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黄柏剥皮再生机理及其影响因素

Research on the Mechanism and Its Influential Factors in Bark Regeneration of Phellodendron Chinense Var. Glabriusculum

【作者】 叶萌

【导师】 罗承德;

【作者基本信息】 四川农业大学 , 森林培育, 2007, 博士

【摘要】 生物的“再生”现象和机制是发育生物学的研究热点。再生在动物领域已经有了突破性进展,可望在不远的将来对于人类的某些疾病实现“人造器官”的移植,实现保障人类健康、延续人类生命的目的。植物领域以细胞的全能性理论和组织培养技术顺利克隆出以几何级数增加的植物种类和数量。广义的剥皮再生包括果树的环剥(0.3~0.5cm)和皮用类林木主干大面积剥皮(>50cm),狭义指后者。有关剥皮再生的理论基础研究缺乏,实践应用难度很大,已有报道的树种有杜仲、关黄柏(P.amurense)、厚朴、肉桂等,再生机理的研究主要集中在解剖学方面,探索了新皮的起源,却未能揭示树干大面积剥皮后不死这一核心问题。因而,研究剥皮再生的机理,对于完善发育生物学中植物再生的相关理论、揭示林木剥皮再生的生命过程具有重要的理论意义。由于皮用类经济林木生长期长(10~15a),资源利用速度远远大于生长速度,剥皮再生技术对于提高皮类林木的生产力,实现资源的可持续利用,稳定退耕还林成果具有重要的现实意义。黄柏(Phellodendron chinense. Schneid.)是中国传统的皮用类中药材和提取小檗碱的主要原料,四川的道地药材和乡土树种,已经有2000多年的药用历史,也是传统的出口商品。黄柏皮为临床常用的清热燥湿的中药,用途广泛,市场需求量大,可制成多种中成药,化妆品、生物农药等,对人民的生活健康具有重要价值。黄柏也被认为是难以剥皮再生的树种,从2002年至2006年,在四川省黄柏栽培现状调查以及实施规范化种植研究的基础上,针对剥皮再生的理论问题和现实问题,设立四川的荥经、雅安、洪雅、沐川、大邑5县(市)为研究区,以发育生物学为理论指导,黄柏(P. chinense)为供试材料,采用定位观察法、光镜和电镜技术、14C同位素示踪技术、现代光合测定技术、叶绿素荧光动力学技术,HPLC法研究了黄柏剥皮后新皮的形态发生和组织发生过程;剥皮对同化物在树冠的分配以及在新生韧皮部的运输;剥皮对光合特性和叶绿素荧光的影响,剥皮对生长和性能的影响,以及影响黄柏剥皮再生的因素,旨在完善植物再生理论,揭示林木剥皮再生机理,构建剥皮再生技术体系并应用于生产。主要研究结果如下。1、黄柏剥皮再生的形态学和组织发生学(1)黄柏剥皮后包裹透明聚乙烯膜,其外观形态经历如下变化过程:刚剥皮的木质部呈黄白色,光滑而湿润,3~4d表面产生乳白色的愈伤组织,7d左右,愈伤组织逐渐变为浅绿色,并逐渐扩展。30d表面形成浅褐色膜状栓质层,其下为绿色组织;60~120d,栓质层逐渐增厚,表面粗糙,凹凸不平,形成褐色保护层,其下为绿色活组织。第2a褐色栓皮增厚,呈块状脱落。第3a再生皮的颜色、裂纹和厚度和原树皮相似,其分界处不明显。因此,黄柏剥皮后在包裹的条件下可再生新皮,再生皮的外观形态和原皮相似。(2)黄柏幼茎和原皮的解剖学特点。幼茎绿色,近圆形,初生结构由表皮、基本组织和维管束三部分构成,其中表皮无表皮毛和气孔,皮层中有分泌囊(精油腔)和成团的石细胞分布,皮层薄壁细胞中含有叶绿体,维管束为典型的外韧维管束,初生韧皮部中有纤维带和成团的石细胞,木质部有木射线,茎中部为髓;8年生树皮由周皮、挤缩的皮层、失去功能的韧皮部和有功能的韧皮部组成,周皮有7~8层,韧皮部也有7~8条与树木年龄相对应带状纤维束,呈年轮状,在无功能的韧皮部和皮层内有成团的石细胞分布。