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Genetic transformation of crop plants using microprojectile bombardment
... The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants (Cai, et al.,1999). Christou (1992) identified three critical variables viz., physical, environmental and biological, which require careful optimization. ...
... Biological: Selection and nature of papaya explant is taken into consideration for microprojectile transformation. Pre and post bombardment culture conditions has to be optimized for different plant species (Christou, 1992). ...
Papaya is the first fruit crop which was not only successfully genetically engineered but also deregulated and commercialized. Pathogenic derived resistance was utilized for harnessing PRSV resistance. Coat protein gene from PRSV was invariably used to confer resistance against papaya ring spot virus. Microprojectile transformation has been the most preferred pathway. However, several reports are also available involving Agrobacterium pathway. Majority of workers found somatic embryos as the explant of choice for genetic manipulation in papaya compared to other explants. This paper highlights the global status of development of genetically engineered papaya for viral resistance.
... This method is also called a gene gun, particle acceleration, or microparticle bombardment for the growth of transformants [27]. This method is useful for both dicot and monocot plants, consisting of bombarding cells or tissues with 0.5 mm gold or tungsten microparticles carrying exogenous DNA-coated projectiles using compressed helium incubated at 30 °C in a special chamber under vacuum conditions ( Figure 2). ...
Genetic transformation has emerged as an important tool for the genetic improvement of valuable plants by incorporating new genes with desirable traits. These strategies are useful especially in crops to increase yields, disease resistance, tolerance to environmental stress (cold, heat, drought, salinity, herbicides, and insects) and increase biomass and medicinal values of plants. The production of healthy plants with more desirable products and yields can contribute to sustainable development goals. The introduction of genetically modified food into the market has raised potential risks. A proper assessment of their impact on the environment and biosafety is an important step before their commercialization. In this paper, we summarize and discuss the risks and benefits of genetically modified plants and products, human health hazards by genetically transformed plants, environmental effects, Biosafety regulations of GMO foods and products, and improvement of medicinal values of plants by the genetic transformation process. The mechanisms of action of those products, their sources, and their applications to the healthcare challenges are presented. The present studies pointed out the existence of several controversies in the use of GMOs, mainly related to the human health, nutritions, environmental issues. Willingness to accept genetically modified (GM) products and the adoption of biosafety regulations varies from country to country. Knowledge about the gene engineering technology, debate between the government agencies, scientist, environmentalist and related NGOs on the GM products are the major factors for low adoptions of biosafety regulation. Therefore, the genetic transformation will help in the advancement of plant species in the future; however, more research and detailed studies are required.
... Plant tissue was immersed in a GUS staining solution (100 mL L −1 sodium phosphate buffer (200 mM, pH = 7), 100 mg L −1 X-Gluc [Thermo Fisher Scientific™, Waltham, Massachusetts, USA) dissolved in 2 mL dimethyl sulfoxide, 0.2 mL L −1 Triton X-100, 4 mL L −1 EDTA (0.5 M, pH = 8), 42 mg L −1 K 3 Fe(CN) 6 )] and incubated at 37 °C for 4 h. The GUS staining solution was then removed and replaced with absolute ethanol to stop the enzymatic reaction (Jefferson et al. 1987;Christou 1992). Images of the embryos, callus and leaf segments were taken using a stereo-microscope (Moticam S3 camera and Motic Microscopy stereo zoom SMZ-171 BLED (Pole Type), Motic®, Hong Kong). ...
We report the development of an efficient and reproducible genetic transformation system for the recalcitrant Spanish elite rice paella genotype, Bomba. Preconditioned embryos derived from dry seeds were bombarded with gold particles carrying a plasmid containing a screenable and a selectable marker. We confirmed integration and expression of hpt and gusA in the rice genome. Transformation frequency was ca: 10% in several independent experiments. We show Mendelian inheritance of the input transgenes and zygosity determination of the transgenic lines in the T1 generation. A unique and critical step for the regeneration of plants from transformed tissue was shading during the early stages of regeneration, combined with a specific cytokinin:auxin ration at the onset of shifting callus to regeneration media.
... The technique of particle bombardment, also known as microprojectile bombardment, biolistics and particle acceleration, has been proved to be a very effective and versatile way for integrating genes into plant genomes. The DNA containing tungsten or gold particles, referred to as microprojectiles are carried by a microprojectile and are accelerated into living plant cells (Christou, 1992;Twyman and Christou, 2004). In eggplant, different explants have been targeted for transformation using the biolistic approach in eggplant but the use of cotyledon as an explant was found advantageous over others in terms of regeneration efficiency and several buds per explant (Sidhu et al., 2014a). ...
Eggplant is a member of the family Solanaceae, and it is commonly cultivated in many parts of the world. Eggplant is
susceptible to a number of biotic and abiotic stresses, therefore, there is a continuous demand for varieties with
disease and insect pest resistance, better nutraceutical capacity, and adaption to climate change.
Biotechnological approaches/tools have helped in the expansion of eggplant ideotype. In this direction, tissue
culture techniques for organogenesis and somatic embryogenesis are standardized in eggplant. Also, plant
transformation techniques like Agrobacterium-mediated gene transfer have been established in eggplant. Even if,
the information on eggplant from a biotechnology perspective is increasing yet there is a lack of knowledge.
