Changes in Growth and Physiological Parameters of × Amarine Following an Exogenous Application of Gibberellic Acid and Methyl Jasmonate

: × Amarine hybrids are attractive ornamental geophytes grown for cut ﬂower production. Their cultivation is limited due to lesser ﬂowering percentages and lesser bulb weight gain. To optimize the growth and propagation of geophytes, plant growth regulators (PGRs) are used, but so far none have been tested in × Amarine . We investigated the e ﬀ ect of gibberellic acid (GA 3 ; 50, 100, and 200 mg dm − 3 ) and methyl jasmonate (MeJA; 100, 500, and 1000 µ mol dm − 3 ) on growth, ﬂowering, bulb yield, and select physiological parameters of × A. tubergenii “Zwanenburg”. PGRs were applied as foliar sprays on the 70th and 77th day after planting. GA 3 treatment at 200 mg dm − 3 exhibited the greatest leaf number, leaf length, bulb weight, daughter bulb number, CO 2 assimilation intensity, greenness index, total sugars, and total protein content in bulbs. GA 3 application at 100 and 200 mg dm − 3 accelerated ﬂowering and at 50 and 100 mg dm − 3 signiﬁcantly increased the bulb ﬂowering percentage. MeJA at all tested concentrations prolonged anthesis time and reduced the bulb ﬂowering percentage. GA 3 at all concentrations and MeJA at 500 and 1000 µ mol dm − 3 stimulated daughter bulbs formation. GA 3 , especially at 200 mg dm − 3 can improve anthesis and increase × A. tubergenii “Zwanenburg” bulb yield.


Introduction
Ornamental plant production is an important horticultural branch. The expansion of the selection with previously unknown species, their hybrids, and new varieties is a key factor for the development of this sector [1]. An important part of flower production is the cultivation and reproduction of bulbous plants [2]. There are more than 800 different botanical genera of ornamental geophytes on the market. Their number systematically increases due to species introduction from natural sites and extensive breeding programs [3,4]. Plants newly introduced to the flower market lack appropriate cultivation technologies and methods of species propagation. Therefore, research is needed to effectively encourage producers to start growing previously unknown plants.
Among bulbous plants, considerable success was achieved in the breeding of Amaryllidaceae interspecific and intergeneric hybrids [5], using for crosses South African species from the genera A. belladonna and N. bowdenii species, from which ×Amarine was obtained, differ in the duration of dormancy and growth and development stages. In A. belladonna, the foliage emerges after anthesis (a hysteranthous growth habit), while in N. bowdenii foliage emerges before anthesis (a synanthous growth habit) [10]. In the Northern Hemisphere, after a dormancy period, ×Amarine bulbs first grow leaves in the spring, followed by floral stems in late summer and autumn. After anthesis, the plants become dormant. During this time, the bulbs should be exposed to decreased temperatures for flower primordia initiation. A serious problem in ×A. tubergenii cultivation is decreased flowering percentages commonly observed in N. bowdenii [11], a parent plant of the hybrid. The reasons for nonflowering in N. bowdenii are complex and result from many independent factors, such as inadequate temperature during bulb dormancy and plant growth, undersized bulbs, or insufficient carbohydrate content [12]. The flowering of ×A. tubergenii hybrids are irregular and extended in time (unpublished data), which limits their widespread use as a cut-flower crop.
In ornamental plant cultivation, plant growth regulators (PGRs) are used. They are active at exceptionally decreased concentrations and participate in the regulation of growth, anthesis, propagation, and physiological and metabolic processes [13,14]. Gibberellins are one of the best known natural phytohormones widely used in horticulture to terminate dormancy [15], stimulate floret formation and development [16], and accelerate or delay plant anthesis [17]. Gibberellins are responsible for stem elongation [18], stimulation of cell division and development of lateral buds [19], and also intensify photosynthesis and respiration [20]. Conversely, jasmonates, including methyl jasmonate (MeJA), are a fairly recently discovered phytohormone class [21]. MeJA is involved in regulating germination [22], morphogenesis [23], and aging [24], as well as the plant response to environmental stresses [25]. The available data from studies on the influence of MeJA on plant growth present divergent results. MeJA shows both growth-stimulating and growth-inhibiting effects [26,27], it can speed up or inhibit anthesis [28,29], and increase or decrease bulbing [30,31].
As there is no information on the use of PGRs in ×A. tubergenii cultivation, we assessed the effect of gibberellic acid (GA 3 ) and MeJA at different concentrations on ×A. tubergenii morphological traits, anthesis, and bulb yield. To obtain information on potential physiological changes induced by GA 3 or MeJA, the study also examined select gas exchange and chlorophyll fluorescence parameters and determined total sugars and total protein content in bulbs. We hypothesized that the applied regulators influenced the growth and physiological condition of ×A. tubergenii plants.

