Abstract
BACKGROUND:
Amelanchier alnifolia is an attractive small fruit difficult to propagate by traditional methods. Moreover, this species can be susceptible to dormancy after transplanting to the soil.
OBJECTIVE:
The aim of this work was to evaluate factors that contribute to effective in vitro rooting and acclimatization of micropropagated shoots of Amelanchier alnifolia.
METHODS:
Different auxins, media concentrations, and sprays containing plant growth regulators were tested. The experimental data were treated by analysis of variance.
RESULTS:
1-naphthalene acetic acid (NAA) was superior for rooting compared to indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Cultivation of shoots on Murashige & Skoog (MS) medium with a half-strength concentration (1/2 MS) led to higher rooting frequencies than on full-strength MS medium. Addition of 1.5 mg l–1 spermidine to cultivation medium did not significantly improve rooting. Gibberellic acid (GA4+7) alone or in combination with 6-benzylaminopurine (BAP) did not effectively break post-rooting dormancy.
CONCLUSION:
The greatest number of actively growing plants was recorded after rooting the shoots on 1/2 MS medium with 1 mg l–1 NAA followed by a spray treatment with 1 mg l–1 BAP. These results are directly applicable for improving rooting efficiency and acclimatization of micropropagated Amelanchier spp. plantlets.
Introduction
Amelanchier alnifolia (Nutt.) Nutt. ex M. Roem. (also called saskatoon or serviceberry) is a fruit-bearing ornamental shrub from the Rosaceae family [1]. Owing to its high yields, frost-resistance, and edible berries rich in biological compounds, saskatoon is considered an attractive small fruit species for consumers [2–4]. The species is propagated vegetatively by softwood and root cuttings, but this technique was found to pose a certain risk of plant stock damage. The timing and developmental stage of the cuttings are the most important factors affecting rooting [5, 6]. Propagation by seeds results in approximately 30% of not true-to-type progeny [7].
Micropropagation offers an efficient way for overcoming these problems. However, it is necessary to mention in this connection that data on micropropagation of Amelanchier spp. are rather scarce. Apical and axillary buds are commonly used as primary explants, shoot cultivation has been carried out on medium with 0.5–3 mg l–1 6-benzylaminopurine (BAP) [7–10] and low concentration (0.01–0.1 mg l–1) of indole-3-acetic acid (IAA) [9–11]. Indole-3-butyric acid (IBA) [12, 13] and 1-naphthalene acetic acid (NAA) [14] have also been reported to be suitable for shoot multiplication. Inconsistencies exist relative to the rooting phase and subsequent acclimatization. Some authors observed rooting of shoots on hormone-free media [9, 15], others reported successful rooting only in the presence of auxin, mainly NAA [10, 16]. The auxin IBA [14, 17] or combinations of auxins [7] were used less frequently. The so-called phenomenon “post-rooting dormancy” often accompanies A. alnifolia propagation [6, 7]. In similar cases rooted plants cease growth shortly after transplantation into the soil. This type of dormancy was also described in beech (Fagus spp.), hornbeam (Carpinus spp.), lilac (Syringa spp.), and oak (Quercus spp.) [18].
Except for the auxins mentioned above, the polyamine spermidine and phytohormones of gibberelline type were shown to play a role in cell division and differentiation and plant micropropagation [19, 20]. In soybean [21] and Withania somnifera [22] spermidine induced micropropagation process and increased shoot multiplication but in both cases substance exerted its inhibitory effect on rooting. At high concentration, spermidine induced rooting of micropropagated cucumber [23]. In A. alnifolia the role of spermidine has not been examined yet. Gibberellic acid (GA4+7) has been tested in several species, including hornbeam, hazelnut, and apple, where it stimulated bud break of rooted cuttings [18]. Likewise, increased growth was observed in Stewartia pseudocammelia and witch-hazel (Hamamelis spp.) cuttings after spraying their leaves with a GA4+7 and thidiazuron (TDZ) solution [24]. Rooted A. alnifolia plantlets receiving spray treatments of GA4+7 alone or in combination with BAP successfully overcame dormancy [7].
