| The phenomenon and mechanism of regeneration has been the focus of the DevelopmentBiology. Scientists have made a breakthrough in the research of animal regeneration and, it ispromising to achieve the transplantation of artificial organ in the near future so as to heal certaindiseases, maintain human health and prolong the life. Based on the theory of totipotency of plantcell and the method of tissue cultivation, species and amounts of plants have been cloned successfullyand that could increase in geometric progressions. In broad sense, the bark regeneration is constitutedof fruit tree girdling (the length of barking ranges from 0.3cm to 0.5cm)together with large-scale trunkbarking of the bark-utilization kind of economical woods(the length can be extended to more than50cm), however, the latter can be attributed to narrow barking. Large proportion of barking onEucormrnia ulrnoides Oily., Phellodendron amurense, Magnolia officinalis Rehd. et Wils. Subsp,Cinnamomum cassia, has already been reported. In fact, these studies of the mechanism inregeneration concentrated on the genesis of secondary bark and its anatomical characteristics,nevertheless the reason of survival for the plant under the condition of large-scale barking on the trunkstill remains to be revealed. Therefore, on the one hand, the study on the mechanism of barkregeneration could have a theoretic significance both in supplementing the correlative theory ofDevelopment Biology and in showing the life process of bark regeneration in trees. On the other hand,because the growth period of the bark-utilization kind of economical woods lasts long (varies from 10years to 15 years) and besides, the growth velocity of Phellodendron chinense has been outpacedby the depleting velocity of itself, consequently, the measure of bark regeneration could also have apractical significance in achieving sustainable resource employment of bark-utilization of economicalwoods as well as in boosting productivity of P. chinense and maintaining the fruits of the project ofCropland Conversion to Forest(CCF).Phellodendron chinense var. glabriusculum Schneid. belongs to the Family ofRutaceae. and falls into the Genus of Phellodendron which is a deciduous arbor and, as utilizing thebark of itself, it has become the main medicinal material for extracting the berberine. Since it is theoriginal medicinal material and indigenous wood in Sichuan, therefore, it is called Chuanhuangbo.(Sichuan P. chinense)Aiming at solving both the theoretic and practical problems, systematic study on the barkregeneration of P. chinense which was based on the Development Biology has been carried. Mystudy method were: 1) The method of localized observation using light and electron microscopeswas applied to the study that revealed the morphological and histogenetic development process of theP. chinense under the pressure of barking. 2) 14C-isotopes Tracing Technology was applied to thestudy that showed the effect of barking on the distribution of assimilates in crown and thetransportation of assimilates in renewed phloem. 3) The method of modern photosynthesis measurmentwas applied to the study that revealed the effect of barking on the photosynthetic traits, i. e.photosynthetic rate and transpiration rate. 4) The Chlorophyll Fluorescence Dynamic Technology wasapplied to the study that revealed the effect of barking on Maximum Quantum Efficiency ofchlorophyll fluorescence, Actual Quantum Efficiency, Photochemical Quenching Coefficient.Furthermore, the factors that influence bark reconstitution was systematic studied. The main results ofthe research are as follows:1. Morphologic and histogenetic study on bark regeneration of P. chinense(1) The barked plants of P. chinenses were wrapped by the transparent polyethylene film. Under the circumstance of high moisture, ivory-white callus appeared on the yellow-white surface of xylem.Gradually, the callus turned into light green and expanded. Light-brown and membranaceous subrinlayer with green tissues beneath had formed since unwrapped the film and then, the subrin layerthickened gradually. Until the end of the first year, it had shaped into protective layer. In the secondyear, the brown cork thickened and fell off in pieces. In the third year, the color and crack as well asthickness of the recovered bark were similar to that of primary bark. Moreover, edges of the girdle couldbe seen indistinctively.(2) The anatomic characteristics of juvenile stem and virginal bark. The color of juvenile stem of P.chinenses is green and close to circular shape. Primary structure of the young stem is constructed ofthe epidermis and indispensable tissues together with vascular bundles, whereas the epigenous hairsand stomas of the epidermis could not be discovered. However, the secretory cavity(cavity of essentialoil) as well as the sclereid in group-shape could be found in cortex, additionally, the chloroplast lies inthe parenchyma cell of the cortex. Virtually, the vascular bundles of the juvenile stem is the typicalcollateral vascular bundle and, the fibrous strips and group-shape sclereid could be found in theprimary phloem. Xylem rays exits and pith locates in the middle of the stem.