Techniques like gene editing have not been tried in eggplant, further, eggplant still remains unexplored from
the molecular farming outlook. In this review, we compile the information regarding tissue culture, genetic
engineering, and genome editing advancements so far accomplished in the eggplant.
... The technique of particle bombardment, also known as microprojectile bombardment, biolistics and particle acceleration, has been proved to be a very effective and versatile way for integrating genes into plant genomes. The DNA containing tungsten or gold particles, referred to as microprojectiles are carried by a microprojectile and are accelerated into living plant cells (Christou, 1992;Twyman and Christou, 2004). In eggplant, different explants have been targeted for transformation using the biolistic approach in eggplant but the use of cotyledon as an explant was found advantageous over others in terms of regeneration efficiency and several buds per explant (Sidhu et al., 2014a). ...
Eggplant is a member of the family Solanaceae, and it is commonly cultivated in many parts of the world. Eggplant is
susceptible to a number of biotic and abiotic stresses, therefore, there is a continuous demand of varieties with
disease and insect pest resistance, better nutraceutical capacity, and adaption to climate change.
Biotechnological approaches/tools have helped in the expansion of eggplant ideotype. In this direction, tissue
culture techniques for organogenesis and somatic embryogenesis are standardized in eggplant. Also, plant
transformation techniques like Agrobacterium-mediated gene transfer have been established in eggplant. Even if,
the information on eggplant from a biotechnology perspective is increasing yet there is a lack of knowledge.
Techniques like gene editing have not been tried in eggplant, further, eggplant still remains unexplored from
the molecular farming outlook. In this review, we compile the information regarding tissue culture, genetic
engineering, and genome editing advancements so far accomplished in the eggplant.
... The technique of particle bombardment, also known as microprojectile bombardment, biolistics and particle acceleration, has been proved to be a very effective and versatile way for integrating genes into plant genomes. The DNA containing tungsten or gold particles, referred to as microprojectiles are carried by a microprojectile and are accelerated into living plant cells (Christou, 1992;Twyman and Christou, 2004). In eggplant, different explants have been targeted for transformation using the biolistic approach in eggplant but the use of cotyledon as an explant was found advantageous over others in terms of regeneration efficiency and several buds per explant (Sidhu et al., 2014a). ...
Eggplant is a member of the family Solanaceae, and it is commonly cultivated in many parts of the world. Eggplant is susceptible to a number of biotic and abiotic stresses, therefore, there is a continuous demand for varieties with disease and insect pest resistance, better nutraceutical capacity, and adaption to climate change. Biotechnological approaches/tools have helped in the expansion of eggplant ideotype. In this direction, tissue culture techniques for organogenesis and somatic embryogenesis are standardized in eggplant. Also, plant transformation techniques like Agrobacterium-mediated gene transfer have been established in eggplant. Even if, the information on eggplant from a biotechnology perspective is increasing yet there is a lack of knowledge. Techniques like gene editing have not been tried in eggplant, further, eggplant remains unexplored from the molecular farming outlook. In this review, we compile the information regarding tissue culture, genetic engineering, and genome editing advancements so far accomplished in the eggplant.
... The technique of particle bombardment, also known as microprojectile bombardment, biolistics and particle acceleration, has been proved to be a very effective and versatile way for integrating genes into plant genomes. The DNA containing tungsten or gold particles, referred to as microprojectiles are carried by a microprojectile and are accelerated into living plant cells (Christou, 1992;Twyman and Christou, 2004). In eggplant, different explants have been targeted for transformation using the biolistic approach in eggplant but the use of cotyledon as an explant was found advantageous over others in terms of regeneration efficiency and several buds per explant (Sidhu et al., 2014a). ...
Eggplant is a member of family Solanaceae, and it is commonly cultivated in many parts of the world. Eggplant is
susceptible to a number of biotic and abiotic stresses, therefore, there is a continuous demand of varieties with
disease and insect pest resistance, better nutraceutical capacity, and adaption to climate change.
Biotechnological approaches/tools have helped in the expansion of eggplant ideotype. In this direction, tissue
culture techniques for organogenesis and somatic embryogenesis are standardized in eggplant. Also, plant
transformation techniques like agrobacterium mediated gene transfer has been established in eggplant. Even if,
the information of eggplant from a biotechnology perspective is increasing yet there is a lack of knowledge.
Techniques like gene editing have not been tried in eggplant, further, eggplant is still remains unexplored from
the molecular farming outlook. In this review, we compile the information regarding tissue culture, genetic
engineering, and genome editing advancements so far accomplished in the eggplant.
... These cells are useful and versatile gene expression systems competent for transfection of exogenous genetic material. Other experimental platforms exist in plants, such as heterologous expression [5,6] and stable and transient transformation by Agrobacteria [7,8] or particle bombardment [9,10]. However, heterologous expression systems can be linked to a caveat of aberrant characteristics [11], and stable transformation requires signi cant resources and can be super uous for some applications including, but not limited to, studies in protein subcellular location and protein-protein interactions. ...
Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.
Results: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.
Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
... These cells are useful and versatile gene expression systems competent for transfection of exogenous genetic material. Other experimental platforms exist in plants, such as heterologous expression [5,6] and stable and transient transformation by Agrobacteria [7,8] or particle bombardment [9,10]. However, heterologous expression systems can be linked to a caveat of aberrant characteristics [11], and stable transformation requires significant resources and can be superfluous for some applications including, but not limited to, studies in protein subcellular location and protein-protein interactions. ...
Background
An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.
Results
Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.
Conclusion
The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
... Over the years, different types of "gene guns" have been developed. First-generation particle bombardment devices were using gunpowder charges, however, improved devices got the use of electrostatic, pneumatic, and compressed gas propulsion forces [31]. Here, we describe some of the most effective particle bombardment devices below. ...
Particle bombardment, or biolistics, has emerged as an excellent alternative approach for plant genetic transformation which circumvents the limitations of Agrobacterium-mediated genetic transformation. The method has no biological constraints and can transform a wide range of plant species. Besides, it has been the most efficient way to achieve organelle transformation (for both chloroplasts and mitochondria) so far. Along with the recent advances in genome editing technologies, conventional gene delivery tools are now being repurposed to deliver targeted gene editing reagents into the plants. One of the key advantages is that the particle bombardment allows DNA-free gene editing of the genome. It enables the direct delivery of proteins, RNAs, and RNPs into plants. Owing to the versatility and wide-range applicability of the particle bombardment, it will likely remain one of the major genetic transformation methods in the future. This article provides an overview of the current status of particle bombardment technology and its applications in the field of plant research and biotechnology.
... These cells are useful and versatile gene expression systems competent for transfection of exogenous genetic material. Other experimental platforms exist in plants, such as heterologous expression in plants like onion or tobacco [5,6] and stable and transient transformation by Agrobacteria [7,8] or particle bombardment [9,10]. However, heterologous expression systems can be linked to a caveat of aberrant characteristics [11], and stable transformation requires signi cant resources and can be super uous for some applications. ...
Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.
Results: Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.
Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
... Les trois prérequis pour la transformation génétique stable sont l'existence d'un tissu (Christou, 1992). L'organogénèse directe (Figure III-1C) à partir de fragment végétaux est moins sensible au génotype (Harshavardhan et al., 2002;Zhong et al., 1998) mais peut aisément conduire à la formation de plantes chimériques (constituées d'un assemblage de cellules qui n'ont pas le même patrimoine génétique) (Sant, 2011 (Hansen et Wright, 1999;Rakoczy-Trojanowska, 2002). ...
Les parois secondaires (PS) des plantes représentent la majorité de la biomasse renouvelable et participent à des fonctions physiologiques des plantes comme le transport des nutriments, la résistance à la verse et aux stress. Le sorgho est une graminée en C4 bien adaptée aux conditions limitantes en nutriments et en eau. L’amélioration des connaissances relatives à la mise en place des PS chez le sorgho a pour objectif de contribuer au développement de variétés adaptées aux filières de valorisation de la biomasse incluant l’alimentation animale, la production énergétique et le développement de bio-matériaux. Les objectifs de la thèse présentée ici sont de contribuer à la compréhension des mécanismes moléculaires de la mise en place des PS par l’identification des réseaux de gènes impliqués et la validation de leur rôle par génétique inverse. Dans un premier temps, une analyse phylogénétique comprenant 9 espèces, nous a permis d’identifier les liens d’homologies au sein des familles de facteurs de transcription (FT) NAC et MYB, qui sont les principaux régulateurs de la mise en place des PS chez Arabidopsis et d’autres espèces. Nous avons ensuite construit les réseaux de co-expression géniques impliqués dans la mise en place des entre-nœuds en développement du sorgho et nous nous sommes plus particulièrement intéressés aux réseaux comprenant des NAC et des MYB ainsi qu’à ceux présentant des enrichissements en gènes liés aux PS. Puis les gènes identifiés par ces approches ont été comparés à ceux situés dans les zones chromosomiques impactant la variabilité de la composition des PS. Dans un deuxième temps, nous avons développé des approches de transformation génétique afin de caractériser le rôle des gènes mis en évidence par génétique inverse.Nous avons identifié 6 modules de gènes co-exprimés enrichis en gènes et en ontologies géniques liés aux parois et en particulier 2 modules dont les gènes sont potentiellement impliqués dans la mise en place des PS. En outre, 122 NAC et 135 MYB ont été identifiés chez le sorgho. Parmi eux, 14 NAC et 25 MYB sont impliqués dans les 6 réseaux de co-expression de gènes fortement enrichis en gènes de paroi. Dix-neuf sont des homologues de gènes déjà validés chez d’autres espèces pour leur rôle dans la mise en place des PS et 20 n’ont pas de rôle connu. La comparaison des gènes faisant partie des 6 modules impliqués dans la mise en place des parois et des régions chromosomiques associées à la composition de la biomasse a mis en évidence 70 gènes en commun et quelques gènes de structure impliqués dans la biosynthèse des parois mais peu de convergence concernant les FT NAC et MYB.Bien que l’optimisation des protocoles de transformation génétique stable n’ait pas abouti, un protocole efficace de transformation transitoire basée sur des protoplastes a été optimisé. Cet outil sera notamment mobilisé pour tester les rôles des FT identifiés dans les réseaux enrichis en gènes de parois.
