Keywords

Callus induction; Dendrocalamus asper; Genetic improvement; Regeneration; Zygotic embryo.

1. Introduction

Bamboos, belonging to Poaceae Bambusoideae with 88 genera and 1400 species, are important forest resources in the tropical and subtropical regions [1]. Bamboos are highly demanded for their great economic and social values in many fields such as agriculture, food, paper, decoration and so on so that their reproduction and breeding become more and more important [2].

Conventional reproduction by cuttings, offsets and rhizomes are inadequate to cope up with the demand of planting stock due to large propagule size, limited availability, seasonal dependence and low multiplication rate [3]. In addition, traditional breeding is largely limited because of its unpredictable, longperiodic, uncontrollable flowering phenomena and low percentage of seed setting [4,5]. Instead, an efficient regeneration system could not only yield thousands of plants with high efficiency, but also be essential to the gene transformation system [6-8].

Since the first report of obtaining regenerated plantlets from the mature embryos of Bambusa arundinacea via somatic embryogenesis, various explants have been utilized to establish an efficient regeneration system for many bamboo species such as Dendrocalamus latiflorus, D. farinosus, Bambusa arundinacea and so on [9-12]. However, the efficiency of bamboo regeneration was still low, conferring difficulties in establishing gene transformation system.

Dendrocalamus asper, a multipurpose tropical clumping bamboo, is highly demanded because of its high economic value for the delicious edible tender shoots and other traits [13]. Callus induction and regeneration of D. asper were established via different explants including nodal segments, leaves and roots [14,15]. In the present study, we developed a regeneration system via the mature embryos and determined the effects of the basal media and the concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) on the callus induction, as well as the effects of plant growth regulators and the times of subculture on the shoot differentiation. The regeneration system we developed could serve as a platform for future genetic improvement in bamboos.

2. Materials and Methods

2.1 Plant materials and tissue culture condition

Seeds of D. asper were collected from Yunnan province, China in 2010. The stripped seeds were rinsed in running tap water for 12 h, sterilized by immersion in 2.5%(v/v) sodium hypochlorite, plus 2 drops/100 ml of Tween-20 for 20 min, washed with sterile distilled water for five times, and then placed in a petri dish with a layer of filter paper. Embryos were removed from surface sterilized seeds under the stereomicroscope and placed with scutellum upwards on the callus induction media. The callus induction and maintenance were conducted in the dark at 25 ± 2℃, while shoot differentiation and root induction were performed under a 16/8 h light/dark photoperiod with light intensity of 30–50 μmol m^-2s^-1 at 25 ± 2℃.

2.2 Callus induction

For callus induction, six basal media including MS (Murashige and Skoog) medium, 1/2 MS medium (half-strength MS macrosalts), B5 medium, NB medium (N6 salts and B5 vitamins), HB medium and N6 medium were used [16-20]. The different concentrations of 2, 4-D (0, 0.3, 1, 3 and 10 mg/l) were supplemented in the MS medium. All media were supplemented with 500 mg/l proline, 500 mg/l glutamine, 500 mg/l casein hydrolysate, 30 g/l sucrose and 8 g/l type A agar. We quantitatively measured the frequency of the callus induction and the frequency of the granular and compact callus induction. They were calculated as follows:

Frequency of the callus induction=Number of calli/ Total explants inoculated.

Frequency of the granular and compact callus induction=Number of granular and compact calli/ Total number of calli.

2.3 Callus growth maintenance

Calli were transferred to the MS medium supplemented with the different concentrations of benzyladenine (BA) (0, 0.1, 0.3 and 1 mg/l) with 0.5 mg/l 2, 4-D, 500 mg/l proline, 500 mg/l glutamine, 500 mg/l casein hydrolysate, 30 g/l sucrose and 8 g/l type A agar. Callus subculture was carried out a 3-week interval. At the beginning, the weight of the calli was recorded. Three weeks later, the calli were subcultured. The final weight of the callus and the percentage of the browning callus were measured and recorded. The proliferation rate was calculated by the final weight of the callus/the initial weight of the callus inoculated. In addition, the subculture times (2, 4, 5) of calli were compared for shoot differentiation ability.

2.4 Shoot differentiation

Calli were transferred to the MS medium supplemented with different cytokinins such as BA (0, 1, 2 and 4 mg/l), and kinetin (KT) (0 and 1 mg/l) with 0.5 mg/l Naphthaleneacetic Acid (NAA), 30 g/l sucrose and 2.5 g/l gelrite. 4 weeks later, the differentiation rate and growth condition of shoots were recorded.

