Patent Application: US-201514738944-A

Abstract:
the present invention relates to a method for authenticating age of longer - lived vascular plants based on microstructural data obtained either destructively or nondestructively and based on implementation of at least one model to determine growth - year of different cultivars .

Description:
the following description and the corresponding embodiments of the present invention are set forth as preferred examples . it will be apparent to those skilled in the art that modifications , including additions and / or substitutions , may be made without departing from the scope and spirit of the invention . specific details may be omitted so as not to obscure the invention ; however , the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation . different cultivars of ginseng were collected respectively in october 2011 and august 2012 . because of the precious resource and the top - down sheet agricultural base , line transect method ( buckland et al ., 2007 ) was used in the present invention . it is found that there are three cultivars which can reflect the different root - types of ginseng in three regions of northeast china shown in table 1 . fig1 is a schematic diagram of the location of sampling in ginseng samples including a taproot view ( left hand side ) and a cross - sectional view ( right hand side ). first of all , taproot section is measured by microscopy . then , the centre of the cross section is used for telomeric terminal restriction fragment ( trf ) measurement . taproot view is drawn according to traditional chinese medicine graphics which includes most of the characters and features of ginseng , and is referenced to the “ wild ginseng character identification technology ” ( shifu fang , 2010 . ); the cross - sectional view is drawn according to the plant microscopic graphics . the samples are cut into small sections of 10 mm , and kept in formalin - acetic acid ( faa ) solution for more than 24 hours . the target site is labeled as “ sampling location ” as shown in the fig1 , and the portion belonging to “ sampling location ” is sliced into many pieces by leica weaving slicer . the sliced pieces are screened for microscopic measurement , followed by permeated with the chloral hydrate , and the average values of four measurements in different directions were processes used to obtain the values of “ r ” and “ b ” shown in the fig1 . ( cheng et al ., 2013 ). cambium lived as a banding between the primary xylem and phloem which are made up by smaller cells of 2 ˜ 3 layers ( banan , 1968 ). the reports of the microscopic structure of the vascular cambium cell showed that it is constituted by a fusiform initial and ray initial , and there is a larger nucleus in the cell ( buvat , 1955 ). the model is based on the prompts of the experiences and findings of telomerase research , described in the previous report , and in which initial theories of plant growth year &# 39 ; s authentication were discussed ( cheng et al ., 2013 ). reference to the reported method ( kimura et al ., 2010 ), as a target in the centre of xylem which is labeled as “ sampling position of trf ” as shown in the fig1 . three separate fresh ginsengs are crushed with liquid nitrogen and used for dna extraction using the method of cetyltrimethylammonium . ( shepherd et al ., 2011 ). dna ( 5 ˜ 10 ug ) is digested by ex - taq ( new england biolabs ( beijing ) ltd . neb ) overnight and fragments are separated by electrophoresis at 60v constant voltage for 4 ˜ 5 hours using 0 . 95 % agarose - gel . the telomere length is measured by southern blot analysis of terminal restriction fragment lengths ( masayuki et al ., 2010 ). the roche hybrid kit is used in washing nylon membrane , closing and photographing development . for the second procedure , the trf average length is calculated by telometric 1 . 2 with the following formula : where ai stands for the absorbance value , li stands for the marker length of the calculation object . the ginseng samples from different cultivars are dried under reduced pressure at − 20 ° c ., over 24 h ., and stored in a dry vessel , until machine testing . small - angle scattering ( saxs ) is used to obtain the projection and background images . the objects are rotated 360 degrees and projection data are obtained at the waist in the institute of high energy physics research ( hepr ), chinese academy of sciences . section and three - dimensional images are reconstructed by dei reconstructor v3 . 6 which is developed by hepr . it is found that the proportion of cambium cells decreased with the age from telomerase research ( cheng et al ., 2012 ). according to previous studies of relational expression : where p : the proportion of cambium area and sectional area ; n : the age ; r : section radius ; b : radius of cambium ; x : the harvest age ; β : coefficient ; α : index , and whereby , in the regression equation , the proportion of cambium cells decreases with age , and three kinds of cultivated ginseng are collected from three different origins . different regression equations are shown in fig2 . the respective regression equation about the proportion y and harvest age x for each of the cultivars is as follows : y = 0 . 0169x − 0 . 657 is obtained to estimate the age of ji &# 39 ; an ermaya ginseng ( fig2 a ); y = 0 . 0157x − 0 . 682 is obtained to estimate the age of fusong ermaya ginseng ( fig2 b ); y = 0 . 0152x − 0 . 763 is obtained to estimate the age of fusong damaya ginseng ( fig2 c ); y = 0 . 0205x − 0 . 467 is obtained to estimate the age of shizhu - ginseng ( fig2 d ). allometric law also exists between the area of phloem and xylem and the growth year in different ginseng cultivates shown in fig3 . plant age and the mass of xylem and phloem growth are also subjects in the allometric law . simulated three - dimensional curved surface equation is based on the xylem and phloem radial data of ginseng in different ages . the dependence of a biological variable y on body mass m is typically characterized by an allometric scaling law of the form : then the functional relationship , growth years ( y ) and the area of phloem ( x 1 ) and xylem ( x 2 ) are described as follows : y =√{ square root over ( a 1 a 2 * x 1 b1 x 2 b2 )} ( 2 )*( 3 ) y = k * x 1 m x 2 n ( 4 ) ( k is a coefficient ) matlab 7 . 