Skip to main content
PMC home page
Plant Biotechnology logoLink to Plant Biotechnology
. 2018 Mar 28;35(1):51–58. doi: 10.5511/plantbiotechnology.18.0130a

Development of an efficient regeneration system for the precious and fast-growing timber tree Toona ciliata

Pei Li 1,2,3, Yuanyuan Shang 1,2,3, Wei Zhou 1,2,3, Xinsheng Hu 1,2,3, Wenmai Mao 1,2,3, Jingjian Li 1,2,3, Juncheng Li 1,2,3, Xiaoyang Chen 1,2,3,*
PMCID: PMC6543735  PMID: 31275037

Abstract

Toona ciliata (Chinese mahogany) is an important timber species and secondary protected plant due to excessive exploitation in China. Here we developed a robust and efficient regeneration system for adventitious shoot induction using hypocotyl explants of T. ciliata. To facilitate plant growth, different regulators were added to Murashige–Skoog (MS) medium (0.5 mg/l 6-BA, 1.0 mg/l KT and 0.1 mg/l IBA). A regeneration frequency of 58.67% with four shoots per explant was achieved by horizontal setting of hypocotyls on MS medium and following a 20-day seeding period. MS medium supplemented with 0.3 mg/l 6-BA and 0.2 mg/l NAA was optimal for shoot multiplication and elongation, with a multiplication coefficient of 3.06. A rooting frequency of 93.33% was achieved using the half-strength MS containing 0.1 mg/l NAA. After acclimatization, plantlets were transplanted to sterilized nutrient soil containing a 2 : 1 ratio of vermiculate with 90% survival frequency. Thus, the regeneration system developed in this study would be useful for genetic transformation and other biotechnology endeavours in T. ciliata.

Keywords: hypocotyl explant, plant regeneration, precious tree, Toona ciliata

Introduction

Toona ciliata belongs to the Meliaceae family and is a precious timber species in China, T. ciliata is primarily distributed in south China (Chen et al. 2014; Feng et al. 2015), and is also sporadically distributed along the east coast of India, Laos, Myanmar, Pakistan and the east coast of Australia (Heinrich and Banks 2005; Li et al. 2017a). Referred to Chinese mahogany, T. ciliata has straight trunks and produces red wood with a desirable grain (Heinrich and Banks 2005; Li et al. 2012; Liang et al. 2011). It has high economic value in wood industry. The impacts of environmental changes, low population renewal rates and over-harvesting of T. ciliata leading to it being an endangered species and listed as a level II national key protected wild plant in the China Plant Red Data Book (Li et al. 2015). Recently, increased attention has been paid to research and use of this tree species.

A broad-leaf tree species that with high hardness, high density and beautiful material appearance and texture can be called precious broad-leaved tree (Jiang 2013). They have special process properties which suitable to produce high-end furniture, high-grade instruments or high value-added terminal high quality arts and crafts products. Therefore, precious broadleaf trees such as T. ciliata have become scarce resources on the market (Luo et al. 2010). T. ciliata is the focus of development and utilisation programs designed to enhance the planting of fast-growing timber species in southern China (Li et al. 2017b; Zou 1994). T. ciliata also has applications in landscaping and potential medicinal uses. Thus, all of these uses of T. ciliata have led to its depletion (Chen et al. 2009; Cheng and Cui 2010; Chowdhury et al. 2003; Zhou et al. 2015).

The sustainability of T. ciliata is challenged owing to its growth characteristics and natural environmental impacts. Due to straight and tall adult tree, artificial seed harvesting is more difficult and the viability of the seeds decline in short time (Liu et al. 2014a; Zhao et al. 2005). The long breeding cycle and labour intensiveness of T. ciliata are disadvantageous for its conventional breeding. Our previous studies also found that T. ciliata is readily damaged by Hypsipyla robusta, which can cause death of T. ciliata. Furthermore, T. ciliata is subjected to freezing injury in some regions, which slows growth and unable to perform the characteristics of the rapid growth. All these influences negatively affect the species growth and practical demand in a large scale. Plant genetic engineering can provide an effective way for genetic improvement (Raza et al. 2017). The establishment of genetic transformation system that is a key technique for genetic engineering can be used for plant conservation (Anjusha and Gangaprasad 2016; Chauhan 2016). But few studies have focused on the development of tissue culture methods for T. ciliata. The stems of mature trees or seedlings have been used as explants; however, low regeneration frequencies and difficulty in disinfecting have limited the methodological development (Angeloni et al. 1992; Chen et al. 2014; Liu et al. 2014a; Mroginski et al. 2003).

