Axillary shoot proliferation and plant regeneration in Euryodendron excelsum H. T. Chang, a critically endangered species endemic to China

Euryodendron excelsum H. T. Chang is a single-type, rare and endangered woody plant unique to China. In this study, young stems were used as explants and cultured on Woody Plant Medium (WPM) supplemented with 5.0 μM 6-benzyladenine (BA), were subcultured for more than 15 times over a total of more than 3 years and finally an efficient axillary shoot proliferation and plantlet regeneration system was established in which one shoot could proliferate an average of 5.1 axillary shoots every 2 months on the medium supplemented with 5.0 μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). Shoots rooted at a moderate frequencies (50.1%) on agarized WPM supplemented with 0.5 μM NAA but 100% of shoots rooted in agar-free vermiculite-based WPM after culture for 2 months. Plantlets, when transplanted to peat soil: vermiculite (1:1), showed the highest 95.1% survival within 1 month.


Results
Effect of subculture period on shoot proliferation. In the early stage, very few axillary shoots were proliferated (1-2 shoots) (Fig. 1a). The subcultures were prolonged on WPM supplemented with 5.0 μM BA for more than 3 years and they generally developed multiple shoot clumps (Fig. 1b). The SPC could reach to 4.7 within 2 months.
As the multiple shoot clumps were transferred to different media, the SPC showed different. 1-10 μM Kinetin (KIN) induced SPC was usually between 2 and 3 ( Table 1), while 1-10 μM BA-induced SPC generally reached 4-5 indicating that BA induction effect of SPC is stronger than KIN. The higher of KIN concentration, the higher the induced SPC. Similarly, the higher the BA concentration, the higher the induced SPC to 2.8 in 1 month (Fig. 1c) and 5.1 in 2 months (Fig. 1d). As BA concentration reached to the level of 5-10 μM, SPC is basically flat. When BA and NAA are combined in the WPM, the SPC seemed improve to some extent, but overall, there is no significant differences.

Discussion
In the early stage, shoot proliferation was usually much low. With the subculture times prolonged, more and more axillary shoots were induced from one shoot node. One shoot node could proliferate for 5.1 shoots every 2 month. This high SPC could never been extravagant wished 2 years before. This maybe that the level of cytokinins in the stem at an early stage is relatively low, so the number of induced axillary shoots is less, as the number of subcultures increased, endogenous cytokinin levels presumably increased, thereby causing to proliferate more axillary shoot buds. Through more than 3 years long term and successive tissue culture, we finally established an efficient shoot proliferation system (Table 1).
In the family Pentaphylacaceae, there was no report about tissue culture. In the former family, Theaceae, tea [Camellia sinensis (L.) O. Kuntze] has a very high economic value. Micropropagation have been reviewed, providing comprehensive accounts of the success and limitations of biotechnological tools applied to tea and its wild relatives 19,20 . The embryogenic callus of Camellia nitidissima Chi. could differentiate into somatic embryos, nodular embryogenic structures and adventitious shoots depending on the PGR used in WPM. BA was best for adventitious buds 21 . The effect of cytokinins and GA 3 as well as different sucrose concentrations (5, 10, 20, and 30 g/L) on axillary shoot multiplication of Camellia japonica L. was investigated. High quality shoots and highest multiplication coefficient (3.4 shoots/basal explant; 2.4 shoots/apical explant) were obtained on WPM medium supplemented with BA, TDZ and GA 3 22 . Through 3 years subculture and test optimal PGRs in axillary shoots and rooting conditions. We established an efficient shoot proliferation, rooting and transplanting system. It may be the most successful establishing an efficient multiple shoot proliferation system in the family Theaceae. This will laid a better foundation for the future proliferation, biotechnology and preservation in E. excelsum.
Vermiculite is sometimes used in tissue culture for rooting and transplanting 23 . In this experiment, vermiculite is much more effective than agar on rooting medium (Table 2). On the vermiculite-based WPM rooting media, rooting percentage could reach 100%. However, on agar-based media, maximum rooting was < 48.5%. Adding vermiculite to transplanted substrates improves rooting percentage and plantlet survival due to increased  www.nature.com/scientificreports/ aeration. In contrast, almost all plantlets in substrates with yellow mud could not survive, so E. excelsum needs a well-aerated substrate for plantlets transplantation and recovery.

Materials and methods
Selection and culture of explants. Young stems of E. excelsum were collected from several 18-year-old trees growing on a mountainside in the Magnolia Garden of the South China Botanical Garden, Guangzhou. The sample seedling trees were brought back from the habitat, Yangchun City and transplanted and appraised by our colleague Prof. Huagu Ye (first author in 6th reference) 6 . The E. excelsum seedlings had been approved by local forestry permission. The stems 7-8 cm long with 2-3 nodes were disinfected in 0.1% mercuric chloride (HgCl 2 ) solution for 12 min then washed five times with sterile distilled water. Then the stems were cut into 2-3 cm long with one node were placed on an ultra-clean workbench and air-dried, and then inoculated onto agar (Solarbio, Beijing)-solidified plant growth regulator (PGR)-free half-strength Woody Plant Medium for new axillary shoot development 24 . In the early stage, every culture jars (12 cm high; 10 cm diameter) contained only one stem (Fig. 1a). After culture in light for a total of 2 months on this medium, the stems developed new axillary shoots and then transferred to new WPM supplemented with 5.0 μM 6-benzyladenine (BA) for multiple shoot proliferation (Fig. 1b). The WPM was supplemented with 20 g/L sucrose and 6.0 g/L agar, and medium pH was adjusted to 5.8-6.0 with 1.0 N HCl or 1.0 N NaOH. All the media was sterilized at 105 kPa and 121 °C for 20 min. Culture jars were placed in a 25 ± 1 °C culture room under a 12-h photoperiod with a photosynthetic photon flux density of 80 μM m −2 s −1 emitted by 40 W fluorescent lights (Philips, Tianjing, China). After that, the culture jar contained 3 multiple shoot clumps were subcultured onto the same WPM every 2 months. The shoots were continuously subcultured for more than 3 years with subculture number increasing to more than 18 times and generally developed multiple shoots. These multiple shoots could begin the following tests.