Advances in Cold Tolerance Genes and Their Application in Genetic Engineering of Plant for Cold Tolerance

Low temperature is one of the main environmental stress factors influenc- ing plant growth and development and crop yield. Cold tolerance genes and progress of their application in genetic engineering of plant for cold tolerance were reviewed comprehensively and systematically from the aspect of genes that are in- volved in biosynthesis of osmotic substances, genes coding fatty acid desaturation enzymes, antifreeze protein genes, genes coding antioxidant enzymes and so on, aiming at laying the foundation for genetic improvement of cold tolerance and breeding of plants.

Chloroplast Genetic Engineering in Higher Plants

Chloroplast genetic engineering, with several advantages over nuclear genetic engineering, is now regarded as an attractive new technology in basic and applied research, including deepening our understanding of plastid genome, engineering plant metabolic system, generating transplastomic plants with higher resistance to insect, disease, drought and herbicide and bioproducing of antibodies and vaccines. In this review, the principle and operating system for chloroplast genetic engineering and its application in higher plants have been discussed.

Crop Resources Ethic in Plant Genetic Engineering and Fortune Transfer Between Generations

The relation between human and crop resources belongs to the ethic of resources exploitation. The purposes of discussing the ethic of crop resources are to protect the ecology and safety of crops, to gain sustainable development, furthermore, to choose and form the production structure that is favorable to saving crop resources and protecting the ecology of crops. Plant genetic engineering is the technology of molecule breeding of rearrangement of inheritance materials at the level of molecule directionally, of improving plant properties and of breeding high quality and yield varieties of crops. The prominent effects of the technology on the crop ecological system are human subjective factors increasing as well as violating the nature and intensifying the conflict between human being and nature. Therefore, in plant genetic engineering, crop resources exploitation should follow certain ethic principles. Under the theory of ethics of natural resources, by the means of biologinal-statistics, the author systematically analyzed the possible model of crop resources transfer between generations as well as the transfer mode of magnitude of real materials and magnitude of value.

Osmoregulation Mechanism of Drought Stress and Genetic Engineering Strategies for Improving Drought Resistance in Plants

Drought, one of the main adverse environmental factors, obviously affected plant growth and development. Many adaptive strategies have been developed in plants for coping with drought or water stress, among which osmoregulation is one of the important factors of plant drought tolerance. Many substances play important roles in plant osmoregulation for drought resistance, including proline, glycine betaine, Lea proteins and soluble sugars such as levan, trehalose, sucrose, etc. The osmoregulation mechanism and the genetic engineering of plant drought-tolerance are reviewed in this paper.

A Comparative Testing of <i>Cucumber mosaic virus</i>(CMV)-Based Constructs to Generate Virus Resistant Plants

Among the viruses Cucumber mosaic virus (CMV) has been rated worldwide as one of the five most important viruses infecting vegetable species. CMV is a tripartite virus with high sequence variability, classified into three subgroups with 80% to 97% identical nucleotides in their coat protein. Due to the absence of natural resistance CMV is the plant virus with longest history in genetic engineering using pathogen induced approaches. However, the transformation and regeneration for some very important crops like chili is difficult. Therefore it will be an advantage to screen in model plants for gene constructs which might be independent of the target of final transformation and other parameters having an influence on the efficiency of a biotechnological approach. In our study we compared the resistance for all combinations of five different antiviral constructs, two different transformation vectors and two model host plants. From these approaches we identified the most effective construct which might also be applicable to transform eventually chili plants.

Virus Movement Protein Gene Mediated Resistance Against Cucumber Mosaic Virus Infection

Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.

Identification of Bt-transgenic Rice Plants for Resistance to Stripe Stem Borer (Chilo suppressalis) and Genetic Analysis of the Transgenes

Resistance to herbicide Basta was used to identify Bt-transgenic rice plants and the progenies of crosses between the Bt-transgenic rice and the rice varieties cultivated in the Huang Huai area of China. The results demonstrated that the Basta-positive rice plants were highly resistant to stripe stem borer (Chilo sup-pressalis) both in the laboratory and field tests. Both cryIA (b) and bar genes were expressed and co-inherited in both self ing and crossing progenies. Mendelian segregation of the marker gene bar was observed in F2 and BC1 progenies. The results implicates that it is possible to transfer cryIA (b) gene into other cultivated varieties through crosses and back crosses.

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.

Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

VIRUS RESISTANCE OF TRANSGENIC TOBACCO PLANTS EXPRESSING TOBACCO MOSAIC VIRUS COAT PROTEIN GENE

An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected after inoculation and they went to flowering and seeding normally. The possible mechanism of this kind of virus resistance could be the inhibition of the uncoating of the invading virus particles at the early stage of infection in the presence of the viral coat protein in plant cells, thus blocking the virus replication cycle.

Nanoparticle-Mediated Genetic Engineering of Plants

Genetic engineering of plants is at the core of sustainability efforts,natural product synthesis,and agricultural crop improvement.The past several decades have brought remarkable progress in biotechnology with the improvement of genome editing and sequencing tools,which stand to advance plant synthetic biology and bioengineering.In agriculture,genetic engineering can be employed to create crops that have in creased yields and nu tritio nal value,are resista nt to herbicides,in sects,diseases,and abiotic stresses,in cludi ng drought and heat .In pharmaceuticals and therapeutics,genetically engineered plants can be used to synthesize valuable small-molecule drugs and recombinant proteins.