As an explant, hypocotyls has the advantages of short cycles, simple culture procedures and adequate repeatability (Shahzad et al. 2014). Hypocotyls are embryogenic organs derived from zygotic embryos with high embryonic activity that undergo division and differentiation via the parenchymal cells of the vascular-forming layer, and hypocotyls have high vitality and the ability to differentiate and regenerate (Liao et al. 2015). Notably, hypocotyls from Vernicia fordii (Mu et al. 2016), Cyphomandra betacea (Kahia et al. 2015), Fagopyrum esculentum (Hou et al. 2015), Tectona grandis (Tambarussi et al. 2017), nectarines (Yue et al. 2007) and Eucalyptus dunnii (Lu et al. 2016) are used routinely to produce regenerated plants. Besides, hypocotyls are excellent for genetic transformation as a result of their high rates of differentiation, convenience of selection, lack of seasonal restrictions and a high rate of infection with Agrobacterium (Huang 2006; Liao et al. 2015; Liu et al. 2017).

In this study, we developed an efficient and stable shoot regeneration system for T. ciliata using hypocotyls as explants. Such an approach provided the necessary technical support to achieve rapid propagation, which allowed identification, isolation and improvement of the desirable traits of T. ciliata.

Materials and methods

Preparation of culture medium and conditions

Murashige and Skoog (MS) medium (Murashige and Skoog 1962) containing 3% sucrose (w/v) and 0.5% agar (w/v) was used as the base medium in all studies, except the rooting studies, in which 1.5% (w/v) sucrose was used instead. All media were adjusted to a pH of 5.8 prior to the addition of agar and were autoclaved at 121°C for 20 min. Cultures were maintained at 25±2°C under white fluorescent light (30 µmol m−2 s−1 photosynthetic photon flux) and a 12/12-h light/dark cycle.

Seed samples and sterilization

Seeds of T. ciliata were collected from healthy mature trees in Pupiao, Yunnan, China (99°06′E, 25°04′N; altitude of 1,513 m; annual average temperature of 14°C) and stored at 4°C in the South China Agricultural University in Guangzhou.

After the wings were removed, the seeds were submerged in sterile water for 3–5 h at 45°C initial temperature. Seeds lacking wings were sterilized in 75% ethanol 60 s and then washed in sterile water for 60 s. Following washing, seeds were sterilized in 0.1% HgCl2 and 10% NaClO for different periods of time. The treatments with 0.1% HgCl2 were 5, 10 and 15 min, while the treatments with 10% NaClO were 10, 15 and 20 min. Seeds were then washed five times for 4 min each with sterilized water.

Sterilized seeds were inoculated on MS medium without growth regulators. Following incubation, 10 seeds from each treatment were placed in a single flask (100 seeds total) and grown for 10 days. Then, the contamination and germination frequencies were assessed.

Adventitious shoot induction

Hypocotyls were cut once the aseptic seedlings had grown to 2–3 cm (Figure 1A) and were inoculated on MS medium supplemented with 0.1 mg/l IBA; 0.3, 0.5 or 1 mg/l 6-BA; and 0.5, 1 or 1.5 mg/l KT. The medium without growth regulators was used as control. The optimal medium composition was determined based on the callus growth and shoot induction of hypocotyls.

Figure 1. Plant regeneration from hypocotyl explants of T. ciliata and acclimatization. A: Hypocotyl segments were cut from aseptic seedling. a: the upper of hypocotyl; b: the middle of hypocotyl; c: the lower of hypocoty. B: Shoots induction from hypocotyl in T. ciliata on MS media containing 0.5 mg/l 6-BA, 1 mg/l KT and 0.1 mg/l IBA. a: the explant that was cultured for 7 days, callus inducted; b: the explant that was cultured for 10 days, callus inducted continuously; c: the explant that was cultured for 20 days, shoots elongated from the calli; d: the explant that was cultured for 35 days, shoots elongated continuously; C: Shoot multiplication and elongation on MS medium containing 0.3 mg/l 6-BA and 0.2 mg/l NAA after 30 days of growth; D: Roots formed in half-strength MS with 0.1 mg/l NAA, 1.5% sucrose (w/v) and 0.5% agar (w/v). E: Acclimatized plantlet after 20-day transplantation. F: Acclimatized plantlet after two month; G: Acclimatized plantlet after four month.

Figure 1. Plant regeneration from hypocotyl explants of T. ciliata and acclimatization. A: Hypocotyl segments were cut from aseptic seedling. a: the upper of hypocotyl; b: the middle of hypocotyl; c: the lower of hypocoty. B: Shoots induction from hypocotyl in T. ciliata on MS media containing 0.5 mg/l 6-BA, 1 mg/l KT and 0.1 mg/l IBA. a: the explant that was cultured for 7 days, callus inducted; b: the explant that was cultured for 10 days, callus inducted continuously; c: the explant that was cultured for 20 days, shoots elongated from the calli; d: the explant that was cultured for 35 days, shoots elongated continuously; C: Shoot multiplication and elongation on MS medium containing 0.3 mg/l 6-BA and 0.2 mg/l NAA after 30 days of growth; D: Roots formed in half-strength MS with 0.1 mg/l NAA, 1.5% sucrose (w/v) and 0.5% agar (w/v). E: Acclimatized plantlet after 20-day transplantation. F: Acclimatized plantlet after two month; G: Acclimatized plantlet after four month.

