Field evaluation and agronomic performance of transgenic wheat

Seven transgenic lines of wheat have been evaluated under field conditions during 2 agonomic years. Four lines contained the transgenes for beta-glucuronidase ( uidA), herbicide resistance ( bar) and for one high-molecular-weight (HMW) subunit, and three lines contained only one transgene for one HMW glutenin subunit and no marker genes. Agronomic traits and yield components were studied in transgenic lines and compared with the non-transgenic parent and null segregant lines. Although phenotypic differences for many traits have been found, only heading date and the number of spikelets per spike showed clear genotypic differences for both field trials. All transgenic lines had a longer heading date than parent lines whereas the number of spikelets per spike in transgenic lines was around that for L88-31 and higher for L88-6 than the corresponding parent lines. No differences were found between lines constitutively expressing the uidA and bar genes from those which only expressed the HMW genes. We conclude that differences between transgenic lines and their parents are small, and could be eliminated by backcrossing transgenic lines with their parents and selecting for the wanted genotype.     

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.     

Assessment of expression and inheritance patterns of three transgenes with the aid of techniques for promoting rapid flowering of transgenic apple trees

Rapid flowering of transgenic Royal Gala apple (Malus×domestica) trees was achieved by growing trees under controlled greenhouse conditions. The expression and inheritance of three transgenes were confirmed in the seedling progeny. Grown as single stems on their own roots, the transgenic apple trees produced 80–110 nodes and were 2 m high on average at the end of the 1st year's growth. In the 2nd year, approximately 20% of these trees flowered around node 80. However, when scions collected from the top of 1-year-old trees were grafted onto the dwarfing rootstock Malling 9, 85% produced flowers and fruit within the next year. The grafted trees continued to produce fruit in the following years. Expression of the transgene uidA was monitored by assaying β-glucuronidase (GUS) activity in leaves, flowers and fruit. Inheritance of three transgenes, uidA, neomycin phototransferase II and acetolactate synthase, were demonstrated through the recovery of GUS-positive, kanamycin-resistant and chlorsulfuron-resistant progeny. Segregation patterns fitted a 1:1 ratio in most lines. However, a detailed analysis in one progeny line revealed a complex T-DNA integration pattern. Received: 8 June 1998 / Revision received: 19 November 1998 / Accepted: 26 November 1998     

Transformation of chicory and expression of the bacterial uidA and nptll genes in the transgenic regenerants

Transgenic chicory plants were obtained from different explantsco-cultured with Agrobacterium tumefaciens. Among tap-root,leaf and cotyledonary tissues, etiolated cotyledons showed thegreatest competence for transformation. The Agrobacterium strainsused contained either pGSGLUC1 or pTDE4 as a vector which carryboth the neomycin phosphotransferase II gene (nptll) for kanamycinresistance and ß-glucuronidase gene (uidA) under thecontrol of different promoters. Transformation was confirmedby NPTII enzymatic assay, histochemical analysis of GUS activityand DNA hybridization. Transgenic plants expressed both markergenes in root and shoot tissues. In leaves, GUS activity wasexpressed in all tissue types, whatever the nature of the promoter.Nevertheless, variable heterogeneous patterns of expressionwere observed in the different root tissues. Differential expression of the GUS fusions controlled by thedual TR or the CaMV 35S promoters are discussed. Key words: Chicory, genetic transformation, GUS activity, kanamycin resistance     

Field decomposition of transgenic Bt maize residue and the impact on non-target soil invertebrates

The release of chemical compounds from plant roots that suppress soil nitrification is termed biological nitrification inhibition (BNI). Determining the environmental factors that control the synthesis and release of BNI-compounds from Brachiaria humidicola (Rendle) Schweick, a tropical pasture grass that thrives on acid soils, is the focus of this investigation. Because the BNI trait is related to the N status of the plant, we investigated the possibility that the expression of this trait would be related to the forms of N found in the root environment. Plants were grown with two sources of N, NH₄⁺ or NO₃⁻ for 60 days and the release of BNI-compounds monitored. Only plants grown with NH₄⁺ released BNI-compounds from roots. The presence of NH₄⁺ and possibly the secondary effect of its uptake (i.e., acidic pH) in the root environment significantly enhanced the release of BNI-compounds. Both the NH₄⁺ and NO₃⁻ grown plants responded to the stimulus from NH₄⁺ in the root environment. BNI-compounds found in root tissue and their release were nearly three times greater in NH₄⁺ grown than from NO₃⁻ grown plants. The BNI-compounds released from roots composed of at least three active components—Type-I (stable to pH changes from 3.0 to 10), Type-II (temporarily loses its inhibitory effect at a pH higher than a threshold pH of 4.5 and the inhibitory effect is reestablished when the root exudate pH is adjusted to <4.5) and Type-III (inhibitory effect is irreversibly lost if the pH of the root exudate reaches 10.0 or above). A major portion of BNI-compounds released in the presence of NH₄⁺ is of Type-I. In the absence of NH₄⁺, mostly Type-II and Type-III BNI-compounds were released. The BNI-compounds inhibited the function of Nitrosomonas europaea through the blocking of both ammonia monooxygenase and hydroxylamino oxidoreductase pathways. These results indicate that the release of BNI-compounds from B. humidicola roots is a regulated function and that presence of NH₄⁺ in the root environment is necessary for the sustained synthesis and release of BNI.     

Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.)

Variation in the inheritance of expression among subclones for an unselected (uidA) and a selected (bar) transgene was analyzed in two individual transformation events in maize. The unselectable gene (uidA) and the selectable gene (bar), on two separate plasmids, were transferred to maize (Hi-II derivative) by particle bombardment of embryogenic calli or suspension cells. A total of 188 fertile T1 plants were obtained from one transformant (transformation event BG which integrated uidA and bar). A total of 98 fertile T1 plants were obtained from a second transformant (transformation event B which integrated bar). Through self-pollination and/or cross-pollination in the greenhouse, approximately 10 000 T2 progeny were obtained from event BG, and more than 1000 T2 progeny were obtained from event B. Segregation of transgene expression was analyzed statistically in a total of 2350 T2 progeny from 40 T1 subclones of event BG and in 217 T2 progeny from six T1 subclones from event B. Variation in the inheritance of expression among subclones for the two transgenes (uidA and bar) was observed in the two transformants. A significant difference was observed between the use of the female or male as the transgenic parent in the inheritance of expression for the two transgenes in event BG. No inheritance through the pollen was observed in two of four T1 subclones analyzed in event B. Co-expression analysis of event BG showed that both transgenes were co-expressed in 67.7% of the T2 plants which expressed at least one of the two transgenes. Of the T2 expressing plants, 30.4% expressed only bar, and 1.9% expressed only uidA. Inactivation of the unselected (uidA) and the selected (bar) transgenes was observed in individual T2 plants.     

A broad exploration of a transgenic population of citrus: stability of gene expression and phenotype

A collection of 70 transgenic citrus plants for the uidA and nptII genes have been maintained under screenhouse conditions over a period of 4–5 years. A detailed scanning of the plants allowed us to detect four phenotypic off-type plants and a large variation of transgene integration and expression patterns among the population. Off-type plants were analysed and characterised as nucellar tetraploids, probably originating from tetraploid starting tissues rather than from somaclonal variation events. Transgene integration and expression analyses revealed that: (1) a significant negative correlation was found between copy number and GUS activity; (2) rearrangements of the T-DNA inserts did not imply low expression levels; and (3) stability of integration and expression of the transgenes was confirmed for all the transformants grown under natural environmental conditions. These combined features validate transformation as a tool for the genetic improvement of citrus. Received: 11 January 1999 / Accepted: 19 January 1999     

Browse characteristics and impact of browsing on trees and shrubs in African savannas

Abstract. Studies on browse and browsing impact on woody plants in African savannas show that browse can be a significant part of the total food for large herbivores. Utilization of browse is little studied and patterns are therefore difficult to examine. The quality of browse has been described mainly in terms of nutrients, fibre and digestibility. Studies on chemical and physical defences of plants against herbivory can add considerably to the understanding of the browse-browser system. The various ways woody plants respond to browsing are little known. Interactions between browsers and woody plants are dynamic, since responses of woody plants to browsing result in changes in feeding behaviour of the browsing animal, both in savannas and in temperate ecosystems.     

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

ABSTRACT: BACKGROUND: The Biocontrol Peptide BP100 is a synthetic and strongly cationic a-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. RESULTS: Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. CONCLUSIONS: Constitutive expression of transgenes encoding short cationic a-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.     

Response of transgenic tobacco overexpressing the CchGLP gene to cadmium and aluminium: phenotypic and microRNAs expression changes

Physiology and Molecular Biology of Plants - Transgenic tobacco (N. tabacum cv. Xanthi nc) expressing Capsicum chinense&nbsp;CchGLP gene that encodes an Mn-SOD, constitutively produces hydrogen...     