(3) The bark of eight-year old plant is integrated by periderm, pressed and shrinked cortex,non-functional and functional phloem. Both the number of fibrous bands(in shape of annual ring) andthe number of layers within the periderm are correspondence with the age of the tree. In particular, thesclereid in group-shape could be found both in cortex and phloem. Histogenetic study on barkregeneration of P. chinense. The position of peeling off the bark was on the boundary between thesecondary phloem and the second xylem, i. e. the zone of vascular cambium. Majority of the cambialcells along with the bark were removed. Immature parenchyma cells, vessels, tracheids and xylem raysof the xylem were on the periphery of the secondary xylem. The wound of the girdled was wrapped bythe transparent polyethylene film. 3～7 days after packing, the immature cells of xylem between thecells of xylem ray and xylem ray began to split into callus; 15～20 days after packing, the cell of 2nd～3rd layers beneath the surface layer transformed into the cork cambium. 30～60 days after wrapping, thezone of vascular cambium with cells of 4th～6th layers formed within the combinative position of thexylem and the callus. Scattered sclereids in group-shape as well as ibriform fibres emerged in thesecondary phloem.The callus beneath the surface layer developed into cork cambium and immature cells within thexylem resplitted into vascular cambium, however, the emergence of the cork cambium was earlier thanthat of vascular cambium. Actually, the anatomical structure of the renewed bark in three-year old wassimilar to that of the primary bark..(4) Mass chloroplasts could be found in the light green callus and the growth stages could be dividedinto three phases, i. e. prophase and metaphase as well as anaphase. In the prophase, most of thechloroplasts aggregated in the cytoplasm and the cell membrane, whereas the grana and the lamellacould be discovered. In the metaphase, the starch grain presented itself and began to expand. The garanaas well as lamella could be saw. In the anaphase, the starch grain swiftly enlarged and the chloroplastswere broke down.The bark regeneration underwent 3 stages: 1) the formation of the renewed cells; 2) cork cambiumas well as vascular cambium splitted into periderm, secondary xylem and secondary phloem. 3)ultimately, recovered bark came to being. Based on the theory of Development Biology, the barkregeneration itself is "epimorphosis" rather than "morphallaxis". For the former, it signifies theredevelopment of the structure and tissue in advanced level. For the latter, however, it means low-level redistribution and reconstitution which depends on the tissues left.2. The effect of barking on the photosynthetic traits and transportation anddistribution of 14C-assimilates(1) The mean carrying velocity of 14C-assimilates was 50.2cm/h that was measured by 14C-isotopesTracing Method. The distribution for 14C-assimilates of the crown leaves was revealed: for thenon-girdled, the middle leaves of the crown was highest, bottom lowest and for the girdled, thedistribution altered, i. e. the top became highest while the bottom remained to be unchanged.Furthermore, the survival reason under the circumstance of large-scale barking on trunk was discovered,i. e. 15 days after barking, the renewed tissues possessed the capability of carrying the photosyntheticassimilates downwards.(2) Under the pressure of bark removing, the Photosynthetic Rate (Pn), Transpiration Rate(Tr),Maximum Quantum Efficiency of chlorophyll fluorescenee(ΦPO), Actual Quantum Efficieney(ΦP,),Photochemical Quenching Coeffieient(qp) were determined by the Li-6400 Photosynthesis MeasuringSystem and the result suggested that the effect of both the photosynthesis and transpiration could besignificantly reduced, however, evident damages of structure and function within the PSⅡdid not occur.The reason for decline in photosynthetic effect in a certain period probably lay in that the channels forcarrying photosynthetic products of the leaves downwards was obstructed and moreover, thephotosynthetic process was affected by the feedback restraints which occurred on the leaves inaccumulative way. The feedback constraints released as soon as the function of carrying the14C-assimilates downwards recovered, subsequently, photosynthesis and transpiration restored to normallevel.3. The effect of barking on the growth and contents of effective drug composition of P.chinense.