Open in a new tab

Hypocotyls were inoculated from 15-, 20- and 25-day-old aseptic seedlings (in optimal medium, as described above), and shoot induction was assessed based on seedling age.

To assess shoot induction based on growth position, hypocotyls were divided into three 1-cm sections: one near the cotyledonary node (the upper) (Figure 1Aa), one in the central section (the middle) (Figure 1Ab) and one near the radicle (the lower) (Figure 1Ac). Sections were inoculated vertically stand on the medium, or placed horizontally, and shoot induction was assessed according to the position and the inoculation method.

Each assessment usually had three replicates, with 10 culture flasks containing five explants each. Shoot regeneration was assessed after 35 days of culture.

Shoot multiplication and elongation

Multiple shoots were placed in MS media supplemented with 0.1, 0.3 or 0.5 mg/l 6-BA and 0.1, 0.2 or 0.3 mg/l NAA. Shoot multiplication and elongation were assessed following growth under light. Each treatment usually had three replicates, with 10 culture flasks containing three explants each. Multiplication frequencies and seedling heights were assessed after 30 days of growth.

Rooting and field acclimatization

Four half-strength MS media containing 0, 0.1, 0.2 or 0.3 mg/l NAA were assessed to optimise the rooting medium for growth. Shoots 3–4 cm in length were grown in each medium, and the rooting frequency was calculated following growth under light for 20 days. Each treatment usually had three replicates, with five culture flasks containing three shoots each.

Culture flasks containing healthy plantlets that were adequately adapted to the culture environment were opened for 1 day, and the plantlets were washed to remove the medium and placed in water for 1 h. Plantlets were then transferred to plastic cups containing sterilized nutrient soil and vermiculite at a 2 : 1 ratio (Zhang et al. 2017). Cups were covered with plastic film for 3 days and watered to maintain moisture. Survival frequencies were evaluated after 20 days.

Statistical analysis

Analysis of variance (ANOVA) to compare mean values was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The means were also compared using Duncan’s multiple range tests. A p-value of ≤0.05 was considered the statistically significant level.

The contamination frequency (%) was calculated using the following formula: number of contaminated seeds/total number of seeds ×100%. The germination frequency (%) was calculated as the number of germinated seeds/total number of seeds ×100%. The shoot regeneration frequency (%) was calculated as the number of hypocotyl explants producing shoots/total number of hypocotyl explants ×100%. The multiplication frequency (%) was calculated as the number of new multiplied shoots/total number of shoots ×100%. The rooting frequency (%) was calculated as the number of shoots with roots/total number of shoots ×100%.

Results and discussion

Establishment of sterile seeds

The large quantity of aseptic seedlings provided sufficient experimental materials to establish the hypocotyl regeneration system. Therefore, seed sterilization was an important step in this study. The germination frequency was higher when sterilization was performed using 0.1% HgCl2 for 5 min or 10% NaClO in all three treatment time (Table 1). Following 3 days of culture, the radicle developed in seeds sterilized using 10% NaClO, while 6 days of culture were required for radicle development in seeds sterilized using 0.1% HgCl2.

Table 1. Effect of different sterilization time on the seed sterilization.

Sterilization agent Sterilization time (min) Contamination frequency (%) Germination frequency (%)
10% NaClO 10 34.67±3.21b 77.67±2.52b
15 28.00±2.65c 88.33±2.08a
20 6.33±2.52e 91.67±1.53a
0.1% HgCl2 5 31.00±4.58bc 79.00±3.61b
10 53.00±4.36a 60.00±3.00c
15 18.00±1.73d 49.33±5.01d
Open in a new tab

Values are mean±standard error. The different letters behind data mean significantly different from each other at p≤0.05 level, according to Duncan’s multiple range test. All the treamnets were after sterilized in 75% ethanol 60 s and then washed in sterile water for 60 s.

Contamination was the lowest among seeds sterilized using 0.1% HgCl2 for 15 min or 10% NaClO for 20 min, compared with all other treatments (Table 1). These data indicated that contamination was reduced with increasing 0.1% HgCl2 treatment time; however, 0.1% HgCl2 affected the germination frequency. This finding was consistent with studies in processing tomato (Tang et al. 2016) and Tung Tree (Vernicia fordii) (Mu et al. 2016). The optimal sterilization treatment was 75% ethanol for 60 s, followed by 10% NaClO for 20 min; 75% ethanol treatment resulted in the lowest contamination without affecting germination. Additionally, cotyledons grew from seeds cultured for 7 days.

