IAWA Bulletin n.s ., Vol. 9 (4), 1988: 375-383

DIFFERENTIATION OF SECONDARY XYLEM AFrER GffiDUNG

by

Li Zhengli (Lee Chenglee) and Cui Kerning Department of Biology, Peking University, Beijing, China

Summary Under favourable growth season and by after. Three years after girdling, the regener• suitable technical means, regeneration and ated bark has the same thickness as nearby continuous growth of new bark after girdling nongirdled trees. has been observed in many trees. Differen• The bark of Eucommia is a very important tiation of the secondary xylem varies after traditional Chinese medicine which has an arteficial treatment. Thus , the authors con• anti-hypertensive and metabolic regulatory ef• sider that (1) under appropriate conditions feet. Thus the adoption of extensive girdling most trees could be girdled on a large scale obviously alleviate the short supply of this with subsequent new bark regeneration and drug. continued growth, (2) after removal of the Since 1979, the authors in collaboration phloem the living cells of the secondary xy• with some other laboratory workers have car• lem, i.e., wood parenchyma cells, may func• ried out various trials on girdling of a variety tion in transporting nutrients from the tree• of trees and woody herbaceous plants and crown downwards, and (3) finally, after gir• have found that under suitable conditions dling or when cultured in vitro, both imma• many trees and woody herbaceous plants can ture xylem and phloem can dedifferentiate be girdled, after which they resume normal into meristematic tissue that further develops growth similar to the nearby upper or lower vascular tissue. bark (Li & Cui 1983; Lu et at. 1987; Li et ai. Key words: Girdling , xylem differentiation, 1988). Eucommia ulmoides. For the past nine years the authors have also investigated the differentiation of sec• ondary xylem after girdling and tentatively Introduction proposed that the xylem can transport nutri• It has been a general concept that a tree, if ents downwards when the bark is removed completely girdled, sooner or later will die over a long part of the stem. In the present eventually due to the blockage of nutrients report, only Eucommia ulmoidesand Brous• from the upper portion of the excision. Al• sonetia papyrifera are emphasised for anato• though some authors have reported that trees mical description. after natural or artificial damage to the bark could grow continuously for a certain period of time (Noel 1970), normal growth after ar• Materials and Methods tificial girdling in a very large number of trees Large-scale girdlings of the trees were car• in China has not been reported previously. ried out within the last ten days of June (Li In recent years, many trees of Eucommia et al. 1981) with the following procedures. ulmoides Oliv. are cultivated for medicinal Firstly two circular incisions were made purpose in some provinces of China, such through the bark about 1 or 2 metres apart. as Shandong, Hubei, Sichuan and Guizhou . Then the bark was vertically slit from the top These trees can all be girdled on a large scale downward (Fig. I), so that the separated bark (1-2 m in length) and about 96% of their was easily removed as a whole piece (Fig. girdled trunk can regenerate new bark in a 2). After the bark was removed , the exposed short period and then grow normally there- surface should be protected from any touch-

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Downloaded from Brill.com10/05/2021 10:27:21PM via free access Li Zhengli & Cui Keming - Xylem differentiation after girdling 377 ing or else the regenerated new bark would together to form a more or less complete fail to grow at the site of touching. Usually zone. This newly formed cambial zone pro• the exposed trunk was loosely wrapped with duced xylem towards the inside and phloem a semi-transparent plastic sheet immediately towards the outside in the same way as nor• after girdling, the upper and lower ends of mal cambium. One year later the newly the wrapped sheet were tied up with strings formed bark of the girdled portion appeared (Fig. 3). similar to the surrounding bark (Fig. 4). Several experimental procedures were also It is really surprising that the main trunk carried out on the girdled tree trunks such as of the Eucommia tree in the Laoshan area excision of the tree crown ; application of could usually be girdled over a length of one ethrel, 2,4-D, NAA, GA and NAA + GA at to two metres and that regeneration of bark various concentrations to the exposed sur• took place normally where plastic sheet wrap• face; application of mechanical injury to the ping was not compulsory. However, in the exposed surface and cultivation of immature Eucommia trees from elsewhere in China and xylem and phloem in vitro etc. All samples in all other species , wrapping with plastic for microscopic observations were taken just sheet immediately after girdling was neces• after girdling and at some time later. Materials sary; otherwise the exposed surface of the were prepared by the conventional paraffin trees usually dried out and eventually caused methods. the death of the whole tree.

