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.     

The Role of Direct Cellular Contact in the Formation of Compatible Autografts inSedum telephoides

In order to determine the importance of direct cellular contactfor the formation of a compatible graft, I have monitored thedevelopment of compatible autografts in Sedum telephoides inwhich porous membrane filters were inserted between the graftingsurfaces. The tensile strengths of these grafts levelled offat approximately 10 g breaking weight per square millimetregraft area baween 2 and 5 d after grafting. Callus proliferationoccurd at both grafting surface by 5 d after grafting. By 21d after grafting, vascular redifferentiation across the graftinterface was complete. These results indicate that direct cellularcontact is not necessary for the (I) cohesion of the stock andscion, (2) callus proliferation or (3) vascular redifferentiationthat occur during the formation of a compatible graft. Sedum telephoides, Crassulaceae, grafting, vascular redifferentiation, compatibility     

In vitro regeneration of Gaillardia pulchella Foug.

Young leaf and internodal stem segments of Gaillardia pulchella, collected from wild species re-established in the greenhouse, were used to initiate callus on Murashige & Skoog medium supplemented with NAA (2.0 mgl⁻¹) and BA (0.4 mgl⁻¹). Callus formed after 10 to 14 days in the dark. Cultures were transferred to fresh medium and placed under lighted conditions where shoot formation occurred approximately 14 to 30 days after initiation. Callus sub-cultured at 14 to 21-day intervals continued to produce primordia for several weeks. Flowers were produced by regenerated shoots maintained on MS medium, but roots did not develop until the plantlets were transferred to soil conditions.     

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.     

Graft formation in Solanum pennellii (Solanaceae)

Three phases of cohesion were observable during the development of compatible autografts in Solanum pennellii. Phase I cohesion 1) lasted 4–5 d after grafting, 2) was characterized by an average increase in tensile strength of 4 g breaking weight (BW) mm^–2 graft area (GA) d^–1, and 3) correlated positively with cellular interdigitation at the graft interface. The fresh weight of the scion increased by approximately 5% d^–1 during the first 2 d after grafting. Phase II cohesion occurred 5–15 d after grafting, during which time 1) the tensile strength of the graft union increased by 14 g BW mm^–2 GA d^–1, 2) vascular differentiation across the graft interface was completed, and 3) the fresh weight of the scion increased by 9% d^–1. Phase III cohesion occurred subsequent to 15 d after grafting, during which time 1) the tensile strength of the graft union leveled off at a value similar to that of an ungrafted internode, and 2) the fresh weight of the scion increased by 14% d^–1. These results are discussed relative to mechanisms underlying the formation of compatible grafts.     

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.     

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。     

STUDIES OF VEGETATIVE COMPATIBILITY-INCOMPATIBILITY IN HIGHER PLANTS. IV. THE DEVELOPMENT OF TENSILE STRENGTH IN A COMPATIBLE AND AN INCOMPATIBLE GRAFT

Three phases of adhesion between the stock and scion are observable during the formation of a compatible autograft in Sedum telephoides and an incompatible heterograft between Sedum telephoides and Solanum pennellii. The first phase of adhesion is similar in both systems in that it 1) lasts 2 to 3 days, and 2) is characterized by an average increase in tensile strength of 1 g breaking weight (BW)/mm² graft area (GA)/day. In the compatible Sedum autograft, the second phase of adhesion lasts from Days 3 to 11 after grafting and is correlated with a 28-fold increase in the tensile strength of the graft union to approximately 56 g BW/mm² GA by 11 days after grafting. The third phase of adhesion in the compatible autograft is characterized by a leveling off of the tensile strength of the graft union at approximately 56 g BW/mm² GA, roughly equal to that of an ungrafted internode. Graft formation is now complete. These results suggest that the ratio of the tensile strength of the graft union : tensile strength of a comparable ungrafted internode provides an estimate of the percent development of compatible autografts. In the incompatible heterograft between Sedum and Solanum, Phase II adhesion 1) lasts from Days 2 to 5 after grafting, and 2) peaks at 12 g BW/mm² GA at 5 days after grafting. Phase III adhesion in the incompatible heterograft occurs subsequent to Day 5 after grafting and is characterized by an average decrease in the tensile strength of the graft union of 0.3 g BW/mm² GA/day. The results of this study are discussed relative to the quantitative contributions of various structural events to the tensile strength of a graft union.     

