Callus Formation at Graft Interface in Ginkgo biloba L.

The early stages of graft union, when male branch was grafted onto female branch in Ginkgo biloba L. by cleft graft, have been observed under light microscope in order to determine the origin of callus cells between the stock and scion. Pith parenchyma cells near the graft interface were the earliest cells in response to such method of grafting. These cells dedifferentiated and then divided within 7～ 12 days after grafting. A large number of callus cells extended from the pith into the space between the graft interface linking the stock with the scion about 18～ 20 days after grafting; and then continued to proliferate and extend outwards along the space. Cambium cells and immature vascular tissue near the graft interface dedifferentiated into callus rather late. Theover all link between the stock and scion was completed in the sites 30 days after grafting. Callus cells were also produced from corticai parenchyma cells, but they were much limited in quantity. In conclusion, the graft interface may be considered as a "natural culture bed" after grafting, in which all undamaged, living cells are capable of dedifferentiation and producing callus cells for compatible graft union. In the case of G. biloba (male/female) it were the pith parenchyma cells that appeared first to form the callus cells and later extend to link the stock with the scion.     

油茶芽苗砧嫁接口愈合过程解剖学研究

采用石蜡切片法对油茶（Camellia oleifera）芽苗砧嫁接口的愈合过程进行组织解剖学研究。观察结果表明：（1）在嫁接后的第4天,嫁接口产生隔离层;在嫁接后的第8天,嫁接口的砧木产生愈伤组织;在嫁接后的第16天,嫁接口的接穗产生愈伤组织;在嫁接后的第22天,嫁接口的砧木与接穗连接;在嫁接后的第29天,嫁接口的形成层分化形成;在嫁接后的第35天,嫁接口的愈伤组织维管束形成,接穗连接成功。（2）油茶芽苗砧嫁接部位愈伤组织形成前,芽苗砧木的解剖结构在短时间内加速发育,逐渐与接穗的组织结构相似。（3）嫁接口的形成层和其它薄壁细胞组织均能产生愈伤组织,但形成层是其主要来源。     

Graft Formation in Sitka Spruce: A Scanning Electron Microscope Study

Low-temperature and conventional scanning electron microscopyhave been used to examine the callus formed at the graft interfacein Picea sitchensis (Bong.) Carr. Callus cells are producedby both cambium and ray parenchyma dedifferentiation and redifferentiationin scion and stock. Adhesion between the cells derived fromscion and rootstock is thought to be my means of pectinaceousbeads at the surface of the callus cells, preceding a more generalfusion of cell walls. The cambia of the two graft componentsare prevented from growing towards each other by the presenceof callus. Instead, the differentiation of new cambium withinthe callus, in the vicinity of the cambia exposed at the preparedsurfaces of the scion and rootstock, links them to form a continuouscambial layer around the combined stem. Callus, cambium, differentiation, grafting, Picea sitchensis     

Histo-Cytological Observations on Callus and Bud Formation in Cultures of Nicotiana tabacum L.

Leaf explants of tobacco were cultured on MS medium supplemented with 2 mg/ l NAA and 0.5 mg/l BA for induction of callus formation, or supplemented with 2 mg/l BA for bud formation. Histocytological observations on callus and bud formation were carried out. Three days after cultivation, mesophyll cells enlarged, the nuclei became more apparent and dark stained, and starch accumulated in the cells. Cell divisions began in the mesophyll cells at the cut ends, in the palisade cells near the vascular bundles and in the vascular parenchyma. Mitotic activity then spreaded over tbc explants, and was most active at the edges of leaf explants. Regular rows of cells appeared as a result of series of transverse divisions in the palisade. The number of chloroplast in the mesophyll cells decreased and degenerated gradually. A number of meristemoids ware initiated in the cultured leaf explants after 7 days of cultivation. They were originated from two kinds of tissues, the mesophyll and vascular bundle, including the phloem parenchyma and vascular sheath. On the medium with NAA and BA, callus formation was induced with vigorous divisions, whereas bud primordia were differentiated from the meristomoids on the medimn with 2 mg/l BA. The buds were developed from both the superficial meristemoids and the meristematic regions deep within the callused leaf explants. The accumulated starch in the cells gradually disappeared as bud formation proceeded.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a Model System for Graft Union Development in Homografts and Heterografts