Observations The effect ofplastic wrapping on new bark formation . Differentiation of newly formed bark after As mentioned above, it is necessary to girdling wrap the exposed trunk of Eucommia trees The girdled trunk of Eucommia grown in with plastic sheet after girdling in most areas Laoshan mountains of Shandong Province, of China. In these girdled Eucommia trees, did not need to be wrapped up with plastic the types of plastic sheet, whether semi-trans• sheet after exposure. Within 24 hours after parent or black, influenced tissue differen• girdling, it formed a surface layer which pro• tiation of the newly formed bark (Li et al, tected the immature xylem immediately un• 1982). After being wrapped, several cell lay• derneath against drying (Li et al. 1981; Li & ers adjacent to the surface of the trunk formed Cui 1983). loose callus cells outwards. New cambium One week later, the ray cells of immature formed about two weeks earlier in the trunk xylem swelled, proliferated and spread later• wrapped with semi-transparent plastic sheet ally under the surface layer. They gradually than it did in stems wrapped with black plas• joined together with the neighbourhood ray tic sheet. However, after either type of plastic cells and were also mixed with some other sheet was removed one month after girdling, cells derived from the immature xylem. Soon the constituents of the regenerated barks as afterwards a cork layer appeared near the sur• they continued to grow was about the same face and a newly formed vascular cambium as that of the original bark. appeared deeply in the callus. Three to four In the tree trunk of Eucommia as well as months after girdling, the cambium cells in other trees, the regenerative processes and divided tangentially and gradually joined tissue differentiation of the newly formed

Fig. 1. Girdling of the bark of Eucommia ulmoides. - Fig. 2. The large piece of bark imme• diately after girdling. - Fig. 3. The girdled trunk of Eucommia wrapped with plastic sheet. • Fig. 4. One year after girdling of the Eucommia trunk. The demarcation of the new bark is arrowed .

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Downloaded from Brill.com10/05/2021 10:27:21PM via free access Li Zhengli & Cui Keming - Xylem differentiation after girdling 379 barks were quite similar after being girdled cambium and acceleration of bark growth, the and wrappedin plastic sheets. Various amounts therapeutic effectiveness of this newly form• of developing xylem cells were tom off dur• ed bark for medicinal use as compared with ing girdling. The ray cells located near the that of the ordinary bark still needs further in• surface became swollen (Fig. 5, r). vestigation. One week after girdling, the swollen ray cells closely grouped near the surface into a Effect of bark grafting on the traumatic shape of a bell rim. Eventually these cells be• girdled trunk came mingled with the other immature xylem Although it is evident that the trunk of cells to form callus (Fig. 6, ca) which gradu• Eucommia can be girdled and can regenerate ally increased and enlarged to a certain extent. a new bark during favourable growth season Two weeks after girdling, cell arrange• and by suitable experimental procedure, the ment near the surface became somewhat reg• exposed surface became traumatic due to ular. This might be considered as a very early poor wheather conditions or careless han• stage of phellogen. Some deep-seated, flat dlings leading to cessation of new bark for• meristematic cells, as seen in cross sections, mation and death of the tree (Li et al. 1984). gradually appear (Fig. 7, arrows). These flat Once the ringed surface was damaged, it was cells might reflect the early vascular cambium possible to rescue the girdled trunk by cov• cells which later produce phloem outwards ering or grafting a piece of new bark taken and xylem inwards. from another Eucommia tree (Li et al. 1983). The differentiation of a cork layer is very conspicuous one month after girdling (Fig. 8, The role of the tree crown in new bark for• p). The cells of the deep-seated vascular cam• mation bium also proliferate forming a zone of cam• The starch grains stored in the ray cells of bium (Fig. 8, c) that produces phloem out• xylem play an important role for the initiation wards (Fig. 8, ph) and xylem inwards (Fig. of new bark formation in the girdled trunk of 8, x). Eucommia. Later, however, material for con• Thereafter, differentiation of this newly stitution and metabolism of the regenerated formed cambium was similar to the cambium bark must be transported from the tree of ordinary bark. Three years after girdling, crown. Excision of the tree crown after gir• the regenerated bark became as thick as the dling greatly hampered new bark formation five-year-old ordinary bark (Figs. 11 and 10 (Lee & Cui 1983). Under such condition the respectively). It seems that the three-year-old girdled trunk formed only incomplete bark regenerated bark had fewer and thinner peri• and the trunk eventually died about two derm and its vascular cambium cells were months later. It seems that the amount of more active than those of the ordinary bark. starch in the secondary xylem is inadequate to Despite the merit of girdling, i. e. promotion meet the need for continuous growth of new of differentiation of newly formed vascular bark.

