柑橘抗寒育种早期鉴定的一种指标

以５个抗寒性不同的柑橘品种或杂交后代：“山金柑”（ＦｏｒｔｕｎｅｌａｈｉｎｄｓｉＳｗｉｎｇｌｅ）、“伏令夏橙”（ＣｉｔｒｕｓｓｉｎｅｎｓｉｓＯｓｂ．ｃｖ．Ｖａｌｅｎｃｉａ）、“伏令夏橙”＋“宁波金柑”（Ｃ．ｓｉｎｅｎｓｉｓｃｖ．Ｖａｌｅｎｃｉａ＋Ｆ．ｃｒａｓｉｆｏｌｉａＳｗｉｎｇ．ｃｖ．Ｍｅｉｗａ）、“Ｐａｇｅ”橘柚（Ｃ．ｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｇｒａｎｄｉｓＯｓｂ．ｃｖ．Ｐａｇｅ）、“Ｍｕｒｃｏｔ”橘橙（Ｃ．ｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｓｉｎｅｎｓｉｓＯｓｂ．ｃｖ．Ｍｕｒｃｏｔ）为材料,研究了低温锻炼期间柑橘原生质体抗寒性的变化及其田间植株的冬季抗寒性,表明离体原生质体抗寒性与田间植株抗寒性之间有显著的正相关,因而前者可以作为衡量后者的指标。     

Establishment of an Index to Appraise Cold-resistant Breeding for Citrus in Early Stage

Cold-resistance of citrus protoplasm and field plants in 5 different varieties was studied. The relative analysis between cold resistance of citrus protoplasts and that of field plants indicates that the cold resistance of citrus protoplasm can be considered as an index of cold resistance of field plants.     

不同品种小麦叶片对拔节期低温的生理响应及抗寒性评价

以黄淮海麦区参加区试的24个小麦品种为材料,分析了拔节期低温胁迫对其叶片相对电导率、可溶性糖含量、可溶性蛋白含量、游离脯氨酸含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)和过氧化物酶(POD)活性的影响。结果表明,低温胁迫后,叶片相对电导率、可溶性糖含量、脯氨酸含量、MDA含量、SOD活性和POD活性均高于对照(P0.01),而可溶性蛋白含量低于对照(P0.01)。小麦叶片各指标存在极显著的品种间差异(P0.01)。以叶片各指标的相对值作为抗寒性指标,采用隶属函数法和极极点排序法计算参试品种的平均隶属度值为0.19—0.63,综合排序值为1.66—4.08。通过K-means聚类,将24个小麦品种聚为5类。其中良星619、丰德存麦1号、B07-4056、石H083-363、山农055843和良星99等6个品种抗寒性最强,宿553、陕农509、A-9、中原6号、徐麦4036、舜麦1718和石麦19等7个品种抗寒性强,尧麦16、C-44、山农05-066、冀麦585和石B05-7388等5个品种抗寒性中等,偃展4110、B-33、B05-6507和石4185等4个品种抗寒性弱,石06-6136和石优20这2个品种抗寒性最弱。相关分析表明,平均隶属度值和综合排序值与相对可溶性糖含量、相对脯氨酸含量、相对POD活性呈正相关(P0.05),与相对MDA含量呈负相关(P0.05)。叶片可溶性糖含量、游离脯氨酸含量、POD活性及MDA含量可以作为拔节期小麦抗寒性的鉴定评价指标。     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Cytological Studies on the Cold Resistance of Plants-Morphological Changes of the Intracellular Structures of Wheat in the Overwintering Period

