An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba?��?P. berolinensis and Populus davidiana?��?P. bolleana

Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT–PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence

Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of l-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of l-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.     

Transposable elements as plant transformation vectors for long stretches of foreign DNA

The production of transgenic plants is now routine for most crops. However, using currently available transformation methods it is still difficult and time-consuming to obtain a collection of transformed individuals containing single or low-copy-number, intact transgenic inserts. Here we describe a set of broad-hostrange transformation vectors based on the Ac/Ds transposition system that improve both transformation efficiency and the quality of transgenic loci. These vectors efficiently deliver long stretches of foreign DNA into the genome, leading to transgenic strains containing an intact single-copy insert of 10kb. This type of vector could be an important additional tool for the production of transgenic plants with the well-defined, foreign DNA inserts required for biosafety approval and commercialisation.     

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the β-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4–10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1–2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra × P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.     

A tissue culture system for different germplasms of indica rice

Agrobacterium-mediated transformation of indica rice has been manipulated in only a limited number of cultivars because the majority of indica varieties are recalcitrant to in vitro response. Establishment of a highly efficient and widely used tissue culture system for indica rice will accelerate the application of transformation technology in breeding programs and the study of the functions of indica-specific genes. By manipulating plant growth regulators, organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds representing different indica rice germplasms. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. The results suggest that this optimized tissue culture system will be widely applicable for the tissue culture of indica varieties. Electronic Supplementary Material Supplementary material is available for this article at The first two authors contributed equally to this work.     

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.     

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

Summary When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.     

Symbiophagy and biomineralization in the “living fossil” Astrosclera willeyana

Representatives of all major metazoan lineages form biominerals. The molecular mechanisms that underlie this widespread and evolutionarily ancient ability are gradually being revealed for some lineages. However, until a wider range of metazoan biomineralization strategies are understood, the true diversity, and therefore the evolutionary origins of this process, will remain unknown. We have previously shown that the coralline demosponge, Astrosclera willeyana, in some way employs its endobiotic bacterial community to form its highly calcified skeleton. Here, using in situ hybridization and immunohistochemistry, we show that an ortholog of ATG8 (most likely a GABARAPL2/GATE-16 ortholog) is expressed in cells that construct the individual skeletal elements of the sponge. In TEM sections sponge cells can be observed to contain extensive populations of bacteria, and frequently possesses double-membrane structures which we interpret to be autophagosomes. In combination with our previous work, these findings support the hypothesis that the host sponge actively degrades a proportion of its bacterial community using an autophagy pathway, and uses the prokaryotic organic remains as a framework upon which calcification of the sponge skeleton is initiated.     

Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl₃ and LaCl₃ leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl₃ and LaCl₃ had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl₃ showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl₃ can be effectively used to improve quantity and quality of transgene integrations.     

Transcriptome Profiling of Barley Cultivar Hua 30 MDEC in Response to <i>Agrobacterium</i> Infection

Agrobacterium-mediated transformation has been widely used in plants. However, the mechanism in plant cells’ response to Agrobacterium infection was very complex. The mechanism of the determinants in host cell remains obscure, especially in barley, which is recalcitrant for Agrobacterium-mediated transformation. In the present study, microspore-derived embryogenic calli (MDEC) from barley elite cultivar were employed as unique subjects to characterize the mechanisms during the Agrobacterium infection process. Hua 30 MDEC can be successfully infected by Agrobacterium. RNA-sequencing at different infection points (0, 2, 6, 12, 24 hpi) was performed. The average expressional intensity of the whole genomics increased from 0 to 2 hpi, and then decreased subsequently. More upregulated than downregulated differentially expressed genes (DEGs) were counted at the same time. GO enrichment analysis showed that protein modification was significantly overrepresented in upregulated DEGs. Chromosome-related biological processes, gene expression and cellular metabolic processes were significantly overrepresented in downregulated DEGs. KEGG analysis showed that plant defense responses, phenylpropanoid biosynthesis and biosynthesis of amino acids were significantly enriched across the infection time course. Nine DEGs related to defense responses were identified. All DEGs were upregulated from 2 to 24 hpi. We speculate that these genes are possibly related to Agrobacterium infection. These findings will provide deep insights into the molecular events occurring during the process of Agrobacterium-mediated transformation.     

Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation.     

Biotechnology Applications for Sugar Beet

Sugar beet (Beta vulgaris L.) is an important industrial crop, being one of only two plant sources from which sucrose (i.e., sugar) can be economically produced. Despite its relatively short period of cultivation (ca. 200 years), its yield and quality parameters have been significantly improved by conventional breeding methods. However, during the last two decades or so, advanced in vitro culture and genetic transformation technologies have been incorporated with classical breeding programs, the main aim being the production of herbicide-and salt-tolerant, disease- and pest-resistant cultivars. Among the many applications of in vitro culture techniques, sugar beet has benefited the most from haploid plant production, protoplast culture, and somaclonal variation and in vitro cell selection. Several genetic transformation technologies have been developed, such as Agrobacterium-meditated, PEG-mediated, particle bombardment, electroporation, sonication and somatic hybridization, the first two being the most successful. Development of herbicide- and salt-tolerant, virus-, pest/nematode-, fungus/Cercospora- and insect-resistant sugar beet has been demonstrated. However, only herbicide-tolerant varieties have been approved for commercialization but not yet available in the marketplace; rhizomania-resistant varieties are being evaluated in field trials. Transgenic plants that convert sucrose into fructan, a polymer of fructose, were also developed. Initial attempts to increase sucrose yields produced promising results, but it still requires additional work. Despite marked progress in improving regeneration and transformation of sugar beet, genotype dependence and low regeneration and transformation frequencies are still serious restrictions for routine application of in vitro culture and, more importantly, transformation technologies. Selected food safety and environmental impact, as well as regulatory and public acceptance issues relating to transgenic sugar beet are also discussed.     