Experimental Location, Plant Materials, and Growth Conditions
The experiment was conducted in an unheated plastic house (25 m in length, 9 m in width, and 4.7 m in total height), covered with a double layer of UV-resistant foil, located at the West Pomeranian University of Technology in Szczecin (53 • 25 N, 14 • 32 E, 25 m a.s.l., sub-zone 7a USDA).

Experimental Design
On the 70th and 77th day after planting we used two growth regulators from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany): gibberellic acid (GA 3 ) at 50, 100, and 200 mg dm −3 , and methyl jasmonate (MeJA) at 100, 500, and 1000 µmol dm −3 . The plants were sprayed in the afternoon with an aqueous solution of GA 3 or MeJA, using about 15 cm 3 solution per plant. The control plants were sprayed with distilled water. Ethanol (0.04%, v/v) was used as a solvent. Directly after spraying, a plastic bag was placed on each plant and removed after two hours. Each experimental variant included 18 plants, six plants per repetition, in a random block system ( Figure 2).

Determination of Plant Growth Parameters
The number of days from bulb planting to the beginning of anthesis was recorded when the first florets in the inflorescence opened. In this phase, we determined the stem length, leaf number, and length and width of the longest leaf. The flowering bulb number (%) was determined in relation to the bulb number planted. When the inflorescences were fully developed, we counted the florets in each. Once the cultivation was complete, we removed the plants from the pots and determined the bulb fresh weight and the daughter bulb number per single planted bulb.

Determination of Gas Exchange Rate, Chlorophyll Fluorescence, and Leaf Relative Chlorophyll Content
The parameters of the gas exchange rate, including CO 2 assimilation intensity (A), transpiration (E), stomatal conductance for water (g s ), and CO 2 concentration in the intercellular spaces of the assimilatory parenchyma (c i ) were measured with a TPS-2 (PP Systems) portable gas analyzer (with standard settings), equipped with a PLC4 measuring chamber operating in an open system. Based on CO 2 assimilation intensity and transpiration, the photosynthetic water-use efficiency (ω W ) was calculated as a ratio of assimilation intensity to transpiration [32].
Chlorophyll fluorescence parameters were recorded using a Handy PEA (Hansatech) spectrofluorometer, based on the standard apparatus procedure. Leaves were shaded for 20 min prior to the measurement with a leaf clip (4 mm in diameter). The following parameters of chlorophyll fluorescence induction were measured and calculated using the spectrofluorometer: initial fluorescence excitation energy loss index in power antennas (F 0 ); maximum fluorescence after reduction of acceptors in photosystem II (PSII) and after dark adaptation (F M ); variable fluorescence, determined after dark adaptation, a parameter dependent on the maximum quantum yield of PSII (F V = F M − F 0 ); maximum potential photochemical reaction efficiency in PSII determined after dark adaptation and after reduction of acceptors in PS II (F V /F M ); PSII vitality index for the overall viability of this system (P I); the surface area above the chlorophyll fluorescence curve and between the F 0 and F M points proportional to the size of the reduced plastoquinone acceptors in PS II (Area) [33].
Leaf relative chlorophyll content (greenness index) on the Soil and Plant Analysis Development (SPAD) scale was measured with the Chlorophyll Meter SPAD 502 (Konica-Minolta cooperation, Ltd., Osaka, Japan).
Non-destructive measurements of the gas exchange rate, chlorophyll fluorescence, and SPAD were carried out on the 91st cultivation day from 09:00 AM to 12:00 PM in the middle part of the adaxial leaf blades. The measurements involved two fully expanded leaves with a length of 48-50 cm and width of 2.0−2.2 cm in two matched for size plants from each repetition. The conditions in the tunnel during the analyses were: temperature 20−22 • C, relative air humidity 70−75%, natural light (PPFD 450 µmol m −2 s −1 ), air CO 2 concentration 600 µmol mol −1 .

