In Vitro Propagation And Rejuvenation of Senescent Maternal Plant of Ardisia Crenata Var. Bicolor (Primulaceae)

Ardisia crenata var. bicolor is an ornamental shrub, owing to its declined wild population, recalcitrant seeds and few high-quality cuttings, the main objective of this study was to optimize an in vitro propagation protocol by using tip shoot and nodal segment as explants from senescent plant. Explants were sterilized and cultured on Muraghige and Skoog medium contained 1.0 mg·L -1 benzylaminopurine and 0.05 mg·L -1 1-naphthaleneacetic acid for shoot initiation. For shoot proliferation, explants were cultured on MS medium with 1.0 mg·L -1 BAP, 0.1 mg·L -1 NAA, and 0.5 mg·L -1 kinetin, and the proliferation coecient were 3.1 and 2.5. Rooting was achieved by two explants in half-strength MS medium containing 0.5 mg·L -1 indole-3-butyric acid + 0.1 mg·L -1 or 0.2 mg·L -1 NAA, and 0.5 g·L -1 activated charcoal. The highest rooting rate were 72.7% and 65.1% with the highest mean number of roots (4.2 and 2.8, respectively). After acclimatization, 83.3% and 81.2% of plants were survived in the greenhouse. The plant can be rejuvenated via in vitro propagation and provide a reference for supplying the planting materials quickly with an uniform genotype.


Introduction
The genus Ardisia Swartz 1 was placed in Myrsinaceae in traditional taxonomy systems, but in APG ( ) and APG (IV), it was included in Primulaceae. Ardisia crenata Sims var. bicolor (Primulaceae) is an evergreen shrub that exists the transitional traits with Ardisia crenata Sims, which leads to no obvious distinction of them. The two plants forms clusters and glossy, drupe fruits 2 and produces red or white fruits 3,4 , the berries could adhere to maternal plant for 12 months or longer in weak-light environment, which is one of the most popular ornamental plants grown in houses or as an undergrowth companion species in gardens. In addition, its phytochemical constituents possess anti-tumor, anti-cancer, and anti-in ammatory properties [4][5][6][7][8] . In China, it has been widely used as a traditional folk herbal medicine for detoxi cation. In recent years, wild sources of A. crenata var. bicolor have been heavily exploited, which has rapidly decreased natural populations, whereas the poor arti cial selection and domestication methods resulted in low quality and mixed germplasm, which has become a bottleneck in its large-scale production.
Generally, the propagation of A. crenata var. bicolor is accomplished sexually by seed and asexually by cutting. The seeds are categorized as recalcitrant-type and thus are highly susceptible to desiccation which can't survive in dry storage 9 . Yang et al. 10 also reported that seeds of A. crenata var. bicolor did not germinate under dry conditions for 90 days, but the germination rate reported by Tezuka et al. 11 was more rapid after long periods of storage at low temperature (approximately 5 ℃). Roh et al. 12 used conventional sowing method, the seeds needed 13 weeks or longer to achieve 80% germination at 25.8 ℃, and 4 years for obtaining commercial high-quality plants, while the cuttings required longer than 45 days to form 76% rooting, although the commercial seedlings were obtained in less than 2 years, only 31-40% of high quality plants. In order to protect A. crenata var. bicolor from extinction and and utilize continuely, although some researchers contributed to the species cultivation and propagation methods, few references reported on the in vitro culture of A. crenata var. bicolor.
In vitro propagation serves an important method to study shoot induction and initiation as it enables shoot to grow rapidly, genetic stability, and supply the planting materials quickly with an uniform genotype. The main objective of this study was to optimize an e cient medium for the regeneration protocol of two types of explants (tip shoot and nodal segment), and include different concentrations of PGRs for obtaining the maximum frequency of shoot and root regeneration. This protocol will provide a reference for the e cient regeneration and contribute to its rapid propagation and germplasm preservation of A. crenata var. bicolor.

Results
Explants sterilization. Different sterilization methods signi cantly in uenced the explants of A. crenata var. bicolor. The contamination rate of the tip shoot were slightly higher than that of the nodal segment using the same disinfection protocol, but the difference was no signi cant ( Table 1). The highest contamination rate of the tip shoot was 58.9 ± 0.1% which was recorded in samples disinfected with 75% ethanol for 30 s and 0.1% HgCl 2 for 5 min (T1). With the extension of HgCl 2 disinfection time, the contamination rate of the two types of explants decreased (T1 to T6). A. crenata var. bicolor was disinfected with 75% ethanol (30 s) and 0.1% (w/v) HgCl 2 (10 min), obtaining contamination rate of 31.1 ± 0.1% and survival rate of 57.5 ± 0.1% for tip shoot explants sampled from 5-6 year old saplings. In addition, the survival rate of explants were also affected by the disinfectant types, the contamination rate of tip shoot and nodal segment decreased with the extension of NaClO disinfection time (T7 to T9). Among them, the highest survival rate of nodal segments was 56.1% and was disinfected in 75% ethanol for 45 s and in 3% NaClO for 10 min, while the other treatments were lower, especially T7, the tip shoot were only 19.5%.
