Morphological, physiological features and differences of Vriesea splendens ’Fire’ plants during in vitro multiplication and rooting (2022): Morphological, physiological features and differences of Vriesea splendens ’Fire’ plants during in vitro multiplication and rooting.

Summary: During in vitro multiplication and rooting of Vriesea splendens ’Fire’, 0.1, 0.2, 0.4 and 0.8 mg l -1 benzyladenine (BAP), benzyladenine-riboside (BAPR), kinetin (KIN), meta-topoline (MT), indole-butyric acid (IBA) and naphthalene-acetic acid (NAA) were added to basal Murashige and Skoog (1962) MS medium. As compared to the hormone-free control, plants developed significantly more shoots on medium supplemented with almost all cytokinins (excepting KIN), especially BAP resulted the highest multiplication up to almost 26 shoots. Enhancement of cytokinin concentrations increased shoot number (and in case of BAP, peroxidase activity) but decreased plant height and rooting parameters. Regarding root production, both auxins were definitely beneficial (0.2 mg l -1 NAA resulted more than 7.5 roots and higher auxin concentrations efficiently stimulate root elongation); however, KIN had similar effects. After a three-month duration time of acclimatization, we observed that plants which were previously cultured on medium containing certain cytokinins (KIN in all doses and 0.1 mg l -1 MT) or both auxins had greater survival, moreover, as negative after-effect, higher cytokinin concentrations reduced the number of survived specimens.


Ördögh, M.
The aim of our study was to find morphological and physiological differences between V. splendens 'Fire' in vitro groups and ascertain the effects of different cytokinins and auxins on the efficiency of proliferation and rooting.

Origin of plant material
1.5-1.8 cm sized, rootless plants originated from an in vitro stock of V. splendens 'Fire', a smaller sized cultivar with 20-25 cm leaves and 30-35 cm flowers stalks (Tillyné & Honfi, 2008) were used for in vitro studies in the laboratory of the Department of Horticultural and Dendrology, Hungarian University of Agriculture and Life Sciences. After multiplication and rooting, the same (but larger, 3-4 cm) specimens were acclimatized in one of the greenhouse of our department.
Three months later, plant height and fresh weight, number of shoots, number and length of roots were examined, additionally, physiological parameters (total chlorophyll content, peroxidase enzyme activity of leaves) were also analysed. Each experiment was repeated twice and 30 plant per treatment was examined. After all measurements (as the end of our in vitro studies), stocks were planted in a mixture (1:1:1) of vermiculite, perlite and peat (Novobalt, Rekyva, Lithuania) and acclimatized in heated greenhouse (170 days later we calculated survival ratios).

Measurement of physiological parameters
For determination of chlorophyll (a+ b), we collect 3 x 0.1 g leaf from each medium. After preparation of leaf samples with the use of approximately 0.5 g of quartz sand + 10 ml acetone (80%) and a 24-hour long refrigeration (+4 o C) period, absorbance of suspensions was measured by GeneSys VIS-10 (Thermo Fisher Scientific Inc., USA) spectrophotometer at 644 and 663 wavelength. Leaf pigment concentration (µg g -1 ) were calculated by formula (20.2 × A644 + 8.02 × A663) × V/w; where V= volume of tissue extract (10 ml), w= fresh weight of tissue (0.1 g), A= absorbance (Arnon, 1949).

Data and statistical analysis
Data of shoot, root number, plant height and fresh weight, chlorophyll and peroxidase parameters were evaluated by SPSS 23.00 (IBM Corp., USA). An analysis of variance (ANOVA) was conducted to calculate the statistical significance of every data presented. In cases of detecting significant differences between treatments, recorded means were separated by Tukey's test at p < 0.05.