Effects of plant growth regulators on shoot induction

Plant growth regulators are important for callus formation and adventitious shoot induction. Hormone levels in plants change continuously in vitro, and exogenous hormones directly impact the endogenous levels (Baskaran et al. 2015; Liu et al. 2014b). The formation of callus and adventitious shoots is influenced by the interactions between endogenous and exogenous plant growth regulators (Guo et al. 2016). Multiple studies have shown that 6-BA influences adventitious shoot differentiation more than do other cytokinins (Al Khateeb et al. 2013; Lee and Pijut 2017; Zhang et al. 2017), but only in the condition of rational configuration of cytokinin and auxin, there is a high frequency induction in explants.

The hypocotyls from aseptic T. ciliata seedlings were used as explants and incubated on MS medium supplemented with different combinations of plant growth regulators. When the concentration of IBA was kept at 0.1 mg/l, the shoot regeneration frequency initially increased with higher 6-BA and KT concentrations (6-BA was in 0.5 mg/l and KT was in 1 mg/l), followed by a decrease with a further increase in concentration (6-BA was in 1 mg/l and KT was in 1.5 mg/l) (Table 2). Thus, effects of the two cytokinins were concentration-dependent.

Table 2. Effect of different concentrations of cytokinin and auxin on shoot induction in T. ciliata on MS after 35 days of culture.

No. Plant growth regulators and concentration Shoot regeneration frequency (%) Number of shoots per explant
6-BA (mg/l) KT (mg/l) IBA (mg/l)
T1 0 0 0 0f 0c
T2 0.3 0.5 0.1 19.33±5.03e 2.00±0b
T3 0.3 1 0.1 26.67±4.16de 2.67±0.58ab
T4 0.3 1.5 0.1 42.00±4.00bc 2.67±1.15ab
T5 0.5 0.5 0.1 34.67±7.02cd 2.33±0.58b
T6 0.5 1 0.1 58.67±5.03a 4.00±1.00a
T7 0.5 1.5 0.1 48.67±4.08b 2.33±0.58b
T8 1 0.5 0.1 21.33±9.24e 2.67±1.15ab
T9 1 1 0.1 42.00±7.21bc 2.67±0.58ab
T10 1 1.5 0.1 24.67±2.31e 2.67±1.15ab
Open in a new tab

Values are mean±standard error in triplicate, each with 50 explants. The different letters behind data mean significantly different from each other at p≤0.05 level, according to Duncan’s multiple range test.

Among the 10 medium compositions containing with different concentrations of 6-BA and KT, calli were observed at the wounds of hypocotyls after 7 days of culture on MS containing 0.5 mg/l 6-BA, 1 mg/l KT and 0.1 mg/l IBA (optimal composition). Callus growth continued gradually following the initial growth. After 20 days of culture, shoots elongated from the calli (Figure 1B). The shoot regeneration frequency reached 58.67%, while the number of shoots per explant reached 4.0 (Table 2).

Optimization of culture conditions

Except plant growth regulator, the differentiation of adventitious shoots from hypocotyls was influenced by seedling age, explant genotype and inoculation method.

Seedling age is important factor for the inducting of adventitious shoots in T. ciliata. Significant differences in regeneration capacity of hypocotyls from 15-, 20- and 25-day-old seedlings were observed (Table 3). The reason for that is seedling age determined the physiological state of the explants, and the young seedlings used in this study reflected the strong differentiation and regeneration capabilities of the parenchymal cells in incision (Gaur and Srivastava 2017; Zhu et al. 2005). Differences in physiological state also existed in the same explant comprised of seedlings of different ages (Compton et al. 1993). Additionally, the highest shoot regeneration frequency was 43.82%, observed in 20-day-old seedlings, in which the number of shoots per explant was 4.67 (Table 3). These data suggest that regeneration of explants younger than 20 days old was less than ideal, and that after 20 days of growth, the regeneration ability decreased with seedling age.

Table 3. Effect of aseptic seedling age and position of hypocotyl on adventitious shoot induction from hypocotyl of T. ciliata.

Effect factors Shoot regeneration frequency (%) Number of shoots per explant
Seedling age 15-day-old 28.37±7.39b 3.33±0.58b
20-day-old 43.82±6.64a 4.67±0.58a
25-day-old 21.49±3.49b 2.67±0.58b
Position Upper 45.19±2.57 3.67±1.54
Middle 43.70±10.02 3.33±0.58
Lower 40.74±20.12 3.33±0.58
Open in a new tab

Values are mean±standard error in triplicate, each with 50 explants. All the culture media contained 0.5 mg/l 6-BA, 1 mg/l KT and 0.1 mg/l IBA. The different letters behind data mean significantly different from each other at p≤0.05 level, according to Duncan’s multiple range test.

