Rapid Micropropagation of Sapium Sebiferum using Segments with Axillary Bud for Forest Bioenergy
Abstract
Sapium sebiferum is one of the four woody oil tree species in China for bioenergy forest. Its seeds have high oil content and are widely used. It is an excellent tree species that can be cultivated as a renewable energy source. An experiment was conducted to study culture in vitro and rapid propagation of S. sebiferum using segments with axillary bud. The results showed that the survival rate could be reached to 50.0% when the explants were disinfected with 70% ethanol for 30 seconds and with 0.1% HgCl for 10 minutes. In induction culture, the lateral buds began to sprout about 5 days later, 2 and after 20 days, the buds gradually differentiated. Among the three basic mediums, WPM medium has the highest effective bud induction rate, the effect of 6-benzyladenine (6-BA) mass concentration on axillary bud induction rate is extremely significant. The optimal medium WPM +6-BA 0.5mg/L+ NAA 0.05mg/L, conducting bud induction, and the induction rate could reach 62.9% after 30 days. Multiplication culture was carried out in the medium MS + 6-BA 1.0 mg/L +NAA 0.5 mg/L+ KT 0.5 mg/L, the proliferation effect was the best, with the multiplication rate of 81.2% and proliferation multiple of 3.2. The results of variance analysis showed that Naphthalene acetic acid (NAA) is the main factor affecting bud proliferation, followed by 6-BA. The rooting rate was up to 93.3% after induction in the medium 1/2 MS+ IBA 0.5mg/L. The establishment of rapid propagation technology system through tissue culture is an important way to solve the shortage of high-quality seedlings and provide technical support for the establishment of large scale S. sebiferum energy forest.
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Introduction
Sapium sebiferum (Roxb.) is a deciduous tree of Sapium in Euphorbiaceae , also known as wood wax tree. S. sebiferum is a drought-tolerant, short-term water-tolerant tree, There is a cultivation history of more than 1400 years in China (Editorial committee of Chinese flora 1980). It is one of the four major woody oil-bearing plants with strong cultivation adaptability, high oil content and wide use. It is an important economic bioenergy tree in China (Li et al. 2011). S. sebiferum is an excellent afforestation tree for timber forest and shelter forest in China with fast growth and long life. Especially as a scenic forest, it has a tall crown and different colors in four seasons, which is of great ornamental value. The root bark, bark and leaves of Sapium sebiferum are important raw materials of traditional Chinese medicine for the treatment of cirrhosis ascites, eczema and dermatitis. The leaf phenol extract has many effects, such as antioxidant, anti-inflammatory and anti ACD (Fu et al. 2015) (allergic contact dermatitis). The seed is covered with a layer of wax skin, which can be used to extract sapium fat (also known as Chinese (vegetable) tallow); the kernel can be extracted sapium oil (Liand Liu 2011). 100 kg seeds can be extracted 23 to 25 kg of sapium fat and 16 to 18kg of sapium oil. The well-operated sapium forest, the oil yield of seeds is as high as 40%, higher than that of Camellia oleifera , and it is also higher than Elaeis guineensis Jacq known as "oil king". The oil extracted from the seeds can be used not only as an edible oil source or an industrial oil source, but also as a power oil source, and is an inexhaustible source of renewable oil(Li et al. 2011; Shi and Guo 2001; Tan 2013; Liand Xu 2006; Peng 2013). With the deepening of biodiesel research and the accelerated development of biomass energy forestry industrialization, As a kind of high oil content substance, S. sebiferum seeds have a quite considerable prospect to be converted into biodiesel. Biodiesel is atypical “green energy” with the advantages of extremely low sulfur content, low aromatic hydrocarbon content, high oxygen content, high cetane content, and low exhaust gas emission(Wu et al. 2011; Helwani et al. 2009; Chen 2006; Campanelli et al. 2010; Hayyan and Mjalli 2010). The importance of wood biomass energy has been raised many times (Venketeswaran et al. 1983; Bajaj 1986). Therefore, it is necessary to carryout the research on the breeding, the rapid propagation, cytochemical composition, secondary metabolites, and the comprehensive utilization technology of seed production biodiesel, which is of great significance to alleviate the energy shortage, improve the ecological environment, and promote the adjustment of rural industrial structure. The establishment of S. sebiferum energy forest requires a large number of high-quality seedlings. Compared with traditional propagation methods (such as seedling planting or grafting), this method can produce plant materials for breeding or other purposes with less labor and lower cost in a shorter time (Bommineni et al. 2001). The establishment of rapid propagation technology system through tissue culture is an important way to solve the shortage of high quality seedlings. With regard to tissue culture, previous researcher used callus to regenerate the plants in vitro (Venketeswaran and Gandhi 1982), and performed in vitro propagation by means of bud proliferation from nodal explants (Siril and Dhar 1997). In this study, stem segments of S. sebiferum were cultured in vitro, and its high-quality seedlings rapid propagation technology system was studied to provide technical support for the establishment of large-scale S. sebiferum energy forest.