Axillary shoot induction. The effects of different concentrations of plant regulators were studied on buds regeneration in MS medium and WPM medium. One week later, the rst shoot was appeared in WPM media, and more shoots were observed after 15 days (Fig. 1C), this implies that the explants derived from A. crenata var. bicolor were suitable. However, the induction rate was signi cant differences among different concentrations of PGRs, some shoots were uneven, most of the buds or nodal segments showed browning.
Based on the phenomenon, three concentrations of BAP (0.5, 1.0, and 1.5 mg·L −1 ) and NAA (0.01, 0.05, and 0.1 mg·L −1 ) were further optimized for shoot regeneration ( Table 2). The obtained results showed that MS medium supplemented with 1.0 mg·L −1 BAP recorded relatively higher shoot induction (T4~T6), which was found to exert the best response that forti ed with 0.05 mg·L −1 NAA (T5), and the highest shoot induction rate of the tip shoot and the nodal segment were 68.58% and 70.53% respectively, followed by the T6, but there was no statistical difference between two treatments (P > 0.05). Although two types of explants cultured on WPM medium could also induce few shoots, the rate of shoot regeneration was much lower than that on MS medium (P < 0.01). The root necrosis with the extension of incubation time, and abnormal characteristics such as ower bud differentiation or albino leaves occurred in WPM medium (Fig. 1D). Shoot multiplication. The explants of A. crenata var. bicolor were inoculated in culture medium with varying concentrations of BAP, NAA, and KT (Table 3). Among them, the highest multiplication coe cient of tip shoot and nodal segment in T4 were 3.1 and 2.5 respectively of BAP-containing (1.0 mg·L −1 ) medium with NAA (0.1 mg·L −1 ) and KT (0.5 mg·L −1 ) (Fig. 1E), while BAP at the lowest and KT and NAA at their highest concentrations in T3 led to the least multiplication coe cient, 1.38 and 1.28 respectively, with yellowish-green shoots. With increasing of BAP concentrations, the multiplication coe cient of shoots rst increased and then decreased, the lowest multiplication coe cient of shoots were inoculated in the medium containing the lowest concentration of BAP (0.5 mg·L −1 ) and the highest concentrations of NAA (0.5 mg·L −1 ) and KT (1.0 mg·L −1 ). When the concentration of BAP was maintained constant, the proliferation coe cient of shoots were less at a lower or higher KT concentration than the optimal T4 medium (0.5 mg·L −1 ), while NAA only affected induction of tip shoot (P < 0.01, Table 4), but had a weak in uence on the nodal segments and the multiplication coe cient (P > 0.05). The proliferated shoots grew rapidly and healthily and elongated gradually within a month, but the average multiplication coe cient of the tip shoot and nodal segment were low (2.03 and 1.66). Based on the ANOVA (Table 4), which con rmed that an optimal concentration of BAP and KT plays a signi cant role in improving the shoot proliferation coe cient (P < 0.01), while NAA affects induction of tip shoot (P < 0.01), but a weak in uence on nodal segment and the multiplication coe cient (P > 0.05), which may be related to the fact that auxin mainly plays a role in apical meristems.   Rooting induction. After shoot multiplication, healthy and well-grown shoots were selected and inoculated into the rooting medium. The effects of different concentrations of IBA and NAA, and 0.5 g·L −1 AC were analyzed to determine the optimal rooting medium. Table 5 shows a broad spectrum of rooting responses.
Root regeneration was produced from the base of a bud directly but not from the callus (Fig. 1F). Of the three concentrations of IBA tested, the half-strength MS medium supplemented with 0.5 mg·L −1 IBA was found to exert the relatively better response on root induction, from which three concentrations of NAA were investigated, half-strength MS medium forti ed with 0.1 mg·L −1 NAA was found to produce the best response from the tip shoot and the highest rooting rate was 72.7% with the mean root number (4.2) in T4 media, while the nodal segment in T5 medium with 0.2 mg·L −1 NAA obtained the highest rooting rate of 65.1% and mean root number of 2.6, which were no signi cant difference with T4 medium (2.8) (P > 0.05). Transplanting. After 5 weeks, all aseptic rooted shoots were planted in the four different proportions of the disinfected matrix (Table 6). Among them, the highest survival rate from tip shoot and nodal segment were 83.33% and 81.2% that planted in peat: vermiculite: perlite = 3:2:1 (v/v) at 75% shading condition (Fig. 1G), which were higher than other treatments. could affect the growth of axillary shoots and roots and their physiological characteristics [13][14][15][16][17] . However, the availability of explants and their aseptic nature, the ability to respond to induction and propagation methods are crucial for the successful establishment of Aidisia regeneration system. The result provided evidence that the tip shoot and the nodal segment subjected to different disinfection methods could be used as inoculated materials, but explant type have a great in uence on the surface sterilization, cell permeability could increase in 75% ethanol for 30-45 s, whereas the cell injury increase and survival rate of explants decrease along the HgCl 2 immersion time.