Time difference in the onset of differentiation was based on the positioning of hypocotyls; however, this finding may have been due to different auxin contents in the sections themselves (Lin et al. 2016), which led to differences in the meristematic cells. Statistical analyses revealed that the regeneration frequency of adventitious shoots did not differ among the positions of hypocotyls after 35 days of culture. The shoot regeneration frequency ranged from 40.74 to 45.19%, and the number of shoots per explant was 3.33 to 3.67 (Table 3). Those data indicated that each section induced adventitious shoots under appropriate culture conditions. This finding is consistent with a previous study on Brassica napus L. (Shi and Zhou 1998), but inconsistent with a study on Platanus acerifol (Liu and Bao 2009) and Solanum melongena (Sharma and Rajam 1995; Zhang et al. 2014). In P. Acerifol, the cotyledon section readily induced adventitious shoots, while polarity phenomenon easy shown near the radicle in S. melongena.

The placement of explants influenced the induction of shoots. Significant increases in differentiation were observed when the explants horizontally placed on the medium. Hypocotyls which placed vertically stand on the medium exhibited poor differentiation, with calli observed only in the wounds that were exposed to the culture medium, the shoot regeneration frequency was just 3.82%. Additionally, calli were brown until explant death. However, horizontal explants expanded rapidly, exhibited transparent calli containing green buds and produced adventitious shoots, the shoot regeneration frequency was 41.64%, and the number of shoots per explant was 3.33.

Effects of plant growth regulators on shoot multiplication and elongation

To achieve normal growth following shoot induction, healthy adventitious shoots were transferred to medium optimized for multiplication and elongation. Compared with the adventitious shoots induction culture medium, this medium had reduced 6-BA but added NAA instead of IBA to promotes cell division and elongation (Campanoni and Nick 2005). The result is shown in Table 4. Assessments of the nine different culture media combinations for multiplication and elongation revealed that increases in NAA concentrations initially increased the multiplication frequency when 6-BA was kept in the same concentration, but then decreased with further as the concentration increased. Shoot heights of 3.87 cm were achieved in plants cultured in MS medium containing 0.3 mg/l 6-BA and 0.2 mg/l NAA, and this height was significantly greater than that of seedlings grown on other media (Figure 1C). Furthermore, a multiplication coefficient of 3.06 was observed in these adventitious shoots. Thus, this medium composition was most suitable for shoot multiplication and elongation.

Table 4. Effects of different concentrations of 6-BA and NAA on the adventitious shoots multiplication and elongation.

Plant growth regulators Multiplication coefficient Shoot height (cm)
6-BA (mg/l) NAA (mg/l)
0.1 0.1 1.36±0.13c 2.31±0.86bc
0.1 0.2 2.06±0.85bc 3.04±0.31abc
0.1 0.3 1.67±0.33bc 2.93±0.80abc
0.3 0.1 2.00±0.33bc 3.15±0.51ab
0.3 0.2 3.06±0.59a 3.87±0.53a
0.3 0.3 1.78±0.63bc 3.24±0.39ab
0.5 0.1 2.28±0.25b 1.92±0.22c
0.5 0.2 1.28±0.25c 2.70±1.46abc
0.5 0.3 1.50±0.17bc 2.86±030abc
Open in a new tab

Values are mean±standard error in triplicate, each with 30 explants. The different letters behind data mean significantly different from each other at p≤0.05 level, according to Duncan’s multiple range test.

Rooting culture

Shoots were cut to lengths of 3–4 cm and transferred to half-strength MS medium containing different concentrations of NAA that played a mainly role in rooting (Wei et al. 2015). After 7 days of culture, adventitious roots were formed. Rooting frequencies and the number of roots were determined after 20 days of culture. As shown in the Table 5, no significant differences in rooting frequencies were observed based on the culture medium used. But many calli were induced on the medium supplemented with more than 0.3 mg/l NAA.

Table 5. Effect of NAA on root induction and growth of adventitious shoots.

No. NAA (mg/l) Rooting rate (%) The number of roots Growth status of roots
TCR1 0 67.22±7.52b 10.33±0.58ab Thin roots without callus at the base
TCR2 0.1 93.33±11.55a 12.33±2.08a Strong roots with few callus at the base
TCR3 0.2 80.56±17.35ab 8.33±0.58b Strong roots with a lot of calli at the base
TCR4 0.3 85.00±13.23ab 8.67±1.53b Short roots with many calli at the base
Open in a new tab

Values are mean±standard error in triplicate, each with 30 explants. The different letters behind data mean significantly different from each other at p≤0.05 level, according to Duncan’s multiple range test.

Treatment with TCR2 generated the highest rooting frequency and average root number, were 93.33% and 12.33, respectively. TCR2 was comprised of half-strength MS with 0.1 mg/l NAA, 1.5% sucrose (w/v) and 0.5% agar (w/v) (Figure 1D). Additionally, roots grown in TCR2 were sufficiently strong for acclimatization and transplantation.