(1) The content of berberine hydroehloride, paimatine hydrochloride, jatrorrhizine hydrochloride inrenewed bark of two-year and three-year old as well as these in primary bark were measured by themethod of High Performance Liquid Chromatography(HPLC).For the content of berberine hydroehloride, the values were 61.3784mg.g-1, 62.6545mg.g-1, 61.8816mg.g-1 respectively, of which suggested the difference in content between the renewed bark and virginalbark was not significant. For the content of jatrorrhizine hydrochloride, the values were 0.4815mg.g-1, 0.4600 mg.g-1, 0.2231 mg.g-1 respectively, of which revealed the content of the recovered barkwas much more significant than that in the primary bark.. For the content of palmatine hydrochloride,the values were 0.1004 mg.g-1, 0.0904 mg.g-1, 0.1621 mg.g-1 respectively, of which indicated thedifference in content was not significant. Therefore, the second-birth bark could be utilized as themedicinal material to extract the berberine.(2) Three years after barking, the thickness of the bark was ranked as the following: secondary bark ofthree-year old＞primary bark＞secondary bark of two-year old＞secondary bark of one-year old. Inparticular, the thickness of secondary bark (3.98mm)of three-year old was much more significant thanthat(3.01 mm) of the primary bark.Bark removing resulted in different degrees of etiolation within leaves, furthermore, the time ofdefoliation was advanced by about one month and, the burgeoning time in the next spring waspostponed by seven to nine days, however, the process of efflorescence and the burliness remained to bein running order. In the third year, the growth of the barked had recovered to normal state.The bark regeneration underwent 3 phases sequentially as following: 1) unwrapped the film and theformation of the callus 2) the suberization on surface layer. 3) the development of renewed periderm and phloem. During the first stage, putrescence and fungal infection would be caused by any touch. Duringthe first and second stage, the depanperateion or infection may be apt to occur on the of periphery of therenewed tissues. Actually, the main cause of the decline in healing rate is fungal infection.4. The factors which influence the bark regeneration of of P. chinense(1)The bark regeneration could be influenced by ten factors of which were wrapping, way of barking,time of barking, time of packing, intensity of barking, the method of barking, the operation of barking,weather condition, the state of the trunk, growth regulator. The result suggested that the regenerationcould be affected significantly by five factors of the referred above, i. e. wrapping, operation, thecondition of the trunk, the time of barking, the time of packing. Furthermore, based on the five factorsin differed extent towards the importance of the regeneration, the five factors could be divided into keyfactor, important factor, sub-important factor by the method of Path Analysis. Wrapping and operationtogether with the condition of trunk were the key factors which acted as the determinative effect in thebark regeneration. The importance of the three in key factors was ranked as following: wrapping＞operation＞trunk condition, i. e. wrapping with film was the precondition of the bark regeneration;extensive operation was the main factor which caused the bark regeneration into failure and also it wasthe key reason which brought on the reduction in concrescent rate; trunk state did indirect influence onthe regeneration, i. e. the rainwater filtered into the gap between the film and the trunk that was led bythe roughness of the latter, hence the rainwater, consequently, callus on the knurr had notformed. Therefore, the trunk condition had indirectly impact on the recovery of the barking surface.(2) Both the barking time and the wrapping time were the important factors, i. e, the healing processdisbenefit from barking too late or too early as well as too long or too short time in wrapping. The daysin wrapping together with the color post-removing had close correlation with the AccumulationTemperature. Besides, higher the temperature, shorter, time required for the wrapping. Contrarily, lowerthe temperature, longer time in need for the packing. Based on the formula of y=0.0495x-1.4328,the time of unwrapping could be predicted by Accumulation Temperature. However, in terms of thesefactors such as season, Phenology, growth etc., the time within the last ten days in May to first ten daysin July may probably be the appropriate occasion for barking. While barking within the last ten days inMay to the first ten days in June, the time required for wrapping is ten to fifteen days; While barkingwithin the last ten days in June to the first ten days in July, five to ten days is in demand for wrapping.In deed, light green color of the barking surface has acted as the key morphological index to determinethe implementation of unwrapping. Therefore, fitting time for unwrapping is jointly determined by theindex of days in wrapping and the index of morphology.