The present study was undertaken because of inconsistencies in previous findings of some authors working on A. alnifolia and owing to repeated occurrence of the species dormancy in our laboratory. The primary objective of the study was to analyze the role of such factors as auxins, medium concentration, spermidine, and GA4+7 in in vitro rooting and acclimatization of this species.
Material and methods
Conditions for in vitro and in vivo cultivation
In vitro shoots of Amelanchier alnifolia var. cusickii were obtained from donor cultures established in vitro in 2015 [13], and used as biological material in all experiments. Briefly, donor cultures were cultivated on Murashige & Skoog (MS) medium [25] supplemented with 30 g l–1 sucrose (Slavus, Bratislava, Slovakia), 8 g l–1 plant agar, 1 mg l–1 BAP, and 0.5 mg l–1 IBA (Duchefa, Haarlem, The Netherlands). The pH of media was adjusted to 5.6 before autoclaving (20 min at 1 kg cm3, 121°C). This composition (plant growth regulators excluded) and pH of cultivation medium were applied for all variants of media used in individual experiments. All cultures were maintained in a growth chamber at 22±2°C with a 16 h photoperiod under cool white fluorescent lights at a photosynthetic photon flux density 50μmol m–2 s–1.
After performing experiments, plants were transferred to pots with soil substrate, watered, and sprayed with tap water to ease their acclimatization. They were maintained in a growth chamber room at 23°C, a 16 h photoperiod under cool white fluorescent tubes at a photosynthetic photon flux density 50μmol m–2 s–1. Further treatments are described in individual sections. Concentrations of growth regulators and spermidine for experiments were chosen according to already published data [7, 27].
Effect of different auxin and medium concentration treatments on in vitro rooting and post-rooting dormancy
Three types of auxin were examined: IBA, NAA, and IAA. Shoots were rooted on MS medium at either full-strength or half-strength concentration (1/2 MS). Hormone-free MS medium was used as a control.
Ten treatments were performed as follows: hormone-free MS medium hormone-free 1/2 MS medium MS medium + 1 mg l–1 IBA 1/2 MS medium + 1 mg l–1 IBA MS medium + 1 mg l–1 NAA 1/2 MS medium + 1 mg l–1 NAA MS medium + 1 mg l–1 IAA 1/2 MS medium + 1 mg l–1 IAA MS medium + 0.5 mg l–1 IAA + 0.2 mg l–1 NAA 1/2 MS medium + 0.5 mg l–1 IAA + 0.2 mg l–1 NAA
Each treatment consisted of 24 explants (six explants/vessel). The percentage of rooted shoots was evaluated at two week interval. Surviving explants were transplanted into soil and observed further for 11 weeks to reveal post-rooting dormancy, if any. Only plants with new leaves or a green, actively elongating shoot-tip were considered as non-dormant.
Effect of spermidine on in vitro rooting
All treatments were performed using a half-strength concentration of MS medium. Hormone-free MS medium was used as a control.
Five treatments were performed as follows: hormone-free 1/2 MS medium 1/2 MS medium + 1.5 mg l–1 spermidine 1/2 MS medium + 1 mg l–1 NAA 1/2 MS medium + 1.5 mg l–1 spermidine + 1 mg l–1 NAA 1/2 MS medium + 1.5 mg l–1 spermidine for 7 days, followed by transfer of explants to 1/2 MS medium + 1 mg l–1 NAA
Treatments consisted of 96 explants (variants 1, 3 and 4), 90 explants (variant 2), or 48 explants (variant 5). The percentage of rooted shoots was evaluated after five weeks.
Effect of acclimatization treatment on post-rooting dormancy
Each treatment from the previous experiment (section 2.3) was divided into four groups and transferred to pots with soil substrate. Each group was subjected to different acclimatization treatments with the growth regulators BAP and gibberellic acid GA4+7, both of which were applied as a spray. Sprays were applied thoroughly on the adaxial surface of plant’s leaves and shoot tip, thrice weekly for a period of 14 days. Watered plants without any further treatment were used as a control.