Figs. 5-9. Transverse section of a portion of Eucommia trunk after girdling; c = cambium; ca = callus; p = periderm; ph = phloem; r = ray cells; x = xylem; x 150. - 5: The exposed surface of the trunk after girdling to show the damaged cells. - 6: One week after girdling, showing the bulged ray cells (r) and callus (ca) around. - 7: Two weeks after girdling, some flat meristem• atic cells (arrowed) appeared in the deep site. - 8: Establishment of vascular tissues one month after girdling . - 9: Four months after square-frame shaped scraping of the exposed surface of the girdled trunk, demonstrating the abnormal vascular tissues formed at the centre of the scraped frame.

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Figs. 10 & 11. A portion of the transverse section of the trunk of Eucommia ulmoides; x 200. • 10: Normal growth of the bark of the 5-year-old tree. - 11: Formation of the regenerated bark 3 years after girdling . - Figures on this page represent peripheral parts to be joined on top of figures on p. 381.

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Influenceofexternalplant hormones on new fugal differentiation of phloem was evidenc• bark formation ed. The vascular tissue was ultimately de• No significant effects on the promotion of generated 6 months after culture in vitro, and callus formation and differentiation of vascu• died eventually. lar tissues were demonstrated when the ex• Inside the peeled bark of Eucommia the posed surface of the girdled trunk of Eucom• immature secondary phloem which included mia was daubed with plant hormones such as ray cells and other living cells of phloem, un• ethrel, 2,4-D, NAA, GA and NAA + GA re• der cultures in vitro, could develop callus spectively in appropriate concentration. How• from which further a discontinuous meris• ever, it was found that in the 2,4-D, GA and tematic zone differentiated or some tracheids NAA + GA treated trunks, initiation of phel• were formed directly. Later on, clusters of logen and vascular cambium in callus was tracheid-like elements appeared. The callus earlier than that in the control. In those treated from the phloem of male trees also differenti• with ethrel, formation of phellogen alone was ated into primordia of adventitious buds and earlier. Initiation of phellogen and differen• roots (Zhang & Li 1984). However, no sieve tiation of vascular cambium in the trunk treat• cell-like elements were developed in the ed with NAA was similar to the control (Li & phloem callus during cultivation in vitro. Cui 1984). It seems possible that action of wound hormones produced by girdling out• Discussion weighed the effect of exogenous hormones. Results from a series of experiments indi• cate that most trees can be girdled over large Effectof mechanical injuryon newbarkfor• stem sections under suitable conditions and that these trees continue to regenerate new mation bark. It is quite possible that the suitable con• Mechanical injury to the exposed surface ditions for girdling vary in different trees, of the girdled trunk of Eucommia ulmoides Therefore, one should attempt to seek for was conducted by scraping frames 10 x 10 other factors or conditions for those trees that ern- in four different shapes . The scraping so far failed to regenerate new bark after gir• edge was about 2 ern in width and about dling, 1 mm in depth. Four months after treatment, Girdling of some tree species with com• periderm was formed at the central portion of mercial barks , such as medicinal barks or all the scraped areas (Fig. 9), but variations fragrant barks, can be achieved successfully. were observed in the thickness, degree of dif• In addition, the trunks of some woody herba• ferentiation and starch distribution of the re• ceous plants, such as egg plants (Li et al. generated bark (Cui & Li 1986). 1987), Jerusalem artichoke (Li & Xu 1988), cotton, pepper etc. when girdled could all re• Immature xylemandphloem cultured in vitro generate new rind and grow normally after a Following a complete ringing of the main certain period of time. However, the initiation stem of Eucommia , regeneration of normal and tissue differentiation of these new rinds new bark has been observed as a subse• were rather different from the regenerated quence of repeated partial dedifferentiation of bark of the trees. On the other hand, her• the immature xylem into cambium. This im• baceous plants after girdling may show ear• mature xylem, when cultured in vitro, show• lier flowering and fruiting . Our studies pro• ed production of callus tissue from the ray vide the possibilities to scale up girdling cells (Zhang & Lee 1981). After 15 to 30 technique from the experimental to the pro• days of continuous culture, meristematic tis• ductivelevel. sue appeared as a discontinuous and more or After girdling trees of Eucommia , Brous• less regularly arranged zone within the callus sonetia (Li et al. 1988) and other genera can tissue . The meristematic cells, unlike the produce callus from the exposed immature elongated fusiform initial cells, are isodia• xylem from which the vascular tissue later metric in shape. This meristem produced tra• differentiates. No doubt, the constituent ma• cheids centripetally. Nevertheless, no centri- terial is obtained from the reserve substances,

