Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

An efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L^?1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).     

根癌农杆菌介导的CG1-400-RNase基因转化创制锦橙转基因再生植株

【目的】为了快速有效地利用基因工程的方法获得柑橘无核新种质,【方法】以我国特色多核柑橘优良品种锦橙实生苗上胚轴切段为外植体,采用根癌农杆菌介导法进行能导致种子败育基因CG1-400-RNase的转化;为快速有效地筛选出转化子,在实生苗上胚轴切段转化再生过程中,根据不同发育阶段的组织或器官对抗生素的敏感程度不同采用不同的选择压。【结果】结果表明,在抗性芽再生过程中卡那霉素质量浓度设定为50 mg.L-1,获得的362个抗性芽转入卡那霉素质量浓度为100 mg.L-1的伸长培养基中进行伸长培养后,进行早期PCR检测,获得28个阳性芽;经过不定芽诱导生根或试管嫁接,获得22株完整植株。【结论】再生植株经PCR和Southern杂交检测,获得2株目的基因以单拷贝的形式插入锦橙基因组的转基因植株,为最终获得具有无核性状且可稳定遗传的柑橘新种质奠定了基础。     

Selection and characterization of a new switchgrass (Panicum virgatum L.) line with high somatic embryogenic capacity for genetic transformation

Switchgrass (Panicum virgatum L.) is used horticulturally as an ornamental and agronomically as an animal feedstock and a putative bio-energy crop. Genetic transformation, using somatic embryogenic (SE) callus derived from mature seeds, is one strategy for improving switchgrass traits. A superior switchgrass line, HR8, was developed in this study using recurrent tissue culture selection from cv. Alamo. Eighty two percent of HR8 seeds germinated after harvest comparing to 26.8% for unselected ‘Alamo’. HR8 seeds that germinated produced 84.9% SE callus. HR8 seeds had higher endogenous abscisic acid (ABA) contents and responded differently to exogenous additions of ABA in culture. Endophytes were isolated from switchgrass seeds and callus. HR8 callus had less endophytic contamination than that of ‘Alamo’ callus. HR8 SE calli were genetically transformable using Agrobacterium. Therefore, HR8 is a superior line for generating SE callus and Agrobacterium-mediated transformation.     

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease.     

Seed germination and seedling vigour of transgenic tobacco (Nicotiana tabacum L.) with increased proline accumulation under osmotic stress

Proline plays important roles in various stages and processes of plant development. However, there are few reports on the effect of endogenous proline accumulation on germination and seedling vigour under suitable conditions of germination and development. The aim of this work was to evaluate the effect of endogenous proline in transgenic tobacco (Nicotiana tabacum L.) constitutively expressing the VaP5CSF129A gene in germination and seedling vigour. Under optimal conditions of germination, the transgenic seeds had high proline content when compared to the non-transgenic plants. However, this higher accumulation did not result in better germination or seedling vigour, contrasting to reported results for exogenous proline application. The pre-treatment by water submersion indicated that the lowest initial seed germination with the highest proline concentration may be due to their greater post-harvest dormancy. When newly germinated seedlings were subjected to osmotic stress (?0.9 MPa), the free proline content increased proportionally in all genotypes and the transgenic events seedlings showed greater root length compared to those of the non-transformed control. This can be advantageous as, in theory, seedlings with longer roots may have a better chance of growing and exploring the different soil layers, allowing the transgenic events to be more tolerant to edaphic constraints.     

番茄叶霉菌及番茄抗性基因分子生物学研究进展

 叶霉菌(Cladosporium fulvum) 是番茄(Solanum lycopersicum ) 叶霉病致病真菌, 严重地威胁着番茄生产; 二者互作遵循“基因对基因”假说, 是目前研究植物与病原菌互作模式系统。本文简要地回顾了番茄叶霉菌和抗性基因(Cf) 研究历史; 概述了叶霉菌侵染特征、效应因子的类型、功能和特性; 对番茄Cf基因的类型、定位、进化、结构和功能研究进展加以综述, 特别是对Cf/Avr互作的分子机制、信号转导和抗性基因自激活等最新研究成果加以重点阐述; 同时, 对今后番茄抗叶霉病研究所面临问题、研究热点和解决策略进行了讨论。以期为番茄抗叶霉病或植物抗真菌病研究提供一些有用信息。     

Lettuce (Lactuca sativa): a species with a high capacity for cadmium (Cd) accumulation and growth stimulation in the presence of low Cd concentrations