Determination of Total Sugars and Total Protein Content in Bulbs
Once the cultivation was complete, four bulbs from each experimental variant were cleaned from the covering scales and roots. The analyses involved bulbs of similar fresh weight. The bulbs were cut longitudinally into four segments with a knife, and then middle scales were cut out from each segment and analyzed. Samples (250−300 g) were taken for the determination of total sugars and total protein. Concentrations of both reducing and invert sugars were determined by extraction with diluted ethanol, clarification of extracts with Carrez solutions, and titration with sodium thiosulphate solution in the presence of Luff-Schoorl reagent according to PN-R-64784:1994 standard. Total protein content was calculated based on nitrogen content determination according to the Kjeldahl method using a mineralization block, copper catalyst, and steam distillation into boric acid according to PN-EN ISO 5983-2:2009 standard. Content determination of the tested components was performed in three repetitions and presented as a percentage of fresh weight.

Data Analysis
The experimental results were statistically analyzed with the one-way ANOVA using Statistica 13.3 (TIBCO Software Inc. Statsoft, Kraków, Poland). To ensure the normality of data distribution, the plant flowering percentages were subjected to the Bliss transformation (arcsin(sqrt(X)) and the analysis of variance. The confidence intervals were calculated based on Tukey's HSD test (p ≤ 0.05).

Effects of Foliar Application of GA 3 and MeJA Solutions on Growth, Flowering, and Bulb Yield
Treatments with exogenous PGRs significantly affected the leaf number and their length, but not the stem length and floret number per inflorescence (Table 1). Plants treated with GA 3 at all applied concentrations and MeJA at 500 and 1000 µmol dm −3 formed significantly more leaves as compared with the control. The plants sprayed with GA 3 at 200 mg dm −3 produced the greatest leaf number, which were also the longest.
In all cases, one bulb produced only one inflorescence. Anthesis time and bulb flowering percentage depended on the phytohormone type and concentration (Table 2). Plants treated with 100 and 200 mg dm −3 GA 3 were the first to start anthesis, while those sprayed with 50 and 100 mg dm −3 GA 3 showed the greatest flowering percentage. MeJA treatment delayed flowering and significantly reduced the flowering plant number, regardless of the concentration applied.
PGR treatments and their concentrations influenced bulb yield (Table 3) MeJA at 500 and 1000 µmol dm −3 ) significantly increased the daughter bulb number in comparison with untreated plants.    Table 4 presents the data on leaf gas exchange parameters depending on the plant hormone and its concentration. The greatest CO 2 assimilation intensity (A) was found in plants sprayed with 200 mg dm −3 GA 3 solution. This parameter in the other variants did not differ significantly from the control. GA 3 or MeJA application resulted in a significant increase in the intensity of transpiration (E), as compared with untreated plants, except for 1000 µmol dm −3 MeJA. The greatest E was recorded in plants sprayed with 100 µmol dm −3 MeJA. The increase was more than twofold vs. the control. Plants treated with 50 mg dm −3 GA 3 exhibited the greatest photosynthetic water-use efficiency (ω W ). The ω W for the other treatments did not differ significantly from the control. Exogenous application of GA 3 and MeJA at all tested concentrations resulted in a significant increase in water stomatal conductance (g S ). Compared to untreated plants, the greatest, more than fourfold increase in g S was recorded after the application of 100 and 500 µmol dm −3 MeJA. Both phytohormones triggered a significant increase in CO 2 concentration in the intercellular spaces (c i ), particularly noticeable in the plants treated with 1000 µmol dm −3 MeJA. Table 4. Effects of GA 3 and MeJA on CO 2 assimilation intensity (A), transpiration (E), photosynthetic water-use efficiency (ω W ), stomatal conductance (g s ), and CO 2 concentration (c i ) of ×A. tubergenii "Zwanenburg".

Effects of Foliar Application of GA 3 and MeJA Solutions on Chlorophyll Fluorescence and SPAD
In comparison with the control plants, GA 3 and MeJA did not significantly affect the chlorophyll fluorescence parameters (F V /F M , PI, and Area), but caused changes in the leaf greenness (SPAD index) ( Table 5). Plants treated with 200 mg dm −3 GA 3 showed the greatest SPAD values. Similarly, an increased greenness index was observed in plants treated with 50 and 100 mg dm −3 GA 3 and 1000 µmol dm −3 MeJA.