Conclusion
AND DISCUSSION The modernization of plant biotechnology began at the beginning of the 20th century, Plant tissue cell culture and regeneration, and cloning of new plants. After that, through molecular breeding to modify their genetic characteristics, including maker-assisted selection, genetic transformation and gene editing, there are likely to be more new programs in the future (Altman 2019). However, as a basic technology, tissue culture has made a great contribution. Among them, micropropagation technology has the advantages of rapid propagation, maintaining in vitro activity and protecting germplasm resources (Amirchakhmaghi et al. 2019). Just to solve the problem that some woody plants are difficult to be sowed or cut propagation and the vitality is easy to degenerate. Difficulty in sowing or cutting propagation. During the cultivation of S. sebiferum stem as explant, the pollution is more serious. After 30 s of disinfection with 70% alcohol and 10 minutes of sterilization with 0.1% mercuric chloride, the pollution rate is still high, which may be related to endophytic bacteria. In many woody plant species, especially in the wild, bacterial contamination is particularly difficult to treat and detect, because bacterial infection is large and often cannot be accurately identified and disposed in plant tissues (Viss et al.1991; Buckley et al. 1995). Antibiotics can be added to the culture medium to prevent the pollution of stem culture, which may needs further study. Ascorbic Acid (VC), Polyviny Pyrrolidone (PVP) or Sodium thiosulfate pentahydrate (SH) can be added to the medium optionally to control browning.
The success of tree tissue culture system largely depends on the selection of medium (Liu et al. 2017). WPM medium is conducive to the induction and growth of axillary buds of woody plants. It has been reported that the possibility of variation in vitro is much lower through axillary bud and adventitious bud as explants (Sharma et al. 2007; Shenoy and Vasil 1992). In this experiment, stem segment with buds of Sapium sebiferum was used as explant, and buds were induced in medium of WPM + 6-BA0.5mg/L + NAA 0.05mg/L. The induction rate was 62.9% after 30 days. In the 3 levels of 6-BAconcentration , when the concentration was 0.5mg/L, the average induction rate was 47.60%, significantly higher than the other two levels, it is also confirmed that 6-BA, at high concentration can stimulate shoot multiplication and inhibit their elongation (Lee and Wetzstein 1990; Jaime and Teixeira 2003). The variety and combination of growth regulators are more comprehensive, which is beneficial to screening of culture conditions conductively. Multiple analysis also be more scientific selection. In the process of proliferation, the medium of MS + 6-BA1.0 mg/L + KT 0.5mg/L + NAA0.5mg/L had the best proliferation effect, and the multiplication multiple was up to 3.2. IBA is the most commonly used for rooting induction (Keresa et al. 2012; Xu et al. 2008). Single bud was inoculated on 1/2MS + IBA 0.5mg/L medium for rooting induction, with the best effect and the rooting rate of 93.3%. These results showed that the concentration of hormones used in the medium significantly affected the regeneration mode and differentiation type of the culture. In our study, axillary bud stem segment was used as explants, there is recent scientific study (Hou 2020) on the establishment of in vitro plant regeneration systems directly derived from leaves and petioles of S. sebiferum, and adventitious bud induction rate is very high. This study has completed the study of aseptic culture of tissues, and the next step is the study of cell suspension culture and secondary metabolites, which is devoted to the identification of chemical components and pharmacological analysis of cell products, and in the hope of contributing to human health and forest bioenergy. LIST OF ABBREVIATIONS Abbreviation Full name 6-BA 6-benzyladenine NAA Naphthalene acetic acid KT Kinten HgCl Mercuric Chloride 2