The present work comprises a study of 6 wheat varieties of various degrees of cold resistance. They are: 2 winter varieties, Nungta 183 and Huapei 187; 2 spring varieties, Nanta 2419 and Piyü; and 2 intermediate varieties (according to their winter hardiness), Pima No. 1 and Pingyüan 50. All these varieties were cultivated under the same natural conditions. Cytological changes of each of the varieties were studied comparatively at different stages of the overwintering period. In addition, certain morphological and physiological features of the above mentioned varieties were also studied. With the temperature gradually falling in autumn, the rate of the cell divisions and the physiological activities of the nucleoli of the winter varieties decreased, the growth of the plants and the development of the apical cones were suppressed, and so more storage materials were left. The less hardy and non-hardy varieties, on the other hand, retained their cytophysiological activities and high growing rate, so that the stored materials were much less than those left in the winter varieties. However the non-resistant varieties could not withstand the winter frost and survived no more. The plants of the less hardy varieties were partly killed by winter frost. When the temperature dropped further in winter, plasmolysis gradually appeared in the cells of the tillering nodes of the cold resistant wheat plants, and it disappeared with the return of the warmer weather. The degrees of the plasmolysis in different varieties were found proportional to their cold hardiness, and no plasrnolysis was observed in the spring wheat. Thus a correlation apparently exists between the plasmolysis and the cold resistance in wheat. In winter, the vacuoles of the young leaf cells and of the cells of the primary meristematic tissues of the apical cones became smaller and smaller, while a dense reticular structure appeared in the cytoplasm. In spring, the vacuoles restored, and the reticular structure disappeared. It seems that the occurrence of the reticular structure in the winter season is also closely correlated with the cold resistance of the wheat plants. It was found that the nuclei and the chloroplasts of the winter varieties were more resistant to cold than those of the less resistant and non-resistant varieties. The stability of these structures increased in hardened condition. Morphological changes of the chloroplasts of the young leaf cells occured from autumn to winter, they were transformed from polymorphic to uniformly globular. The chloroplasts of some cells were balling together, however, they actually retained their individualities. The chondriosomes increased in size and quantity during the hardened period. They became short and thick, some were in globular form or they clumped together in severe winter, and gradually restored when warmer weather arrived in the spring. The correlations of the mitosis, the plasmolysis, the appearance of the reticular structure in the cytoplasm and the stability of the nuclei with the cold resistance of the wheat were discussed.     

Cytological Studies on the Cold Resistance of Plants the Changes of the Intracellular Materials of Wheat in the Overwintering Period

In the literature, the information on the correlation between the material changes and the cold resistance of plants is full of contraditions, especially in regard to the changes of the sugar and protein contents in connection with the development of the cold resistance in hardened condition. The present work was undertaken with an attempt to study the changes of the intracellular materials of wheat in the overwintering period with cytochemical methods. The wheat varieties used in this study were the same as used in our previous work (Chien and Wu, 1965), these are, 2 winter varieties (Nungta 183 and Huapei 187), 2 spring varieties (Nanta 2419 and Piyü), and 2 intermediate ones (Pima No. 1 and Pingyaian 50). The plants of the different varieties were cultivated under the same environmental conditions. Samples were collected and fixed at the same time (twice a month from October to March). For making microscopical preparations, the sections of materials for comparative studies were mounted on the same slide, so that they all were subjected to exactly the same treatment. The results obtained are summarized as follows: 1. The starch content in the cells of the tillering nodes increased, as the temperature fell in autumn. The starch grains gradually disintegrated when the plants came in hardened period (late October to mid-November), and completely disappeared in severe winter (January). They were regenerated in mid-February. 2. The soluble sugar gradually increased with the falling of the temperature in late autumn and steadily increased until the temperature approached 0 ℃. However, when the temperature dropped further, the soluble sugar content decreased and finally in March reached the level which it assumed before winter. 3. It was determined by cytochemical reactions that the cytoplasmic proteins and RNA and the nuclear basic proteins increased from late autumn to early winter. The degree of these changes showed apparent difference between the different varieties. Based on the phenomena observed, the authors discussed their correlation with the cold resistance of wheat plants. 4. The fat and lipids were also tested, but there was no indication of the occurrence of these substances in the winter season. The study on the DNA content revealed no correlation of any kind with the development of the wheat hardiness.     