NAA is routinely used in rooting studies, and therefore, we only added it to our rooting medium. Also, as was found with Syzygium alternifolium (Khan et al. 1999), Vernicia fordii (Lin et al. 2016), Crassocephalum crepidioides (Opabode et al. 2017) and Couroupita guianensis aubl. (Shekhawat and Manokari 2016), the sucrose and nutrient contents (used half-strength MS) were decreased to increase rooting.

Acclimatization and transplantation

Washed plantlets were transplanted to sterilized nutrient soil containing vermiculite at a 2 : 1 ratio. Using this mixture, the 20-day survival frequency was greater than 90%. After 20 days of growth, the root systems were developed, and the leaves were dispersed with new leaves growing (Figure 1E). Plant heights were greater than 30 cm in two months after transplantation, then grew healthy (Figure 1F, G).

Conclusions

T. ciliata is an important and precious tree species in China and abroad. Since this species is exposed to pests and cold temperatures, production is often impeded. Genetic engineering has been actively promoted to increase its production; however, this has not been systematically investigated, and the transgenic technologies for T. ciliata are lacking.

In this study, we optimised transplantation methods and culture conditions for adventitious shoot induction, multiplication and elongation, and rooting using hypocotyl explants. We investigated multiple factors that affected regeneration. The regeneration system established in this study could provide experimental and technical references for future genetic improvements, conservation and production of T. ciliata.

Acknowledgments

This study was founded by public welfare projects of the National Forestry Bureau (201004020), and National Key Research Projects, Forestry Resource Cultivation and Utilization Technology Innovation (Ref. No.: 2016YFD0600606), People’s Republic of China.

Conflict of interest statement

The authors declare no conflict of interest.

Author Contribution Statement

CX designed the experiments. LP and SY performed them and analyzed the data. LP wrote the manuscript. ZW, MW, LJ and LJ carried out the shoot multiplication and elongation and field acclimatization experiment. All authors read and approved the final manuscript.

Statement

The researchers only collected the seeds from Toona ciliata and didn’t harm the tree body. The collected behavior through the consent and supervision of the local protection agency and the forestry bureau. In this study, we get the help from Baoshan forestry bureau. Hereby declare.

