Introduction
The family Hydrangeaceae includes mainly woody plants and comprises 17 genera and about 170 species (Mabberley 1990). Hydrangea macrophylla is one of the most well-known species in the genus and is known by the name of Hortensia. It has been widely cultivated as a garden and potted plant. In addition, cut flower cultivars have been developed and widely grown in the gardens (Schiappacasse et al. 2014). In commercial practice, hydrangea is propagated either by seeds or stem cuttings. However, seed plants exhibit variability and do not always produce the desired characteristics or forms (Hartmann et al. 1997). Because of the clonal uniformity, most of commercial propagation of H. macrophylla is done by root cuttings (Hartmann et al. 1997; Jacobs et al. 1990). Then, plants from cuttings are slow to establish and lack of good basal branching. Moreover mother plants requirement is high in multiplication by cutting for the commercial production. Therefore tissue culture methods for vegetative propagation of plants have become increasingly important (Thomas and Heuser 1987).
Tissue culture method is part of biotechnology that is used for mass propagation especially for horticulture crops and ornamental plants. So many factors such as growth regulator, plant and explants type, environmental condition influence organogenesis and in vitro multiplication. Among these factors, growth regulators have the most effect on plant in vitro micropropagation (Jain 2002). Abou Dahab (2007) set up a protocol for micropropagation of H. macrophylla for commercial production. The experiments on salts compositions (MS or B5 full or half strength) on different cytokinins (BAP or TDZ) were performed to optimize adventitious shoot regeneration in H. macrophylla Thunb. ‘Nacthtigall’ (Doil et al. 2008). Cytokinins such as BA, 2iP or zeatin are also used for micropropagation of Hydrangea spp. ‘Blaumeise’ is supposed to be blue flowering variety and one of the best of the blue hydrangeas. ‘Vanilla Sky’ is vanilla white color flat mophead hydrangea and which possess strong stem. Both cultivars have high commercial value for cut flowers and flower pots. Therefore, this work evaluated the effects of cytokinins (BA) and auxins (IBA) testing with different treatments to enhance shooting and rooting and thus improve the production of H. macrophylla plantlets through micropropagation and acclimatize plantlets into a greenhouse for mass production of two commercial varieties. This updated method will facilitate the in vitro propagation of interspecific hybridization in Hydrangea breeding program.
Materials and Methods
This investigation was carried out in the Ornamental Plant Science Laboratory in Chonnam National University, during the years 2016 and 2017.
Plant material
In this experiment, two varieties of Hydrangea macrophylla species ‘Blaumeise’ and ‘Vanilla Sky’ were used as explants. Shoot tips and internodes were taken from mature plants grown in a greenhouse.
Sterilization of explants
Different explants (shoot tip and nodes) of H. macrophylla were washed under running water for 30 minutes. After washing the explants were dipped in ethanol 70 (%) for 15 minutes then were r insed with sterilized distilled water. Then the explants were placed in mercuric chloride (HgCl2) at concentration of 0.2 (%) for 5 minutes then were rinsed in sterilized distilled water. After t hat explants w ere sterilized with s odium hypochlorite (NaOCl) solution with the concentration 1 (%) for 10 minutes. Tween 20 (polyoxythylene sorbitan monolaurate) was used as a wetting agent at the rate of one drop per 100 mL of the sterilization solution. Sterilized explants were rinsed three times with sterilized distilled water to remove all traces of sterilizing substances.
Initial source of explant
Vigorous shoots of H. macrophylla were initial culture in vitro onto full strength of B5 medium according to Gamborg (1968), supplemented with 2 % sucrose, and solidified with 8 g･L-1 of agar. After 15 days of incubation, survival percentage and contamination percentage were estimated.
Culture condition
All of this experiments the pH of the medium was adjusted to 5.8 and autoclaved at 121˚C for 20 minutes. All the cultures were incubated in growth room at 25 ± 2˚C temperature under 16 hours photoperiod using cool-white fluorescent lamps.
Multiplication and shooting stage
Effect of different levels of BA on shooting behavior of Hydrangea macrophylla
For shoot multiplication, used the explants of successful initial culture stage and cultured on full strength B5 medium supplemented with the following concentration of BA (0.0, 0.5, 1.0, 1.5, and 2.0 mg･L-1). This experiment consisted of 5 treatments and each treatment consisted of 4 replicates, with each replicate consisting of 3 explants. After 4, 8, and 12 weeks (3 subcultures) of incubation the following data were recorded: number of shoots per explant, shoot length (cm), and number of leaves per explant.
After shoot multiplication stage, shoot elongation stage was started with the explants of well developed H. macrophylla and cultured in vitro on half strength of B5 medium supplemented with BA each at concentrations of 0.5, 1.0, 1.5, and 2.0 mg･L-1, in addition to control explants grown on a hormone-free B5 medium. Each treatment consisted of 4 replicates, with each replicate consisting of 3 explants. After 8 weeks the following data were recorded: number of shoots per explant, shoot length (cm), and number of leaves per explants.
