Risk assessment of outcrossing of transgenic rapessed to related species:

Summary The risk for a gene dispersal is reported for reciprocal crosses between a transgenic rapeseed variety resistant to the herbicide phosphinotricin and five related species. The first stages after pollination were cytologically observed and fertilized ovaries were established in in vitro culture for the production of interspecific hybrids. A similar classification was observed for the index of pollination compatibility and embryo yield. From the 243 embryos produced, 109 plantlets were obtained in a greenhouse. All the interspecific combinations tested were able to produce hybrid plants. A higher number of hybrids was obtained when rapeseed was used as the female parent. The hybrids had the expected triploid structure except for two amphidiploid, B. napus × B. oleracea, and one amphidiploid, B. napus × S. arvensis, plants with 56 chromosomes. The triploid hybrids were sterile or partially fertile but two of the amphidiploid plants, B. napus × B. oleracea, were fully fertile. The cytoplasm source did not seem to affect the fertility of the hybrids.     

Interspecific hybridization between Cicer arietinum and C. pinnatifidum

An interspecific hybrid between Cicer arietinum cv. GL 769 and a wild species C. pinnatifidum was obtained after emasculation, pollination and application of growth regulators. Ovules were cultured and embryos were later dissected to obtain hybrid plants. These plants were albinos and morphologically resembled C. pinnatifidum. Shrivelled seeds were also obtained in 2% of the crosses, which on germination gave rise to albino plants. These plants did not survive beyond 20 days. The hybrid nature of these plants was confirmed by esterase isozyme studies. Hybrid shoots obtained from germinating embryos were cultured on modified ML-6 medium with BAP 2 mg/1, IAA 0.5 mg/1, where they turned green after 3–4 weeks. Transmission electron microscopy (TEM) studies on leaf sections from green hybrid shoots showed an improvement in the chloroplast structure, with better organized grana.     

Congruity backcrossing as a means of creating genetic variability in self pollinated crops: seed morphology of Phaseolus vulgaris L. and P. acutifolius A. Gray hybrids

Summary Seeds of early generations of three reciprocal congruity-backcross (CBC) pedigrees, developed by backcrossing Phaseolus vulgaris-P. acutifolius hybrids to each of the parent species in alternate generations, exhibited a preponderance of traits (size, shape, color, and pattern) of the cytoplasmic parent. The large size of Red Cloud (V₁), the P. vulgaris parent common to all of the pedigrees, dominated pedigrees with V₁ as the cytoplasmic parent, while the small size and rounded or square shapes of the tepary parents, wild P. acutifolius var. acutifolius PI 263590 or G400445 (A₁₉), wild P. acutifolius var. latifolius PI 406622 (A₁₀), or cultivated P. acutifolius var. latifolius Serowi PI 319443 (A₉), were the majority phenotypes when P. acutifolius was the cytoplasmic parent. Continuing through the second cycle of CBC, that is the second backcross with each of the parent species or the fourth backcross, began an amelioration of the apparent cytoplasmic effect on gene expression, as reciprocal pedigrees became more alike, usually with intermediate expression of parental traits or the appearance of new traits. The large seed size of V₁ was recovered in hybrids with P. acutifolius cytoplasm and the kidney shape of V₁ became rare in hybrids with P. vulgaris cytoplasm. Although the tepary-bean parents represented two subspecies and both cultivated and wild P. acutifolius, the three sets of reciprocal-hybrid pedigrees with P. vulgaris Red Cloud are surprisingly similar. It may be that the exotic parent used to develop a CBC pedigree should be selected more for combining ability in the interspecific cross than for specific economic traits. While the number of generations (six or more) required to produce fertile, intermediate CBC hybrids (that did not require embryo rescue) may preclude routine use of this method by practical plant breeders, the crossability of advanced hybrids with both parental species and the amount of variability apparent in advanced-hybrids progenies suggests that CBC would be valuable for maintaining exotic germplasm in immediately useful forms.     

Opioid requirements after locoregional anaesthesia in dogs undergoing tibial plateau levelling osteotomy: a pilot study

ObjectiveTo determine the intraoperative and early postoperative opioid requirement after ultrasound-guided sciatic and/or femoral nerve block or epidural anaesthesia in dogs undergoing tibial plateau levelling osteotomy (TPLO).Study designProspective, masked, pilot, randomized, clinical trial.AnimalsA total of 40 client-owned dogs undergoing TPLO.MethodsEach dog was randomly assigned to group SF (combined sciatic and femoral nerve block), group S (sciatic nerve block), group F (femoral nerve block) or group E (epidural anaesthesia). A total of 0.3 mL kg^–1 of ropivacaine 0.5% was administered to each nerve or in the epidural space. Intraoperatively, fentanyl (2 μg kg^–1) was administered intravenously when heart rate, mean arterial pressure or respiratory rate increased by >30% compared with baseline values. Postoperatively, a visual analogue scale (VAS) and a modified German version of the French pain scale (4AVet) were used to assess pain every 30 minutes for 150 minutes and again once the morning after surgery. Methadone (0.1 mg kg^–1) was administered intravenously if the VAS was ≥ 4 cm [maximal value 10 cm; median (interquartile range)] or the composite pain score was ≥5 [maximal value 15; median (interquartile range)]. Significance was defined as p ≤ 0.05.ResultsGroups SF and E required less total intraoperative and early postoperative opioid doses compared with groups S and F (p = 0.031). No dogs in group SF had a block failure or required postoperative methadone. A reduced methadone requirement was found in group SF compared with all the other groups up to 150 minutes after recovery (p = 0.041).Conclusions and clinical relevanceCombined sciatic and femoral nerve block and epidural anaesthesia lead to less cumulative consumption of perioperative opioids than single nerve blockade. Sciatic or femoral nerve block alone might be insufficient to control nociception and early postoperative pain in dogs undergoing TPLO.     