The timescale of phenotypic plasticity and its impact on competition in fluctuating environments

Although phenotypic plasticity can be advantageous in fluctuating environments, it may come too late if the environment changes fast. Complementary chromatic adaptation is a colorful form of phenotypic plasticity, where cyanobacteria tune their pigmentation to the prevailing light spectrum. Here, we study the timescale of chromatic adaptation and its impact on competition among phytoplankton species exposed to fluctuating light colors. We parameterized a resource competition model using monoculture experiments with green and red picocyanobacteria and the cyanobacterium Pseudanabaena, which can change its color within approximately 7 days by chromatic adaptation. The model predictions were tested in competition experiments, where the incident light color switched between red and green at different frequencies (slow, intermediate, and fast). Pseudanabaena (the flexible phenotype) competitively excluded the green and red picocyanobacteria in all competition experiments. Strikingly, the rate of competitive exclusion was much faster when the flexible phenotype had sufficient time to fully adjust its pigmentation. Thus, the flexible phenotype benefited from its phenotypic plasticity if fluctuations in light color were relatively slow, corresponding to slow mixing processes or infrequent storms in their natural habitat. This shows that the timescale of phenotypic plasticity plays a key role during species interactions in fluctuating environments.     

High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile

Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.     

木本植物非结构性碳水化合物变化及其影响因子研究进展

非结构性碳水化合物(NSC)是植物新陈代谢过程中重要的供能物质,NSC库的动态变化不仅反映了植物体内的碳收支状况,还影响着植物的生理代谢活动.为了预测树木甚至整个森林生态系统对未来气候变化的响应和适应能力,本文综述了树木NSC研究的最新进展,重点介绍了NSC库的季节和区域性变化,分析总结了影响树木NSC含量和分配格局变化的主要环境因素.最后还对未来气候变化背景下树木NSC库可能的响应策略和适应状况进行了讨论,展望了未来树木NSC领域的研究方向.     

Alloparents in the Mongolian gerbil: impact on long-term reproductive performance of breeders and opportunities for independent reproduction

The presence of alloparents in cooperatively breeding groupsmay enhance the reproductive performance of the breeding pairbut tests of this hypothesis in mammalian cooperative breedershave typically examined short periods in the reproductive lifespan of breeders. The present experiment was designed to evaluatethe long-term effects of the presence of alloparents on reproductiveperformance under laboratory conditions in Mongolian gerbils(Mertones unguiculalus), and to evaluate the possibility thatalloparents may attempt independent reproduction during theirtenure as alloparents. Pairs were established with either 0or 4 alloparents and breeding performance was monitored overa 13.5-month period. The presence of alloparents did not enhancereproductive rates in breeding pairs, and neither enhanced nordelayed developmental rates in offspring. Variation in littersize, offspring survival, pup growth, and interbirth intervaldid not vary significantly as a function of the breeding females'reproductive tenure. Seven of 49 female alloparents of breedingage in family groups produced offspring. Alloparent breedingwas associated with long interbirth intervals in the breedingadult female. Neonatal survival in litters born to alloparentswas higher in litters that were temporally synchronized withlitters produced by the older breeding female than in asynchronouslyproduced litters. Under the conditions in this experiment, then,few indirect benefits accrue to helpers as a consequence oftheir alloparental effort, and immediate (the opportunity forindependent reproductive attempts) and delayed (parental skillsacquisition) direct benefits may account for helping behaviorin this species.     

Effect of selectable gene to reporter gene ratio on the frequency of co-transformation and co-expression of uidA and hpt transgenes in protoplast-derived plants of tall fescue

Forty-six independent transformed plants were regenerated under hygromycin selection from cell-suspension derived protoplasts of Festuca arundinacea (Schreb.) after PEG-mediated transformation. Protoplasts were co-transformed with varying molar gene ratios (0.7:1–6:1) of a marker -glucuronidase (uidA) gene and a selective hygromycin (hpt) resistance gene. Logistic regression analysis indicated that, as expected, the proportion of co-transformed plants tended to increase as the proportion of the marker gene was increased. However, although the proportion of plants co-expressing both genes tended to increase up to a molar ratio of 4:1, it appeared to fall at a molar ratio of 6:1. No statistically significant differences were found in the average copy number of the integrated uidA or hpt transgenes, either in GUS expressing, or in non-GUS expressing plants at the different molar ratios. When using naked-DNA gene transformation methods most authors use a molar ratio of 1:1; our data suggest that adding non-selected and selected transgenes at a higher Molar Gene Ratio would probably improve the proportion of plants regenerated which express both transgenes.     

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

The biopharmaceutical industry has invested considerably in the implementation of single‐use disposable bioreactors in place of or in addition to their stainless steel‐counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene‐film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035–0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label‐free LC‐MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547–1558, 2016     

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns,expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) -endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R₀ and R₁ plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.