Rooting stage
Effect of IBA on rooting behavior
Rooting stage was done by the well grown plants from shoot elongation stage and shoots were cut off the length of 1 cm and cultured on B5 (at half salt strength) and different IBA concentrations (0.5, 1.0, 1.5, and 2.0 mg･L-1), and without IBA on root formation. In this experiment, the 5 treatments were replicated 4 times, with each replicate consisting of 3 explants. After 45 days from culturing the explants, the following data were recorded: the number of roots per shootlets, root length (cm) and plant height (cm).
Hardening stage
Plantlets which produced in vitro were washed under tap water to remove agar from the roots then dipped with IBA solution (1 mg･L-1) and transplanted to peat pellets (Jiffy 7) growing media and the plantlets were gradually exposed to normal greenhouse conditions.
Statistical analysis
The layout of all experiments was a completely randomized design, with two factors. Statistical analyses were performed using Statistix 8 Analytical Software version 2.0. Data were subjected to analysis of variance (ANOVA) and comparisons between the mean values of treatments were made by the least significant difference (LSD) test calculated at the confidence level of p ≤ 0.05.
Results and Discussion
Effect of BA concentration on shooting behavior of Hydrangea macrophylla
The results presented in Table 1 indicated that supplementing the B5 medium with different BA concentrations (0.5, 1.0, 1. 5, and 2 . 0 mg･L-1), significantly increased the number of shoot per explant, compared to the control in ‘Blaumeise’ cultivar. Data show that BA 1.0 mg･L-1 was the best concentration on shoot number and leaf number; they were recorded (12.5) and (93.3) respectively. The results can also be found clearly in Fig 1A. The variety ‘Vanilla Sky’ found that BA 1.5 mg･L-1 gave the highest number of shoots (4.3) and number of leaf (29) (Table 2). But they were not significantly different between the treatments. A significant increase in shoot number and leaf number were recorded with increasing the number of subcultures (Table 1 and 2). High BA concentration caused the reduction in number of shoots per explants due to formation of callus instead of shoot induction.This result agrees with Doil et al. (2008) reported that 6-benzyladenine (BA) rather than TDZ could induce higher regeneration rates for H. macrophylla. Sacco et al. (2012) also found that BA at any concentration, induced the highest multiplication rate (over 7 shoots per explant) and shoot height was not affected by the cytokinin used in each treatment.
In shoot length elongation, there were significant differences between half strength B5 combine with different concentrations of BA (Table 3). In ‘Blaumeise’, the longest shoot (2.1 cm) and in ‘Vanilla Sky’ the longest shoot (1.4 cm) had been obtained at zero level of BA. Result showed without any growth regulators was the best treatment for shoot elongation in both cultivars (Fig. 1B). When compare with both cultivars, the growth of ‘Blaumeise’ was better than ‘Vanilla Sky’ in vitro propagation. This may be due to morphological characters of these varieties were different and ‘Blaumeise’ grow more vigorously under the same green house environment. This result agrees with Azza et al. (2017) reported that the longest shoot (3.0 cm) was obtained at MS medium containing zero level of BA. Using a half strength B5 medium is suitable for increasing the shoot length of H. macrophylla. Similar conclusions were found by Douglas et al. (1986) on the same species.
Effect of IBA on rooting behavior
Effect of half-strength B5 medium containing different concentration of IBA on rooting stage of H. macrophylla in w as s ignificantly d ifferent b etween t he t reatments. The results shown in Table 4 revealed that the mean number of roots varied from 5.0 to 11.0 roots per plantlet in ‘Blaumeise’ and 2.5 to 6.5 roots per plantlet in ‘Vanilla Sky’ depending on the IBA concentration. The number of roots increased significantly by increasing the concentrations of IBA from 0 t o 1. 0 mg･L-1 for both cultivars. Thus, the highest number of roots 11 and 6.5 roots per plantlet were found on IBA 1.0 mg･L-1 in ‘Blaumeise’ and ‘Vanilla Sky’ respectively. Raising IBA concentration from 1.0 to 2.0 mg･ L-1 caused a significant reduction in number of roots per plantlet. This may be due to formation of callus induction in the rooting zone. The same result found by Azza et al. (2017) reported that the most number of roots (4.67roots/plantlet) was obtained at the concentration of BA 1.0 mg･L-1 in H. macrophylla.
Hardening stage
Well rooted plantlets (2 - 3 cm length with 8 - 10 leaves) which produced in vitro were washed under tap water to remove agar from the roots which might be a source of contamination, then dipped with IBA solution (1.0 mg･L-1) and transplanted to peat pellets (Jiffy 7) growing media which containing sphagnum peat moss and wood pulp, these are 100% biodegradable. In order to maintain high humidity in culture environment, the plantlets were placed in container and covered with a light plastic cover. After 2 - 3 weeks, remove the plastic cover and transferred to plastic pots and the plantlets were gradually exposed to normal greenhouse conditions.
Conclusion
Results showed that effects of BA and IBA concentration on shooting and rooting behavior of Hydrangea macrophylla were significantly different between the treatments and without any growth regulators were best treatment for shoot elongation. The effect of BA concentration on plant growth in vitro was d ifferent depends upon varieties. The present study provided useful information for micropropagation of H. macrophylla species and further commercial varieties are needed to propagate by using tissue culture technique for mass production.