... Optimum TM GLY canola is an HT commercial GM canola variety produced through the biolistic transformation of microspores (OGTR 2016). This technique is highly useful for recalcitrant plant species for which Agrobacterium -mediated transformation is not a viable option (Christou 1992). However, high cost, gene rearrangement, multiple insertions lead to high copy numbers through the biolistic approach, which often limits its use for plant genetic transformation. ...
... Many problems and delays associated with conventional breeding brings the role of genetic engineering into play, considering its ability to circumvent the shortcomings of sexual reproduction and rapid regeneration of fertile transgenic crops from sterile and low yielding varieties. The most commonly used methods for soybean transformation are particle bombardment of shoot meristems and Agrobacterium-mediated genetic modification using cotyledonary nodes as explant in in-vitro culture [8][9][10] . According to some researchers 11-14 , these techniques still provide plenty of room for improvement, especially due to the low frequencies of transformation, genotype specificity and very poor reproducibility. ...
This review paper provides a succinct discussion on the potential use of in-planta transformation technique as an alternative for soybean genetic improvement. It discusses beneficial points, simple and straight forward procedures using Agrobacterium tumefaciens without the use of plant tissue culture conditions. Although, in-planta transformation does not regenerate multiple transgenic micro shoots, as often observed under in-vitro culture conditions, the approach has proved to significantly improve the number of glufosinate resistant soybean plants. This approach has been far neglected, with very few reports compared to in-vitro based transformation which is typically achieved through plant tissue culture. The in-vitro based procedure follows several steps including explant tissue preparation, explant infection, co-cultivation, regeneration, selection and acclimatisation of transformed plantlets. It requires longer culture periods, aseptic conditions and the procedure is often challenged by problems such as bacterial overgrowth, poor regeneration frequencies, genotype specificity and contaminations. Some of these steps remain entirely optional and often unnecessary for plant transformation in-planta.
... One approach to overcome these difficulties involves the bombardment of apical meristems from which transformed shoots and plants can be derived [5][6][7]. This method bypasses regeneration procedures and the associated problems, thereby reducing the time required to produce transformed plants for subsequent evaluation [8]. Of several devices that have been reported in the literature [9], only one is commercially available from Bio-Rad (PDS 1000/He), and this device is widely used in many laboratories around the world. ...
Biolistic transformation of cotton (Gossypium hirsutum L.) meristems, isolated from mature seed, is detailed in this report. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with a marker gene (uidA or “GUS”) into the shoot meristem. The penetration of gold particles was dependent on bombardment parameters, and it was mostly one- to two-cell layers deep. Stable transformation of epidermal L1 layer was consistently observed in approximately 5% of the seedlings. Germ line transformation was observed in up to 0.71% of bombarded meristems by several laboratories. Using this method identification of germ line transformation is laborious and time-consuming. However, the protocol described here represents a simple and efficient method for generating germ line transformation events. In addition, this procedure offers a quick method to evaluate gene constructs in cotton tissues (embryos, cotyledons, leaf) especially fibers which originate as single cells from the maternal epidermis layer.
... Biolistic transformation allows direct gene transfer to specific or nonspecific tissues with high efficiency and limitations on host choice. It does not require any intrinsic vector (or vector backbone), meaning linear transgene DNA of any size can be directly bombarded into host cells (Christou 1992). Biolistic transformation requires expensive equipment and skilled personnel. ...
Sugarcane is an important worldwide cash crop used for both sugar and ethanol production. Improvement of sugarcane through conventional breeding practices has been limited by its complex polyploid genome. Considerable improvements have been made with sugarcane through transgenic technology. Biolistic and Agrobacterium transformation methods have been regularly employed to develop transgenic sugarcane containing key agronomic traits. Transgenic sugarcane has been approved for commercial cultivation in both Indonesia and Brazil. Hightransformation efficiencies are on the horizon due to improved methods. Recent advances using CRISPR (clustered regularly interspaced short palindrome repeats)/Cas9 (CRISPR-associated) genome-editing systems require a robust transformation method. Efficient sugarcane transformation protocols will be vital in harnessing the potential of this cash crop. This chapter describes recent advances in sugarcane transformation and highlights novel improvement strategies to enhance target gene expression.
... Two general strategies are used for plant transformation: Agrobacterium-based transformation and a group of unrelated techniques collectively referred to as 'direct DNA transfer' (Kohli et al. 2003). Direct DNA transfer methods such as particle bombardment (Christou 1992) often result in transgenic loci with a high transgene copy number (often more than 40) (Kohli et al. 2003;Latham et al. 2006). Agrobacterium-based transformation procedures produce lines with less complex transgenic loci, but still, the integration of multiple T-DNA copies into a limited number of loci is common (De Buck et al. 2009;Bhat & Srinivasan 2002). ...
