An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars

Background

Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation.

Results

Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected.

Conclusions

The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.     

Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.     

根癌农杆菌介导寒富苹果转化体系的建立

以寒富苹果组培苗叶片为外植体,利用植物表达载体上含潮霉素抗性基因的根癌农杆菌EHA105(pCAMBI-A1301)和含卡那霉素抗性基因的根癌农杆菌EHA105(pCAMBIA2301)对影响寒富遗传转化效率的因素进行系统研究。结果表明,寒富叶片对潮霉素反应敏感,在附加潮霉素的培养基上寒富叶片褐化较为严重。潮霉素和卡那霉素适宜的筛选质量浓度分别为4 mg.L-1和25 mg.L-1。农杆菌介导寒富苹果转化体系的建立以MS+TDZ 2.0 mg.L-1+NAA 0.5 mg.L-1培养基为叶片离体再生芽培养基。适宜的转化条件为:菌液浓度D 600nm=0.5、叶片外植体在菌液浸泡8 min、共培养时间为3～4 d、推迟4 d进行筛选培养。抗性基因对转化效率具有明显的影响,EHA105(pCAMBIA2301)的平均抗性芽再生率(0.96%)比EHA105(pCAMBIA1301)的平均抗性芽再生率(0.58%)高出几乎50%,EHA105(pCAMBIA2301)的抗性芽再生率最高达1.87%。GUS组织化学染色和PCR鉴定结果表明本研究获得了寒富苹果转基因植株。     

梨果实中石细胞含量测定及与果实品质相关性的研究

对梨果实中石细胞含量的测定方法进行了改进，克服了他法中石细胞壁上附带的果肉和测定中步骤复杂的弊病；同时对６个梨品种系统地进行了品质分析。结果表明：梨果肉中石细胞含量的多少不是影响口感的主要因素；果肉中石细胞的含量与其ｐＨ值大小成显著正相关。     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Background  

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.     

An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

Background

Mitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substantial chloroplast DNA (cpDNA) and nuclear DNA (nDNA) and its end use efficiency can be reduced. Clearly, an effective mitochondria isolation method is warranted with wider applicability and with minimum contamination from cpDNA and nDNA.

Results

Here we reported an improved method for isolating mitochondria from dry wheat seeds and its extension to dead seeds, viable seeds, etiolated leaf tissue and several other plant species: oat, Arabidopsis, flax, and yellow mustard. The isolated mitochondria were successfully used to extract mtDNA with QIAamp DNA mini kit (Qiagen). The extracted mtDNA from the assayed samples of these species was intact in large quantity and showed little contamination from nDNA, cpDNA, RNA, and proteins. The mtDNA extracted from dead wheat seeds was also substantial, but more degraded and less intact when compared to those from viable seeds and other tissues.

Conclusion

The improved method was successfully applied to isolate mitochondria and extract mtDNA from several different tissues and plant species. The major advance in the improvement lies in its wider application with the same mitochondria extraction medium to different tissues and species. The improvement is significant, as it helps to widen the scope of future plant mitochondria research.

     

An improved allele-specific PCR primer design method for SNP marker analysis and its application

ABSTRACT: BACKGROUND: Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3'end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge. RESULTS: In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3rd nucleotide from the 3' end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species. CONCLUSION: The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.     

An improved method to develop cell culture model of intermittent hypoxia

AIM: To establish and validate a novel model of cultured cells for imitating intermittent hypoxia. METHODS: In a chamber with experiment cabin and simulated air control cabin, the frequency and duration of the intermittent hypoxia model according to the time of hypoxia and reoxygenation were evaluated. The A549 cells were randomly divided into 7 groups, named as control (Con) group, 6 h intermittent hypoxia (6IH) group, 9 h intermittent hypoxia (9IH) group, 6 h simulated air control (6AC) group, 9 h simulated air control (9AC) group, 4 h sustained hypoxia (4SH) group, 6 h sustained hypoxia (6SH) group, respectively. When the model was established, the cellular morphology was observed under inverted microscope. The mRNA expression of hypoxia-inducible factor (HIF)-1α was detected by real-time PCR. The protein expression of HIF-1α was analyzed by immunohistochemistry. RESULTS: The intermittent hypoxia cycle (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) was established. The damaged A549 cells were observed in 6IH group, 9IH group and 6SH group. Compared with 6IH group, the expression of HIF-1α at mRNA and protein levels was significantly increased in 9IH group (P<0.05). The expression of HIF-1α at mRNA and protein levels in 6IH group and 9IH group was higher than that in 4SH group and 6SH group, respectively (P<0.05). No significant difference among the control group, 6AC group and 9AC group was found. CONCLUSION: The model (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) can simulate the pathological process of obstructive sleep apnea hypopnea syndrome. This model is suitable for studying intermittent hypoxia in adherent cells.     

