An efficient protocol for regenerating shoots from paper mulberry (Broussonetia papyrifera) leaf explants

2.1 Plant materials, explant sources, and growth conditions

Seeds were collected from the Guangzhou cultivar in a field at South China Agricultural University (genotypes not included). Seeds were soaked in concentrated sulfuric acid for 9 min and washed with tap water for 5 min to soften the seed coat. After sterilization with 75% (v/v) ethanol for 50 s and 25% (w/v) NaClO for 15 min, the seeds were rinsed four times with sterile water and inoculated onto the MS medium [26]. When the seeds grew to seedlings after 4 weeks, seedling leaves were used as explants for experiments. All cultures were kept under cool white light (approximately 50 µmol/m²/s) with a 12 h photoperiod at 24 ± 1°C.

2.2 Induction and growth of shoot-buds

To compare the PGRs influencing the culture response, BA (0.0, 1.0, 1.5, 2.0, and 2.5 mg/L), indole-3-butyric acid (IBA; 0.0, 0.01, 0.05, and 0.10 mg/L), and kinetin (KT; 0.0, 0.5, and 1.0 mg/L) were used in combination in the MS basal medium. To investigate the effect of explant age on shoot regeneration, seedlings were divided into three sections from top to bottom: Section 1, top 1–3 leaves; Section 2, 4–6 leaves in the middle; and Section 3, bottom 7–9 leaves. To investigate the effect of explant orientation on shoot regeneration, we performed an experiment to examine the effects of contact of the proximal and distal ends with the medium. Four tree cultivars were used to examine the influence of genotype on shoot regeneration: NC-1, NC-2, TS-1, and ZQ-1. The percentage induction of shoot-buds and the number of shoot-buds per explant were recorded after 5 weeks of culture.

2.3 Rooting and acclimatization

The shoots were cut from the tissue and transferred onto the MS basal medium supplemented with NAA (0.0, 0.1, 0.2, 0.4, and 0.8 mg/L) to facilitate root initiation and the growth of intact plantlets. Plantlets with well-developed roots were removed from the culture bottles and washed under running tap water to remove any remaining medium. Then, the plantlets were placed in plastic pots filled with planting substrate in a greenhouse at high humidity. After acclimatization for 3 weeks, the plantlets were ready for transplantation in the field.

2.4 Data collection and statistical analysis

The experiments were conducted in a randomized complete block design; each treatment consisted of ten explants and was repeated three times. Analysis of variance (ANOVA) was carried out using SPSS 22.0. Duncan’s multiple range test was used to detect differences among means, with a significance level of 0.05.

3.1 Effect of PGRs on shoot-bud induction

The seedlings (Figure 1a) were removed from the culture bottles aseptically, and only the leaves from Section 1 were used as explants for the experiment. The leaves were excised, cut into 5 × 5 mm² pieces, and inoculated on the MS medium supplemented with various concentrations and combinations of BA, IBA, and KT for shoot induction. In the in vitro regeneration process, leaves began to expand gradually, the thickness gradually increased, the color changed from green to light, and the leaves were wrinkled and curled in 1 week. Shoot-buds appeared at 3 weeks (Figure 1b). Only parts of the induced shoot-buds elongated to form shoots.

Figure 1

Shoot regeneration from paper mulberry leaf explants and acclimatization of the regenerated plantlets. (a) 2 week-old seedling; (b) numerous shoot buds developed from leaf explants in the MS medium with 2.0 mg/L BA and 0.05 mg/L IBA in 3 weeks; (c) 2–3 cm-high shoots recovered from leaf explants in the MS medium with 2.0 mg/L BA and 0.05 mg/L IBA for 4 weeks; (d) root systems of regenerated shoot buds in the MS medium supplemented with 0.4 mg/L NAA; (e) an intact regenerated plantlet; and (f) an acclimatized plant transferred to a plastic pot filled with planting substrate.

The experimental results are shown in Tables 1–3. It is clear that PGRs played a vital role in shoot regeneration, and the proliferation at different concentrations was much better than when the PGRs were used alone (Tables 1 and 2). Although BA was more effective at promoting paper mulberry shoot regeneration than IBA, the best results were obtained when they were used together (Tables 1 and 2). KT had an inhibitory effect; the best results were obtained without KT (Table 3). The greatest efficiency was achieved when the MS basal medium was supplemented with 2.0 mg/L BA and 0.05 mg/L IBA: the shoot induction efficiency was 80.33% and there were 4.33 regenerated shoots (Tables 1–3).

Several regeneration protocols using leaves for explants have been reported for B. papyrifera. For example, Huang et al. [31] found that culturing paper mulberry leaf explants on the MS medium supplemented with 2.0 mg/L BA and 0.1 mg/L NAA had the best induction efficiency (81.7%), the regeneration efficiency was similar to this experiment, but the results did not show the number of shoots per explant, which was not rigorous. Liu et al. [30] found that regenerating shoots with leaf explants on the MS medium containing 1.5 mg/L BA and 1.0 mg/L NAA created the maximum number of shoots (3.88 shoots per explant), which was less than this experiment, and the experimental results did not show the regeneration efficiency, which was imperfect. However, Yu et al. [28] achieved a shoot induction efficiency of 36.4% propagating paper mulberry using leaf explants only, which was low. The effects of BA in organogenesis are supported by similar reports in other plants [33,34]. These results suggest that cell activities during shoot-bud regeneration are controlled by various internal factors including cytokinins, which may cause internal chemical and structural differences, leading to different responses when present in the medium. It is worth mentioning that NAA was used for auxin in the above studies, and IBA was used in this study. Obviously, the results of this study are better, and similar results have been reported in hemp [35]. However, the specific roles of BA and IBA remain unclear [26].