2.5 Root induction

Shoots (about 5 cm long) were transferred to the 1/2 MS medium supplemented with 30 g/l sucrose, indole-3-butyric acid (IBA) (0, 1, 3 and 10 mg/l) and 2.5 g/l gelrite for 4 weeks. The plantlets with strong roots were then transferred to potting soil for hardening in the greenhouse.

2.6 Statistical analysis

All treatments in this study were repeated at least twice. Experimental data were analyzed using SPSS ver.17.0 systems software for statistical analyses. Difference between the means was tested using Duncan’s multiple range tests [21]. Sigmaplot ver. 8.0 (systat software) was used to create figures.

3. Results

3.1 Callus induction from mature zygotic embryos

The mature zygotic embryos of D. asper were inoculated on different basal media containing 3 mg/l 2, 4-D. Three types of calli were observed in all the treatments. TypeⅠcalli were compact, granular, and creamy-yellow (Figure 1a), and can differentiate into plantlets and be selected for subculture. Type Ⅱ calli were watery, soft and white (Figure 1b). Type Ⅲ calli were hard, and non-embryogenic (Figure 1c). TypeⅠcalli demonstrated a great capability to proliferate and differentiate, while Type II and Type III calli were difficult.

Figure 1a-1j: Callus induction and plant regeneration from the mature zygotic embryos of D. asper(Callus induction and plant regeneration from the mature zygotic embryos of D. asper. a compact, granular and creamy-yellow calli (TypeⅠ), b watery, soft and white calli (Type II), c hard, non-embryogeniccalli (Type Ⅲ), d green spots on the surface of calli, e green shoots formed from embryogeniccalli, f buds and roots formed simultaneously on the surface of calli, g plantlets development via organogenesis, h plantlets with strong roots, i albino plantlets, j the regenerated plant grew in a soil pot).

As shown in Figure 2, the frequency of callus induction showed no significant difference on the all media, but the level of compact and granular callus induction was the highest on the MS medium than the other media, suggesting that MS medium was optimal for the callus induction from the mature zygotic embryos of D. asper.

Figure 2: Effect of different basal media on the callus induction from mature embryos of D. asper (Effect of different basal media on the callus induction from mature embryos of D. asper. Mature zygotic embryos were cultured on six basal media (MS, 1/2MS, B5, HB, NB and N6) supplemented with 500 mg/l proline, 500 mg/l glutamine, 500 mg/l casein hydrolysate, 30 g/l sucrose and 8 g/l Type A agar. Each treatment consisted of three replications of 20 explants. Values followed by the same letter are not significantly different within the same column according to the least significant difference at P<0.05[21]. All data were collected 4 weeks after the initiation of treatment with basal media).

MS medium supplemented with the different concentrations of 2, 4-D was used. As revealed in Figure 3, the concentration of 3 mg/l showed the highest level in callus induction as well as compact and granular callus induction. Therefore, 3 mg/l 2, 4-D was the best for callus induction.

Figure 3: Effect of the different concentrations of 2, 4-D on the callus induction of D. asper (Effect of the different concentrations of 2, 4-D on the callus induction of D. asper.Mature zygotic embryos were cultured on the MS medium supplemented with 500 mg/l proline, 500 mg/l glutamine, 500 mg/l casein hydrolysate, 30 g/l sucrose and 8 g/l Type A agar, and treated with the different concentrations of 2, 4-D (0, 0.3, 1, 3 and 10 mg/l). Each treatment consisted of three replications of 20 explants. Values followed by the same letter are not significantly different within the same column according to the least significant difference at P<0.05 [21]. All data were collected 4 weeks after the initiation of treatments with 2, 4-D).

3.2 Effect of BA on callus proliferation

We determined the effects of the different concentrations of BA on callus proliferation. Calli obtained in the previous step were inoculated on the MS medium supplemented with 0.5 mg/l 2, 4-D in combination with the different concentrations of BA. All the treatments showed a higher browning rate than that without BA, but they did not exhibit a significant difference in proliferation rate compared with that without BA (Table 1). Our data indicated that BA negatively affected callus proliferation, and MS containing 0.5 mg/l 2, 4-D was a suitable medium for maintaining callus proliferation.

Table 1: Effects of BA concentrations on callus proliferation of D. asper.

3.3 Shoot differentiation

Green spots (Figure1d) were induced from calli 10 days after calli were transferred to a differentiation medium. These green spots were then developed into shoots and plantlets (Figures 1e-1h), and occasionally albino plantlets (Figure 1i). The effects of different hormones on shoot differentiation and development in D. asper were shown in Table 2. Calli cultured on the MS medium supplemented with 2 mg/l BA and 1 mg/l KT exhibited the highest rate of shoot regeneration (43.35%) with fascicled buds compared with those on the other treatments.