The four treatments were performed as follows: watering the plants with tap water watering the plants with tap water, spraying with 0.25 mg l–1 GA4+7
watering the plants with tap water, spraying with 1 mg l–1 BAP watering the plants with tap water, spraying with 0.25 mg l–1 GA4+7 and 1 mg l–1 BAP
The number of plants analyzed in individual groups for each treatment was: 12, 12, 11 and 12 (variant 1) 10, 9, 9 and 8 (variant 2) 16, 15, 15 and 15 (variant 3) 10, 9, 10 and 10 (variant 4) 6, 6, 7 and 6 (variant 5)
The number of growing plants in each treatment combination was recorded two weeks after the spray treatment ended.
Statistical analysis
The results from individual experiments were statistically evaluated by analysis of variance (ANOVA). Data were subjected to a Duncan test at p value ≤0.05 as well as a Pearson’s chi-square test at the same level of significance and evaluated using software STATISTICA, version 8.0.
Results
Auxin and medium concentration effect on in vitro rooting of A. alnifolia
Statistically significant differences were revealed between all medium types (Table 1). With the exception of hormone-free MS medium, rooting was recorded in all the variants tested (range 21–100%). The results varied greatly with respect to the concentration of media. Rooting did not exceed 30% of explants when full-strength MS medium was used, regardless of the auxin or auxin combination used. Comparison of the auxin treatments indicated that IBA and NAA were superior to IAA and the IAA–NAA combination, but these differences were not significant. Treatments with a half-strength concentration of MS medium led to significantly higher rooting efficiency, as well as to a higher mean number of rooted shoots. Interestingly, hormone-free half-strength MS medium led to higher rooting efficiency than the application of any auxin into full-strength MS medium. The best quality roots and 100% rooting were observed in explants cultivated on half-strength MS supplemented with 1 mg l–1 NAA. Again, IBA was superior to IAA and IAA–NAA, but the differences were not significant. Treatments with the same auxin or auxin combination, but different medium concentration were statistically different. Irrespective of plant growth regulators, the treatments with half-strength MS medium always led to higher rooting efficiency as well as longer and stronger roots (Fig. 1). In general, the plants from variants two – ten (Table 1) were in good condition, showing no signs of chlorosis, necrosis, or growth retardation. Plants from variant one died, most probably because of lack of growth regulators in the medium and were discarded. ANOVA analysis indicated that auxins and medium concentration as the main contributing factors were statistically significant for rooting the species (p < 0.0001 and p = 0.0324, respectively), but their interaction was not significant (data not shown).
The efficiency of different auxin growth regulators and medium concentration on in vitro rooting and acclimatization of A. alnifolia.
The efficiency of different auxin growth regulators and medium concentration on in vitro rooting and acclimatization of A. alnifolia.
Values followed by the same letter are not different at p≤0.05 according to Duncan test.
Explants of A. alnifolia after 14 days of cultivation on MS (1) or half-strength MS (2) media supplemented with 1 mg l–1 NAA. The diameter of Petri dishes was six cm.
The growth of plants was observed for 11 weeks to evaluate dormancy (Table 1). All plants from hormone-free MS (full- and half-strength concentration) died within several weeks after transplanting to pots. The abundance of dormancy among plants from half-strength MS variants containing IBA, NAA, and the IAA–NAA combination was always lower relative to plants from the full-strength MS variants. Plants previously cultivated on full-strength MS medium possessed more such impaired traits as shoot-tip necrosis, occasional leaf necrosis, and chlorosis. Under the influence of IBA, less than 25% of plants, regardless of media concentration, were actively growing after 11 weeks. In this case, dormancy was not effectively broken and most of the plants ceased growth after transplanting to the soil. The lowest abundance of dormancy was observed in plants cultivated on half-strength MS with 1 mg l–1 NAA where active growth was registered in 63% of plants. Growth rates of plants on full-strength MS media with NAA was similar to plants cultivated on full-strength MS media with IBA, suggesting that rather than auxin, the concentration of the culture medium was the key factor determining dormancy occurrence.