Effect of GA 3 and MeJA on Growth, Flowering, and Bulb Yield
To improve ornamental geophyte quality, various plant growth and development regulators are used in horticultural practice [34,35]. ×Amarine tubergenii "Zwanenburg" treated with GA 3 (50, 100, and 200 mg dm −3 ) and MeJA (500 and 1000 µmol dm −3 ) exhibited a significantly increased leaf number (Table 1). Moreover, at the greatest GA 3 concentration, the leaves were much longer. More leaves after GA 3 application were observed by Ramzan et al. [36] in Tulipa gesneriana L. The activity of gibberellin family growth regulators involves the stimulation of plant cell mitotic division [37], which can lead to intense growth and production of more leaves. Diallo et al. [38] showed a stimulating effect of MeJA on leaf count in Triticum aestivum L. The researchers suggested that the beneficial effect of MeJA on the leaf count may be because MeJA maintained plants in the vegetative phase longer, so that plants continued their intense growth instead of proceeding to the generative phase. Similarly, we showed MeJA treatment delayed anthesis time ( Table 2).
Neither of the phytohormones used in this study affected the inflorescence morphology, including the floret number (Table 1). The lack of PGR effects on the floret number may stem from the fact that both phytohormones were applied after the inflorescence initiation inside of the bulb. In Nerine bowdenii the time between flower primordia initiation and anthesis is 2-3 years, and in Amaryllis belladonna over a year [6,10].
Foliar treatment with both GA 3 and MeJA strongly influenced the anthesis time and the flowering percentage, but GA 3 worked differently than MeJA ( Table 2). The plants sprayed with GA 3 at 100 and 200 mg dm −3 began anthesis faster. Earlier flowering following GA 3 application at 150-300 ppm was also observed in A. belladonna [39]. However, in Nerine flexuosa GA 3 bulb-dip treatment at 100 mg/L did not accelerate flowering [40]. In Nerine, many inflorescences did not elongate and became desiccated, thus no flowering occurred [41]. It may be assumed that in ×A. tubergenii "Zwanenburg" GA 3 stimulates the final stages of stem elongation and anthesis. GA 3 treatment at 50 and 100 mg dm −3 not only accelerated anthesis, but also positively affected the inflorescence yield by increasing the bulb flowering percentage ( Table 2). The increased number of ×A. tubergenii "Zwanenburg" flowering plants following GA 3 treatment is probably because of the faster execution of the flowering phase. Our results are highly practical, as GA 3 makes more plants flower at an earlier stage, which allows for harvesting the inflorescences of field or unheated tunnel-grown plants before the autumn frosts.
MeJA, regardless of its concentration, delayed flowering and decreased the number of flowering ×A. tubergenii "Zwanenburg" plants (Table 2). Similarly, Zhai et al. [42] reported an inhibitory effect of jasmonic acid on Arabidopsis thaliana flowering. Maciejewska and Kopcewicz [28] observed that treatment with MeJA reduced the flower bud number in Pharbitis nil (L.) Roth. Conversely, 50 µM MeJA application in Brassica napus L. moved the flowering time forward and increased the flower number [29]. Jasmonates control individual plant ontogenesis phases, but their exact mode of action in the regulation of growth and flowering induction is not known.
A common problem in bulbous plant propagation is their low propagation rate, which can be increased using PGRs [43]. Bulb yield analysis in ×A. tubergenii "Zwanenburg" clearly showed that GA 3 increases the bulb weight and daughter bulb number (Table 3). In Allium karataviense Regel GA 3 spraying increased the bulb weight and total bulb yield [44]. Allium sativum L. treatment with GA 3 positively affected axillary bud formation, clove number per bulb, bulb weight, and bulb volume [45]. The stimulating effect of GA 3 on bulbing may be because gibberellins enhance gene expression correlated with cell elongation necessary for cell development and differentiation, and induced lateral bud formation [46].
MeJA at concentrations of 500 and 1000 µmol dm −3 had a similarly stimulating effect on the propagation rate as GA 3 application. Moreover, bulbs obtained from plants treated with the greatest MeJA dose displayed significantly greater weight (Table 3). In vitro jasmonates application increased the bulb number and improved bulblet quality parameters in A. sativum [47] and Narcissus triandrus L. [48]. Nojiri et al. [49] postulated that both jasmonic acid and MeJA are bulbing hormones, as they both stimulate bulbing through microtubles disruption.