Relationship of B blood group locus to skin allograft in chickens

Skin grafts were exchanged among 21 genotypic pairs of B blood group locus in the non-inbred chicks of White Leghorn at 5–7 days of age. The mean percentages of B locus compatible pairs were 94.7, 84.2 and 56.8 at the 11th, 15th and 19th days after grafting, respectively. These percentages of survival grafts were significantly higher than those of incompatible pairs. The effects of three B alleles were investigated but the the differences of effects of them were not found in this experiment. Two of the prolonged survival grafts survived for 35 days after grafting and all of the incompatible grafts were rejected the 20th day after grafting. The results of skin graft provided evidence that the B blood group locus was a histocompatibility locus or closely linked to such a locus.     

Effects of hyperprolactinemia on activities of 17-hydroxylase, 17 beta-ol-dehydrogenase and 5 alpha-reductase in neonatally grafted and host testes in mice

Male and female (WB X C57BL/6)F1 hybrid mice were used. Two pituitaries from 60-80-day-old female mice were grafted under the capsule of the left kidney of 60-80-day-old male mice. One week after grafting, 2 testes from neonatal mice were grafted under the capsule of the right kidney of the grafted mice and 70-90-day-old intact male mice. The grafted and host testes, in groups of 10-26, were removed 15, 30, 40, 60 and 120 days after transplantation of the neonatal testes. Testicular homogenates were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione, and enzyme activities per g tissue were estimated. Significantly elevated prolactin levels, slightly lower LH levels and normal testosterone levels were found in the mice with pituitary grafts, compared with those in the mice without pituitary grafts. Activities of 17-hydroxylase and 17 beta-ol-dehydrogenase increased clearly with age in the grafted testes in the mice without pituitary grafts, though the increases were inhibited significantly by the pituitary grafts. However, the pituitary grafts had no significant effect on activities of 17-hydroxylase and 17 beta-ol-dehydrogenase in the host testes under similar gonadotrophic stimulation. 5 alpha-Reductase activities in the grafted and host testes were unaffected by the pituitary grafts. These results show that hyperprolactinemia may directly inhibit increases in activities of 17-hydroxylase and 17 beta-ol-dehydrogenase with testicular age in neonatally grafted testes in mice.     

嫁接植株形成过程中接合部组织学和生长素含量的变化

本文应用植物激素间接酶联免疫技术（ＥＬＩＳＡ）,第一次定量检测了嫁接植株形成过程中生长素（ＩＡＡ）的动态变化。结果表明：嫁接植株发育的前期,亲和性与非亲和性组合其ＩＡＡ含量的变化相似。在后期,不亲和组合ＩＡＡ含量急骤减少,而亲和性的组合在第八天即维管束桥分化形成的这一天,可见到ＩＡＡ高峰值的出现。     

嫁接植株形成过程中接合部组织学和生长素含量的变化

本文应用植物激素间接酶联免疫技术(ELISA),第一次定量检测了嫁接植株形成过程中生长素(IAA)的动态变化。结果表明：嫁接植株发育的前期,亲和性与非亲和性组合其IAA含量的变化相似。在后期,不亲和组合IAA含量急骤减少,而亲和性的组合在第八天即维管束桥分化形成的这一天,可见到IAA高峰值的出现。     

冠状动脉旁路移植术桡动脉桥家兔模型的建立

目的建立一个能模拟冠状动脉旁路移植术桡动脉桥情况的模型。方法50只新西兰兔,股动脉与颈总动脉行端侧吻合,两吻合口之间的颈总动脉予以结扎。术后1、3、7、14、56d分别取完整动脉桥,进行肉眼观察,HE染色观察病理变化,弹力纤维染色,计算机测算血管内膜厚度、新生内膜中膜比指数;电镜观察血管内皮细胞变化。结果50只兔成功建立动脉桥,无手术及围手术期死亡,桥血管总通畅率为86％,对通畅的桥血管作形态学观测发现血管移植后7d起至56d内膜增厚有统计学差异。结论本动物模型可以较好地模拟冠状动脉旁路移植术桡动脉桥的情况。     