In plants cultivated on media with IAA, the normal growth ranged between 47–60%. Only the plants cultivated with IAA on full-strength MS medium exhibited higher growth rates relative to plants on half-strength MS medium. The effect of IAA in overcoming dormancy was superior to IBA. However, rooting efficiency of shoots under the influence of IAA was lower compared to shoots on media with NAA. The IAA–NAA combination resulted in less than 43% of growing plants, regardless of media concentration. The influence of these two auxins on dormancy exceeded the effect of IBA but was inferior to NAA and IAA.
NAA was the most suitable auxin for promoting newly transplanted A. alnifolia plantlets to overcome dormancy. Analysis with Pearson’s chi-square test (Table 2) indicates that there was a statistically significant difference between full-strength MS and half-strength MS media for acclimatizing and prolonging plant growth (p < 0.0001). Plant dormancy was not altered by auxin type independent of medium concentration (p = 0.0872). When considering only the plants grown on full-strength MS medium, the effect of different auxins was not significant (p = 0.5452). However, auxins varied in their effect on dormancy when plants were grown on half-strength MS medium (p = 0.0258).
The number of dormant and growing A. alnifolia plants grouped by medium concentration and auxin treatments
The number of dormant and growing A. alnifolia plants grouped by medium concentration and auxin treatments
Tests of independence of row and column classifications in the frequency tables were conducted with Pearson’s chi-square tests (df = degrees of freedom).
Adding of spermidine into cultivation medium did not stimulate root formation. The mean number of rooted shoots in spermidine treatments was lower compared to the control, though the difference was not statistically significant (Table 3). The best results were obtained with cultivation on medium with NAA (66.67% of shoots rooted) and addition of spermidine significantly decreased the rooting efficiency (43.75%). The last variant included a short exposure to spermidine followed by NAA which however has not differed significantly from the variant with simultaneous effects of both compounds. Thus neither joint nor individual influence of spermidine boosted the formation of roots. The explants cultivated on media containing spermidine exhibited thinner roots compared to the thicker, calli-originated roots of explants cultivated on media with NAA (Fig. 2).
The efficiency of spermidine treatment on in vitro rooting of A. alnifolia shoots cultivated on half-strength MS medium
The efficiency of spermidine treatment on in vitro rooting of A. alnifolia shoots cultivated on half-strength MS medium
Values followed by the same letter are not different at p≤0.05 according to Duncan test.
Explants of A. alnifolia after five weeks of cultivation on half-strength MS medium supplemented with 1.5 mg l–1 spermidine (1) or 1 mg l–1 NAA (2). The plant’s height was approximately five cm.
There were no differences among spermidine and other treatments for overcoming dormancy (p = 0.1523, Table 4). However, acclimatization treatments affected post-rooting dormancy (p < 0.0001, Table 4). The highest proportions of growing plants (76.92%) were obtained after a spray treatment with 1 mg l–1 BAP and in the control water treatment (75.93%). There was not a significant difference between these two groups (p = 0.0938, Table 4). After treatment with BAP, at least 70% of plants showed signs of active growth, regardless of their origin (plants from hormone-free medium vs. plants cultivated under the influence of NAA or spermidine) (Table 5). Good results were observed in the control group where at least 80% of growing plants were recorded, with the exception of those first cultivated on medium with 1.5 mg l–1 spermidine, which resulted in 30% of actively growing plants only (Table 5). Visually, plants from both the control group and BAP treatment were similar with large dark-green leaves and only minimal leaf dropping or shoot-tip drying (Fig. 3A, C).
The number of dormant and growing A. alnifolia plants grouped by different rooting and acclimatization treatments
The number of dormant and growing A. alnifolia plants grouped by different rooting and acclimatization treatments
Tests of independence of row and column classifications in the frequency tables were conducted with Pearson’s chi-square tests (df = degrees of freedom).