Effect of GA 3 and MeJA on Physiological Parameters
Numerous studies have shown that the course and intensity of the most important plant physiological processes can be regulated with exogenous growth regulators [50,51]. In our study, GA 3 regardless of its concentration enhanced transpiration, stomatal conductance for water, CO 2 concentration in the intercellular spaces of the assimilatory parenchyma, and SPAD in ×A. tubergenii "Zwanenburg" (Tables 4 and 5). A significant increase in CO 2 assimilation intensity was found only in plants sprayed with GA 3 solution at 200 mg dm −3 . These plant leaves showed the greatest chlorophyll fluorescence and SPAD index values. It can be assumed that as a result of increased photosynthesis, plants treated with GA 3 in the greatest dose produced the greatest leaf number and daughter bulbs, and had the longest leaves and bulbs with the greatest fresh weight. Increased photosynthetic efficiency results in faster vegetative growth and leads to increased biomass production [52].
MeJA applied in all tested concentrations increased the stomatal conductance and leaf CO 2 concentration without affecting CO 2 assimilation intensity and assessed fluorescence parameters (Table 4). Moreover, MeJA application at 100 and 500 µmol dm −3 increased transpiration, and at 1000 dm −3 enhanced leaf greenness (SPAD; Table 5). Our results partially confirm those of Ahmadi et al. [53], who found that spraying B. napus with 100 µM MeJA significantly increased the CO 2 compensation point and respiration rate. A positive effect of MeJA on photosynthetic parameters was observed in two Prunus dulcis Mill. rootstocks [54]. The effect of exogenous MeJA on the plant photosynthetic apparatus is complex and depends on many factors, including species, concentration, and environmental conditions [21,23,25].
Geophytes accumulate reserve substances in their bulbs that determine the correct course of dormancy, growth, and flowering [55]. Both PGRs significantly increased total sugar content in ×A. tubergenii "Zwanenburg" bulbs, especially when applied at 100 and 200 mg dm −3 (GA 3 ) and 1000 µmol dm −3 (MeJA) (Figure 3a). Moreover, bulbs obtained from plants treated with GA 3 or MeJA in the greatest concentrations featured significantly more total protein (Figure 3b). Wakchaure et al. [56] found an increased total soluble sugar content and total protein content in Allium cepa L. bulbs treated with GA 3. Jasmonates promote starch accumulation in tubers, as it was apparent in Solanum tuberosum L. subsp. tuberosum [57]. In ×A. tubergenii "Zwanenburg", the greatest total sugars content and total bulb protein was found in plants sprayed with GA 3 at 200 mg dm −3 (Figure 3). This could have been related to increased CO 2 assimilation intensity, and as a result, better bulb supplementation with photosynthesis products. Plant bulbs treated with GA 3 at the greatest dose showed the greatest fresh weight (Table 3). Most probably, increased bulb weight and a greater nutrient content, especially sugars, improves plant flowering in the subsequent growing season. In the case of N. bowdenii, the parent species of the nothogenus ×Amarine, inflorescence quality and flowering percentage is related to bulb size and carbohydrate content in fleshy leaf bases and fleshy scales [11,12]. Decreased sugar level in bulbs during gynoecium development is implicated in inflorescence abortion [58]. Therefore, it seems advisable to conduct further research on the residual impact of PGRs on the growth and anthesis of ×A. tubergenii. We intend to observe bulb performance in the following year, and to dissect them to observe flower primordia and their development after external PGR application, and monitor how it affects floral initiation and development.

Conclusions
We examined the possibility of using PGRs in the cultivation of ×A. tubergenii, an ornamental geophyte with great floricultural potential. Plant growth and physiological condition depended on the PGR type and its concentration. The influence of GA 3 and MeJA on anthesis time was opposite, as GA 3 accelerated and MeJA delayed the beginning of anthesis. Additionally, GA 3 increased and MeJA decreased the bulb flowering percentage. All GA 3 concentrations and MeJA at 500 and 1000 µmol dm −3 stimulated daughter bulb formation. Among all treatments, GA 3 at 200 mg dm −3 seemed to most favorably affect the leaf number, their length, bulb weight, daughter bulb number, photosynthesis rate, greenness, total sugar, and total protein content in bulbs. This treatment could be used as a method for improving ×A. tubergenii "Zwanenburg" plant growth, anthesis, and bulb yield in commercial production.