Experimental design

A factorial experiment was arranged in a randomized complete block design with three factors and three replications. The first factor was hormone treatment time (before sowing, four-leaf stage and two months after sowing), the second factor was the progesterone hormone rate (no progesterone, 5 mg·L^-1 and 10 mg·L^-1), and the third factor was gibberellic acid (GA) (no GA, 100 mg·L^-1 and 200 mg·L^-1). The experiment was carried out with Zinnia elegans variety Dreamland seeds in a greenhouse in Astaneh-ye Ashrafiyeh, Iran, located at 37° 16’ 8.39’’ LN and 49° 56’ 55.47’’ LE.

The solutions were prepared in the laboratory on March 15, 2016. Then, 54 seeds were soaked in the predetermined hormones and 108 seeds in distilled water for 24 h. After soaking for one day, all 162 seeds were sown in a greenhouse according to the plot map on March 16. The second stage spraying was carried out 22 days later, i.e. when plants were at four-leaf stage. The third stage spraying was carried out on all flower plants two months after sowing.

Morphological traits

The distance from the last leaf to the flower, from the crown to the first leaf, from the crown to the first branch and plant height were measured. The number of nodes, branches, flowers, shoots and leaves were obtained in the greenhouse. To obtain the dry weight, the shoots and roots were dried in an oven at 105 °C for 24 hours. Leaf area was determined according to the formula reported by ^{Palaniswamy and Gómez (1974)} (Equation 1), for which the length and width of all leaves of the plant were measured in four stages (April 21, May 10, May 18 and June 13).

where W is maximum leaf width, L is leaf length and the adjustment factor K is equal to 0.74.

Chlorophyll content

Chlorophyll a, b and total chlorophyll were measured using the method described by ^{Mazumdar and Majumder (2003)}; first, 50 g of the plant fresh sample were weighed and poured into a porcelain mortar. Then, 20 mL 80 % acetone were added, after which the mixture was ground and filtrated. The remaining extract was placed in a container. A part of the extract was poured into a cuvette and the absorption was read separately at 660 nm for chlorophyll a and at 643 nm for chlorophyll b by spectrophotometer (PD-303, Apel, Japan). Then, the quantities of chlorophyll a, chlorophyll b and total chlorophyll were expressed in mg·g^-1 fresh weight:

Statistical Analysis

The results were examined by means of an analysis of variance and Tukey’s test (P ≤ 0.05) using the MSTATC software package.

Morphological traits

The analysis of variance (Table 1) shows that treatment time is the only factor that has a significant and highly significant effect (P ≤ 0.05 and P ≤ 0.01, respectively) on all variables evaluated. Additionally, all study factors and their interaction had a significant effect (P ≤ 0.05) on chlorophyll a and b production. For the most part, the factors studied did not have a significant effect on the variables analyzed.

The treatment with hormone application two months after sowing showed the highest shoot fresh weight; however, it did not differ statistically (P ≤ 0.05) from some progesterone treatments (0 and 10 mg·L^-1), while the application of hormones before sowing and in the four-leaf phase had the lowest shoot fresh weight. On the other hand, the highest root fresh weight was obtained with 10 mg·L^-1 progesterone, as well as with 0 and 100 mg·L^-1 GA; the rest of the treatments were statistically similar (Table 2). As for shoot dry weight, the application, two months after sowing, of 10 mg·L^-1 and 0 mg·L^-1 GA produced the highest and statistically similar value (7.07 and 6.63 g, respectively) (Table 2).

Accuracy in the mean comparison of GA treatments revealed that no GA treatment had the highest shoot dry weight; in fact, of the treatments with this hormone, the 200 mg·L^-1 rate had the lowest value for this variable, although this same treatment produced the highest root dry weight (Table 2). GA affects plant growth and development through influencing stem length, germination, the transition from vegetative to reproductive phase, internode spacing and the number of internodes and leaves (^{Arun, Ashok, & Rengasamy, 2000}; ^{Khoskhoy, Shibani, Rouhani, & Tafazzoli, 2010}; ^{Shekari et al., 2005}).

The application of 1 ppb (part per billion) epi-brassinolide improved root growth of pea by 25-256 % (^{Singh, Nakamura, & Ota, 1993}). It was found that the highest number of nodes was related to plants treated with hormone two months after sowing and the lowest one to those plants prior to sowing.

On the other hand, it was observed that the crowns were more distant from the first branch and the first leaf when 10 mg·L^-1 progesterone and 0 mg·L^-1 GA, respectively, were applied (Table 2).