小麦不同品种上麦蚜及其天敌的数量变动

试验结果表明小麦品种 (系 )的抗性对麦蚜种群数量影响很大 ,百株蚜量随着小麦品种抗性增强而下降。而同一小麦品种对不同种蚜虫的抗性存在质的差异 ,铭贤 1 69品种 ,蚜高峰期百株蚜量麦长管蚜 63 0头 ,禾谷缢管蚜只有 1 1 5头 ,两者相差 5 5倍。另一方面 ,小麦品种抗性对麦田天敌的种群数量影响不大 ,而对天敌的发生期有些影响。因此 ,小麦品种抗性、天敌对麦蚜的自然控制能力 ,可把小麦中后期的蚜虫虫口密度控制在经济损失允许水平之下。     

雪花莲凝集素基因转化小麦及转基因小麦抗蚜性的研究

雪花莲凝集素对具有刺吸式口器的同翅目害虫具有毒杀作用。用基因枪法将1个新的雪花莲凝集素(GNA)基因转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中,分别获得了转基因小麦植株。抗蚜实验证明,转化gna基因的小麦植株对我国北方冬麦区的主要麦蚜——麦长管蚜和禾谷缢管蚜的抗性效果不尽相同。对禾谷缢管蚜,在接种当代即表现出明显的毒杀作用。对麦长管蚜,则表现为虫体发育减缓并且降低了其所生产的若蚜成活率。在自然放养条件下,gna基因则对这两种麦蚜的取食均起到了一定的抑制作用。     

Stably transformed callus of wheat by electroporation-induced direct gene transfer

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.     

HRGP在小麦抗寒锻炼过程中的变化及其与抗寒性的关系

强抗寒小麦品种(R-025、中品94-19、品83-1、品83-2、品83-3、米罗诺夫808)经抗寒锻炼后,其幼苗体内的游离脯氨酸、细胞壁结合的羟脯氨酸和糖蛋白含量发生了明显的变化.游离脯氨酸含量比未经抗寒锻炼处理时增加5～32倍,细胞壁结合的羟脯氨酸含量比对照增加1.77～2.17倍,糖蛋白含量比对照增加4.68～9.72倍,而不抗寒小麦品种(中国春、冬103)增加量较小.脯氨酸积累进程各个品种间差异比较大,品83-1、品83-2积累较快,抗寒锻炼第21d时达到最高峰,而R-025在第56d达到最高峰.脯氨酸含量与小麦品种抗寒性相关不显著(相关系数为0.3462),而羟脯氨酸含量、糖蛋白含量与小麦品种抗寒性相关显著,相关系数分别为0.6491和0.7039.从小麦细胞壁纯化得到了2种伸展蛋白Extensm1和Ex-tensin2,其含量都和小麦品种抗寒性呈正相关.Extensin1是分子量为28kD、羟脯氨酸为主要成份(32mo1%)的富含羟脯氨酸糖蛋白.     

Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

Resistance to pink snow mould, caused by Microdochium nivale, was investigated in four resistant winter wheat lines from the USDA World Cereal Collection (CI9342, CI14106, PI173440 and PI181268) and three Nordic wheat lines (Bjørke, Rida and V1004). Pink snow mould resistance was tested in non‐hardened and cold‐hardened plants incubated under artificial snow cover and in detached leaf segments mounted on water agar and incubated at either 3°C in darkness or at room temperature with light during the day. The wheat lines CI9342, CI14106 and PI181268 were more resistant than the Nordic lines, both before and after cold hardening. Thus, although cold hardening strongly increases the level of snow mould resistance in all the wheat lines, some resistance mechanisms are also present prior to cold hardening in some of the resistant lines. CI9342, CI14106 and PI181268 also had a higher level of resistance than the other lines in the detached leaf assay, indicating that these lines have some resistance mechanisms acting in the leaves. The resistance of PI173440 was expressed only in intact hardened plants and not in non‐hardened plants or in detached leaves. This indicates that this line relies on cold hardening‐related changes in the crown for its resistance. In the detached leaf assay the rate of lesion development varied greatly between leaves of different order. The highest correlation with the whole plant test was obtained when using secondary leaves and incubation at 3°C in the dark.     