Homografting of Arabidopsis thaliana scions on stocks of A. thaliana and heterografting on other species were used to study the compatibility and the ontogeny of graft union formation. Highly compatible homografting with scions of young leafy inflorescence stems was obtained on stocks of inflorescence stems growing from large 3-month-old A. thaliana plants. Histologic analysis revealed four developmental stages of graft union formation in Arabidopsis homografting: (1) development of a necrotic layer, (2) callus proliferation in the grafted scion, (3) differentiation of new vascular tissues within the scion, and (4) a full vascular graft union formation between the scion and the stock. Vascular connections were formed within the callus bridge between rootstocks and scions 15 days after grafting. Heterografts of Arabidopsis on two members of Brassicaceae, cabbage (Brassica) and radish (Raphanus), showed partial incompatible interaction with a lower level of vascular differentiation. Arabidopsis grafting on tomato (Solanaceae) rootstock showed complete incompatibility and limited noncontinuous differentiation of new vascular tissues that did not cross the scion/stock boundary. Although lacking scion/stock vascular connections, Arabidopsis scions grafted onto tomato rootstock flowered and produced seeds. This may indicate some nonvascular functional connections between the two plants, probably of parenchyma cells, further emphasizing the usefulness of Arabidopsis as a model plant for studying various levels of the complicated scion/stock relationships expressed in grafting biology. Experiments with dye transport in the xylem showed that although in general there was an agreement between the histologic study and dye transport, in Arabidopsis homografts water transport frequency was lower than functional and histologic compatability. We conclude that homografting and heterografting of Arabidopsis inflorescence stems is a convenient and reproducible method for studying the fundamental cellular genetic and molecular aspects of grafting biology.     

南瓜属植物离体茎段嫁接维管组织的发育过程

在离体条件下,采用同瓜属植物的下胚轴节段进行嫁接,光镜观察发现：异种嫁接西葫芦／南瓜和南瓜自体嫁接嫁接后６－８ｄ在接穗、砧木的薄壁细胞和嫁接面处愈伤组织细胞中分化出管状分子和筛分子,边接接穗和砧木的质部和韧皮部桥在嫁接后８ｄ形成。随后随发育天数的增加其数目增加,用６（５）ＣＦ作为筛管输导的示踪剂检验了不同发育时期砧木和接穗间的连通情况,发现嫁接后８ｄ从接穗引入的６（５）ＣＦ可以输导到砧木。     

Phloem translocation in regenerating in vitro- heterografts of different compatibility

Phloem translocation of [¹⁴C]-sucrose and 5/6-carboxyfluorescein(CF) from scion into the stock was studied in in vitro-heterograftsof Lycopersicon on Solanum (L/S) and Vicia on Helianthus (V/H)at various stages of regeneration. Autografts of all partnersserved as controls. Corresponding with the translocation experimentsnewly formed sieve-tube connections between the graft partnerswere counted. ¹⁴C-translocation experiments with [¹⁴C]-sucrose revealed anage-dependent increase of radioactivity in the stock of allcombinations. In L/S and all autografts the major increase of¹⁴C-label in the stock occurred 5–10 d after grafting.In V/H, however, import of label into the stock remained lowthroughout the regeneration period. In L/S grafts, increasesin the numbers of sieve-tube connections parallel the increasingrate of ¹⁴C-transport, indicating functioning sieve-tube connectionsin the graft union. In contrast, V/H grafts did not show thisstrong correlation between structure and function of wound repairphloem. This suggested the existence of non-transporting sieve-tubesbetween the graft partners. Similar results were obtained withCF-transport, showing that effective phloem translocation acrossthe graft interface occurred in L/S, but not in V/H grafts.The observed differences in phloem translocation are discussedwith regard to compatibility/incompatibility phenomena in heterografts. Key words: Compatibility/incompatibility, in-vitro-heterografts, phloem transport ([¹⁴C]-sucrose, carboxyfluorescein), wound phloem     