Plants are surrounded by diverse microbial communities inhabiting the plants rhizosphere, the leaf surface area but also inner parts of a plant. These endophytic bacteria are tolerated by the plant immune system and seem to form a tight mutualistic relationship with its host. Endophytic bacteria are believed being important for plant growth, plant health and plant resistance, estimated from inoculation experiments with single strains. In order to unravel the effect of their natural abundance, the colonization needs to be influenced within a plant. The wild tobacco Nicotiana attenuate is a non-domesticated, ecological model plant that can be genetically manipulated and allow heterologous gene expression. For this thesis I transformed N. attenuata for the expression of antimicrobial peptides with the goal to establish a toolbox for plant microbe interaction studies. Antimicrobial peptides (AMPs) are small cysteine-rich peptides with a broad spectrum activity against different bacteria or fungi, and part of the innate immunity of plants and animals. Constitutive expression within a transgenic plant could be used to manipulate the natural abundance of endophytic bacteria, if a transgenic plant shows high AMP accumulation and in planta activity under the physiological conditions of the plant apoplast.
I selected a comprehensive set of 11 different AMPs from various AMP families for ectopic expression in N. attenuata and developed an optimized screening method for the selection of suitable plant lines. Several transgenic plants showed gene expression loss and epigenetic gene silencing, and the analysis of the promoter methylation status allowed the selection of plant lines with trans-generational stable gene expression. I further showed that the augmentation of the methylation levels occurred independently from a generational change. Plants developed epigenetic changes solely during normal plant development and showed rapid methylation increase in somatic cells (more than 3% per day!). A secondary callus regeneration step could avoid somatic transgene methylation and was used to recover gene expression in the affected lines. To assess the localization and stability of the expressed AMPs direct within the plants, a universal nanoUPLC-MSE method was developed allowing an absolute peptide quantification and comparison of various AMPs from the intercellular fluid (ICF) of different transgenic plant lines. The results revealed desired high peptide accumulation in the apoplast of the LEA lines (expressing a lipid-transfer protein from motherwort) and the ICE lines (expressing a knottin from the common ice plant). Both AMPs have reported in vitro activity against gram-positive bacteria.
The analysis of the non-culturable bacterial community from N. attenuata plants from a wild population in the Great Basin Desert in Utah, showed that most bacteria belong to gram-positive groups, and Streptomyces and Bacillus dominated the root associated community. I tested the transgenic plants using a leaf infiltration method for in planta antibacterial activity against various bacterial strains. Only the ICE lines showed the ability to reduce infiltrated Bacillus pumilus, and were further characterized in their activity spectrum using culture collection strains (DSMZ) and native endophytic bacteria, previously isolated from N. attenuata. They showed consistent reductions of most native endophytic Bacillus spp. isolates, but no effects on Proteobacteria and most Actinobacteria. B. megaterium isolates showed strong heterogeneity among the isolates with highly distinct susceptibilities.
AMPs were in the past only used to increase the resistance of crop plants against phytopathogens, and potential effects on beneficial endophytic bacteria were usually ignored or not examined. The relevance of endophytic bacteria for natural plant growth is still largely unknown, since it was not possible to grow an aposymbiotic plant under natural conditions. The ICE lines showed in planta activity against endophytic bacteria and stable and high peptide expression which makes them valuable tools for the exploration of fitness effects of endophytic bacteria on N. attanuata.
... Particle bombardment-mediated transformation is one of the most common transformation methods; it is usually highly efficient and genotype independent [24]. Previous studies have shown that RNA and protein molecules can be transferred to plant cells by particle bombardment [25,26]. Recently, CRISPR/Cas9 RNA was delivered into wheat embryos by this method, and genome-edited wheat plants were obtained [27]. ...
... Biolistics, or microprojectile bombardment, transfers exogenous DNA attached to metal particles into explants for stable inheritance [75][76][77]. Cells and tissues that have the ability to divide and differentiate could be used as explants for biolistics, such as embryogenic cultures and meristematic cells and so on [78]. ...
Bermudagrass is widely distributed as a warm-season turf and forage grass in the temperate and tropical zones around the world. Its strong vegetative reproduction and ability to withstand drought make it an ideal species as a most used warm-season forage grass and turfgrass. Genetic transformation is an important tool for the study of gene function and for germplasm improvement in bermudagrass. This paper attempts to present a recent review on genetic resources, plant regeneration and genetic transformation in bermudagrass. We first review the various genetic resources and collection of bermudagrass. Then the explants, basal medium and the effect of different cultivars and plant growth regulators on plant regeneration in bermudagrass are also summarized. Last, we outline the main areas of progress in genetic transformation with either the biolistic or Agrobacterium-mediated method in bermudagrass, and discuss various factors that influence Agrobacterium-mediated transformation. However, the question that still remains is why there have been no genetic modification reports on bermudagrass for 10 years. © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
... Particle bombardment, also referred to as the biolistic system, is a physical method of gene transfer which employs high velocity metal particles (gold or tungsten) to deliver genetic material to prokaryotic and eukaryotic cells, including plants and animals (1). The method was first described by Standford et al. (2) and later became one of the most effective methods in transferring biologically active DNA molecules into plant cells (3). In plant transformation studies, the particle bombardment method has been accepted as a breakthrough since genetic transformation with this method became almost a routine process in many important crop species, including cereals and legumes, which are known to be recalcitrant for transformation with other techniques. ...