References

  1. Al Khateeb W, Bahar E, Lahham J, Schroeder D, Hussein E (2013) Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR). Physiol Mol Biol Plants 19: 157–164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angeloni PN, Mroginski LA, Rey HY, Flachsland EA, Inda M (1992) Establecimiento in vitro de especies de los géneros Gleditsia, Prosopis, Toonay Cedrela. FACENA 9: 135–150 [Google Scholar]
  3. Anjusha S, Gangaprasad A (2016) In vitro propagation and anthraquinone quantification in Gynochthodes umbellata (L.) Razafim. & B. Bremer (Rubiaceae): A dye yielding plant. Ind Crop Prod 81: 83–90 [Google Scholar]
  4. Baskaran P, Kumari A, Naidoo D, Staden JV (2015) In vitro propagation and biochemical changes in Aloe pruinosa. Ind Crop Prod 77: 51–58 [Google Scholar]
  5. Campanoni P, Nick P (2005) Auxin-dependent cell division and cell elongation: 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid activate different pathways. Plant Physiol 137: 939–948 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chauhan RS (2016) Biotechnological approaches for conservation of rare, endangered and threatened plants. Int J Sci Res Pop 6: 10–14 [Google Scholar]
  7. Chen HD, Yang SP, Wu Y, Dong L, Yue JM (2009) Terpenoids from Toona ciliata. J Nat Prod 72: 685–689 [DOI] [PubMed] [Google Scholar]
  8. Chen LW, Shi Q, Liang G, Cai L, He GZ (2014) Tissue culture of precious timber species Toona ciliata Roem. Subtrop Plant Sci 43: 164–167 [Google Scholar]
  9. Cheng DS, Cui TL (2010) Utilization value and cultivation techniques of Toona sureni. For Prod Spec China 4: 39–40 [Google Scholar]
  10. Chowdhury R, Hasan CM, Rashid MA (2003) Antimicrobial activity of Toona ciliata and Amoora rohituka. Fitoterapia 74: 155–158 [DOI] [PubMed] [Google Scholar]
  11. Compton ME, Gray DJ, Gary WE (1993) A simple protocol for micropropagating diploid and tetrapioid watermelon using shoot-tip explants. Plant Cell Tiss Org 33: 211–217 [Google Scholar]
  12. Feng LX, Chen R, Zhu CS, Yang SY, Liao YH, Mai KL, Su FB, Ceng JY, Yang SH, Luo SA (2015) Age structure and spatial distribution pattern of Toona ciliata population in northwestern Guangxi. J Northwest Forest Univ 30: 46–50 [Google Scholar]
  13. Gaur A, Srivastava DK (2017) Effect of Thidiazuron on in vitro regeneration potential of cotyledon and hypocotyl explants of cauliflower (Brassica oleracea L. Var. botrytis). Indian J Biotechnol 16: 126–132 [Google Scholar]
  14. Guo TT, Zhou XY, Zhang W, Chen F, Peng Q, Chen S, Zhang JF (2016) The optimization of the regeneration system of Brassica napus L. Jiangxi Agri Sci 44: 76–79 [Google Scholar]
  15. Heinrich I, Banks JCG (2005) Dendroclimatological potential of the Australian red cedar. Aust J Bot 53: 21 [Google Scholar]
  16. Hou SY, Sun ZX, Linghu B, Wang YG, Huang KS, Xu DM, Han YH (2015) Regeneration of buckwheat plantlets from hypocotyl and the influence of exogenous hormones on rutin content and rutin biosynthetic gene expression in vitro. Plant Cell Tiss Org 120: 1159–1167 [Google Scholar]
  17. Huang XQ (2006) Advances of regeneration in vitro system of cotyledon and hypocotyl and their genetic transform in rape. Southwest China J Agri Sci 19: 152–158 [Google Scholar]
  18. Jiang XM (2013) Development of the concept, policy, technical problems of ValuableBroad-leaved tree species in Jiangxi province and countermeasures. For Tech Jiangxi 1: 3–8 [Google Scholar]
  19. Kahia J, Sallah PK, Diby L, Kouame C, Kirika M, Niyitegeka S, Asiimwe T (2015) A novel regeneration system for Tamarillo (Cyphomandra betacea) via organogenesis from hypocotyl, leaf, and root explants. HORSCIENCE 51: 449–457 [Google Scholar]
  20. Khan PSSV, Hausman JF, Rao KR (1999) Effect of agar, MS medium strength, sucrose and polyamines on in vitro rooting of Syzygium Alternifolium. Biol Plantarum 42: 333–340 [Google Scholar]
  21. Lee JH, Pijut PM (2017) Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra. Plant Cell Tiss Org 130: 335–343 [Google Scholar]
  22. Li P, Que QM, Wang F, Li JC, Zhu Q, Liao BY, Chen XY (2017a) Optimization and primers selection of SRAP-PCR system in Toona ciliata Roem. For Res 30(1): 10–17 [Google Scholar]
  23. Li P, Que QM, Wu LY, Zhu Q, Chen XY (2017b) Growth rhythms of Toona ciliata seedlings from different provenances. J South China Agri Uni 38: 96–102 [Google Scholar]
  24. Li P, Zhan X, Que QM, Qu WT, Liu MQ, Ouyang KX, Li JC, Deng XM, Zhuang JJ, Liao BY, et al. (2015) Genetic diversity and population structure of Toona ciliata Roem: Based on Sequence-Related Amplified Polymorphism (SRAP) markers. Forests 6: 1094–1106 [Google Scholar]
  25. Li ZH, Li BH, Qi CJ, Yu XL, Wu Y (2012) Studies on importance of valuable wood species resources and its development strategy. J CS Univ For Tech 32: 1–8 [Google Scholar]
  26. Liang RL, Liao RY, Dai J (2011) Endangered causes and protection strategy of Toona ciliata. Guangxi For Sci 40: 201–203 [Google Scholar]
  27. Liao ZQ, Wu XM, Sun J, Guan CY (2015) One-step shoot regeneration culture established from hypocotyl of Brassica napus L. and applied to transformation. Mol Plant Breeding 13: 793–799 [Google Scholar]
