Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA.

BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

Control of two insect pests by expression of a mismatch corrected double-stranded RNA in plants.

RNA interference (RNAi) has emerged as an efficient technology for pest control by silencing the essential genes of targeted insects. Owing to its nucleotide sequence-guided working mechanism, RNAi has a high degree of species-specificity without impacts on non-target organisms. However, as plants are inevitably under threat by two or more insect pests in nature, the species-specific mode of RNAi-based technology restricts its wide application for pest control. In this study, we artificially designed an intermediate dsRNA (iACT) targeting two ß-Actin (ACT) genes of sap-sucking pests Bemisia tabaci and Myzus persicae by mutual correction of their mismatches. When expressing hairpin iACT (hpiACT) from tobacco nuclear genome, transgenic plants are well protected from both B. tabaci and M. persicae, either individually or simultaneously, as evidenced by reduced fecundity and suppressed ACT gene expression, whereas expression of hpRNA targeting BtACT or MpACT in transgenic tobacco plants could only confer specific resistance to either B. tabaci or M. persicae, respectively. In sum, our data provide a novel proof-of-concept that two different insect species could be simultaneously controlled by artificial synthesis of dsRNA with sequence optimization, which expands the range of transgenic RNAi methods for crop protection.

Production Technologies for Recombinant Antibodies: Insights into Eukaryotic, Prokaryotic, and Transgenic Expression Systems.

Recombinant antibodies, a prominent class of recombinant proteins, are witnessing substantial growth in research and diagnostics. Recombinant antibodies are being produced employing diverse hosts ranging from highly complex eukaryotes, for instance, mammalian cell lines (and insects, fungi, yeast, etc.) to unicellular prokaryotic models like gram-positive and gram-negative bacteria. This review delves into these production methods, highlighting approaches like antibody phage display that employs bacteriophages for gene library creation. Recent studies emphasize monoclonal antibody generation through hybridoma technology, utilizing hybridoma cells from myeloma and B-lymphocytes. Transgenic plants and animals have emerged as sources for polyclonal and monoclonal antibodies, with transgenic animals preferred due to their human-like post-translational modifications and reduced immunogenicity risk. Chloroplast expression offers environmental safety by preventing transgene contamination in pollen. Diverse production technologies, such as stable cell pools and clonal cell lines, are available, followed by purification via techniques like affinity chromatography. The burgeoning applications of recombinant antibodies in medicine have led to their large-scale industrial production.

Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer-promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well-characterized insulators and binding factor(s). In this study, we isolated a 1.2-kb RS2-9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2-9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2-9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1-binding sites in RS2-9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC-binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1-RS2-9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes.

A wheat WRKY transcription factor TaWRKY17 enhances tolerance to salt stress in transgenic Arabidopsis and wheat plant.

WRKY transcription factors are essential to plant growth, development, resistance, and the regulation of metabolic pathways. In this study, we characterized TaWRKY17, a WRKY transcription factor from wheat, which was differentially expressed in various wheat organs and was up-regulated by salt, drought, hydrogen peroxide (H2O2) and abscisic acid (ABA) treatment. To analyze TaWRKY17 function under salt stress, we obtained stable T3 generation transgenic Arabidopsis and wheat TaWRKY17 overexpression plants. TaWRKY17 overexpression in Arabidopsis and wheat caused a significant plant salt-stress tolerance enhancement. Under salt stress, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activities were elevated in transgenic Arabidopsis and wheat plants compared with the wild type (WT), whereas H2O2 and malondialdehyde (MDA) accumulation was reduced in the transgenic lines. Moreover, ABA/reactive oxygen species (ROS)-related, and stress-response genes were regulated in the transgenic wheat plants, increasing tolerance to salt stress. The transgenic wheat plants were highly sensitive to ABA during seed germination and early seedling growth. In addition, TaWRKY17 virus-induced gene silencing (VIGS) decreased salt tolerance. These results showed that TaWRKY17 enhances salt tolerance by regulating ABA/ROS-related, and stress-response genes and increasing anti-oxidative stress capabilities. Therefore, this gene could be a target for the genetic modification of wheat.