The possibility of transferring genes to mature wheat embryos (Triticum aestivum L. and T. durum Desf.) via accelerated and DNA-coated tungsten particles was investigated. Mature embryos isolated from bread (cv. Atay) and durum (cv. Çakmak) wheat were utilised as targets for bombardment. DNA in the form of circular plasmid (pBSGUSINT) was precipitated on tungsten particles (ca. 2 μm diameter) using the calcium nitrate method. Mature embryos were bombarded by a microprocessor-controlled particle delivery instrument (GENEBOOSTER™) driven by compressed nitrogen gas. Bombardment was carried out at various gas pressure and in a chamber vacuum. The target material was subjected to histochemical GUS staining 24 hours after bombardment or 7 days after the initial germination of the embryos. Almost 80% of the bombarded embryos expressed the transferred GUS gene observed through blue colour formation on the embryos. Negative controls (non-bombarded embryos or embryos bombarded with bare tungsten particles) did not exhibit GUS activity. Seven-day-old seedlings which had emerged from bombarded material also exhibited patches of GUS staining, indicating the integration and expression of the transferred GUS gene to the genome of some tissue segments. The optimised bombardment method is currently in use to obtain transgenic wheat cultivars.
... The methods based on Agrobacterium tumefaciens are the most simple and efficient, but practically limited to dicots, except for a few, as recalcitrant (LACORTE and MANSUR, 1993). The option for the genetic manipulation of these species and monocots is often found in the direct transfer of techniques such as micro bombardment and electroporation, through which it has been already achieved, for example, transgenic corn, cane sugar and rice (CHRISTOU, 1992). ...
World’s ornamental plant market, including domestic market of several countries and its exports, is currently evaluated in 107 billion dollars yearly. Such estimate highlights the importance of the sector in the economy of the countries, as well as its important social role, as it represents one of the main activities, which contributes to income and employment. Therefore a well-structured plant breeding program, which is connected with consumers’ demands, is required in order to fulfill these market needs globally. Activities related to pre-breeding, conventional breeding, and breeding by biotechnological techniques constitute the basis for the successful development of new ornamental plant cultivars. Techniques that involve tissue culture, protoplast fusion and genetic engineering greatly aid conventional breeding (germplasm introduction, plant selection and hybridization), aiming the obtention of superior genotypes. Therefore it makes evident, in the literature, the successful employment of genetic breeding, since it aims to develop plants with commercial value that are also competitive with the ones available in the market.
... Serkan et al. (2005) and Haliloglu (2006) reported that on the basis of efficient plant regeneration protocol, biotechnology can be apply successfully in crop improvement. Legumes are the most recalcitrant to in vitro manipulation but with great interest routine protocols are obtained for stable transformants for the major grain legumes such as the common bean (Phaseolus vulgaris), soybean (Glycine max), pea (Pisum sativum), peanut (Arachis hypogea), and alfalfa (Medicago sativa), as well as the model legume, barrel medic (Medicago truncata) (Christou 1992;Puonti-Kaerlas et al. 1990;Russell et al. 1993). To date many types of primary explants have been used for soybean regeneration via direct organogenesis. ...
Regeneration through mature cotyledonary node has set rapid regeneration of plants directly from explants
which is more time-saving and presented as an effective strategy so we have developed regeneration protocol through
single shoot using cotyledonary node a rapid and efficient protocol for three Indian soybean cultivars. Two explants
were collected from single cotyledonary node and cultured on medium containing N6
-benzylaminopurine (BAP) for
germination, BAP and indole-3-butyric acid (IBA) for shoot induction, Gibberellic acid (GA3) for shoot elongation and
IBA for rooting of explants. The best combination of hormones for all genotypes were obtained as germination of seeds
on half B5 medium supplemented with 1.0 mgl-1 of BAP, shoot induction on full B5 medium having BAP 1.0 mgl-1 and
IBA 0.2 mgl-1 and shoot elongation on GA3 0.75 mgl-1 in full MS medium. Under these conditions, the plantlets could
be raised within 40-45 days. It was observed that selection of proper medium for regeneration of soybean can overcome
genotype associated problems. This regeneration system can be used for soybean transformation.
Key word: Soybean genotype, transformation, regeneration, cotyledonary node.
Abbreviations: BAP – 6-benzyl-aminopurine, IBA - Indolebutyric acid, GA3 - Gibberellic acid, MS - Murashige and
Skoog.
... The limiting factors in developing transgenic ornamental bulbs can be overcome by direct DNA transfer methods; thus bypassing the barriers imposed by Agrobacterium-host specificity and monocotyledonous plant cell constraints (Christou, 1995). Some advantages offered by this system include: transformation of organized tissue, rapid discovery of transformed T 1 seeds, transformation of recalcitrant species and also offering the basis for studying many plant developmental processes (Christou, 1992). This technique has been applied to obtain transgenic plants of tulip (Wilmink et al., 1992), L. longiflorum and Ornithogalum dubium (Cohen et al., 2004). ...
Genetic improvement of ornamental geophytes especially the monocotyledonous type; is often restricted by failure of Agrobacterium to reach competent cells as well as a lack of efficient regeneration systems. Despite all these limitations, it has recently been shown that the use of efficient promoters, super-virulent strains, and the utilization of systems such as an agrobacterial monolayer, Agrobacterium-mediated pollen and seed transformation, floral dip method and sonication-assisted Agrobacterium-mediated transformation (SAAT) will ensure success in the genetic transformation of these ornamentals in the near future. This article outlines factors affecting transformation of monocotyledonous geophytes. Special emphasis is laid on measures that have been employed to alleviate various difficulties. The need to develop somatic embryogenesis protocols for the ease of transformation is highlighted. In addition, perspectives in view of future research are also given. This information is crucial for biotechnological improvement of ornamental geophytes that are proving difficult to transform.