0 is used to create the 3 - d curved surface mathematical model , and the results are shown in table 2 . and the 3 - d curved surface mesh shown in fig4 . according to the value of r 2 , the 3 - d curved surface mathematical model may be more accurate for age authentication . different traits of different varieties and origins can be grasped easily from the type of the 3 - d curved surface mesh shown in fig4 . there is different growth completion in different cultivars showing in fig5 . relative growth rate of xylem of different cultivars , which is expressed as shows that phloem and xylem growth are relatively uniform in different cultivars of damaya and ermaya which were produced in fusong in northeast china &# 39 ; s jilin province ; in contrast , there is an obviously different growth rate with the same cultivar of ermaya which were produced in different origins fusong and ji &# 39 ; an in northeast china &# 39 ; s jilin province . so , as show in fig5 , it illustrated that the growth rate of phloem is lower than the xylem in all cultivars , and the significant results shown in fusong cultivars . in addition , the established models are constructed with a large amount of microscopy data , cultivar identification becomes possible using the same mathematical model . however , due to the large individual differences in the same cultivar , it is very important to obtain a wide range of samples in different growth - year for detection accuracy . the average telomeric terminal restriction fragment ( trf ) analysis can be applied to plant age because it is a mature and recognized method to obtain telomere length parameters by southern blots of trfs in other species ( kimura et . al ., 2010 ). two kinds of plant are chosen in the present invention for trf analysis : one longer - lived and one shorter - lived , which are p . ginseng and p . lactiflora ( cultivar of shizhu ), respectively . according to the previous findings of telomerase test ( cheng et . al ., 2013 ), a special sampling site is considered to be the most representative of age shown in fig1 , which is the xylem centre in taproot , 1 ˜ 2 cm from the reed head . the results are shown in fig6 . the results show that the average trf decreases with an increase in age in p . lactiflora ; there is no clear linear relationship between average trf and age in p . ginseng ( cultivars of shizhu ). it is known in the art that the age can be easily identified by bone age ( walters , 2012 ). the same strategy has been used in the plant age identification , the annual ring or growth ring . it could also be useful for millennium trees . however , it is eclipsed long - lived herb , e . g ., ginseng . because trees and animals share similarities , and the age can be determined by the genetic material , there are different mechanisms of the age - increasing in different species ( watson et . al ., 2011 ). both in animals or at cellular level , many reports have provided that the biological age can be estimated based on the telomere shortening ( chen et al ., 2011 ; watson et . al ., 2011 ). a recent study showed that trfs of ji &# 39 ; an ginseng increases with age , which was tested with 6 stages ( liang et al ., 2015 ). in the above example of the present invention , however , it shows that trf decreases with age in shizhu ginseng , and there is no significant linear relationship exists in the stages of age but only a significant shortening in p . lactiflora . thus , great difference exists in different species and cultivars when identification of plants &# 39 ; age relies on the trf measurement . therefore , specific sampling points ensure the trf shortening trend , but no significant linear relationship exists to build any mathematical models in the present invention . 2 . 4 growth year authentication without damage using micro - ct and dei reconstruction nondestructive method is realized based on dei reconstruction or micro - ct . it proved that the empirical formula can be used in ginseng age estimates . for example , in fig7 . one taproot of shizhu - ginseng , after micro - ct , is obtained with the microstructural data of r = 9 . 75 mm , b = 6 . 90 mm , a clear cross - sectional view is shown in fig1 . referencing to the formula the harvest years x = 13 . 49 can be identified ; referencing to the formula y = k * x 1 m x 2 n , the harvest years y = 13 . 49 can also be identified . these results show that the age can be realized without damage by x - ray using the two mathematical models provided in the present invention . as a result , microscopy is more superior to molecular recognition for the age estimate in vascular plants . it can easily identify ginseng &# 39 ; s age by two kinds of mathematical models . however , the establishment of microscopic identification method is based on the complex distribution of the telomerase in the main root section ( cheng et al . 2013 ). this is an important theoretical evidence to prove the reasonableness of the mathematical model . in spite of the fact that the models can be used to identify growth - years of ginseng , the high growth - years of samples , which are over 40 years old or even 100 - 150 years old , are very scarce . therefore , conventional models have not yet been rigorously corrected for old - wild - ginseng , and the universal principles about age authentication in vascular plants cannot be summarized according to these models . perhaps study of more samples including herbs of high age will make us find “ bone age ” in vascular plants . even so , the microscopic analysis proves engaging and useful . in particular , a method for plant species identification and age authentication without damage under the same mathematical model at the same time comprising using the 3 - d curved surface mathematical model and the curved surface mesh is provided in the present invention . growth - year authentication has extraordinary significance for plant growth and development research , and has important applications in value assessment of economic crops . in the present invention , a systematic method of the growth - year identification theory of ginseng is provided . this is a great deal of progress in material selection for plant research and a key progress for the orientation of breeding based on the root type in ginseng cultivars . in particular , patterns in the taproot structure will create growth and development of visualization ; mathematical model based on the taproot structure development is a great deal of progress in ginseng industry , which makes the age identified without damage using x - ray micro - ct , and meets the traditional experience ; 3 - d mathematical model can identify the growth - year under microscopy , as well as identify different cultivars in the same time , this will save a huge research resources and reduce testing costs . moreover , the result of molecular techniques ( trfs ) using different plant species in the present invention proves that the microanalysis based on microscopy is more reliable in growth - year authentication . thereby , “ bone age ” of plant will become an important medium for plant growth and structure . thomas , s . c . 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