and planted on a fresh multiplication medium (previously determined to be the most suitable for propagation) in Erlenmeyer flasks closed with cellulose stoppers. Explants were placed in darkness in a growth chamber at 5 °C (cold storage, CS) and their viability was examined after three, six, and nine months. After the respective periods of CS, the cultures were transferred to a growth chamber for seven days and the viability of shoots for further propagation (percentages of fully viable shoots, partially viable shoots and fully necrotic shoots) was determined. Each treatment was performed with three Acta Agriculturae Serbica, 25 (50), 141‒147, 2020 143 replicates of five culture vessels with five uniform shoots (75 plants for each treatment). After each CS period, survived shoots were subcultured for three consecutive four-week cycles under standard growing conditions. The number of shoots per explant and their lengths were recorded at the end of the third subculture. After multiplication, cold-stored shoots were rooted on the above-described rooting media, and rooting parameters (rate of rooting, number and length of roots, and height of rooted plants) were determined. Statistical analysis All data were analyzed by ANOVA, followed by Duncan’s Multiple Range Test (P < 0.05) for means separation. The data presented in percentages were subjected to arcsine transformation. 3. Results and discussion Microbial contaminants present a major challenge in in vitro culture technology. Although most of the sterilizing agents used for the initiation and maintenance of viable in vitro cultures show toxicity to plant tissues, it is possible to minimize explant loss and achieve high survival rates by optimizing the concentration of sterilants and the duration of explant exposure to them. In our material, 70% ethanol in combination with 10% bleach, as the source of sodium hypochlorite, proved effective in sterilizing explants taken from open field-grown plants (Fig. 1a). The use of a two-step sterilization procedure has proved beneficial in certain plant species including fruit tree species (Ružić et al., 2010). However, we obtained a markedly higher rate of leaf rosette initiation (68.8%; Fig. 1a and 1b) and a lower contamination rate (8.3%) in comparison with the rates obtained by Ružić et al. (2010) in three vegetative rootstocks for cherry, plum and pear (28.3–46.9% and 48.1–71.7%, respectively), although they took initial explants from screenhousegrown plants. Possible reasons for better results in our experiment are a slightly prolonged bleach treatment (15 min in comparison with 12 min) and treatment of mother plants with fungicides just before taking initial explants. a b Figure 1. Establishment of aseptic culture (a) and initiated leaf rosettes (b) After the establishment of aseptic culture, shoots of ʻCrvena Rankaʻ were multiplied on the MS medium of constant PGR composition, previously determined to be the most optimal for the multiplication of other plum genotypes (Vujović et al., 2018). Monitoring of the regeneration ability of shoots in five successive subcultures, expressed through the multiplication index and lengths of axial and lateral shoots, revealed the increase in shoot formation capacity over repeated subcultures (Table 1). Table 1. Shoot multiplication parameters of ‘Crvena Ranka’ in five successive subcultures after rosette initiation on the MS medium containing 1.0 mg l-1 BA, 0.1 mg l-1 IBA and 0.1 mg l-1 GA3 Subculture Multiplication index Length of axial shoots (mm) Length of lateral shoots (mm) 1st 2.0 b1 9.5 5.7 c 2nd 2.1 b 10.2 6.5 abc 3rd 2.4 a 10.1 7.3 a 4th 2.2 ab 10.2 6.2 bc 5th 2.5 a 10.0 6.8 ab P < 0.05 ns P < 0.05 1Mean values of multiplication parameters followed by the same lowercase letters within the same column are not significantly different according to Duncan's Multiple Range Test A significant increase in the shoot number formed (2.4) occurred in the third subculture and remained constant afterwards. Similarly, Debnath (2004) noticed that, in dwarf raspberry, the mu