New capabilities for Mycosphaerella graminicola research

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens‐mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS‐51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild‐type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the β‐tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.     

Suppression of OsMADS7 in rice endosperm stabilizes amylose content under high temperature stress

High temperature significantly alters the amylose content of rice, resulting in mature grains with poor eating quality. However, only few genes and/or quantitative trait loci involved in this process have been isolated and the molecular mechanisms of this effect remain unclear. Here, we describe a floral organ identity gene, OsMADS7, involved in stabilizing rice amylose content at high temperature. OsMADS7 is greatly induced by high temperature at the early filling stage. Constitutive suppression of OsMADS7 stabilizes amylose content under high temperature stress but results in low spikelet fertility. However, rice plants with both stable amylose content at high temperature and normal spikelet fertility can be obtained by specifically suppressing OsMADS7 in endosperm. GBSSI is the major enzyme responsible for amylose biosynthesis. A low filling rate and high expression of GBSSI were detected in OsMADS7 RNAi plants at high temperature, which may be correlated with stabilized amylose content in these transgenic seeds under high temperature. Thus, specific suppression of OsMADS7 in endosperm could improve the stability of rice amylose content at high temperature, and such transgenic materials may be a valuable genetic resource for breeding rice with elite thermal resilience.     

Transformation of <Emphasis Type="Italic">Spirulina platensis </Emphasis>Strain C1 (<Emphasis Type="Italic">Arthrospira</Emphasis> sp. PCC9438) with Tn5 Transposase–Transposon DNA–Cation Liposome Complex

Spirulina platensis is one of the most commercially important species of microalgae. Thus, it is an attractive candidate for genetic manipulation and the development of novel practical applications. However, this process is hampered by the absence of a stable gene transfer system, specifically the limited number of suitable vectors and transformation methods available for this organism. Artificial transposon systems developed by extracting the essential elements from natural transposons have been extensively studied, and recently a mutated transposase and transposon system was reported to improve transformation efficiency by electroporation. We applied a modified transformation strategy using a natural Tn5 transposon, transposase, and cation liposome complex by electroporation to improve the transformation efficiency for Spirulina platensis strain C1 (Arthrospira sp. PCC9438). Aggregation of cells became visible after 3 weeks during 2.0 g/ml chloramphenicol selection, and growth continued for more than 12 months. Transfected chloramphenicol acetyltransferase (CAT) genes were detected in the genomic DNA by Southern hybridization. Transformed cells demonstrated CAT activity, but non-transformed cells did not.     

On the Chemical Nature and Origin of Teleonomy

The physico-chemical characterization of a teleonomic event and the nature of the physico-chemical process by which teleonomic systems could emerge from non-teleonomic systems are addressed in this paper. It is proposed that teleonomic events are those whose primary directive is discerned to be non-thermodynamic, while regular (non-teleonomic) events are those whose primary directive is the traditional thermodynamic one. For the archetypal teleonomic event, cell multiplication, the non-thermodynamic directive can be identified as being a kinetic directive. It is concluded, therefore, that the process of emergence, whereby non-teleonomic replicating chemical systems were transformed into teleonomic ones, involved a switch in the primacy of thermodynamic and kinetic directives. It is proposed that the step where that transformation took place was the one in which some pre-metabolic replicating system acquired an energy-gathering capability, thereby becoming metabolic. Such a transformation was itself kinetically directed given that metabolic replicators tend to be kinetically more stable than non-metabolic ones. The analysis builds on our previous work that considers living systems to be a kinetic state of matteras opposed to the traditional thermodynamic states that dominate the inanimate world     

马尾松林向香樟林改造林下植物功能多样性研究

在生物多样性研究中,功能多样性比物种多样性可以更直接地反映生态系统的结构和功能,从而在群落生态学研究中受到越来越多的推崇。马尾松次生纯林是一种亟待改造的森林类型,而在亚热带地区香樟是马尾松林改造的理想树种之一。为了完善马尾松林向香樟林改造的研究,以更好地指导马尾松林改造的实践,本文用空间代替时间的方法,选取马尾松林向香樟林改造过程中4种不同阶段群落为研究对象,对林下灌木层和草本层植物的功能性状及其多样性展开了研究。结果表明:(1)随着森林改造的进行,林下植物的物种数目、功能性状多样性均表现为先上升后下降的变化趋势,其中功能丰富度和功能均匀度在改造前期达到峰值,而物种数目和功能离散度则在改造中期表现最高。(2)比较森林改造的4个时期,林下植物功能多样性的综合表现大致为:改造前期改造中期改造后期未改造时期。(3)森林改造过程中,林下植物"光响应性状"和"繁殖性状"的功能多样性变化趋势相似,但前者比后者对改造的响应更敏感。(4)在森林改造过程中,林下植物的生长、发育和扩散受到林下光照资源的影响,并在长时间的生物竞争中逐渐表现为物种种类、多度和空间分布格局的变化,最终影响生态系统功能。(5)马尾松林向香樟林的改造加速了群落的演替进程,提升了群落的功能,但仍存在林下植物功能多样性下降等问题。在对马尾松次生纯林改造过程中,应选择合适的造林密度和混交林的改造模式。     

High efficiency transformation by electroporation of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of F. lipolytica, we report here that pretreatment of F. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 10¹⁰/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform F. lipolytica that will be able to yield an efficiency of 2.1 × 10⁴ transformants/ug for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for F. lipolytica transformation.