Plant genetic engineering and genetically modified crop breeding: history and current status

This review charts the major developments in the genetic manipulation of plant cells that have taken place since the first gene transfer experiments using Ti plasmids in 1983. Tremendous progress has been made in both our scientific understanding and technological capabilities since the first genetically modified(GM)crops were developed with single gene resistances to herbicides, insects, viruses, and the silencing of undesirable genes. Despite opposition in some parts of the world, the area planted with first generation GM crops has grown from 1.7 Mhm^2 in 1996 to 179.7 Mhm^2 in 2015.The toolkit available for genetic modification has expanded greatly since 1996 and recently Nobel Laureates have called on Greenpeace to end their blanket opposition,and plant scientists have urged that consideration be given to the benefits of GM crops based on actual evidence. It is now possible to use GM to breed new crop cultivars resistant to a much wider range of pests and diseases, and to produce crops better able to adapt to climate change.The advent of new CRISPR-based technologies makes it possible to contemplate a much wider range of improvements based on transfer of new metabolic pathways and traits to improve nutritional quality, with a much greater degree of precision. Use of GM, sometimes in conjunction with other approaches, offers great opportunities for improving food quality, safety, and security in a changing world.

Genetic Manipulation of Non-Classic Oilseed Plants for Enhancement of Their Potential as a Biofactory for Triacylglycerol Production

Global demand for vegetable oil is anticipated to double by 2030. The current vegetable oil production platforms, including oil palm and temperate oilseeds, are unlikely to produce such an expansion. Therefore, the exploration of novel vegetable oil sources has become increasingly important in order to make up this future vegetable oil shortfall. Triacylglycerol （TAG）, as the dominant form of vegetable oil, has recently attracted immense interest in terms of being produced in plant vegetative tissues via genetic engineering technologies. Multidiscipline-based ＂-omics＂ studies are increasingly enhancing our understanding of plant lipid biochemistry and metabolism. As a result, the identification of biochemical pathways and the annotation of key genes contributing to fatty acid biosynthesis and to lipid assembly and turnover have been effectively updated. In recent years, there has been a rapid development in the genetic enhancement of TAG accumulation in high-biomass plant vegetative tissues and oilseeds through the genetic manipulation of the key genes and regulators involved in TAG biosynthesis. In this review, current genetic engineering strategies ranging from single-gene manipulation to multigene stacking aimed at increasing plant biomass TAG accumulation are summarized. New directions and suggestions for plant oil production that may help to further alleviate the potential shortage of edible oil and biodiesel are discussed.

Science Letters:Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.

TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modern high expression transgenic Bt plants possible, with an emphasis on the reasons for the low level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.

Defensive Role of Plant Hormones in Advancing Abiotic Stress-Resistant Rice Plants

Consistent climatic perturbations have increased global environmental concerns, especially the impacts of abiotic stresses on crop productivity. Rice is a staple food crop for the majority of the world’s population. Abiotic stresses, including salt, drought, heat, cold and heavy metals, are potential inhibitors of rice growth and yield. Abiotic stresses elicit various acclimation responses that facilitate in stress mitigation. Plant hormones play an important role in mediating the growth and development of rice plants under optimal and stressful environments by activating a multitude of signalling cascades to elicit the rice plant’s adaptive responses. The current review describes the role of plant hormone-mediated abiotic stress tolerance in rice, potential crosstalk between plant hormones involved in rice abiotic stress tolerance and significant advancements in biotechnological initiatives including genetic engineering approach to provide a step forward in making rice resistance to abiotic stress.

Hairy Root and Its Application in Plant Genetic Engineering

Agrobacterium rhizogenes Conn. causes hairy root disease In plants. Hairy root-Infected A. rhizogenes Is characterlzed by a high growth rate and genetic stability. Hairy root cultures have been proven to be an efficient means of producing secondary metabolites that are normally biosyntheslzed In roots of differentiated plants. Furthermore, a transgenlc root system offers tremendous potential for introducing additional genes along with the RI plasmld, especially with modified genes, into medicinal plant cells with A. rhizogenes vector systems. The cultures have turned out to be a valuable tool with which to study the biochemical properties and the gene expression profile of metabolic pathways. Moreover, the cultures can be used to elucidate the Intermediates and key enzymes Involved In the biosynthesis of secondary metabolites. The present article discusses various appllcations of hairy root cultures in plant genetic engineering and potential problems aseoclsted with them.

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Abstract: Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.

Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality

Nutrient deficiencies in crops are a serious threat to human health,especially for populations in poor areas.To overcome this problem,the development of crops with nutrient-enhanced traits is imperative.Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components.Compared with agronomic practices and conventional plant breeding,plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients,phytonutrients,and/or bioactive components in crops.In this review,we discuss recent progress in the field of plant synthetic metabolic engineering,specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways,with a focus on improving traits related to micronutrients,phytonutrients,and bioactive components.Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.