Comparison of different application times of the growth regulators indicated that the highest number of branches, flowers and leaves was obtained when applied two months after sowing. In almost all cases, the lowest values were obtained with application before sowing and in the four-leaf phase (Table 2).

Khoshkhoy et al. (2010), in evaluating the effect of growth regulators on bulbs, observed that they induced the appearance of the floral stalk and flower development, as well as affected plant height and the number of florets per inflorescence. On the other hand, treatment with gibberellins increased the number of flowers and accelerated the flowering of Zantedeschia ‘Black Magic”, which was accompanied by the loss of flower size (^{Brooking & Cohen, 2002}). This type of hormone has an impact on plant traits, such as germination rate, plant height, floral stalk stem length and flower diameter (^{Ahmadpour & Zarghami, 2009}).

The hormonal treatment, two months after sowing, produced the highest plant height and number of leaves. Among the different hormone concentrations, 10 mg·L^-1 progesterone had the highest plant height, followed by the treatment without GA application (Table 2).

Low concentrations of some growth regulators have numerous impacts on plant growth and yield. In addition, these substances are involved in many growth-related processes like flowering and rooting (^{Shekari et al., 2005}). According to ^{Bhattacharya and Gupta (1981)}, 17-beta-estradiol and progesterone (0.25 μg·plant^-1) improved the shoot growth of sunflower seedlings but inhibited their root growth, although root elongation was enhanced by 0.1 μg·plant^-1 progesterone. In a study by ^{Ahmadi-Lashaki, Sedaghathoor, Kalatehjari, and Hashemabadi (2018)}, no specific effect was observed for the application of progesterone on the physiological and growth traits of Petunia hybrida, Tagetes erecta, and Calendula officinalis.

Chlorophyll content

The highest value of chlorophyll a was obtained when GA and progesterone were applied two months after sowing (Table 2). In comparing hormonal treatments, it can be seen that the highest value of chlorophyll a, b and total was obtained with 5 mg·L^-1 progesterone plus 200 mg·L^-1 GA; however, when only 200 mg·L^-1 GA were applied, the lowest value of chlorophyll a and total was recorded (Table 3). In general, treatments without progesterone had the lowest values for chlorophyll a, b, and total (Table 2).

Chlorophyll plays an essential role in photosynthesis through radiation absorption. ^{Fahimi (2014)} indicates that plant growth regulators affect photosynthesis directly through chlorophyll biosynthesis and decomposition. A study on the effect of GA and BA on Zantedeschia aethiopica pot plant showed that the highest chlorophyll was obtained from the treatment of 200 ppm gibberellins (^{Majidian, Naderi, Khalighi, & Majidian, 2012}). A study on the effect of GA and benzyladenine on Zantedeschia elliottiana showed that the highest value of chlorophyll was obtained with 200 ppm of gibberellins (^{Majidian, Naderi, Khalighi & Majidian, 2012}). In this sense, ^{Janowska and Jerzy (2003)} found that foliar application of gibberellins in Zantedeschia elliottiana inhibits chlorophyll deterioration.

Interactions between different levels of progesterone and GA showed that treatments without progesterone or GA and 5 mg·L^-1 progesterone without GA produced the largest leaf area, while 5 mg·L^-1 progesterone plus 200 mg·L^-1 GA had the smallest leaf area (Table 3).

Among the interactions between treatment application time and GA concentration, the largest leaf area occurred without GA two months after sowing, whereas the smallest leaf area occurred with the application of 100 mg·L^-1 GA prior to sowing (Table 4). Something similar happened with the progesterone treatments, where the application of 5 mg·L^-1 of this hormone two months after sowing resulted in the largest leaf area, and the same progesterone concentration but applied prior to sowing yielded the lowest value in this variable (Table 5).

Mean comparisons for the interaction of all treatments for leaf area showed that the largest leaf area was produced with the application of only 5 mg·L^-1 progesterone two months after sowing, and the smallest area was obtained with the application of 200 mg·L^-1 GA prior to sowing (Table 6). In comparing the effect of the different gibberellin levels on Z. elliottiana leaf area, it was found that this hormone affected it significantly, and that the largest leaf area was related to the treatment of 500 ppm gibberellins and the lowest one to the control (no treatment) (^{Majidian et al., 2012}). On the other hand, ^{Bedour, Award, EL Tayeb, Habba, and Metwally (2012)} found that the use of the combination of helium, neon and progesterone had an increasing effect on the structure of the gerbera leaf.