Roles of plant growth regulators on flowering of rose (Rosa hybrida L.’Red Rose’)

Rose is the most popular ornamental flower all over the world, which is used as garden plants and cut flowers. In the case of Rosa hybrida L. ’Red Rose’, flowering provides the major developmental transition from the vegetative to the reproductive stage, and reproduction is one of the most important phases in an organism’s life cycle. In this study, the morphological and physiological changes during the flower development of rose, which is planted in the garden, and roles of plant growth regulators on the flowering of in vitro vegetative shoots of rose were analyzed. The development of a flower includes three stages: the shoot apical meristem, floral meristem, floral bud. Levels of cytokinin, auxins, and gibberellins increased in the transition of meristem from the shoot apical meristem to the floral meristem stage. Plant growth regulators have important effects on the shoot apical meristem cell division and flowering. The combination of 0.5 mg.L−1 GA3, 0.1 mg.L−1 NAA, 2.5 or 3.0 mg.L−1 BA to Murashige and Skoog (MS) medium induces the floral transition of the in vitro vegetative shoots with the highest percentage (41%) as well as growth and development in comparison to the other treatments after 10 weeks. Then, the in vitro floral meristem continuously developed into a flower bud after 12 weeks.


INTRODUCTION
Nowadays, the rose belongs to the Rosaceae family with about 200 species all over the world [1]. Roses with a huge variety of characteristics such as flower shape clour and fragrance are used as garden ornaments. They are also an enormous demand in the perfume and cosmetic industries. The rose has been grown for millions of years, is an important cut flower crop, and the plant adapted to diverse climatic conditions. The transition from vegetative stage to reproductive stage of growth is a significant critical event in the life of a plant. This transition depends on numerous factors, including both internal and external elements. In vitro flowering is influenced by plant growth regulators, such as auxins and cytokinins, has been early studied on flowering in tobacco in vitro by thin cell layers method [2]. In some other reports, the role of cytokinins on in vitro floral morphogenesis is also studied, as observed in rose (hybrid tea) cv. "First prize", the highest percentage of flowering (45%) was obtained from the shoots culture on MS medium containing 3.0 mg/L BA, 0.1 mg.L -1 NAA, and 30 mg.L -1 sucrose after 12 weeks [3]. Another study reported that in vitro flowering induction of Rosa hybrida L. cv "Red Masterpiece" was observed on MS medium containing 2.0 mg/L BA after 9 weeks of culture [4]. Floral organogenesis in rose flowers of these plants has four concentric whorls of organs that are specified by the development from the outside to the center of the flower, in the sequence as follows: sepals, petals, stamens, and carpels followed by the genetic ABC model, that  [5]. However, the effect of plant growth regulators on the in vitro flower development of rose are still obscure, in overcoming abnormal phenomena of flowering rose culture in vitro. Therefore, this study aims to understand the shoot development in in vitro conditions and rose quality, controlling important rose traits, and flower initiation development.

Material
Floral shoots of rose (Rosa hybrida L. 'Red Rose') in the stage before fully blooming with dormant buds were collected from rose gardens in Dalat (Vietnam).

Observation of morphological changes
The developmental flowers were observed by eyes, that appear during stages. Morphological changes of shoot apical meristem during flower development were observed under a stereomicroscope and optical microscope (CKX41, Olympus, Japan) after the longitudinal cut through the shoot apical meristem and dyed with double staining carmine aluminum and iodine green.

Extraction, isolation, and quantitative analysis of endogenous phytohormones
The plant hormones, including auxin, cytokinin, gibberellin, and abscisic acid in shoots at different stages of flowering from the garden, were extracted by using methanol and diethyl ether. The plant hormones were isolated by using silica gel thin-layer chromatogram (60 F254, 105554, Merck), at 29 °C with solvent chloroform: methanol: acetic acid (80:15:5 in volume). The plant hormones were detected under ultraviolet light [6]. Plant hormone level was measured by bioassay technique: Lettuce hypocotyl (Lactuca sativa L.), cucumber cotyledons (Cucumis sativus L.), Oryza coleoptile sections (Oryza sativa L.) were used to evaluate activities of gibberellin, zeatin, auxin, and abscisic acid, respectively. The hormonal level in each sample was measured in three replicates [7].