抗病抗寒无核葡萄胚挽救研究

【目的】为了培育抗病抗寒无核葡萄新品种,【方法】以2个种子败育型无核品种‘波尔莱特’、‘红无籽露’作母本,以抗病抗寒的中国野生山葡萄株系‘黑龙江实生’、‘双优’及欧山杂种‘北醇’、‘00-1-10’(‘玫瑰香’ב黑龙江实生’)分别作父本杂交,授粉51 d后将胚珠分别接种于ER和MM4培养基上进行胚挽救,培养60 d后在WPM+BA 0.2 mg.L-1培养基上诱导成苗。【结果】结果表明,2个母本品种胚挽救的适宜培养基不同,‘波尔莱特’作母本适合于ER培养基,‘红无籽露’作母本适合于MM4培养基。2个母本基因型对胚珠的发育率和成苗率的影响差异不大,‘波尔莱特’略优于‘红无籽露’;但4个父本基因型对胚挽救效果影响较大,以欧山杂种‘00-1-10’和‘北醇’作父本的杂交组合胚株的发育率和成苗率明显高于山葡萄‘黑龙江实生’、‘双优’作父本的杂交组合。共获得无核葡萄胚挽救新种质50个株系。【结论】在胚挽救过程中,不同基因型的无核葡萄适宜于不同的基本培养基,欧山杂种比山葡萄更适宜于作杂交的父本。     

Interspecific hybridization between sunflower and wild perennial Helianthus species via embryo rescue

Interspecific crosses were made between the cultivated sunflower inbred line HA89 and accessions of five wild perennial Helianthus species (H. giganteus L., H. laevigatus T. & G., H. resinosus Small, H. pauciflorus Nutt. and H. decapetalus L.) resistant to broomrape ( Orobanche cernua Loefl.). Using the genetic male-sterile isogenic version of that line as female, successful reciprocal crosses were also obtained with all these wild species except with H. decapetalus. Five-day-old hybrid embryos were excised and cultured in vitro. In all cases, few mature plants were obtained from embryos in early developmental stages (early heart and globular) but up to 28% mature plants were obtained from embryos in later stages of development. Very immature embryos were difficult to excise without damage. Hybrid embryos and mature plants were obtained from all five wild species. Interspecific hybrid embryos from different wild species showed distinct developmental potentials, the proportion of hybrid embryos in different developmental stages varying among species. Differences in the proportion of hybrid embryos at the several developmental stages were also obtained for the reciprocal crosses (cultivated × wild), which showed higher proportion of fully developed embryos. Hybrids involving H. giganteus and cultivated sunflower were difficult to obtain without the use of embryo culture. Embryo culture proved to be an useful tool to overcome post-zygotic hybrid incompatibility in different Helianthus spp., and facilitated interspecific transfer of resistance to O. cernua. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

多花黑麦草和四倍体苇状羊茅属间杂种F#-1及其双倍体的细胞遗传学研究

本研究获得了多花黑麦草（Lolium multiflorum Lam 2n=2x=14）和四倍体苇状羊茅（Festuca arundinacea var.glaucescens Boiss 2n=4x=28）的属间杂种。Lmx Fa.g杂种在形态上呈中间型,而反交杂种则倾向于母本。杂种的细胞学研究表明：多花黑麦草的染色体组（A）与四倍体苇状羊茅的两组染色体（B和B′）亲和性差;相对来说,羊茅属的细胞质有利于A、B、B′三组染色体的配对。杂种F#-1花药不开裂,花粉畸形败育。合成的双倍体细胞学上不稳定,花粉KI-I#-2可染率极低。本文还讨论了利用杂种的途径和实践意义。     

猪圆环病毒2型感染性克隆构建

基于PCV2滚环复制原理,在分析PCV2不同基因亚型代表毒株基因组结构和序列的基础上,设计2对引物用于PCR扩增PCV2基因组序列。以从收集到的疑似PCV2感染的10份猪血清样品中提取的DNA为模板,用设计的引物扩增目的片段,并进行测序分析,1株为PCV2a型,4株为PCV2b型,5株为PCV2d型。每种基因型毒株中各选取一个样本,利用上述2对引物进行PCR扩增,并将扩增产物克隆至pEASY-Blunt simple载体中,通过双酶切连接2个PCR片段,构建含有约1500 bp重叠序列的PCV2基因组的质粒。通过脂质体转染法将含PCV2全基因组的质粒转染至PK-15细胞进行病毒拯救,经PCR和IFA两种方法验证,证明成功拯救出3个基因型的PCV2毒株。通过对PCV2全基因组结构及序列分析,该方法同样适用于PCV2g、PCV2h基因型病毒的感染性克隆构建。本研究建立了一种基于反向遗传学技术的PCV2感染性克隆的构建方法,为开展PCV2的病原学研究提供了技术支持。