Application of Electrical Wave Transmission in Basic Research of Grafting

Electrical wave (EW) transmission from scion to stock across the grafting interface was related to the histological changes during the development of graft union. Variation wave (VW) could not be transmitted to stock from scion before isolation layer broken and callus interdigitation. As plasmodesmata formed secondarily at the interface between stock and scion where the isolation layer had disappeared, VW could be transmitted from scion to stock, but its velocity was not rapid until the vascular bridges form between two partners of graft union. Hence, the authors could deduce the degree of graft union formation by measuring whether VW could be transmitted from scion to stock or not and its velocity. EW trans mission method was a new tool for quickly detecting the formation of graft union.     

金桔组织培养中愈伤组织和芽形成的组织细胞学观察

用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。     

电波传递在嫁接基本理论研究中的应用

研究发现电波从接穗跃过嫁接面向砧木传递与嫁接的组织学变化相关。砧木和接穗愈伤组织细胞突破隔离层互相嵌合之前,接穗中产生的电波不能传到砧木。砧木和接穗细胞间产生次生胞间连丝后,电波即可沿接穗传至砧木,但此时传递速度慢。当砧木和接穗间产生维管束桥时,电波下传的速度加快。由此可见,通过检测电波跃过嫁接面与否及下传的速度,就可推断嫁接接合部组织学变化状况。电波传递法是一种快速鉴定嫁接植株发育过程的新方法。     

The Function of Phloem Connections in Regenerating In Vitro-Grafts*

In order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant-grafts after application of ¹⁴C-sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls. A segmental analysis was used to determine the tissue distribution of ¹⁴C-sucrose in a graft. The ¹⁴C-profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound-phloem bridges (shown as files of aniline-blue-positive sieve plates) traverse the graft interface. In 7-d-old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, ¹⁴C-profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose. Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound-phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro-grafts is directly shown.     

翅果油树茎段愈伤组织和芽发生的组织学研究

本文对翅果油树大宫灯型茎段培养在ＭＳ附加６－ＢＡ较高、ＮＡＡ较低浓度的培养基上培养０～３０ｄ的组织学变化进行了研究。创伤对其愈伤组织的形成有明显的刺激作用,培养３～４ｄ切口处的皮层细胞、形成层细胞、韧皮部薄壁细胞以及髓组织细胞,甚至表皮细胞均脱分化开始分裂;培养８～１１ｄ,切口明显膨大,起源于髓及维管组织周围薄壁细胞的愈伤组织突起大;培养１２～２０ｄ愈伤组织块中出现了分生组织和维管组织结节;培养２１～３０ｄ,愈伤组织表层和近表层细胞分化出芽原基,但与维管组织结节无直接联系。     

A Study of the graft union in in vitro micrografted apple

Light microscopy was used to study graft union formation in in vitro micrografts of tissue cultured apple (Malus domestica. Borkh). Micrografts were constructed using horizontal incisions to form the grafting surface, and placing the cut ends of rootstock and scion into sterile silicon tubing to permit graft formation to occur.The outer morphological and histological development was similar for different stock-scion micrograft combinations but graft union formation was slower in heterografts than in autografts. Initial leaf expansion at the scion shoot apex occurred in all micrografted plantlets within 1–4 days and was not indicative of graft success. Progressive scion growth and development could be used as an indication of graft success by ten to fourteen days after grafting and probably was related to establisment of cell to cell contact at the graft interface. Microscopy showed initiation of callus proliferation in the vascular cumbium and the pith ten days after grafting. Differentiation was observed subsequently and this was reflected in scion development. Longitudinally orientated cambial cells began to differentiate between twenty and forty days after grafting, and formed a bridge between the vascular cylinders of scion and rootstock. The scions at this stage had as many as eight newly expanded leaves and micrografts were strong enough to permit silicon sleeve removal without damage. Continuity of new vascular elements in rootstock and scion was established around forty days. New vascular elements curved slightly in towards the pith to form a c shaped bridge across the graft union. Vascular development continued until it reached completion after six months.     