... Particle bombardment (also known as biolistics) physically shoots DNA and its carrier (heavy metal particles) into target cells, acting as the most popular direct method in plants (Christou 1992;Klein et al. 2011). Using this method, Kartha et al. (1989) transiently expressed the chloramphenicol acetyltransferase (CAT) gene in cultured cells and immature zygotic embryos of barley. ...
Barley (Hordeum vulgare L.) is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.
... Most methods of direct gene transfer, such as the introduction of DNA via electroporation, (Riggs and Bates, 1986;Christou et al., 1987;Shimamoto et al., 1989). PEG-mediated DNA uptake, (Hayashimoto et al., 1990;Torres et al., 1997), protoplast fusion with liposomes containing DNA (Caboche, 1990), biolistics (Christou, 1992) or microinjection (Schnorf et al., 1991), require the regeneration of plants from protoplasts. The recalcitrance of many plant species for efficient regeneration from protoplasts, elaborate protocols and prolonged tissue culture phases, are a disadvantage. ...
Diseases caused by microorganisms are currently the major factor limiting crop production worldwide. In addition to negative effects on yield, diseases can also influence the post-harvest quality of food. Due to high cost, efficacy and environmental concerns, much research is presently aimed at expression of transgenes that can confer significant levels of disease resistance. The genetic manipulation of plants has been going on since the dawn of agriculture, but until recently this has required the tedious process of cross-breeding varieties. Genetic engineering promises to speed the process and broaden the scope of what can be done. To date, most interest has been focused on developing virus resistant transgenic plants, but through biotechnology to confer resistance to fungi, bacteria, or nematodes has also been gaining great consideration. Although recent introductions of plant products for control of insect pests have been highly successful, transgenic plants exhibiting resistance to fungal or bacterial diseases have yet to reach the marketplace. This book chapter mainly focusses on the novel strategies that are being manipulated for the development of disease resistant transgenic plants.
... A common criticism is that these processes are imprecise. In both processes, the insertion site of the new DNA is random (Altpeter et al., 2005;Wilson et al., 2006) and more than one copy of the DNA fragment may be inserted into the target genome (Christou, 1992;Gasson, 2003). This can affect gene expression in a positive or negative manner, for example, by causing gene suppression or gene silencing (Altpeter et al., 2005;Dai et al., 2001). ...
The aim of this review is to examine the relationship between genetically modified (GM) crops and health, based on histopathological investigations of the digestive tract in rats. We reviewed published long-term feeding studies of crops containing one or more of three specific traits: herbicide tolerance via the EPSPS gene and insect resistance via cry1Ab or cry3Bb1 genes. These genes are commonly found in commercialised GM crops. Our search found 21 studies for nine (19%) out of the 47 crops approved for human and/or animal consumption. We could find no studies on the other 38 (81%) approved crops. Fourteen out of the 21 studies (67%) were general health assessments of the GM crop on rat health. Most of these studies (76%) were performed after the crop had been approved for human and/or animal consumption, with half of these being published at least nine years after approval. Our review also discovered an inconsistency in methodology and a lack of defined criteria for outcomes that would be considered toxicologically or pathologically significant. In addition, there was a lack of transparency in the methods and results, which made comparisons between the studies difficult. The evidence reviewed here demonstrates an incomplete picture regarding the toxicity (and safety) of GM products consumed by humans and animals. Therefore, each GM product should be assessed on merit, with appropriate studies performed to indicate the level of safety associated with them. Detailed guidelines should be developed which will allow for the generation of comparable and reproducible studies. This will establish a foundation for evidence-based guidelines, to better determine if GM food is safe for human and animal consumption.
... Efficient protocols for the genetic transformation of alfalfa, white clover and Medicago truncatula using A. tumefaciens have been established over the last decade (Kalla et al. 2001;Zhang et al. 2005;Balance and McManus 2006;Crane et al. 2006;Wright et al. 2006;Xie et al. 2006;Montague et al. 2007;Rosellini et al. 2007;Barone et al. 2008). Cell suspension cultures serve as ideal target material for a biolistic transformation (Christou 1992), as in a relatively short period of time it can generate a large number of homogeneous cells (Ivic and Smigocki 2003) and has been used successfully to transform several monocotyledonous and dicotyledonous species (Kamo et al. 1995). The technique was first successfully applied to the suspension cells of Nicotiana tabacum (Klein et al. 1987(Klein et al. , 1988a and then Zea mays (Klein et al. 1988b). ...