For the most perennial plants, the quality of explants decreased with decrepit plant, which can be rejuvenated via in vitro culture 18, 19 . Tip shoot or nodal segment, in most cases, have differentiated rst into callus, and then take longer into adventitious shoots 20,21 . Ardisia crenata var. bicolor is a perennial plant, it is di cult to induce shoots from the tip shoot or nodal segment, but the bud propagation is time-saving and more reliable way to obtain plantlets from tissue culture, which could shorten the period of propagation. The WPM medium is known for good results with subcultured of woody species and produced more shoots 22 . Zhang et al. 23 reported maximum response for induction frequency, mean number of shoots and rooting rate of two dogwoods achieved on WPM medium, but this was not the case for A. crenata var. bicolor. In our study, WPM medium performances proved to be inferior to MS medium and MS medium gave better results. MS medium was used to induce the shoot initiation and proliferation of the tip shoot and the nodal segment, and halfstrength MS medium was bene cial for rooting. So, the composition of media and suitable concentration plays important role in establishing tissue culture regeneration system in A. crenata var. bicolor.
Plant growth regulators play important roles in regulating cell differentiation, division, and morphogenesis 24 .
In most shoot induction studies, high level of cytokinin has been used together with low level of auxin, which induced synergistic effect on shoot regeneration and leaf induction 25  Wang et al. 27 reported that NAA has a weak in uence on buds, which suggested a signi cant role of optimal KT in increasing the proliferation rate. However, with increasing cytokinin concentrations negatively affected the shoot regeneration frequency, which was consistent with Rejthar et al. 28 . In addition, it has been con rmed that a synergistic effect of different cytokinins can also result in effective regeneration rate 29 , which indicates that KT and BAP in combination were necessary to induce shoot proliferation.
The quality of the root induction is affected by the composition and concentration of the media, most species require auxin to induce rooting and shoot promotion 30 , but the root induction rate was decreased with increasing concentration of IAA 31 . Some research showed that IBA was found to be more effective in rooting induction compared to NAA and IAA 25,32 . Our results showed that the rooting rate and the root number of tip shoot and nodal segment increased rst and then decreased with an increase in the IBA concentration, the appropriate concentration of IBA and NAA could promote rooting signi cantly. The best concentration optimized for root induction was on half-strength MS medium augmented with IBA 0.5 mg L −1 + NAA 0.1 or 0.2 mg L −1 showed 72.7% and 65.1% root induction e ciency of tip shoot and nodal segment respectively.
The highest survival rate of transplanting plantlets were 83.3% and 81.2% which were slightly lower than its related species of A. crenata (86.6%) 33 , which might be related to the difference of the genetic characteristics and culture environments of A. crenata var. bicolor, but the aseptic seedlings grew well. Approximately, 6months duration was required from initial culture to regenerate into whole plant and provided a supply of propagule free of main pathogens. We established a successful regeneration protocol of A. crenata var. bicolor by tip shoot and nodal segment, which might need to be further optimization, but it is a far more e cient method of propagating commercial seedlings than by conventional cuttings.

Conclusion
The Root induction. Elongated shoots, dissected to approximately 1.5 cm in length, were cultured in the rooting medium, which were designed based on 3×3 orthogonal array and comprised different concentrations of IBA (0.2, 0.5 and 1.0 mg·L −1 ) and NAA (0.1, 0.2, 0.5 mg·L −1 ). All of these combinations were added to a halfstrength MS medium consisting of 2% sucrose, 0.8% agar, and 0.5 g·L −1 AC to prevent browning. Statistics were made according to rooting rate and shoot growth.
Transplanting. The in vitro plantlets were moved to a greenhouse before transplanting, opened each tube cap and added 2 ml aseptic water for 3 to 4 days. Subsequently, the plantlets were allowed to adjust to new growth conditions for 2 days and were carefully removed from the tubes and rinsed with sterile water to eliminate media remnants on roots. The clean plantlets were transplanted into seedling-raising plates or bags which were lled with four different proportions of the mixed matrix (peat: vermiculite: perlite=1:1:1; 2:1:1; 3:2:1; 4:2:1) and were then sprayed with 0.5% carbendazim solution. The seedbed was covered with transparent plastic lm and 75% or 50% sunshade nets to maintain a constant temperature (25 ± 2°C) and relative humidity (approximately 85%). The survival of transplanting was determined after 5 weeks.
Statistical analysis. All experiments data were recorded by visual observation of successful shoot and root induction and were subjected to statistical analysis. All percentage data converted to arcsine square root (√P) 35 to normalize error distribution. Analysis of variance (ANOVA) was used to analyze the data, and means were compared using Duncan's post-test at a 5% probability level. All analyses were conducted using the SPSS (version 21.0) software. All data are presented as means ± standard deviations.