Grafting micropropagated tea [Camellia sinensis (L.) O. Kuntze] shoots on tea seedlings – a new approach to tea propagation

Tea microshoots excised from well-established multiple shoot cultures grown in vitro and 8-week-old, three- to five-leaved seedlings from a local chinery stock (Banuri-96) and UPASI-9 (from southern India) were selected as scions and root stocks, respectively, for grafting. In addition, 4-month- and 12-month-old seedlings of Banuri-96 were also used as root stocks. Cut ends of root stocks and scions were pretreated with varying concentrations of BAP and NAA for 10 min. A treatment of BAP (5 mg/l) and NAA (5 mg/l) to both scion and stocks in water renewed foliar development at a relatively early stage (40–60 days). The grafted plants were kept in hardening chambers with CO₂-enriched air. No significant difference was observed between autograft (scion and root stock of Banuri clone) and heterograft (scion of the Banuri clone and root stock of UPASI-9). Of the three types (in terms of age) of seedling-raised root stocks employed, grafts on young tea (4-month-old) performed the best (88.33%). Grafts made in early summer established relatively faster and at a high rate of success. The percentage survival of plants transferred to the field was 88.33%. Received: 21 May 1998 / Revision received: 17 December 1998 / Accepted: 15 January 1999     

八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

杨树叶薄层培养中不定芽形态发生的细胞组织学研究

将杂种杨树(Populus nigra var.betulifolia×P.trichocarpe)NE299叶主脉用振动切片机横切成400μm或800μm的薄切片,培养在附加0.2mg/L BA和0.01mg/L NAA的木本植物培养基上。培养后,位于主脉维管束两侧中上部的维管束鞘薄壁细胞首先启动分裂。几乎同时,与其邻接的一些栅栏组织细胞也分裂,并很快形成胚性分生细胞团。主脉的愈伤组织主要由维管束鞘薄壁细胞,以及与其邻接的一些栅栏组织细胞和韧皮部的薄壁细胞分裂而来。不定芽通常发生在愈伤组织的周边区,也可以起源于维管组织结节(vascular nodules)周围的形成层状细胞。侧脉的维管束鞘细胞分裂活动很强,可不经愈伤组织直接长成不定芽。杨树叶主脉处的维管束鞘薄壁细胞在与叶肉组织相邻接的细胞中,通常含有少量较小的叶绿体,而位于背腹面的细胞中含有贮藏的淀粉。对形态发生的特定部位及其细胞进行了讨论。     

Histology of Callogenesis and Somatic Embryogenesis Induced in Stem Fragments of Cork Oak (Quercus suber) Cultured In Vitro

Calluses able to produce somatic embryos were formed duringin vitro culture of shoot fragments of cork oak (Quercus suberL.).Histological monitoring of these fragments during cultureshowed that it was the cortical parenchyma cells which underwentdedifferentiation before calluses were formed by repeated divisions.The calluses consisted of parenchyma cells surrounded by a fewlayers of meristematic cells. Proembryos formed in groups aroundthe edge of some calluses. Histological examination showed thatthey were produced by the evolution of two different categoriesof cell: one category had the appearance of ‘embryogenic’cells with very thick walls, a small vacuole rich in starchand a well-developed nucleus with a prominent nucleolus. Theother cells were very bulky with large vacuoles; their morphologywas similar to that of suspensor cells encountered in embryogenesisin gymnosperms. The ontogenic stages were similar to those describedin zygotic embryos of the genus Quercus. Nevertheless, mostof the embryonic structures deviated from normal developmentand at all stages produced secondary proembryos. Cork-oak, Quercus suber L, histology, callogenesis, somatic embryogenesis, embryogenic cells, starch, secondary embryogenesis     