The effect of acclimatization treatment on the growth of A. alnifolia plantlets from different rooting media
A. alnifolia plants two weeks after a spray treatment with plant growth regulators. A – untreated plants (height 6–14 cm); B – plants sprayed with 0.25 mg l–1 GA4+7 (height 9–17 cm); C – plants sprayed with 1 mg l–1 BAP (height 4–12 cm); D – plants sprayed with a combination solution of 0.25 mg l–1 GA4+7 and 1 mg l–1 BAP (height 13–18 cm). Plants are arranged according to the rooting media on which they originated. 1 – hormone-free half-strength MS; 2 – half-strength MS with 1.5 mg l–1 spermidine; 3 – half-strength MS with 1 mg l–1 NAA; 4 – half-strength MS with 1 mg l–1 NAA and 1.5 mg l–1 spermidine; 5 – half-strength MS with 1.5 mg l–1 spermidine followed by transfer on half-strength MS with 1 mg l–1 NAA.
After the spray treatment with GA4+7 and combined treatment with GA4+7 and BAP, only 19.61% and 37.25%, respectively, of plants showed active growth. Dormancy was not broken effectively, though the two groups were significantly different (p = 0.0482, Table 4). The phenotype of gibberellin-treated plants differed greatly from control and BAP-treated plants. Plants in both gibberellin groups (GA4+7 and GA4+7 + BAP) had thin stems with elongated internodes (Fig. 3B, D). Leaves were smaller relative to the control and BAP-treated groups, they often dropped and occasionally developed chlorosis. Shoot-tips dried extensively. The fragility of stems often resulted in deformations. Comparing individual groups, the proportion of gibberellin-influenced plants that grew never exceeded 50%. A higher frequency was detected in groups treated with both GA4+7 and BAP (Table 5). Although BAP could not reverse the effect of gibberellin on the phenotype of plants, it contributed likely to the lower abundance of negative traits, such as shoot-tip drying improving growth rate.
The rooting stage is of great importance when preparing regenerated plants for their acclimatization and further distribution through micropropagation [28]. Our results indicate that the concentration of culture medium is the primary factor determining rooting efficiency and possible dormancy in A. alnifolia micropropagation. This is consistent with the results of other authors [29] who observed higher rooting efficiency of A. alnifolia shoots on hormone-free 1/8 MS medium (38%) compared to full-strength MS medium (0%). Similar to our findings, they also reported better results with 2 mg l–1 NAA (67% of shoots rooted) compared to the same concentration of IBA (33%). Some other authors [7] analyzed several types of auxins and their effect on rooting in four A. alnifolia cultivars. The best rooting rates (75–97%) were achieved after cultivation on medium with IAA–NAA combination (0.5 and 0.2 mg l–1, respectively). However, in our experiment, this combination resulted in less than 21% of shoot rooting and was therefore inferior to both NAA and IBA used individually. One possible explanation is a genotype-dependent response since rooting efficiency differed among individual cultivars in the contrasting study. In addition to auxins and medium concentration, there are other potential factors influencing the final outcome. For example, the differences were observed in rooting of A. canadensis ‘Rainbow Pillar’ shoots with variable lengths on MS medium with 0.5 mg l–1 IBA [17]. Longer shoots (3–5 cm) rooted at a higher rate than shorter shoots (less than 2 cm) when the same multiplication medium was used (97 and 17%, respectively).
The role of polyamines on root formation has been examined previously [19, 31]. One study found that 27% shoots of Malus domestica ‘MM106’ rooted when cultivated on half-strength MS medium supplemented with 10μmol l–1 spermidine [27]. Rooting efficiency with spermidine was higher in our study (40%) suggesting a species-depending response. When the effects of spermidine on shoot multiplication and rooting in cucumber (Cucumis sativus L.) were examined, lower concentrations of spermidine (34–68μM) were beneficial for shoot growth and length, while rooting was induced only with the highest concentration (136μM) [23]. These findings indicate that in addition to genotype, spermidine concentration also modulates the final response. The effects of spermidine, spermine, and putrescine were compared in different stages of soybean regeneration [21]. Spermidine was best for shoot multiplication (approximately 15–17 shoots per explant), spermine led to the greatest elongation of shoots, and putrescine was the sole polyamine able to induce rooting (73–94%). Putrescine also positively affected rooting for swallow root (Decalepis hamiltonii) [30] and teak (Tectona grandis L.) [31]. These results demonstrate that spermidine is more suitable for multiplication stages other than rooting. Our results from in vitro rooting of A. alnifolia partially support this statement, though more thorough research is certainly required.
