| The resinous heartwood of Aquilaria and Gyrinops trees,known as Agarwood or Aloeswood,are precious spice and traditional medicinal material,which is presently facing problems of depletion of wild resources and inefficiency of artificial production.Agarwood contains characteristic components like sesquiterpenes and chromones,thus the studies on the biosynthesis of these components in plants will contribute to the understanding of agarwood formation.In vitro root culture system has been shown to be suitable for secondary metabolic research in many important medicinal plants.Therefore,in vitro root culture should be a potential system for the study of sesquiterpene and chromone metabolism in Agarwood-producing plants,laying the basis for exploring and explaining the formation process of Agarwood.1.ObjectiveThe aim of this study is to establish a stable and efficient in vitro root culture system of Aquilaria sinensis(Lour.)Gilg,the unique plant in China producing Agarwood,and evaluate its potential in production of 2-(2-phenylethyl)chromone after induction.2 Methods2.1 Optimization of in vitro root culture systemThe effects of auxin hormone type and concentration,auxin treatment time,inorganic salt concentration and initial inoculation amount on the growth of Aquilaria sinensis were studied by using single factor designs.The root culture system was selected from the rooting rate,lateral root density and lateral root length.Based on this,time-course analysis of root growth was done.2.2 Continuous subculture of in vitro rootsThe effects of continuous subculture on the regeneration of lateral roots and the growth of lateral roots were observed under the original culture conditions.Taking the rooting rate,lateral root density and lateral root thickness as the index,the possibility of long-term cultivation of Aquilaria sinensis roots was evaluated.2.3 MeJA treatment on in vitro roots35d old roots were induced with methyl jasmonate at different concentrations for different treatment time.The effects of methyl jasmonate on the regeneration of root system were observed by observing the regeneration rate and lateral root growth of in vitro roots.The cultures were collected for analysis of 2-(2-phenylethyl)chromones by GC-MS.The influences of methyl jasmonate concentration and treatment time on the determination of 2-(2-phenylethyl)chromones were investigated.3 Results3.1 Optimization of in vitro root culture systemThe results of single-factor experiments showed that induction with 1.5 mg/L 3-indolebutyric acid(IBA)for 7 days produced a large number of well-developed lateral roots;a reduction in the MS medium concentration(1/4,1/2,and 3/4x MS)was favorable for lateral root induction,whereas the normal concentration(lx MS)was favorable for lateral root growth;and the best initial inoculum size was 15 roots per bottle.Observation of the in vitro root development showed that lateral root primordia formed on the 6th day in most cases,and in some cases,lateral root primordia began to break through the main root epidermis on the 9th day,followed by rapid growth of lateral roots.The root multiplication coefficients calculated with dry weight reached a maximum on the 21st day and decreased gradually.3.2 Effects of long-term subculture on the regeneration of in vitro rootsTo observe lateral root primordia under microscope,the roots were cleared by soaking in aqueouschloral hydrate for 24 h.The results showed that the growth curve of the lateral root primordia induced by IBA was the same as that of the single gene in the 6th generation culture.But continuous sub-generation will affect the lateral root density.The lateral root density of the first 3 generations remained stable during the continuous subculture,and the lateral root density increased rapidly in the 4th generation and returned to the same level as the previous three generations.The final generation of the main root can be stable on the formation of 12-15 lateral roots.In addition,the diameter of lateral roots in the continuous culture process gradually decreases with the increase of culture algebra.3.3 Effects of MeJA on the growth and metabolism of in vitro rootsWith the increase of the concentration of methyl jasmonate,the rooting rate of the roots was decreased.Different concentrations will have different effects on lateral root growth.High concentration(400μmol/L)inhibited the growth of lateral roots.Medium concentration(100,200 μmol/L)will limit the lateral root density to some extent,promote lateral root elongation.Low concentration(50 μmol/L)of methyl jasmonate stimulation will increase lateral root density and promote lateral root elongation.The results of GC-MS showed that MeJA could induce 2-(2-phenylethyl)chromone in vitro root culture of Aquilaria sinensis.The results of GC-MS showed that there were positive correlations between the species of chromones and the concentration and time of treatment of exogenous jasmonate.With the increase of MeJA treatment time(0,1,3,5,7d),the detectable chromone species were from 0 to 2 species.With the increase of MeJA treatment concentration(0,50,100,400 u mol/L),the detectable chromones were from 1 to 3 species.4 ConclusionsIn this study,a stable and efficient system of in vitro root culture system of Aquilaria sinensis was established by optimizing the culture parameters.After MeJA treatment,in vitro root of Aquilaria sinensis could produce a couple of chromone substances,most likely in time-effect,and dose-effect correlation.The developed culture system provides an important platform for in vitro studies of Aquilaria sinensispertaining tometabolic process of Agarwood chromones. |
