Axillary shoot proliferation
The axillary shoots of E. excelsum were subcultured in South China Botanical Garden (Guangzhou, China) on Woody Plant Medium38 supplemented with 1.0 mg/L 6-benzyladenine (BA) and 0.2 mg/L α-naphthaleneacetic acid (NAA) for over four years18. The earlier seeds and seedlings were fetched and identified by Prof. Huagu Ye and planted in SCBG. He has collected available specimen (number: 0276615-0276621) in SCBG herbarium. The axillary shoots originated from nodes culture from the stems of younglings which grew in South China Botanical Garden and it has been supported by our institute permission and all the collection comply with relevant institutional, national, and international guidelines and legislation and all methods performed in this study are in accordance with the relevant guidelines and regulations18.
As the stems developed, new axillary shoots were cut and transferred to fresh WPM with 1.0 mg/L BA, 0.2 mg/L NAA, 20 g/L sucrose and 6.0 g/L agar (Solarbio, Beijin, China) (pH 6.0) to proliferate shoots (Fig. 1a). All media were 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) 18. Each culture jar contained three multiple shoot clusters that were subcultured onto the same WPM every two months, and continuously subcultured on fresh WPM for over three years, allowing sufficient stock material to be produced (over 20-fold) in that period, for the following assays.
Effect of plant growth regulators on induction of callus and shoots
Leaves and petioles excised from axillary shoots were inoculated onto several WPM-based media to induce callus and shoots (Table 1). After culture for 6 weeks, the induction of callus, shoots or SELSs was investigated. In the latter two, numbers were assessed per callus clump of the same size. Each treatment contained 10 jars with three leaf or petiole explants per jar. For WPM with 1.0 mg/L 2,4-D treatment, the number of leaf and petiole explants was increased to more than 180 for each treatment since callus derived from this treatment was used for subsequent experiments. After culture for 6 weeks, the induction of callus, shoots or somatic embryos was investigated.
Callus differentiation
Leaf- and petiole-derived callus originating from culture on WPM with 1.0 mg/L 2,4-D (Table 1) for 6 weeks was transferred to WPM-based media in an attempt to differentiate it (Table 2). Each treatment contained 10 jars with three callus clumps of the same size per jar. After 4 weeks, callus differentiation was investigated under a stereomicroscope (Nikon SMZ745T, Tokyo, Japan).
Recovery of somatic embryos
Somatic embryos were cultured on PGR-free WPM with half-strength (micro- and macro-nutrients) (½WPM) or on ½WPM supplemented with 0.2 mg/L NAA or for 4 weeks to allow the somatic embryos to further differentiate and develop. Once SELSs rooted and attained 2 cm in length, they were transferred to plastic bags (12 cm high; 10 cm diameter) with peat: sand (1:1, v/v) and placed in plastic tetragon trays (50 cm length; 30 cm width; 10 cm height) in the controlled greenhouse with temperature at 20-28oC, the greatest nature light intensity at 200 μM m−2 s−1 in the noon and relative humidity at 60-95%. The trays were watered with tap water once daily, every morning. One and three months after transplantation, survival percentage was calculated as (number of plantlets that survived / total transplanted plantlets) × 100%.
Rooting of adventitious shoots
Callus clumps from which adventitious shoots developed were transferred to WPM with 1.0 mg/L BA and 0.2 mg/L NAA for 6 weeks. Single shoots (3 cm high) were excised and inoculated onto WPM with 0.5 mg/L NAA and 11.0 g/L vermiculite per jar (5 shoots/culture jar).
Acclimatization and transplantation
Plants that formed from adventitious shoots and that rooted well in vermiculite-based culture were transferred to plastic bags (12 cm high; 10 cm diameter) filled with peat: sand (1:1, v/v), with one plantlet per bag, and placed in a greenhouse under the conditions indicated above. Plastic bags were placed in plastic tetragon trays (50 cm length; 30 cm width; 10 cm height). Over 100 bags were sprayed daily with 100 mL of tap water at 8:00 a.m. One and three months after transplantation, survival percentage was calculated as indicated above.
Statistical analyses
Experiments were repeated in triplicate. Experimental data were statistically analyzed in SPSS 19.0 software (IBM, New York, NY, USA). After separating means – represented in tables as the mean ± standard errors (SE) – by analysis of variance, Duncan’s multiple range test (DMRT) was used to assess significant differences between means (P≤0.05).