Production, expression, and function of dual-specific monoclonal antibodies in a single plant.

MAIN CONCLUSION: LSC CO17-1AK and anti-HER2 VHH-FcK can be produced in a single plant and exhibit anti-tumor activities comparable to those of their respective parent antibodies. Recombinant monoclonal antibodies (mAbs) which can be applied to treat various cancers, are primarily produced using mammalian, insect, and bacteria cell culture systems. Plant expression systems have also been developed to produce antibodies. Plant expression systems present several advantages, including a lack of human pathogenic agents, efficient production costs, and easy large-scale production. In this study, we generated a transgenic plant expressing anti-colorectal cancer large single chain (LSC) CO17-1AK and anti-human epidermal growth factor receptor 2 (HER2) VHH-FcK mAbs by cross-pollinating plants expressing LSC CO17-1AK and anti-HER2 VHH-FcK, respectively. F1 siblings expressing both LSC CO17-1AK and anti-HER2 VHH-FcK were screened using polymerase chain reaction and Western-blot analyses. The cell enzyme-linked immunosorbent assay (Cell ELISA) confirmed the binding of LSC CO17-1AK and anti-HER2 VHH-FcK to target proteins in the SW620 human colorectal cancer and the SKBR-3 human breast cancer cell lines, respectively. The wound healing assay confirmed the inhibitory activity of both antibodies against SW620 and SKBR-3 cell migration, respectively. In conclusion, both LSC CO17-1AK mAb and anti-HER2 VHH-FcK can be produced in a single plant, achieve binding activities to SW620 and SKBR-3 cancer cells, and inhibitory activity against SW620 and SKBR-3 cell migration similar to their parental antibodies, respectively.

Overexpression of maize ZmLOX6 in Arabidopsis thaliana enhances damage-induced pentyl leaf volatile emissions that affect plant growth and interaction with aphids.

Pentyl leafy volatiles (PLV) are C5 volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C6 volatiles (GLV, green leafy volatiles), little is known about the biosynthesis and physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused a mild chlorotic phenotype, and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting that airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced a new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

Lactoferrin and its role in biotechnological strategies for plant defense against pathogens.

Agricultural crops are susceptible to many diseases caused by various pathogens, such as viruses, bacteria and fungi. This paper reviews the general principles of plant protection against pathogens, as well as the role of iron and antimicrobial peptide metabolism in plant immunity. The article highlights the principles of antibacterial, fungicidal and antiviral action of lactoferrin, a mammalian secretory glycoprotein, and lactoferrin peptides, and their role in protecting plants from phytopathogens. This review offers a comprehensive analysis and shows potential prospects of using the lactoferrin gene to enhance plant resistance to various phytopathogens, as well as the advantages of this biotechnological approach over existing methods of protecting plants against various diseases.

Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Role of carbon nanotubes (CNTs) in transgenic plant development.

Carbon nanotubes (CNTs) are nanostructures, allotropes of carbon which are made up of graphene sheets wrapped around it forming cylindrical structures. CNTs have been regarded to have interesting and attractive physical and chemical properties and have been tremendously used in genetic engineering. Understanding the role of CNTs in development of transgenic plants, review of research papers in the field was done. CNTs are classified into two categories: the single-walled and multiwalled (MWCNTs) structures. They are valuable vectors in various biomedicine fields such as Gene delivery, Drug delivery, Immunotherapy, Tissue engineering, and Biomedical imaging and also, they deliver the DNA without damaging the cells. Based on recent studies, the functionalization of CNTs when combined with some other suitable molecules can drastically subside their toxic effects. Having unique properties such as small size, larger surface area is useful in delivering DNA into mammalian cells as well. Modifications in CNTs can make nucleic acids adhere to them even more efficiently. Also, MWCNTs are crucial in delivery DNA into the cytoplasm. Based on other methods, the CNTs-DNA are a preferred choice and the inclination toward double-stranded DNA is used over single-stranded DNA in gene delivery shows effective results. The only downside of CNTs is that they are hydrophobic and are difficult to form an aqueous solution, thus limiting their applicability. This review will aid you in comprehending useful knowledge related to a general overview of topics related to CNTs.