Increased phenylalanine incorporation in regenerating skeletal muscle grafts

Skeletal muscle regenerates following grafting, but little is known about protein synthesis and its regulation during regeneration. We determined the sequence of changes in protein synthesis in rat extensor digitorum longus (EDL) muscle by the measurement of phenylalanine (Phe) incorporation into muscle protein at various times after grafting. Compared with control EDL, Phe incorporation in grafts doubled in 1 day, was four- to eight-fold greater from days 2 to 10 after grafting, and then subsided. Tissue mass (wet weight) increased rapidly from days 7 to 20 in EDL grafts. The maximal increase in protein synthesis occurred 7-10 days after grafting, whether or not the nerve was left intact. Autoradiography indicated that incorporated radioactivity was associated with regenerating muscle fibers on day 10. Deficiencies of insulin, pituitary or testicular hormones, or chronic in vivo administration of insulin, growth hormone, testosterone, or tri-iodothyronine did not substantially alter the elevation in incorporation of the Phe into muscle protein 10 days after grafting. The breakdown of EDL protein, measured in vitro simultaneously with protein synthesis, was increased five-fold, and overall protein degradation was elevated six-fold 10 days after grafting. These findings indicate that Phe incorporation is rapidly elevated following grafting of the EDL, and that by days 7-10 reflects synthesis in regenerating muscle fibers. The increase in protein synthesis associated with muscle regeneration at this time appears to be independent of innervation and anabolic hormones.     

The early revascularization of membranous bone

The experimental finding that membranous onlay bone grafts maintain volume and viability to a greater extent than do endochondral grafts may be related to the more rapid vascularization of membranous bone. Microangiographic techniques were used to study the rates of vascularization of membranous and endochondral bone grafts in adult white New Zealand rabbits at 1, 3, 7, 14, and 21 days after bone grafting. Vascularization patterns were quantified microscopically using a modified point-counting technique. At 3 days, membranous bone grafts demonstrated vessel ingrowth from both soft tissue and host bone. Little ingrowth was seen in endochondral grafts. By day 7, 2.5 vessels per square were identified entering membranous grafts, while an average of 0.6 vessels per square were counted for endochondral bone grafts. At day 14, there was an average of greater than 20 vessels per square for membranous grafts versus 1.8 for their endochondral counterparts. At 21 days, the endochondral grafts demonstrated persistent avascular central areas not seen in membranous grafts. Membranous onlay bone grafts in the rabbit are more rapidly vascularized than endochondral grafts. This factor may affect the greater volume maintenance seen in experimental membranous grafts.     

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.     

Remodeling of heat-treated cortical bone allografts for posterior lumbar interbody fusion: serial 10-year follow-up

We have selected heat-treated bone allografts as the graft material since the Tokai Bone Bank, the first regional bone bank in Japan, was established in 1992. In this study, we examined changes in bone mineral density (BMD), and morphology observed by magnetic resonance imaging (MRI), and histological findings of bone grafts in cases followed up for 7-10?years after bone grafting to grasp the remodeling of heat-treated cortical bone allografts for posterior lumber interbody fusion (PLIF). BMD of bone grafts was reduced by half at 10?years after grafting. MRI revealed that bone grafts were indistinguishable initially in only 22.2% of cases, whereas after a lengthy period of 10?years distinguishable in many cases. Histologically, new bone formation at the graft-host interface was observed earlier, at 1?year after grafting, than that at the periphery of canals in the specimens. The laminated structure of the cortical bone eroded over time, and fragmented bone trabeculae were observed in the specimens at 8?years or longer after grafting, though necrotic bone still remained in some sites.     