3.2 Effect of explant age on shoot-bud regeneration

Explant age affected the frequency of shoot-bud regeneration and the mean number of shoots per explant (Table 4). Explants from Section 1 had the greatest regeneration frequency (86.67%) and produced 4.35 shoot buds per explant. Therefore, the explant age plays an important role in shoot regeneration. Similar results were obtained in other plants. For example, Vaidya et al. [36] found that the age of peppermint shoot tip explants affected regeneration frequency; Singh [37] observed that the regeneration efficiency of Jatropha curcas was affected by the age of leaf explants; explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas beyond 30 days, the regeneration response decreased with an increase in the age of seedlings. Younger explants have greater morphogenic potential than older explants, as they might have more metabolically active cells due to hormonal, nutritional, and other physiological conditions that are responsible for increased organogenesis [33]. However, Zhang et al. [23] found that for Moringa, the explants were either too young or too old with unsatisfactory results. This may be due to the different meristematic capabilities of different plants. Each plant only has enough metabolically active cells if it grows to a specific age.

3.3 Effect of explant orientation on shoot-bud propagation

The orientation of the explants significantly influenced the shoot-bud induction response. When the distal end of the leaf explants contacted the medium, the shoot-bud regeneration rate reached 73.33%. However, this rate decreased to 13.67% when the proximal end of the explant contacted the medium. The number of induced shoot buds per explant increased from 1.02 to 3.58 after placing the distal end of the leaf explants contacted the medium (Table 5). In the cultures, shoot buds were mostly induced from the vein and edge of the cut surface of the distal end, as has been reported for sugarcane [38]. Similarly, Kumar et al. [39] found that Salvadora oleoides had a higher regeneration efficiency when the distal end of leaf explants contacted the medium. When studying the effect of explant orientation on regeneration efficiency, not only leaf explants, Liu et al. [40] found that for petiole explants of Jatropha curcas L., the method of inoculating the explants horizontally on the medium was more beneficial to the regeneration than inoculating the explants vertically. These results may be related to the transport of polar auxin, because the intensity of auxin polar transport is inversely related to the sensitivity of rooting [41]. Moreover, the logical explanation for the effects of orientation of the explants may be the oxygen tension in the meristematic cells at the explant–medium interface [38]. However, the specific reasons are not clear, and further research is needed.

3.4 Effect of explant genotype on shoot-bud regeneration

To examine the adaptability of the regeneration protocol to different paper mulberry trees, four cultivars with different genetic backgrounds were tested and their regeneration efficiencies and numbers of shoot-buds per explant were compared. As shown in Table 6, the regeneration efficiency and number of shoot-buds per explant varied greatly among the genotypes grown under the same conditions. Explants from ZQ-1 and NC-2 had higher regeneration frequencies of 83.33% and 78.58%, respectively, while explants from NC-1 and TS-1 fared much worse, with regeneration frequencies of 56.67% and 46.67%, respectively. The reason for this difference may be that the endogenous hormone levels varied amongst calli derived from different genotypes and explant types [42]. The differential behavior can be related to different mechanisms for control of the endogenous PGRs metabolism and/or contents. The regeneration capacity of calli is strongly correlated with the concentration of endogenous hormone; recently, Kudo et al. [43] suggested that the endogenous hormone levels affect the regeneration ability of calli. Similar results have been reported in other plants, including Mucuna bracteata DC. ex Kurz [44], Punica granatum L. [45], conifers [46], J. curcas [47], and jojoba [48].

3.5 Rooting and acclimatization

Shoots 2–3 cm in height (Figure 1c) were placed on rooting medium after 4 weeks. The percentage of rooting and the number and length of roots of each seedling increased with the concentration of NAA added to the MS basal medium (Table 7). The greatest rooting percentage (Figure 1d and e) achieved was 70.96% when shoots were placed on the MS medium containing 0.4 mg/L NAA. However, roots induced on the MS medium with higher concentrations of NAA (0.8 mg/L) developed more, but shorter, weaker roots. A similar observation has been observed in Simmondsia chinensis [49].

The rooted shoots survived well after transfer to plastic pots filled with planting substrate (Figure 1f). The acclimatization of the rooted shoots was accomplished and more than 90% of the plants were successfully transferred to plastic pots in greenhouse conditions. The results indicated that the regenerated shoots had good environmental adaptability. Similar results have also been obtained in other plants. Cheng et al. [35] found that 75% of the hemp-rooted shoots survived after acclimation. Similarly, Liu et al. [40] observed that more than 80% of J. curcas were successfully transferred to plastic pots. These results suggest that most of the regenerated seedlings can survive well after transfer to plastic pots, but the survival rate of different plants is different, probably because their environmental adaptability is different.