(3) Sub-important factors which are length of removing, weather condition, tree age have lesssignificant impact upon the bark regeneration. Actually, the length for barking not only depends on thespecification of the medicinal material which is required by the market, but also the convenience of theoperation.(4) The results also suggests that barking can be operated under these weather conditions, i. e. sunny,cloudy, slightly rainy. Besides, the bark regeneration of P. chinense has not been influenced by the ageof itself, i. e. the manipulation of barking can be carried out on these plants aged two to twelve. Underthe condition of wrapping, the healing rate could be achieved by the two ways of barking, i. e. overallbarking, bark left in lengthways. However, concrescent conditions of barking surface differedcompletely. For full barking, above 90% of the barking surface was covered by the callus in shortterm(5 to 15 days); moreover, the trunk remained in straight as well as the morphology of the renewedbark was similar to that of the primary; however, in this way, the healing condition was mainly influenced by the operation and the trunk state. For the way of bark left in lengthways, less than 50%of the wounded surface was took up by the callus in short period, nevertheless the secondary barkexpanded gradually; in this way, without removing the channels, hence the recovery of the barkingsurface was slightly affected by the operation and the stem condition; however, the method wouldresulted in the abnormality of the trunk and xylem together with the secondary bark.In addition, the bark regeneration was probably affected by the plant growth regulator to a certaindegree, however, this must be tested further.5. The technological system of bark regeneration of P. chinense.(1) Multi-spot and multi-year experiments of barking in the main producing regions of Sichuan(Yingjing, Yaan, Hongya, Muchuan, Dayi) had been carried out and the results suggested that thesurvival rates in the 4 counties(Yingjing, Hongya, Muchuan, Dayi) could reach above 80%. Of thereferred 5 regions in Sichuan, the highest survival rate, i. e. 88.7% could be obtained in Muchuan,however, the rate in Yaan was ranked as the lowest, i. e. 50.0%. The main cause to the sharp distinctionof the survival rates between Yaan and Muchuan were the evident differences in the management. InYaan, the scarcity of pruning and fertilization during cultivation led to frank trunk with more knurrs.Less tightly in wrapping as well as the film in tearing or even high infectious rate were induced by thelow height under the branch, consequently, it was difficult for the formation of the callus within theknurrs. However, in Muchuan, the plants of the P. chinense are owned by the farmers and are adjacentto the houses of themselves, therefore, the trees are supervised well. In comparison with those in Yaan,the arbors are kept straight and strong. Besides, the conditions of light and soil as well as nutrients arewell maintained, furthermore crops under the woods are cultivated.(2) The chief running measures of bark regeneration are consisted of non-contaminated environment,site conditions, management, time of barking, way of barking, time of unwrapping. These are outlinesof above: 1) The environment should be non-contaminated 2) the way of cultivation should accord tothe Standard Operating Procedure(SOP) 3)The decorticated should be straight and strong as well as befree of diseases and insects .4)Use the "three-time cutting" and, the depth of cutting should be exactlyget to the surface of xylem and, lancet as well as hand should not touch the barking surface. 5)Removethe bark in appropriate time(between May and July), wrap the cutting position with polyethylene filmtightly, determine the time for unwrapping according to the current temperature and the season, in whichthe barking was done. 6)One week before barking, carbamide weighs 50g and SSP weighs 250g orcompound fertilizer(N:P:K=15:15:15) should be fertilized on each plant. The concrescence rate shouldbe checked one month after barking. If it is low than 60%, cut the baked plant but leave the stake inlength of 20cm. and also leave a new-birth branch. If it is infected by diseases, the speckle of diseaseshould be removed by lancet and spray the 800 times liquid of 70% thiophanate-methyl as well as 800times liquid of 80% Carbendazim on the infected.The research suggests it is comparatively difficult for the P. chinense, to achive barkregeneration, however, with good management and right operation, the survival rate in the end of thatyear could reach above 80%. The improvement of farmer's scientific consciousness and the enactmentof SOP regulations can help to the popularize this technic in yielding region of P. chinense, so as toachieve sustainable resource employment in bark-utilization species of trees.
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