  28. Lin Q, Li Z, Zhang L, Tan XF, Long HX, Wu LL (2016) High-efficiency regeneration of seedlings from hypocotyl explants of tung tree (Vernicia fordii). Int J Argic Biol 18: 370–376 [Google Scholar]
  29. Liu BC, Mu JQ, Zhu JB, Liang ZQ, Zhang L (2017) Saussurea involucrata SIDhn2 gene confers tolerance to drought stress in upland cotton. Pak J Bot 49: 465–473 [Google Scholar]
  30. Liu GF, Bao MZ (2009) Factors affecting shoot regeneration from leaf and hypocotyl explants of Platanus acerifolia Willd. Acta Horticul Sci 36: 399–404 [Google Scholar]
  31. Liu JL, Yang LL, Liu Q, Wu NZ, Zhou X, Chen J (2014a) Tissue culture and plant regeneration of Toona ciliata. For Sci & Tec 39(6): 1–15 [Google Scholar]
  32. Liu SY, Gao W, Xia HF, Yao D, Guan SY, Liu YY, Wang PW (2014b) Effects of plant growth regulators on callus induction and secondary culture of soybean. Jiangsu Agri Sci 42: 40–42 [Google Scholar]
  33. Lu RS, Han ML, Liang Z, Qin JL (2016) Factors affecting system establishment of adventitious bud regeneration from hypocotyls of Eucalyptus dunnii. Acta Agri Jiangxi 28: 31–35 [Google Scholar]
  34. Luo WY, Luo P, Liu YJ (2010) Choice and development of the fine and valuable hardwood tree species in tropical and south subtropical regions of China. Chinese J Trop Agr 30: 15–21 [Google Scholar]
  35. Mroginski E, Rey HY, Mroginski LA (2003) In vitro plantlet regeneration from Australian red cedar (Toona ciliata, Meliaceae). New Forest 25: 177–184 [Google Scholar]
  36. Mu Y, Hou JY, Chen J, Tang CG, Ni J, Chen M, Wu LF (2016) Establishment of direct regeneration system from hypocotyls of tung tree (Vernicia fordii). Mol Plant Breeding 14: 1821–1826 [Google Scholar]
  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assayswith tobacco tissue culture. Physiol Plant 15: 473–497 [Google Scholar]
  38. Opabode JT, Ajibola OV, Lamidi T (2017) In vitro Propagation of crassocephalum crepidioides: An endangered African traditional leaf vegetable and molecular analysis of micropropagated plants. Int J Veg Sci 23: 18–30 [Google Scholar]
  39. Raza G, Singh MB, Bhalla PL (2017) In vitro plant regeneration from commercial cultivars of soybean. BioMed Res Int 2017: 1–9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shahzad U, Jaskani MJ, Ahmad S, Awan FS (2014) Optimization of the micro-cloning system of threatened Moringa oleifera LAM. Pak J Agri Sci 51: 449–457 [Google Scholar]
  41. Sharma P, Rajam MV (1995) Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant (Solanum melongena L.) [J]. J Exp Bot 46: 135–141 [Google Scholar]
  42. Shekhawat MS, Manokari M (2016) In vitro propagation, micromorphological studies and ex vitro rooting of cannon ball tree (Couroupita guianensis aubl.): A multipurpose threatened species. Physiol Mol Biol Plants 22: 131–142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shi SW, Zhou YM (1998) High frepuency of shoot regeneration from hypocotyl explants in Brassica napus L. China J Oil Crop Sci 20: 1–6 [Google Scholar]
  44. Tambarussi EV, Rogalski M, Galeano E, Brondani GE, de Fatima De Martin V, Silva LAD, Carrer H (2017) Efficient and new method for Tectona grandis in vitro regeneration. Crop Breed Appl Biot 17: 124–132 [Google Scholar]
  45. Tang YP, Wang BK, Yang BS, Li N, Yang T, Patigu Wang Q, Yu QH (2016) Establishment of hypocotyls regeneration system for processing tomatoes. Xingjiang Agri Sci 53: 785–790 [Google Scholar]
  46. Wei Q, Cao JJ, Qian WJ, Xu MJ, Li ZR, Ding YL (2015) Establishment of an efficient micropropagation and callus regeneration system from the axillary buds of Bambusa ventricosa. Plant Cell Tiss Org 122: 1–8 [Google Scholar]
  47. Yue HY, Han MY, Zhang MR, Tian YM (2007) Factors affecting regeneration from hypocotyls of nectarine (Prunus persica var. nectarina). J Northwest A & F Uni (Nat Sci Ed) 35(11): 157–160 [Google Scholar]
  48. Zhang JJ, Yang YS, Lin MF, Li SQ, Tang Y, Chen HB, Chen XY (2017) An efficient micropropagation protocol for direct organogenesis from leaf explants of an economically valuable plant, drumstick (Moringa oleifera Lam.). Ind Crop Prod 103: 59–63 [Google Scholar]
  49. Zhang MH, Chen YH, Liu FZ, Zhang Y, Lian Y (2014) Optimization on regeneration protocol for genetic transformation of eggplant. J Changjing Veg 14: 15–20 [Google Scholar]
  50. Zhao RY, Li GY, Xu JM (2005) Breeding and afforestation technology of Toona ciliata. Guangxi For Sci 34: 155–156 [Google Scholar]
  51. Zhou H, Chen S, Fu C, Xie CB, Li YJ, Wang Y (2015) Research progress in cultivation and pharmaceutical chemicals of Toona ciliata and Toona ciliata var. pubescens. Agri Sci Tech 16: 722–726 [Google Scholar]
  52. Zhu LH, Zhang CQ, Sheng XG, Zhu YL (2005) Studies on high efficient system for in vitro shoot regeneration from hypocotyls of Chinese cabbage. J Wuhan Bot Res 23: 427–431 [Google Scholar]
  53. Zou GS (1994) The study of introdution and cultivation on valuable fast-growing species of Toona ciliata and Toona ciliata var. J Fujian Coll For 14: 271–276 [Google Scholar]

Articles from Plant Biotechnology are provided here courtesy of Japanese Society for Plant Biotechnology

RESOURCES