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background  

Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.     

Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana

Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination.     

Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)

Background  

We previously developed a high-throughput system called 'Ice-Cap' for growing Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method.     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

Development of a transformation protocol for Tecomella undulata (Smith) Seem from cotyledonary node explants

A protocol was optimized for genetic transformation of Rohida (Tecomella undulata) from cotyledonary node tissues using Agrobacterium tumefaciens strain GV2260 harboring binary vector pBinAR containing osmotin and nptII gene under control of CaMV35S promoter. This is the first report on the transformation of T. undulata. The effective concentration of selectable marker antibiotic used for screening of transformants was 85.97 μM in shooting media and 42.98 μM in rooting media. PCR and Southern blot confirmed integration of osmotin gene into genome of Rohida.     

农杆菌介导霞多丽葡萄胚性细胞系遗传转化条件的优化

为建立葡萄(Vitis vinifera)遗传转化技术体系,以霞多丽葡萄(Chardonnay)胚性细胞系为靶组织,采用GUS检测法,对影响农杆菌介导葡萄遗传转化效率的主要因素进行了研究。结果表明,超声波处理时间长短对转化效率有较大影响,在所试的0.5、1、5、10 min 4种不同时间的超声波处理中,以5 min时转化效率较好,平均达到9.11个蓝色斑点;AS浓度对转化效率有明显差异,当浓度为50μmol/L时,平均蓝色斑点数为8.89个,100μmol/L时,达到12.44个;DTT质量浓度对转化效率也有较大影响,在所试的3种质量浓度(1,2,3 mg/L)中,以3 mg/L为最佳,有16.67个蓝色斑点。通过GUS瞬时表达检测,确立了农杆菌介导葡萄胚性细胞系遗传转化的几个最适影响因素,从而为葡萄遗传转化技术体系的建立奠定了基础。     

A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

Characterizing plant genetic resources and their response to the environment through accurate measurement of relevant traits is crucial to genetics and breeding. Spatial organization of the maize ear provides insights into the response of grain yield to environmental conditions. Current automated methods for phenotyping the maize ear do not capture these spatial features. We developed EARBOX, a low-cost, open-source system for automated phenotyping of maize ears. EARBOX integrates open-source technologies for both software and hardware that facilitate its deployment and improvement for specific research questions. The imaging platform consists of a customized box in which ears are repeatedly imaged as they rotate via motorized rollers. With deep learning based on convolutional neural networks, the image analysis algorithm uses a two-step procedure: ear-specific grain masks are first created and subsequently used to extract a range of trait data per ear, including ear shape and dimensions, the number of grains and their spatial organisation, and the distribution of grain dimensions along the ear. The reliability of each trait was validated against ground-truth data from manual measurements. Moreover, EARBOX derives novel traits, inaccessible through conventional methods, especially the distribution of grain dimensions along grain cohorts, relevant for ear morphogenesis, and the distribution of abortion frequency along the ear, relevant for plant response to stress, especially soil water deficit. The proposed system provides robust and accurate measurements of maize ear traits including spatial features. Future developments include grain type and colour categorisation. This method opens avenues for high-throughput genetic or functional studies in the context of plant adaptation to a changing environment.     

An improved surface disinfection method for shoot expiants from Iris rhizomes infected with bacterial soft rot (Erwinia carotovora subsp,carotovora)

Summary

The standard method (protocol 1) for disinfecting shoot expiants used for initiation of plant tissue cultures failed when Iris rhizomes were infected with bacterial soft rot (Erwinia carotovora subsp, carotovora), Two new disinfection protocols were therefore developed, They involved keeping stock plants without irrigation for two months prior to excision of expiants; alcohol disinfection of the expiants prior to excision from the rhizome; dipping of the expiant in alcohol immediately after excision; soaking in 1 % (v/v) sodium hypochlorite (protocol 2) or saturated calcium hypochlorite (protocol 3); and soaking in mercuric chloride, With protocols 2 and 3 up to 90% and 62% respectively of expiants taken from infected Iris rhizomes showing soft-rot symptoms remained free of detectable microorganisms in vitro.     

枇杷属植物基因组DNA提取方法的改进及其应用

以枇杷属的普通枇杷和多种野生枇杷的叶片为材料,对枇杷的DNA提取进行了研究。针对枇杷富含多酚类 等次生物质的特点对枇杷DNA的提取步骤进行了改良处理:采用CTAB-蛋白酶K法加重复抽提,去除枇杷材料中 的相关次生物质,获得了高质量的DNA。所提取的DNA完全能满足PCR、RAPD、AFLP等一些分子生物学实验要 求。