Effect of plant growth regulators on in vitro flowering from vegetative shoots
All nodal sections in positions from 3 to 5 (from flower buds down) containing vegetative shoots were excised. The nodal sections were washed out under running water in 30 minutes, then sterilized with 0.1 % HgCl 2 for 15 minutes. Thereafter, the explants were rinsed 3 times with sterile distilled water. Then, damaged tissues were aseptically separated from the nodal sections. The shoots with 0.5 cm in height were placed on MS [8] medium supplemented with 0.5 mg.L -1 BA, 0.1 mg.L -1 NAA, 0.5 mg.L -1 GA 3 , and 30 g.L -1 sucrose [9].
After 30 days, the shoots approximately 2.0 cm in height were obtained from the shoot cluster and cultured to MS medium supplemented with 0.1 mg.L -1 NAA, 0.5 mg.L -1 GA 3 , BA at different concentrations (0.5; 1.0; 1.5; 2.0; or 3.0 mg.L -1 ), and 30 g.L -1 sucrose. A few rose traits such as plant structure, flower development observing in all treatments.

Experimental design and statistical analysis
All experiments were set up under the controlled condition of fluorescent light with an intensity of 27 μmol.m -2 .s -1 , temperature of 23 ± 2 °C, relative humidity (60 -70 %), and photoperiod 12 h/day. The experiment was repeated 3 times and each treatment was replicated in ten culture vessels. The data were analyzed by using Statistical Package for the Social Sciences (SPSS), version 16.0 for Windows. After cutting rose branches, shoots develop and form a flower bud. There are three main stages in the development includes (Figure 1): (1) Vegetative shoot: the shoot apical meristem is formed about 14 days after cutting rose branches. At this stage, the shoot apical meristem (SAM) mainly produces leaves (Figure 1a, b). (2) Flowering initiation: day 14 through 21, the SAM becomes the inflorescence shoot apical meristem. At this stage, the flower primordium becomes meristem maintenance and determinacy (Figure 1c, d). (3) Floral bud: from day 21 to day 31, branch rose continues to lengthen relatively. The floral bud as well as a floral meristem that appears during this stage. The sepals grow to completely cover the floral bud, petals, and sepals continue expanding until SAM has disappeared (Figure 1e, f).

The changing of plant growth regulators in shoots during flower development of the flowers from
the garden. In the transition of shoots from the vegetative growth to the flowering initiation stage, the level of auxins, cytokinins, and gibberellins of the shoot were increased. In the floral bud stage, the level of auxin continues increasing, cytokinin and gibberellin activity maintains the same level at this stage. The ratio of cytokinin/auxin in each stage was decreased. Meanwhile, the level of abscisic acid did not change (Table 1).

Influence of plant growth regulators on in vitro flowering from vegetative shoot cultures.
After 10 weeks of culture, the results showed that floral bud was the highest proportion significantly when using BA 2.5 mg.L -1 and 3.0 mg.L -1 , which higher compared to 1.5 mg.L -1 and 2.0 mg.L -1 (25.27%; 35.37 %, respectively). BA 0.5 mg.L -1 in this experiment did not improve flowering efficiency, which is no floral bud (Figure 2). In terms of the height of shoot, the treatment with the ratio of BA/NAA 5 and 10 was the highest number ( Table 2). The BA/NAA ratio was an important factor for in vitro flowering and BA was the key in the transition of shoots from the vegetative to the reproductive stage of buds. (-), Not shoot regeneration.
Differentiated shoots were obtained after 10 weeks at all the treatments. On MS medium containing The BA and NAA with the ratio of 25 or 30, the flower primordia established the sepals and petal primordia were at 6 weeks of culture, which is in setting up organ identity early in flower development, compared to other treatments in the experiment. After 8 weeks of culture, flower morphogenesis of floral organs with sepal, petal, stamen, and carpel (Table 3). After 10 weeks, flowers comprise four different organs of concentric circles. In addition, lower ratios of BA and NAA (10, 15, and 20) had delayed flower organogenesis, compared with high rates of BA and NAA (25 or 30) after 8 weeks (Figure 4 b, c, d). In vitro flowering has petal numbers, and red color, which was less than ordinary flowers in the garden (Figure 3, 4).