Shoot number
BAP enhanced effectively the shoot multiplication, and higher (0.4 and 0.8 mg l -1 ) concentration resulted significantly the largest values more than 20 shoots. Additionally, higher BAP, BAPR and MT dosages also increased shoot production, thus, plants with the largest shoot-clusters were grown on the highest cytokinin levels, excepting the use of KIN. We observed the lowest averages on medium supplemented with KIN, plantlets developed usually not more than 6 shoots in these groups. During in vitro proliferation of V. scalaris, also BA was more effective than KIN, because the latter cytokinin resulted only around 1-2 shoots instead of almost 8 (da Silva et al., 2009). Also low values (chiefly between 6-8 shoots) were recorded on medium with IBA or NAA. In general, auxins rather stimulate root development than shoot proliferation, however, in case of V. gigantea and V. philippocoburgii, NAA helped formation of both shoots and roots (Droste et al., 2005). Other species such as V. cacuminis or V. heliconioides produced the best multiplication (approximately 7 or 9 shoots) when high (2.5 or 3.4 mg l -1 ) dose of BAP combined with 0.2 or 0.9 mg l -1 NAA (Resende et al., 2016;Hernández-Meneses et al., 2018). In case of V. reitzii, intensified shoot development was achieved after 2 months, mainly on medium supplemented with 0.4 mg l -1 NAA and 0.4 mg l -1 2-iP (Dal Vesco & Guerra, 2010). In another study, the same species' multiplication was optimal (with 60 shoots/g nodule cluster) when plants were cultured on a cytonikin mixture with BAP, KIN and 2,4-D (Alves et al., 2006).

Plant height
Every concentration of all cytokinin increased plant height, principally, 0.4 and/or 0.8 mg l -1 BAP, BAPR and MT resulted significantly shorter (mostly, 24-26.5 mm) plants if we compared other stocks cultured on hormone-free medium. On the other hand, in cases of the auxin-treated groups, we observed considerably the highest (at least 33 mm) specimens on medium containing 0.2-0.8 mg l -1 NAA; thus, negative correlation can be presumed between the number of shoots and height of plant. Hernández-Meneses et al. (2018) experienced that 0.3 mg l -1 GA3 efficiently elongated V. heliconioides stocks, which reached 76 mm after 12 weeks, but hormone-free medium also resulted higher plants around 60 mm. V. reitzii in vitro plantlets also required GA3 treatment with different concentrations in order to gain similar sizes, however, the absence of this hormone similarly promoted adequate growth with at least 50 mm height after 10 weeks culturing (Dal Vesco & Guerra, 2010). Overall, it could be that the use of GA3 advance better the elongation of V. splendens 'Fire' plants's, anyway, as Guerra and Dal Vesco reported (2010), the utilization of this plant growth regulator was necessary to develop higher V. splendens hybrids specimens originated from media containing thidiazuron (TDZ) which usually has cytokinin activity.

Fresh plant weight
We found the heaviest plants on medium supplemented with NAA, most concentrations of this auxin resulted at least 1 g of weight. The lowest values (around 0.4-0.5 g) were observed in the case of KIN and MT. In addition, these plant growth regulators decreased the number of shoots and/or plant height, thus, these parameters usually correlated with their weight. The supplementation of certain accessories (such as B5 vitamins) increased shoot proliferation and fresh weight of V. scalaris, specimens with larger number shoots also had larger weight (Da Silva et al., 2009). In case of V. flammea, higher sucrose concentrations (up to 60 g l -1 ) promoted larger fresh mass (Sasamori et al., 2020).
Numeric data and noticeable morphological differences were shown on Table 1 and Figure 1.

