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
]. 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 Amaryllis
W. Aiton, Haemanthus
L., and Nerine
Herb. These plants are cultivated for their attractive flowers [6
] and as a source of valuable alkaloids with therapeutic effects [7
]. Breeding resulted in the nothogenus ×Amarine tubergenii
Sealy, a hybrid between Amaryllis belladonna
L. and Nerine bowdenii
]. Inflorescence of ×A. tubergenii
sets on a long leafless stem and consists of helicoid cymes inflorescence, each with several pink florets (Figure 1
a,b). The leaves are dark green, ensiform, form a rosette, and grow directly from bottle-shaped perennial bulbs covered with brown scales (Figure 1
c,d). ×A. tubergenii
inflorescences demonstrate very good post-harvest durability and are a desirable commodity on the cut flower market [9
]. As a result of further intergeneric crosses, many ×Amarine
varieties were obtained, differing in floret color, size, and flowering time. Research is lacking on ×Amarine
cultivation, which is an obstacle to the wider spread of this prospective ornamental plant.
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
], 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
]. 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
], it can speed up or inhibit anthesis [28
], and increase or decrease bulbing [30
As there is no information on the use of PGRs in ×A. tubergenii cultivation, we assessed the effect of gibberellic acid (GA3) and MeJA at different concentrations on ×A. tubergenii morphological traits, anthesis, and bulb yield. To obtain information on potential physiological changes induced by GA3 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.
2. Materials and Methods
2.1. 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).
Dormant ×A. tubergenii “Zwanenburg” bulbs with a 12–14 cm circumference and an average fresh weight of 39.0 g, imported from the Netherlands by Ogrodnictwo Wiśniewski Jacek Junior (Góraszka, Poland), were stored for 3 weeks in dark at 5–8 °C until planting. Before planting, sorted for disease-free bulbs were treated for 30 min in a fungicide mixture containing 0.7% (w/v) Topsin M 500 SC (Nippon Soda, Tokyo, Japan, active ingredient: thiophanate-methyl) and 1% (w/v) Kaptan 50 WP (Organika-Azot Chemical Company, Jaworzno, Poland, active ingredient: Captan). On 14 April, the bulbs were planted individually into black round PVC pots with 15 cm diameter and a 1.5 dm3 capacity, filled with deacidified peat (Kronen, Cerkwica, Poland) (pH 6.3; 16 mg dm−3 N-NO3, 42 mg dm−3 P, 19 mg dm−3 K, 1550 mg dm−3 Ca, 101 mg dm−3 Mg and 27 mg dm−3 Cl) mixed with Hydrocomplex fertilizer (Yara International ASA, Oslo, Norway) containing 12% N, 11% P2O5, 18% K2O, 2.7% MgO, 8% S, 0.015% B, 0.2% Fe, 0.02% Mn, and 0.02% Zn at a dose of 3 g dm−3. The pots were placed in 60 × 40 × 19 cm plastic boxes, six pots per box, which were placed in a tunnel on white non-woven fabric. The air temperature was regulated with air vents, which opened automatically when the temperature exceeded 22 °C. The average monthly maximum/minimum air temperature and average relative humidity (RH) in the plastic house were respectively: April 22.7 °C/6.8 °C, 70.9% RH; May 25.0 °C/6.9 °C, 76.9% RH; June 27.3 °C/13.3 °C, 70.6% RH; July 32.5 °C/17.6 °C, 69.4% RH; August 25.6° C/14.7 °C, 78.5% RH; September 25.9 °C/13.0 °C, 80.4% RH; October 18.6 °C/7.1 °C, 90.2% RH; and November 14.9 °C/5.5° C, 95.7% RH. The plants were cultivated until 15 November under natural day/night conditions (without shade nets or artificial lighting). The photosynthetic photon flux density (PPFD) in the plastic house on a sunny day ranged from 420 to 1151 μmol m−2 s−1 (as per Radiometer-Fotometr RF-100, Sonopan, Białystok, Poland).
2.2. Experimental Design
On the 70th and 77th day after planting we used two growth regulators from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany): gibberellic acid (GA3
) 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 GA3
or MeJA, using about 15 cm3
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
2.3. 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.
2.4. Determination of Gas Exchange Rate, Chlorophyll Fluorescence, and Leaf Relative Chlorophyll Content
The parameters of the gas exchange rate, including CO2
assimilation intensity (A), transpiration (E), stomatal conductance for water (gs
), and CO2
concentration in the intercellular spaces of the assimilatory parenchyma (ci
) 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 CO2
assimilation intensity and transpiration, the photosynthetic water-use efficiency (ωW
) 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 (F0
); maximum fluorescence after reduction of acceptors in photosystem II (PSII) and after dark adaptation (FM
); variable fluorescence, determined after dark adaptation, a parameter dependent on the maximum quantum yield of PSII (FV
); maximum potential photochemical reaction efficiency in PSII determined after dark adaptation and after reduction of acceptors in PS II (FV
); PSII vitality index for the overall viability of this system (P I); the surface area above the chlorophyll fluorescence curve and between the F0
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 CO2 concentration 600 µmol mol−1.
2.5. 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.
2.6. 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).
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 GA3 and MeJA on anthesis time was opposite, as GA3 accelerated and MeJA delayed the beginning of anthesis. Additionally, GA3 increased and MeJA decreased the bulb flowering percentage. All GA3 concentrations and MeJA at 500 and 1000 µmol dm−3 stimulated daughter bulb formation. Among all treatments, GA3 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.