Trends in PHA Production by Microbially Diverse and Functionally Distinct Communities.

Along with the wide applications of conventional plastics, they have a large number of disadvantages like their non-biodegradable nature, dependency on fossil fuels and the release of large amounts of toxic materials in the environment. Therefore, to resolve these problems, a number of bioplastics are studied, out of which polyhydroxyalkanoates are considered as the best alternatives. Polyhydroxyalkanoates (PHAs) are produced by microorganisms as intracellular granules during stressful conditions. Though a wide range of organisms can naturally produce PHAs, only a few of them can be used for commercial production. Therefore, more diverse organisms that accumulate a considerable amount of PHAs and also reduce the production cost need to be exploited. Transgenic plants, recombinant bacteria, algae and extremophiles are some diverse organisms that produce a high amount of PHAs at a low cost. So, if potential organisms are used for PHA production, bioplastics will be able to completely replace petroleum-based polymers. Therefore, our review mainly focuses on production of PHAs using potential organisms so that amount of PHAs produced is high and cost-effective which would further help in the commercialization of PHAs.

Genome-wide analysis of growth-regulating factor genes in grape (Vitis vinifera L.): identification, characterization and their responsive expression to osmotic stress.

KEY MESSAGE: Identification, characterization and osmotic stress responsive expression of growth-regulating factor genes in grape. The growth and fruit production of grape vine are severely affected by adverse environmental conditions. Growth-regulating factors (GRFs) play a vital role in the regulation of plant growth, reproduction and stress tolerance. However, their biological functions in fruit vine crops are still largely unknown. In the present study, a total number of nine VvGRFs were identified in the grape genome. Phylogenetic and collinear relationship analysis revealed that they formed seven subfamilies, and have gone through three segmental duplication events. All VvGRFs were predicted to be nucleic localized and contained both the conserved QLQ and WRC domains at their N-terminals, one of the typical structural features of GRF proteins. Quantitative real-time PCR analyses demonstrated that all VvGRFs, with a predominant expression of VvGRF7, were constitutively expressed in roots, leaves and stems of grape plants, and showed responsive expression to osmotic stress. Further growth phenotypic analysis demonstrated that ectopic expression of VvGRF7 promoted the growth and sensitivity of transgenic Arabidopsis plants to osmotic stress. Our findings provide important information for the future study of VvGRF gene functions, and potential gene resources for the genetic breeding of new fruit vine varieties with improved fruit yield and stress tolerance.

Functional characterization of glycine oxidase from Bacillus licheniformis in Escherichia coli and transgenic plants.

OBJECTIVES: To find glycine oxidase genes that can be applied to the breeding of glyphosate resistant crops. RESULTS: The glycine oxidase (GO, EC 1.4.3.19) gene (GenBank No: KC831746) from Bacillus licheniformis (B. licheniformis) was chemically synthesized and transformed into glyphosate-sensitive Escherichia coli (E. coli). The GO gene was transformed into Arabidopsis and rice through Agrobacterium-mediated transformation. The test results confirmed that transgenic plants containing GO genes are more resistant to glyphosate than wild-type plants. On solid Murashige and Skoog (MS) (Murashige and Skoog1962 ) medium containing 200 µM glyphosate, transgenic Arabidopsis thaliana grew normally, while wild-type plants were stunted and root growth was restricted. In a solution containing 500 µM glyphosate, wild-type rice showed severe yellowing, while transgenic rice grew normally. In addition, when sprayed with 10 mM glyphosate solution, wild-type rice withered and died, while transgenic rice grew well. The function of GO gene in glyphosate resistance and the application value of GO gene in the cultivation of glyphosate-resistant crops is proved. CONCLUSIONS: The glycine oxidase gene from B. licheniformis enhances the resistance of E. coli, Arabidopsis and rice to glyphosate.