Gibberellic acid, especially GA3, is a useful additive to culture media that promotes shoot multiplication in several species [32, 33]. The effects of GA4 + 7 are mostly reported within the context of breaking seed dormancy [34, 35]. However, this gibberellin is also able to break dormancy and induce bud break in several species [7, 24]. Gibberellin application resulted in successful bud break and stem elongation with rooted cuttings of Carpinus, Corylus, and Malus spp. when applied as a spray treatment of GA4+7 at concentrations of 250–500 mg l–1 [18]. A mix of GA4+7 and 10 mg l–1 BAP decreased bud break frequency in Carpinus betulus ‘Fastigiata’ and Corylus colourna, but not in Malus ‘Spring Snow’. Addition of BAP also reversed stem elongation, with the exception of Malus spp. where the stem length increased. These results indicate that individual species react differently to the same post-propagation treatment. Our results partially support these findings since BAP did not reverse the effect of GA4+7 on A. alnifolia plant length, but the mix solution led to a higher number of growing plants relative to the GA4+7 solution alone. In addition to BAP, TDZ can also be used in combination with GA4+7 to overcome dormancy, as shown for rooted cuttings of Stewartia pseudocammelia, Hamamelis vernalis, and H. virginiana. However, bud break was only observed after application of both growth regulators in the spray solution together because TDZ itself caused mild growth inhibition in most of the studied species. No such observation was reported after application of BAP in our study, suggesting that the choice of growth regulator is an important factor for overcoming dormancy. Another study compared the effect of 400 ppm BAP, 100 ppm GA4+7, and their combination on dormancy of newly rooted A. alnifolia plantlets [7]. Although the gibberellin treatment restored the growth of plants within 48 hours, no increase in stem nodes was observed after 10 weeks. Compared to plants from other treatments, GA4+7 led to significantly longer internodes and reduced development of leaves while application of BAP resulted in compact and bushier plants [7]. In our study, we observed similar phenotype of treated plants. The combination of BAP and GA4+7 was the most promising in the study of above mentioned authors [7], although the number of stems calculated after 10 weeks differed among individual cultivars. Our study did not support these results because we observed the highest number of growing plants after application of 1 mg l–1 BAP. The cultivar used in our study may be more sensitive to the analyzed growth regulators or the concentrations of GA4+7 were simply not sufficient to break dormancy efficiently.
In some aspects, our results are in contradiction with previous findings regarding the positive effects of GA4+7 on bud dormancy. However, the final outcome depends on several factors, such as the model plant used, conditions during the rooting phase, and the particular suitability of growth regulators selected. Despite the wide use of IBA in tissue cultures [36], available evidence suggests that it can reduce or inhibit bud break of apple [37] and rose [38], and that this effect is probably applicable to other species as well. In our study, the highest number of dormant plants was always observed after cultivation on IBA-supplemented media when half-strength MS medium was used.
In conclusion, the concentration of rooting medium and a suitable choice of auxin are the most important factors in preventing “post-rooting dormancy”. Recommendations presented herein are not applicable to all species, though they may provide valuable recommendation for the micropropagation of plants that are difficult to root or acclimatize.
Conflict of interest
All authors declare that they have no conflicts of interest.
Funding
The authors report no funding.
Footnotes
Acknowledgments
This scientific work was co-funded by Research Centre AgroBioTech built in framework of European Community project Building Research Centre “AgroBioTech” ITMS 26220220180 and by the Slovak Grant Agency VEGA (project 2/0052/17).