(3)黄柏再生皮的组织发生学。黄柏树皮剥离时大部分形成层细胞随树皮剥离。木质部外缘为未完全发育成熟的木质部薄壁细胞、导管、管胞及木射线,以及残存的形成层细胞。3~7d木射线细胞和木射线之间的未成熟木质部细胞开始分裂形成愈伤组织;15~20d,表层下2~3层细胞转化为木栓形成层细胞;30~60d,在木质部与愈伤组织的结合部位形成具有4~6层细胞的维管形成层区。新生韧皮部中出现散生的石细胞团和韧皮纤维。表层下的愈伤组织形成木栓形成层;未成熟木质部细胞恢复分裂能力形成维管形成层;木栓形成层的发生早于维管形成层;3a再生皮和原皮的解剖结构基本相似。(4)在浅绿色愈伤组织的细胞内有大量叶绿体,叶绿体发育分为三个时期:初期、中期和晚期。初期叶绿体多集中在细胞质膜处,看不到基粒和片层;中期出现淀粉粒,并开始增大,可见基粒和片层;后期,淀粉粒迅速增大并撑破叶绿体,叶绿体裂解。黄柏剥皮后在一定条件下可再生新皮。其新皮的再生经历了新细胞的形成,分生组织的重新建构,由新的木栓形成层和维管形成层分裂形成周皮、次生木质部和次生韧皮部,最后形成新树皮的过程。其再生不是简单的依靠存留组织的重新分配和构建的“变形再生”,而是在高级层次上的结构和组织重新发育的“新建再生”。研究结果充实了植物的再生理论;从解剖学角度揭示了剥皮再生的机理。2、剥皮对黄柏光合特性和14C同化物运输和分配的影响(1)黄柏剥皮后光合速率(Pn)显著下降,第15d达到最低水平,蒸腾速率(Tr)在剥后1d迅速下降至最低水平,在第20d都基本恢复至正常水平;叶绿素荧光在暗适应条件下,最大量子效率ΦPO实际量子效率ΦP-和光化学猝灭系数qP。均在剥后15d达到最低水平,在20d恢复或接近正常水平。表明剥皮显著降低光合作用和蒸腾作用,但未对光系统Ⅱ造成明显的结构和功能性破坏;剥皮后光合作用在一定时间内下降,其原因是由于剥皮阻断叶片光合产物向下运输,在叶片上积累产生的对光合过程的反馈抑制,一旦光合碳同化物向下运输的功能恢复,反馈抑制随即解除,光合、蒸腾作用迅速恢复到正常水平。(2)黄柏14C同化物的平均运输速率为50.2 cm/h。剥皮显著增加了光合同化物在树冠上的积累。未剥皮黄柏树冠不同部位的功能叶14C同化物分配为:中部功能叶>上部功能叶>下部功能叶;剥皮后,同化物分配规律发生改变:上部功能叶>中部功能叶>下部功能叶。剥皮后15d,14C同化物经剥皮部位的新生组织向下运输到根系。研究结果揭示了“树不怕剥皮”的实质,即剥皮虽然暂时降低了黄柏的光合作用,阻断了同化物的下行,改变其在树冠上的分配,但是当剥面长出新组织时,同化物经新生的韧皮部输送到根系,黄柏的光合功能和同化物分配恢复到剥前状态,树体的生命活动自然能够正常进行。3、剥皮对黄柏生长和性能的影响(1)黄柏2a和3a再生皮与原皮的盐酸小檗碱含量与原皮差异不显著,分别为61.3784mg·g-1,62.6545mg·g-1和61.8816 mg·g-1。盐酸药根碱含量分别为0.4815 mg·g-1、0.4600 mg·g-1和0.2231 mg·g-1,再生皮含量极显著大于原皮;盐酸巴马汀含量分别为0.1004 mg·g-1、0.0904 mg·g-1和0.1621 mg·g-1,三者差异不显著。因此,再生皮可作为提取小檗碱的原料和药材。(2)黄柏剥皮后导致叶片不同程度黄化,落叶提前1个月左右;次年春季萌芽延迟10d左右,仍能正常开花结实;第3a生长即恢复正常。剥皮后第3a,树皮的厚度依次为3a再生皮>原皮>2a再生皮>1a再生皮。3a后再生皮厚度极显著大于原皮。(3)在刚剥皮至愈伤组织阶段,任何微小触碰都将造成剥面组织坏死或真菌感染,这两种情况是导致解膜后愈合率下降的主要原因。