Autonomic innervation and plasma estradiol-17β and progesterone levels in rats with subcutaneous ovarian autografts

Summary Ovaries were removed from female rats and immediately autografted into a subcutaneous pouch in the flank in order to quantitate the relationship of graft re-innervation, steroid secretion and vaginal smear pattern. Animals were killed at three time periods: three days after grafting, on the first day a cornified vaginal smear appeared and at the first metestrus. In addition, control animals were killed at metestrus. Plasma samples were obtained from all rats and analyzed for estradiol-17 and progesterone concentration by radioimmunoassay.At the first day of vaginal cornification after grafting, plasma estradiol-17 (45.8±4.0 pg/ml) was elevated in comparison to controls at metestrus (24.0±2.6 pg/ml), but plasma progesterone (21.5±4.0 ng/ml) was not different (30.6±1.7 ng/ml). Subsequently, at the first metestrus following grafting, plasma estradiol-17 (23.0±3.5 pg/ml) was comparable to control values. In contrast, progesterone was decreased (17.5±1.9 ng/ml). A definite correlation was detected between the vaginal smear and plasma levels of steroid hormones in the castrated female rat with subcutaneous ovarian autografts.Histochemical techniques were used to study the adrenergic and cholinergic innervations of grafts three days after grafting, at the first day of vaginal cornification, and at the first metestrus. No correlation was shown between density of adrenergic or cholinergic innervation and plasma levels of estradiol-17 and progesterone or onset of a cycling vaginal smear.Supported in part by USPHS Grant T01-DE00241-04The authors wish to thank J. Canale, S. Hemelt, E. Schwartz and J. Skaggs for their technical and secretarial assistance. Anti-estradiol-17 antibody was obtained from Dr. I.H. Thorneycroft, University of Southern California School of Medicine, and anti-progesterone antibody from Dr. D. Tulchinsky, Harvard Medical Center     

Muscle grafts overloaded by ablation of synergistic muscles

We hypothesized that the mass and maximum tetanic tension (Po) of nerve-intact grafts overloaded by ablation of synergistic muscles would be greater than that of standard nerve-intact grafts or of control soleus muscles. Soleus muscles were grafted orthotopically and bilaterally in 35 female rats. Control soleus muscles were obtained from 30 age-matched cohorts. Twenty-eight days following grafting, gastrocnemius muscles were ablated bilaterally in half of the animals. Comparisons were made between 28 and 112 days following grafting. By 112 days the wet mass of the overload nerve-intact grafts was 138% of the standard grafts and 152% of the control soleus muscles, whereas the Po was 161% and 107%, respectively. Specific tension stabilized at approximately 19 +/- 1 N/cm2 for both types of grafts, significantly lower than the value of 24 +/- 1 N/cm2 for control soleus muscles. Ablation of synergistic muscles resulted in a significant and sustained increase in mass and Po in regenerating skeletal muscle autografts. We conclude that provided the appropriate conditioning stimulus small grafts (100-200 mg) are capable of achieving the values for the mass and Po of control muscles.     

High activity of 17 alpha-oxidoreductase in neonatally grafted mouse testes in adult female mice

Male and female (WB-C57BL/6)F1 hybrid mice were used. Two testes from neonatal mice were grafted into the spleen of adult male and female mice, and the grafted testes were removed 30 and 60 days after grafting. Normal testes from 30- and 60-day old mice were also used. Testicular homogenates were incubated with [14C]4-androstene-3,17-dione or [3H]progesterone, and enzyme activities per g wet tissue and progesterone metabolism were examined. Activity of 17 alpha-oxidoreductase in the grafted testes in females (20 nmol/g/h) was approx. 10 times the activity in the grafted testes in males or in the normal testes, whereas 17 beta-oxidoreductase activity in the grafted testes in females was the lowest among these testes. The bilateral ovariectomy performed 1 month before the grafting of neonatal testes, artificial cryptorchidism performed at 20 days of age, and estrogen treatment for 10 days by diethylstilbestrol pellets resulted in no significant changes in 17 alpha-oxidoreductase activities in 30- and 60-day old grafted, cryptorchid or normal testes. The major 17-hydroxy-C19-steroids formed in vitro from progesterone by the grafted testes in female mice were testosterone and 17 alpha-hydroxy-4-androsten-3-one (epitestosterone), but the formation of epitestosterone was insignificant in the normal testes. The present results demonstrate for the first time that epitestosterone is formed as one of major C19-steroids in neonatally grafted mouse testes in females but not in those in males or in normal mouse testes. However, the mechanisms remain unexplained.