Summary

Cadmium (Cd) is a metal pollutant that accumulates in cultivated soils and has detrimental consequences in terms of food safety. Lettuce (Lactuca sativa) can be characterised as having a high capacity to accumulate Cd in its tissues. An analysis of Cd tolerance and Cd accumulation was carried out using two varieties of lettuce (‘Divina’ and ‘Melina’). A wide range of CdCl₂ concentrations was used (0.0, 0.1, 0.6, 3.0, and 15.0 µM CdCl₂). The lowest concentration (0.1 µM CdCl₂) stimulated growth, while the two highest concentrations resulted in a reduction in biomass. Cadmium concentrations were found to be twice as high in roots as in shoots. ‘Divina’ displayed lower concentrations of Cd than ‘Melina’ in nearly all treatments. A strong negative correlation was observed between Cd concentration and Cd tolerance in the roots and shoots (R² > 0.87) of both ‘Melina’ and ‘Divina’. Lettuce grown in the presence of 15.0 µM CdCl₂ had leaf Cd concentrations that were 100-fold higher than the legal maximum level for vegetable products marketed for human consumption, but showed no symptoms of dehydration, chlorosis, or necrosis. This result represents an important alert for lettuce consumers and growers.     

大白菜的马铃薯蛋白酶抑制剂基因转化及抗菜青虫性的鉴定

 以大白菜(B．campestris ssp．pekinensis)‘北京80号’生长3 d无菌苗的带柄子叶为外植体，通过根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘法导人马铃薯蛋白酶抑制剂基因(pinⅡ)。通过抗性株的真叶在不定芽诱导培养基上的再生， 证实了嵌合体的存在，并通过此方法来消除嵌合体。获得的转基因植株经PCR、PCR-Southern杂交以及植物基因组Southern杂交证实，目的基因已经整合入植物基因组中。对菜青虫(Pieris rapae L．) 进行了转基因大白菜叶片的连续离体饲喂，结果表明：受试的转基因大白菜对菜青虫的生长发育有较明显的抑制作用。     

优质高抗核桃新品种‘洛核1号’的选育

‘洛核1号’是从‘彼特罗’ב中林5号’杂交子代中选育的高抗晚熟核桃新品种。坚果长椭圆形,大,平均单果质量16.46 g,单个核仁质量8.38 g,壳厚1.30 mm,出仁率达50.81%。壳较光滑,纵径47.33 mm,横径35.50 mm,侧径41.00 mm。缝合线略凸起,结合紧密。核仁饱满,黄色,口感浓香,无涩味,腹缝线结合紧密。果实生育期126 d,在洛阳地区9月15号左右成熟,侧芽结果率80%。‘洛核1号’对核桃炭疽病和核桃细菌性黑斑病表现为抗病,耐干旱、耐瘠薄能力较强。在河南省广大地区可进行生产性栽植,栽植当年可见雌花,第二年见果,高接大树第4年666.7 m^2平均产量203.72 kg。     

优质高产抗病黄秋葵新品种‘石秋葵2号’的选育

‘石秋葵2号’是采用系统选育方法从‘12-5’与‘13-2’的杂交后经多代自交培育而成的黄秋葵新品种。石家庄地区4月下旬采用地膜覆盖露地种植,该品种生长整齐一致,植株节间短,平均株高1.67 m,茎粗4.0 cm,节间长3.0 cm,始花节位5~6节。叶片掌状5裂,绿色,叶面有硬毛,叶柄长36 cm,叶长22 cm,叶宽29 cm。花后4~6 d采摘最佳,嫩果五棱,果翠绿色且富有光泽,萼片不易脱落,果长10~12 cm,果肩1.5~1.7 cm,平均单果质量15.6 g。种子球形,灰绿色至褐色,表面被细毛,千粒重约68 g。生长势旺盛,抗病性强,丰产性好,生育期190 d,分批采摘,平均667 m^2产量2487.0 kg。适宜石家庄地区及生态条件相似的地区种植,2020年9月通过了河北省科技成果转化服务中心成果评价。     

Isolation and expression analysis of a cobalamin-independent methionine synthase gene from the parasitic plant Orobanche ramosa

Broomrapes (Orobanche spp) are holoparasitic weeds that cause devastating losses in many economically important crops. The branched broomrape (Orobanche ramosa) represents a real threat for many vegetable crops including tobacco, tomato and potato.     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.