Table 2: Effects of different hormones on shoot differentiation of D. asper.

The times of subculture may affect the ability of calli to differentiate shoots. As illustrated in Table 3, the rate of shoot differentiation and the quality of shoots all decreased with the increase times of callus subculture. The highest shoot differentiation (45%) and the best quality of the shoots were exhibited at the second time of subculture.

Table 3: Effects of different callus subculture times on shoot differentiation of D. asper.

3.4 Rooting of differentiated shoots

Plantlets (about 5 cm in length) were transferred to the rooting media supplemented with the different concentrations of IBA. As demonstrated in Table 4, the rooting rate and the numbers of roots were all elevated. With increasing concentrations of IBA, the induced roots became stronger, but their lengths shorter. Taken together, our data suggested that IBA at 3 mg/l was optimal for root formation in the plantlets, yielding a 90% rooting rate. The obtained plantlets with thick and long roots showed up to 95% survival after they were transferred and grew in the greenhouse (Figure 1j).

Table 4: Effects of different concentrations of IBA on shoot rooting of D. asper.

4. Discussion

Callus induction and plant regeneration are basic technologies in the transgenic breeding of plants. High regeneration efficiency is commonly required for gene transformation, which is influenced by many factors. In this paper, a stable and reproducible regeneration system of D. asper with high callus induction and shoot differentiation rates was established using the mature zygotic embryos as the explants. The system is beneficial for subsequent genetic transformation in bamboos with high economic and social values.

Basal media and plant growth regulators are known to play important roles in callus induction and plant regeneration in bamboos. The MS basal medium was usually used in bamboos, but other basal media such as B5, modified MS, and N6 were also used in bamboo organogenesis and embryogenesis [22,23]. In this study, six basal media (MS, 1/2MS, B5, HB, NB, and N6) were tested for callus induction and plant regeneration in D. asper. Our results suggested that the MS medium was more optimal than the other tested basal media.

Auxins like 2, 4-D is generally used to induce callus formation, but their optimal concentrations turned out to be variable and depended on bamboo species. We found that 3 mg/l 2, 4-D was the best concentration to induce callus formation in D. asper. The ability of callus differentiation is a key factor for plant regeneration. Auxins and cytokinins play important roles in callus differentiation [24,25]. In our study, regenerated shoots occurring on the MS medium supplemented with 2 mg/l BA and 1 mg/l KT showed the best condition. Moreover, the shoots would generate stronger roots in the medium with 3 mg/l IBA, and thus could develop into complete plantlets.

More calli may be obtained after continuous subcultures. If the calli can be continuously subcultured and maintained in good capability to proliferate and differentiate, there is no need to re-induce the calli frequently, which offers great advantage for research purposes such as gene transformation. However, serial subcultures and maintaining in the medium with 2, 4-D for a long time often result in browning calli and less differentiation [24]. In this paper, we found continuous subculture do reduce the differentiation ability in the calli of D. asper, and the second time of subculture was optimal for keeping the callus differentiation rate and high quality of the shoots in D. asper.

At present, there are few reports on bamboo transgenic research due to the relatively low regeneration efficiency [26,27]. More plant growth regulators in combination with different organic addictive may be applied in future to increase the frequencies of callus induction and differentiation as well to improve the quality of the inducted callus and differentiated shoot in D. asper.

5. Conclusion

An efficient plant regeneration system of D. asper by using the mature zygotic embryos was established. Callus formation induced on MS medium supplemented with 3 mg/l 2, 4-D, and 0.5 mg/l 2, 4-D was optimal for the callus proliferation. In addition, MS medium supplemented with 2 mg/l BA, 1 mg/l KT, and 0.5 mg/l NAA was optimal for the callus differentiation and subsequent shoot growth. It was worth noting that the calli maintained high differentiation rate (45%) after twice subculture. The rate of the root formation was 90% in the 1/2 MS medium supplemented with 3 mg/l IBA. Rooting plantlets showed a 95% survival rate when they were transferred and grew in the greenhouse. As a conclusion, the approach for plant regeneration of D. asper developed in the present study provides a tool to manipulate bamboo species for transgenic improvement.

Acknowledgement

This study was supported by the National Natural Science Foundation of China [Grant no. 31270677], the China Scholarship Council to X Lin, and the Natural Science Foundation of Zhejiang Province [Grant No. Z3100366].

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