Wheat curl mite resistance: interactions of mite feeding with wheat streak mosaic virus infection

The majority of plant viruses are dependent on arthropod vectors for spread between plants. Wheat streak mosaic virus (family Potyviridae, genus Tritimovirus, WSMV) is transmitted by the wheat curl mite, Aceria tosichella Keifer, and this virus and vector cause extensive yield losses in most major wheat (Triticum aestivum L.)-growing regions of the world. Many cultivars in use are susceptible to this vector-virus complex, and yield losses of 10-99% have been documented. wheat curl mite resistance genes have been identified in goat grass, Aegilops tauschii (Coss) Schmal., and transferred to hexaploid wheat, but very few varieties contain effectively wheat curl mite resistance, due to virulent wheat curl mite populations. However, wheat curl mite resistance remains an effective strategy to reduce losses due to WSMV. The goal of our project was to identify the most effective, reproducible, and rapid method for assessing wheat curl mite resistance. We also wanted to determine whether mite resistance is affected by WSMV infection, because the pathogen and pest commonly occur together. Single and group wheat curl mite infestations produced similar amounts of leaf rolling and folding on wheat curl mite-susceptible wheat varieties that were independent of initial wheat curl mite infestation. This finding will allow accurate, efficient, large-scale screening of wheat germplasm for wheat curl mite resistance by infesting plants with sections of wheat leaf tissue containing mixed stages of wheat curl mite. The wheat curl mite-resistant breeding line 'OK05312' displayed antibiosis (reduced wheat curl mite population development). The effect of WSMV infection on wheat curl mite reproduction was genotype-dependent. Mite populations increased on infected wheat curl mite- and WSMV-susceptible plants compared with uninfected plants, but WSMV infection had no significant effect on wheat curl mite populations on resistant plants. OK05312 is a strong source of wheat curl mite resistance for wheat breeding programs.     

通过组织培养筛选小麦抗赤霉病突变体的研究

选用中抗赤霉病的春小麦品种和品系的花药进行离体培养,以小麦赤霉病29号菌株产生的致病培养滤液为选择剂,结合物理诱变处理,进行抗病鉴定,用赤霉病菌分生孢子直接接种在愈伤组织和再生植株筛选抗病突变体。在83块愈伤中有53块抗病。在11株的再生植株中有9株均比未经培养滤液处理的对照提高了抗病性,从中选出4株抗病性接近或超过“苏麦3号”品种。     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

The problem of durable resistance of plants to different pests

Different hypotheses concerning durable plant resistance against different pest were tested: 1) resistance is weak and polygenetically controlled; 2) resistance depends on "residual effect" of oligogenes that were overcome by pests. Contrast pair of plants and pests were used in experiments: wheat, barley--facultative parasite Bipolaris sorokiniana Shoem., wheat--obligate parasite Puccinia recondita Rob. ex Desm., sorghum--greenbug Schizaphis graminum Rond. Differential interaction between parasite and host plant resulted in their increased compatibility under long reproduction of parasite on resistant varieties were regarded as criteria of quick overcoming of resistance. The results did not support any hypothesis. The rate of adaptation of B. sorokiniana to the resistant varieties of wheat and barley did not depend on the level of resistance expression (weak, moderate or strong) and genetic control (oligogenic, polygenic or cytoplasmatic). It was shown by hybridological analysis that "residual effect" of oligogenes of sorghum resistance against greenbug depended on small resistance genes, that can be independent or weakly connected with marker oligogene. These data allows to doubt in phenomenon of "residual effect" of oligogenes. It was shown that non specific pathogenicity of parasitic fungi increased during their reproductions on sensitive varieties of plants. Thus, cultivation of sensitive varieties causes damage of crop culture non only because of their own losses, but also by increasing the infection of moderately resistant varieties.     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA

普通小麦"济南177"(Triticum aestivum L.cv.Jinan177)经继代培养和选择,形成了性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质具有旺盛的分裂能力,但不能分化,称为Cha9;另一种具有一定能力,但游离原生质体分裂能力低,称为176.二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株.而将源于Cha9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株.再生的愈伤组织及植株经表现型、细胞学、有工酶、RAPD分析,证明了其杂种性质,用不麦叶绿体特异的简单得复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况.在不同的杂种克隆中,同时带有Cha9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株.     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA(英文)

普通小麦“济南177”(Triticum aestzvum L.cv.Jinan 177)经继代培养和选择,形成了两种性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质体具有旺盛的分裂能力,但不能分化,称为Cha 9;另一种具有一定分化能力,但游离的原生质体分裂能力低,称为176。二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株。而将来源于Cha 9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株。再生的愈伤组织及植株经表型、细胞学、同工酶、RAPD分析,证明了其杂种性质,用小麦叶绿体特异的简单重复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况。在不同的杂种克隆中,同时带有Cha 9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株。     

Effect of low temperature stress on the expression of sucrose synthetase in spring and winter wheat plants. Development of a monoclonal antibody against wheat germ sucrose synthetase.

A monoclonal antibody against wheat germ sucrose synthetase is developed and characterized. Its use in studying the effect of cold acclimation on the expression of sucrose synthetase in winter and spring wheat plants is described. The antibody shows cross-reactivity with sucrose synthetase from maize and pea plants, as well as carrot cells. A gradual accumulation of the enzyme as a function of time spent at 2 degrees C is observed in both wheat varieties. In contrast, an initial sharp rise in the mRNA level is observed, which agrees with the previously reported response of maize plants subjected to anaerobic stress.     

Influence of Low Temperature on Spatial-temporal Changes of Ca2+ in Winter or Spring Wheat

Stained with Fluo-3/AM and investigated under the laser scanning confocal microscope (LSCM), the spatial-temporal changes of ［Ca2+］cyt(the free Ca2+ concentration in the cytoplasm) in the protoplasts of mesophyll cells of wheat (Triticum aestivum L.) cultivars with different cold-hardiness under resting and temperature decreasing conditions were compared. The results showed that under the resting condition, the fluorescence intensities in the protoplasts of both cold-sensitive and cold-resistant wheat manifested no significant changes, implying that ［Ca2+］cyt could keep at a stable level under the resting condition although different wheat cultivars were different. However, different dynamics appeared with temperature decreasing from 15℃ to 2℃. In particular, for the cold-resistant wheat, ［Ca2+］cyt level at first increased, followed by drop to the resting level at 2℃, and then increased further when the temperature was below 2℃. On the contrary, the ［Ca2+］cyt level of the cold-sensitive spring wheat increased continuously to the maximal level within the whole range of temperature changes. Based on the above observations, it could be reasonably inferred that different dynamics of ［Ca2+］cyt determines decisively different cold acclimation abilities of plants. Furthermore, the results provide a new supporting evidence for the hypothesis that Ca2+ plays a key role as a primary physiological transducer upon chilling”.     

超声波辅助农杆菌介导的小麦遗传转化初步研究

报道了一种改进的小麦转化方法——超声波辅助的农杆菌转化法。发芽的小麦种子经超声波处理20min后再经农杆菌浸染,浸染后的种子按正常播种和栽培管理至结实,对收获的成熟种子进行卡那霉素抗性筛选。结果表明,受试的4个小麦品种均得到抗性后代,PCR扩增结果证明这些抗性植株是转化植株。GUS染色结果表明不经超声波处理的种子几乎看不到转化的细胞,但经过超声波处理的种子,幼苗基部的细胞转化成功。研究表明,利用超声波辅助的农杆菌转化方法转化小麦能产生可遗传的后代。