Heterologous codA Gene Expression Leads to Mitigation of Salt Stress Effects and Modulates Developmental Processes.

Transgenic tobacco plants overexpressing the choline oxidase gene from A. globiformis showed an increase in resistance at the level of primary and secondary biosynthesis of metabolites, removing the damage characteristic of salinity and stabilizing the condition of plants. We used 200 mM NaCl, which inhibits the growth of tobacco plants at all stages of development. Leaves of transgenic and wild-type (WT) plants Nicotiána tabácum were used for biochemical, cytological and molecular biological analysis. However, for transgenic lines cultivated under normal conditions (without salinity), we noted juvenile characteristics, delay in flowering, and slowing down of development, including the photosynthetic apparatus. This caused changes in the amount of chlorophyll, a delay in the plastid grana development with the preservation of prolamellar bodies. It also caused changes in the amount of sugars and indirectly downstream processes. A significant change in the activity of antioxidant enzymes and a change in metabolism is probably compensated by the regulation of a number of genes, the expression level of which was also changed. Thus, the tolerance of transgenic tobacco plants to salinity, which manifested itself as a result of the constitutive expression of codA, demonstrates an advantage over WT plants, but in the absence of salinity, transgenic plants did not have such advantages due to juvenilization.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

Modifying the Expression of Cysteine Protease Gene PCP Affects Pollen Development, Germination and Plant Drought Tolerance in Maize.

Cysteine proteases (CPs) are vital proteolytic enzymes that play critical roles in various plant processes. However, the particular functions of CPs in maize remain largely unknown. We recently identified a pollen-specific CP (named PCP), which highly accumulated on the surface of maize pollen. Here, we reported that PCP played an important role in pollen germination and drought response in maize. Overexpression of PCP inhibited pollen germination, while mutation of PCP promoted pollen germination to some extent. Furthermore, we observed that germinal apertures of pollen grains in the PCP-overexpression transgenic lines were excessively covered, whereas this phenomenon was not observed in the wild type (WT), suggesting that PCP regulated pollen germination by affecting the germinal aperture structure. In addition, overexpression of PCP enhanced drought tolerance in maize plants, along with the increased activities of the antioxidant enzymes and the decreased numbers of the root cortical cells. Conversely, mutation of PCP significantly impaired drought tolerance. These results may aid in clarifying the precise functions of CPs in maize and contribute to the development of drought-tolerant maize materials.

Effect of ipt Gene Induction in Transgenic Tobacco Plants on Hydraulic Conductance, Formation of Apoplastic Barriers and Aquaporin Activity under Heat Shock.

Cytokinins are known to keep stomata open, which supports gas exchange and correlates with increased photosynthesis. However, keeping the stomata open can be detrimental if the increased transpiration is not compensated for by water supply to the shoots. In this study, we traced the effect of ipt (isopentenyl transferase) gene induction, which increases the concentration of cytokinins in transgenic tobacco plants, on transpiration and hydraulic conductivity. Since water flow depends on the conductivity of the apoplast, the deposition of lignin and suberin in the apoplast was studied by staining with berberine. The effect of an increased concentration of cytokinins on the flow of water through aquaporins (AQPs) was revealed by inhibition of AQPs with HgCl2. It was shown that an elevated concentration of cytokinins in ipt-transgenic plants increases hydraulic conductivity by enhancing the activity of aquaporins and reducing the formation of apoplastic barriers. The simultaneous effect of cytokinins on both stomatal and hydraulic conductivity makes it possible to coordinate the evaporation of water from leaves and its flow from roots to leaves, thereby maintaining the water balance and leaf hydration.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

Using an RNA Aptamer to Inhibit the Action of Effector Proteins of Plant Pathogens.

In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.

Cell-Penetrating Peptides for Use in Development of Transgenic Plants.

Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens-mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.