The Effect of Scion Water Potential on Graft Success in Sitka Spruce (Picea sitchensis)

The effect of scion storage on graft success in Sitka sprucehas been examined. Scions kept in cold store for up to 7 d werefound to graft successfully to active rootstocks in about 95%of cases. From 7 d, however, there was a marked decline in successuntil, after 14 d of storage prior to grafting less than 40%of grafts survived. Scion leaf-water potential decreased continuouslyduring storage and fell dramatically during the first 3 d aftergrafting. In successful grafts, scion water potential then recovered,reaching a stable level after 3 weeks. No recovery occurredin grafts, which eventually failed. The relationship betweenscion leaf water potential and graft success rate is discussedwith reference to the role of callus development in graft formation. Picea sitchensis, water potential, graft success     

In vitro morphogenesis patterns from shoot apices of sugar cane are determined by light and type of growth regulator

The regeneration patterns of shoot apices derived from in vitro plants of four varieties of sugar cane in response to different growth regulators and light were evaluated. The cellular origin of the regeneration processes was also investigated. Explants cultivated on medium supplemented with NAA and incubated under light showed direct bud regeneration from cells of external layers of the ground parenchyma of the stem. Explants cultivated in the dark on medium supplemented with low concentrations of picloram (PIC) or 2,4D (4.0 and 4.5 μM, respectively) showed callus formation derived from the ground parenchyma of stem and development of preembryogenic masses derived from bundle sheath cells facing the phloem tissue of immature leaves. Somatic embryos at further developmental stages were visible following transfer to medium devoid of growth regulators and incubation under light. When incubated under light since the begining of the experiment, explants cultivated in the presence of higher PIC or 2,4D concentrations (40 and 22.6 μM, respectively) first displayed direct organogenesis from external layers of the ground parenchyma of the stem, followed by the development of organogenic calluses. Preembryogenic masses were also observed from bundle sheath cells of immature leaves. However, in contrast to the cultures pre-incubated in darkness for 30 days, the subsequent stages of embryo development were not detected. The regeneration efficiency of calluses induced by 2,4D and PIC was generally increased following desiccation in laminar flow or incubation on medium solidified with phytagel.     

STUDIES OF VEGETATIVE COMPATIBILITY-INCOMPATIBILITY IN HIGHER PLANTS. I. A STRUCTURAL STUDY OF A COMPATIBLE AUTOGRAFT IN SEDUM TELEPHOIDES (CRASSULACEAE)

Initial adherence of the cut surfaces occurred by 24 hr after grafting and was correlated both with a pronounced dictyosome activity along the graft interface and with callus proliferation in both the stock and scion. A necrotic layer of one or two collapsed cells in thickness initially extended as a continuous barrier between the stock and scion, but the layer was fragmented by 2–3 days after grafting as the callus proliferation continued. Graft incision also induced a mild senescence in cells at the graft interface characterized by a reduced staining intensity of the cytoplasm, replacement of the large primary vacuole by numerous smaller vacuoles, and the occurrence of flocculent material throughout the cytoplasm. Starch accumulated during the first day after grafting but disappeared by 2–3 days after grafting. The cellular senescence never proceeded beyond an early, nonlethal stage, and cells along the graft interface completely recovered by 3 wk after grafting. Procambial differentiation occurred across the callus bridge by 10 days after grafting, and mature vascular continuity was established by 14 days. The results of this study are discussed relative to cellular recognition phenomena and to proposed mechanisms for plant graft compatibility-incompatibility.