Downloaded from Brill.com10/05/2021 10:27:21PM via free access Li Zhengli & Cui Keming - Xylem differentiation after girdling 383 mainly the starch grains, in the living pa• -&- 1984. Abnormal development of renchyma cells, mostly ray cells, of the regenerated bark in Eucommia ulmoides. xylem. The starch grains conspicuously de• Acta Bot. Sinica 26: 252-257. (In Chi• crease in number and eventually disappear nese, English abstract.) following callus formation. Hereafter, the -& -1985. The effects of several exoge• constituent materials must be supplied by nous hormones on bark regeneration after materials synthesised in the tree crown and girdling in Eucommia ulmoides. Acta Bot. transported through the xylem. It is sug• Sinica 27: 1-6. (In Chinese, English ab• gested that after the phloem is removed with stract.) the bark, the xylem parenchyma cells and - ,- & Liu Qinghua. 1987. Anatomical other living elements may be forced to trans• studies on regeneration of eggplant stem port nutrients downwards, simultaneous with after girdling . Scientia Sinica ser.B, 1987: its transport of water upwards in the adjoin• 824-831. ing tracheary elements. This is indirectly evi• - ,- & Lu Pengzhe. 1988. Studies on re• denced in the experiment of tree-crown exci• generation after girdling of Broussonetia sion after girdling (Lee & Cui 1983). papyrifera. Acta Bot. Sinica 30: 236-241. When immature xylem or phloem of Eu• - ,- & Luo Zhengrong. 1984. Tissue ne• commia is cultured in vitro, both of them can crosis of the regenerated bark of Eucom• produce callus which later differentiates into mia ulmoides. Acta Bot. Sinica 26: 456• meristematic tissue that forms tracheids and 458. (In Chinese.) other xylary elements only . It is suggested - , - , Yu Chunsheng & Cheng Xiaolin. that both the immature xylem and phloem cul• 1981. Anatomical studies of regeneration tured in vitromay regain the character of me• after ringing of Eucommia ulmoides. Acta ristematic tissue. The phenomenon that im• Bot. Sinica 23: 6-11. (In Chinese, Eng• mature phloem cultured in vitro may change lish abstract.) its ability to differentiate into tracheids has - ,-, Yu Chunseng & Cheng Xiaolin. not been reported. The mechanism which 1982. Effect of plastic sheet wrapping triggers such transformation warrants further upon girdled Eucommia ulmoides. Scien• study. tia Sinica 25: 367-375. The bark of many trees are widely utilized -, -, Yuan Zhengdao & Liu Shijin . 1983. in traditional medicine in China. The employ• Regeneration of re-covered bark in Eu• ment of the girdling technique to increase commia ulmoides. Scientia Sinica 26: 33• production shows great economic potential. 40 . Furthermore, girdling has shown to be an ex• -& Xu Xin. 1988. Tissue differentiation of cellent approach in the study of formation and the regeneration rind in Jerusalem arti• differentiation of secondary xylem. choke stem. Acta Bot. Sinica 30 (in press), Lu Pengzhe, Cui Kerning & Li Zhengli (Lee References Chenglee). 1987. Preliminary investiga• Cui Kerning & Li Zhengli (Lee Chenglee). tions of the regeneration of 14 seed plants 1986. Influences of special damage upon after girdling. Acta Bot. Sinica 29: 111• exposed surface of girdled trunk in Eu• 113. (In Chinese.) commia ulmoides. Acta Bot. Sinica 28: Noel, A. R. A. 1970. The girdled trees. Bot. 27-32. (In Chinese, English abstract.) Rev. 36: 162-195. Lee Cheng lee & Cui Kerning. 1983. The ef• Zhang Xinying & Lee Chenglee. 1981. His• fect of new bark formation after removal togenesis of xylem of Eucommia ulmoides of tree-crown in Eucommia ulmoides. cultured in vitro. Acta Bot. Sinica 23: Acta Bot. Sinica 25: 208 -214. (In Chi• 339-343. (In Chinese, English abstract.) nese, English abstract .) -& Li Zhengli (Lee Chenglee). 1984. Stud• Li Zhengli & Cui Kerning. 1983. Vascular ies on the phloem of Eucommia ulmoides cambium formation in immature xylem . in vitro. Scientia Sinica ser. B. 27: 671• Kexue Tongbao, Special Issue 1983: 247• 678 . 249 .

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