Root number and length, rooting ratio
Definitely, the highest averages (usually more than 6 roots with at least 20 mm length) were recorded in NAA-treated groups; 0.2 mg l -1 NAA resulted the most, 7.58 roots and the longest ones (almost 27 mm) developed in the case of 0.8 mg l -1 NAA. Furthermore, only this agent promoted 100% rooting ratio. The other hormones (including IBA) had less effect, and we obtained the lowest values especially on medium supplemented with higher dosages of BAP, BAPR or all concentration of MT ( Table 2). In the case of a 3-month-period of in vitro rooting of V. cacuminis (Resende et al., 2016), the addition of NAA also stimulated root development more efficiently than the other auxins (IAA, IBA), the best results (2.3 roots and 14 mm length) was achieved in medium with 0.04 mg l -1 NAA. Not every Vriesea taxa preferred auxins; the use of IBA decreased rooting of V. scalaris (Da Silva et al., 2009), and the highest rooting ratio (40%) was detected in the absence of this hormone, however, lower rooting connected with higher number of adventitious shoots. For V. heliconioides, 0.3 mg l -1 GA3 was optimal, averagely four roots were found on the basal parts of the in vitro plants (Hernández-Meneses et al., 2018). Sometimes, modifying sucrose concentration affected root parameters; for example, lower dosages (averagely 17 g l -1 instead of 20-60 g l -1 ) enhanced in vitro root growth of V. inflata (Freitas et al., 2015), but V. flammea required higher levels (usually more than 30 g l -1 ) for better root production (Sasamori et al., 2020). Rooting differences shown (based on type and concentrations of auxins) on Figure 2.

Total chlorophyll content
Compared with the control, most of the plant growth regulators resulted lower (in general, not higher than 1500 µg g -1 ) chlorophyll values; only 0.1 and 0.2 mg l -1 IBA, 0.2 and 0.8 mg l -1 NAA enhanced leaf pigment above 1750 µg g -1 , overall, higher means were obtained on medium containing auxins (Figure 3). It is worth mentioning that IBA and NAAtreated plants with more (and longer) root usually had higher chlorophyll contents in their leaves. In another trial, enhancement of sucrose dose increased this parameter, during in vitro multiplication of V. flammea (Sasamori et al., 2020).

Peroxidase activity
Especially NAA, IBA and higher concentrations of BAP resulted significantly higher enzyme activities, around 0.2 U mg -1 (Figure 4). Probably, certain hormones that are effectively stimulate shoot/root development or plant growth also increase physiological processes, which cause enhanced enzymatic reactions, however, unfavourable conditions, such as extreme temperatures (Duarte et al., 2019) or higher sucrose concentrations (Martins et al., 2020) can also induce stress in Vriesea hybrids.

Survive the acclimatization
Most cases, plants that were previously cultured on medium supplemented with 0.1 mg l -1 MT and every dosage of KIN, IBA or NAA survived better the 170-day-length acclimatization; these groups generally produced more and longer roots in higher ratios in vitro, and had at least 70 % survival ex vitro. As negative after-effect, enhancement of cytokinin (especially MT) concentration during in vitro multiplication decreased plant's survival (Figure 5). Similarly, lower concentrations (of MS macronutrients or sucrose) gave better acclimatization results of V. flammea (Sasamori et al., 2020) and V. inflata (Freitas et al., 2015). Main phases of acclimatization were represented on Figure 6.

Conclusions
The use of BAP promoted the best proliferation and a positive relationship was found between the cytokinin concentrations and shoot number, however, higher dosages definitely decreased rooting parameters, and respectively, plants' survival chance during the acclimatization. For rooting, NAA was more suitable than IBA in every concentration; and especially 0.2 mg l -1 NAA proved to be optimal, having regard to not only the trend of root development, but also the plant height and chlorophyll content. After almost half-year acclimatization period, we observed positive after-effect of both auxins and certain cytokinins (particularly KIN); these plant growth regulators previously resulted the best rooting parameters that facilitated better adaptation for ex vitro conditions.   Murashige & Skoog (1962) basal medium containing different plant growth regulators in 0.1-0.8 mg l -1 concentrations. Data represented by mean ± standard deviation (SD). Means with different letters are significantly different according to Tukey's test at p < 0.05.  Murashige and Skoog (1962) basal medium containing different plant growth regulators in 0.1-0.8 mg l -1 concentrations. Data represented by mean ± standard deviation (SD). In cases of root number and length, means with different letters are significantly different according to Tukey's test at p < 0.05.