4、影响黄柏剥皮再生的因素影响因素有包扎、剥皮方式、剥皮时间、包膜时间、剥皮强度、剥皮操作、剥皮天气、树龄、树体状况和生长调节剂共10项。经统计分析检验,包扎、操作、树干状况、剥皮和包膜时间共5项因素对再生有显著或极显著的影响,据此建立多元线形模型。至于树龄、剥皮强度和剁皮天气对再生的影响则不显著。影响剥皮再生的10项因子,根据其重要程度划分为关键因素、重要因素和次要因素。(1)剥皮再生的关键因素包括包扎、操作、树干状况,其重要性依次为:包扎>操作>树干状况:薄膜包裹是黄柏剥皮再生的前提;粗放操作是剥皮再生失败的主要因子,也是导致愈合率下降的关键原因;由于树干不平滑使薄膜和树干间有缝隙,造成雨水渗入,节疤处不能形成愈伤组织等等而间接影响剥面的愈合。(2)重要因素有剥皮和包膜时间,剥皮过早或过迟,包膜时间太短或太长均不利于伤口愈合。包膜天数和剥皮后的颜色与积温关系密切,气温越高,所需包膜时间越短,反之亦然。结合季节、物侯和生长发育等多因素,剥皮时间以5月下旬~7月上旬为宜。若5月下旬~6月上旬剥皮,包膜10~15d,而6月下旬~7月上旬剥皮,则需5~10d。剥面的浅绿色是解膜的关键形态指标,还应结合该指标确定适宜的包膜时间。(3)剥皮强度、剥皮天气树龄因素为次要因素,可根据药材规格以及操作便利确定剥皮长度;晴天、阴天和微雨天均可剥皮;黄柏从2年生到12年生均可剥皮。(4)在包膜的条件下,全剥和纵向留树皮两种方式均能获得好的愈合率,但它们的剥面愈合情况完全不同。全剥在短时间内愈伤组织覆盖剥面90%以上,以后逐渐下降,树干保持顺直,新皮形态与原皮相似,但受操作和树干状况影响较大。纵向留树皮剥皮在短时间内愈伤组织覆盖剥面不到50%,以后新皮面积逐渐增加。由于留树皮保留了运输通道,故剥面愈合受人工操作和树干状况的影响较小,但此法导致树干和新皮发育畸形。5、黄柏剥皮再生技术体系剥皮再生技术体系主要包括多点试验和剥皮再生经营措施。(1)多点试验。荥经、雅安、洪雅、沐川、大邑5个黄柏主产区的剥皮成活率均在80%以上。沐川的成活率最高,达88.7%,雅安最低,仅50.0%,造成雅安和沐川黄柏成活率差异的主要原因是管理水平。雅安黄柏栽植后未进行修枝、施肥等管理,导致树干不直,节疤多,枝下高较低,包膜不易严密,造成渗水感染,节疤处难以形成愈伤组织或戳破薄膜等。沐川黄柏属于农户自有,长于农户附近,树下多种植农作物,以耕代抚,管理较好,树体健壮端直。(2)剥皮再生经营措施包括环境、立地条件、管理、剥皮时间、剥皮方式、解膜时间六个方面。其要点:剥皮的黄柏应满足无公害产地环境条件,按规范化种植技术规程(SOP)种植;树干应通直健壮、枝下高3m左右,无病虫害;采用“三刀法”,刀具和手勿触碰剥面,切口深度刚及木质部,透明聚乙烯膜包裹并扎紧,适宜时间(5~7月)剥皮,根据剥皮季节和温度确定解膜时间(5~15d);剥皮前一周按每株施入尿素50g+过磷酸钙250g或复合肥50~100g(氮:磷:钾=15:15:15),剥后一个月检查愈合率,低于60%,采用留桩20cm砍伐,保留一枝新梢更新,病害感染用小刀刮除病斑,喷70%甲基硫菌灵800倍液或80%多菌灵800倍液。研究表明,黄柏属于剥皮再生较困难的树种,但在良好的管理和操作下可再生新皮,当年末成活率可达80%以上;农民科技意识的提高和剥皮再生操作技术规程(SOP)的制定将推动该技术在黄柏产区推广,促进皮类林木资源的可持续发展。

【Abstract】 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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