Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Effect of Exposure Temperature on Over-All Efficiency of Autoradiographs and on Fogging of Emulsion

Systematic investigations of the influence of various temperatures of exposure on the autoradiographic efficiency and on the rate of fogging of Kodak AR 10 stripping film were carried out. The oxygen content and humidity of the exposed autoradiographs as well as the time of exposure were constant. Exposure of autoradiographs or storage of film at room temperature caused an increase of overall autoradiographic efficiency by approximately 40%, but although the rate of film fogging was also increased, it did not exceed the safe values of background. Both autoradiographic efficiency and rate of fogging were not altered significantly when a temperature of 4 C or -18 C for exposure was used. Therefore, room temperature of exposure is recommended for ordinary autoradiography. For a special autoradiographic technic when low temperature of exposure has to be used, the temperature -18 C may be safely applied without significant impairment of autoradiographic efficiency.     

Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Flower buds of `Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix ¹⁴CO₂ into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total ¹⁴C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the ¹⁴C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO₂ per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO₂ into different compounds suggests that this CO₂ assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.     

Formation of Adventitious Buds in Decapitated Citrus Seedlings and the Effect of Some Growth Regulators

Young seedlings of various citrus species give rise, after decapitation,to adventitious buds at the cut end of the epicotyl. With suchtreatment, more buds formed on seedlings of sweet orange, sourorange, and grapefruit than of lemon, mandarin, and calamondin.Anatomical observations at the cut stem end show formation ofbuds occurring from tissues close to the cambium. Growth regulatorsapplied at the cut epicotyl end modified the patterns of budformation. 6-Benzylaminopurine (BAP) promoted bud formation;indole-3-butyric acid (IBA), abscisic acid (ABA), and gibberellicacid (GA₃) inhibited it. BAP reversed the inhibitory effectof GA₃. Gibberellic acid caused a typical elongation of thesprout axis. The use of decapitated seedlings as a tool forstudying stem differentiation in citrus is suggested.     

Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.     

Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium

The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway.     

三价铁螯合物还原酶在香橙和枳中的表达

用耐缺铁的香橙(Citrus junos Sieb. ex Tanaka)和极不耐缺铁的枳(Poncirus trifoliata (L.) Raf.),在铁胁迫条件下对根的三价铁螯合物还原酶活性变化和酶基因的表达情况进行了研究.离体根的酶活性测定表明,在铁胁迫4周时,香橙根的酶活性增强约20倍,枳仅增强约3倍.用拟南芥的三价铁螯合物还原酶基因作探针进行组织印迹的Northern杂交检测香橙和枳三价铁螯合物还原酶的mRNA,在铁胁迫2周时,香橙吸收根、幼茎和新叶中均检测到强烈的表达信号,而枳相同器官的表达信号则极其微弱.实验结果表明,三价铁螯合物还原酶活性在缺铁胁迫下被诱导强烈增加是香橙耐缺铁的重要原因,该酶活性的调控发生在转录水平上,而且该酶基因在诱导条件下在根、茎和叶中均有表达.     

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells^1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells³. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)^4-5. Sufficient numbers of cells can be obtained from one individual''s tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation^4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic^5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.     

Non-Enzymatic Reduction of Nitrite by Means of Ascorbic Acid in Citrus and Other Higher Plant Tissues

It has been proved that the nitrite reduction in the leaves and other plant tissues of citrus and other green plants is partly or mainly a non-enzymatic chemical process, and a heat-stable factor present in these tissues is responsible for this reduction. It is suggested that ascorbic acid plays a major role in this chemical reaction since the reduction is inhibited by ascorbic acid oxidase. A significant association was also found between the ascorbic acid content and the nitrite reduction capacity of citrus leaves. Evidence has been presented that this non-enzymatic chemical reduction of nitrite occurs also in vivo as undetached citrus leaves on branches placed in NaNO₂ solution have shown diminution of their ascorbic acid content along with the absorption of nitrite. Stronger accumulation of nitrite in these leaf tissues was observed under dark conditions, apparently due to the inhibition of the biosynthesis of the ascorbic acid.     

天津市彩色红外相片在研究植被与大气污染关系中的应用

利用彩色红外相片来研究城市植被与大气污染的关系,这是遥感技术应用于环境动态研究中的一个重要课题。在研究过程中,使用了中国科学院遥感所提供的天津市1／5000和1／10000比例尺彩色红外相片。我们首先将显示在相片上的植被与大气污染的遥感信息进行了解译,然后取样分析该地植物体中污染物质的含量,最后再到实地验证。结果表明,彩色红外相片记录了当时植被与大气污染的真实状况,具有丰富的信息,植被信息特征能够反映大气污染的基本情况。同时还可发现一些抗性强、吸毒能力大的绿化效应好的树种和绿化结构,可为城市建设、城市环境质量评价提供科学依据。     

Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

Variations of Flavonoid Composition and Antioxidant Properties among Different Cultivars,Fruit Tissues and Developmental Stages of Citrus Fruits

A large number of biologically active compounds are present in ripe citrus fruits. However, few studies have been focused on the changes in flavonoids and the evolution of antioxidant activity during citrus fruit growth. In this study, fruits of five citrus cultivars cultivated in China were sampled at 60–210 days post‐anthesis (DPA) at intervals of 30 days. The amounts of main flavonoids in the peel and pulp were analyzed by HPLC and their activities were studied by DPPH, ABTS and FRAP. The results showed that the contents of hesperidin, diosmin, eriodictyol, rutin and nobiletin increased before 90 DPA and then decreased with the growth and development of fruits, but an opposite tendency was observed for naringin and narirutin. The antioxidant activities in citrus peel and pulp were found to be significantly correlated with some flavonoids. The results may be of guiding values in citrus production and utilization of citrus fruit by‐products.     

Infusion of a Lipid Emulsion Modulates AMPK and Related Proteins in Rat Liver,Muscle, and Adipose Tissues

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate‐buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca²⁺/calmodulin‐dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCβ, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARγ‐co‐activator 1α (PGC1α) protein levels were also increased in LE‐treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCα, PPARα, AdipoR1, AdipoR2, and sterol regulatory element–binding protein‐1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Rhythmic Growth and Optimization of Micropropagation: The Effect of Excision Time and Position of Axillary Buds on in vitro Culture of Citrus aurantium L.

We wanted to ask whether the rhythmic pattern of shoot elongationshould be taken into account for the micropropagation ofCitrusaurantium L. and whetherin vitro culture altered the rhythmicproperties. It has been established in the present work that(1) where and when the axillary buds are excised with respectto the elongation cycle are of importance to optimize micropropagationand (2) the infradian rhythmic growth pattern of micropropagatedplants is maintained with a period of 45.8d not significantlydifferent from that of plants derived from seeds (42.4d). Citrus aurantium L.; excision-timing; infradian rhythm; micropropagation; rythmic growth; sour orange     

Growth-Regulating Substances and the Growth of Tiller Buds in Barley; Effects of Cytokinins

An hypothesis was set up from which it was predicted that applicationof cytokinin to barley seedlings grown without mineral nutrientswould lead to rapid growth of the coleoptile and first leaftiller buds. Application of cytokinins to the leaves was ineffective,but supplying a number of known cytokinins by steeping the rootsof 4 d old seedlings in solution for 4 h led to significantgrowth of the coleoptile bud. Adenine and cytokinin analogueshad no effect. Supplying cytokinins through the roots also furtherenhanced the growth of buds of plants given mineral nutrients.Cytokinin treatment reduced root dry matter, with small reductionsin mean axis length and number of lateral roots. For plantsnot given mineral nutrients reduction in root weight was compensatedby an increase in weight of the aerial parts; however, for plantssupplied with mineral nutrients this was not so and the lowerroot weight resulted in a smaller total plant dry weight. An interpretation of tiller bud growth in terms of control byinteracting effects of mineral nutrition, assimilate supply,and cytokinin availability is proposed.     

节瓜茎尖多胺含量和比值与花性别分化的关系

以4个不同基因型的节瓜为材料,通过两个发育时期（10、19片叶展平）茎尖取样,研究了多胺（PA）含量和比值与植株花性别分化的关系。结果表明,节瓜茎尖4种多胺含量差异显著,两个取样时期都是亚精胺（Spd）〉腐胺（Put）〉尸胺（Cad）〉精胺（Spm）。10片叶展平时期多胺含量与节瓜花性别分化之间没有明确的相关性;19片叶展平时期,节瓜茎尖Put、Spd和多胺总量与植株雌花分化比例呈极显著的正相关,而Cad则与雌花分化比例呈极显著的负相关。在两个取样时期,复合指标Spd/PA都与植株雌花分化比例呈显著的正相关,而（Put＋Cad）/（Spd＋Spm）均与之呈显著的负相关,可以较好地预测节瓜的花性别分化状况。     

化橘红生理落果与若干生理生化指标的关系

通过测定正常果与脱落果若干生理生化指标的变化,探讨化橘红生理落果的机理.结果显示,N、P、K、Ca、Mg含量正常果一致地高于脱落果;脱落果总糖含量呈下降趋势,正常果则先升后降;果实中生长促进类内源激素(GA3、IAA、ZT)含量正常果显著高于脱落果,而ABA/(GA3 LAA ZT)值则是脱落果显著地高于正常果;三类水解酶活性均为脱落果显著高于正常果.说明化橘红果实的脱落与果实内部的矿质元素、总糖、内源激素含量和水解酶活性密切相关.     

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.