Introgression of genes from alien species into crop plants could be achieved through distant hybridisation aided by tissue culture-based embryo rescue techniques. Beside this, in vitro mutagenesis, gametoclonal/somaclonal variation and transgenesis are the other tools which can generate additional variability. However, all these tissue culture-based tools require totipotent tissues. The direct regeneration of plants from an explant without a callus stage via organogenesis or somatic embryogenesis is the quickest path for micropropagation. Because of their speed and low costs of culture phase and the fidelity of the genotype in the cloned progeny, systems with direct somatic embryogenesis or organogenesis are often recommended and subjected to transformation. On the other hand, most micropropagation procedures with a callus stage can be applied as a basis for transformation, and the fresh friable calli can be directly used as the transformation target. Cell and microspore suspension cultures have also been seen as the ideal targets for genetic transformation due to the large amount of homogenous material, easy selection of the targeted cells and less chances of chimeric regeneration, while protoplasts due to exposed plasma membrane can introduce foreign DNA very easily and therefore form the ideal targets for generating unique and novel plants. This chapter discusses various plant regeneration methods and the factors affecting them towards achieving alien gene transfer in crop plants.
Plant molecular farming is a nascent but promising biotechnology-based industry. It is an alternate system for the development of pharmaceutical and non-pharmaceutical products cost-effectively in a bulk amount and in short timescale as compared to already established expression systems. This chapter covers the different strategies used for plant molecular farming (PMF) such as stable or transient transformation methods and what advantages they offer, how PMF can help the developing countries to eradicate the different health-related problems they face, what problems are encountered during the establishment of plant molecular pharming as local industry, and what steps should be taken to solve them. We also discuss how PMF helped and is helping the world in different sectors of life globally. Further, the current status of the products that are made via PMF is also discussed, including antibodies, enzymes, growth factors, other pharmaceutical products, and non-pharmaceutical or industrial products, which have the greatest impact on health, economy, poverty, and industry of developed and developing countries.
The human population has reached 7.8 billion by 2020 and is estimated to reach 9.9 billion by the end of 2050 which is nearly 25% increase in total world population. With tremendous increase in world population, plants which are the main food source must be produced at higher rate to fulfil the food demand. The agricultural lands are limited, so, there is need to find out some other alternative route to fulfil the food requirement. In the past, traditional plant breeders use only individuals of the same or closely related species to propagate plants with desirable traits. Through the classical breeding techniques, breeders were not able to obtain desired traits which were not present within the gene pool of their target plants. However, with recent use of genetic techniques such as vector mediated transgenesis and direct gene transfer methods, we can now able to produce plants with desired traits from a completely different species. Vector mediated gene transfer methods are based on Agrobacterium plasmid and plant virus, whereas direct gene transfer methods are based on physical (electroporation, microinjection, particle gun bombardment methods) and chemical methods (lipofection, polyethylene glycol (PEG)-mediated, etc.). In the development of a transgenic plant, there are certain steps such as isolation of desired gene, vector constructions, transgenesis methods, transgene integration and inheritance of transgene, need to be carefully monitored during transgenesis. Production of transgenic plant will allow us to fulfil the food requirement of growing population but future of GM crops remains a point vital debate.KeywordsAgrobacteriumTransgeneGene gunTransgenic plantGene cloning
Solanum melongena L., commonly called as brinjal/eggplant, occupies an important position in vegetable rearing across the globe and has been regarded as the poor man’s crop. The estimated production goes over 52,309,119 metric tonnes annually. Traditional plant breeding techniques have played a vital role in developing new cultivars, thereby improving the overall crop production that catered to the needs of the global requirement. However, in the long run, the requirement has risen enormously due to the rapidly growing population. Simultaneously, the reduction in the yield due to various factors including soil quality, environmental vagaries, diseases and pest attacks posed new challenges in the production-consumption landscape. Of all the factors, the threat of the notorious insect pest, Leucinodes orbonalis, commonly known as brinjal shoot and fruit borer (BSFB) which belongs to the phylum Arthropoda and to the order Lepidoptera stood as the greatest challenge to counter as it withstood several broad range insecticides. This situation demanded for BSFB-resistant varieties of brinjal, eventually leading to the development of the genetically modified Bt brinjal. The development of such an insect-resistant variety has been a landmark in brinjal production. The present chapter focuses on transgenic brinjal with improved agronomic traits, particularly insect-resistant Bt varieties, the basic biology of Bt and the major methodologies, the mechanism of action involved in the development of the Bt brinjal.
The biosynthesis of biologically active secondary metabolites in plant tissues is frequently related to their differentiation. From this point of view, the hairy roots are a suitable plant in vitro system for producing bioactive substances due to their degree of differentiation and metabolic profiles identical to or similar to that of the root systems of the intact plants from which they are derived. In addition, they are grown in growth regulators-free nutrient media, which is essential for the subsequent application of their metabolites in variety of nutritional and pharmaceutical systems. They are characterized by high genetic and biochemical stability, making possible the scale-up of the cultivation processes in bioreactors. This provides complete control and management of the biosynthetic process and results in high yields of target metabolites for a short period of time, regardless of seasons. Salvia plant species are widely used in traditional medicine. The major biological activities they possessed are antioxidant, anti-inflammatory, anti-tumor activities and etc. These activities are consequence of biosynthesized biologically active substances: sterols, various polyphenol structures, di- and triterpenes, etc. Data about Salvia hairy roots in the scientific literature are limited. Nevertheless, it is clear that they biosynthesized mainly diterpenes and phenolic compounds. Current progress of investigation of Salvia hairy roots is limited to the phytochemical profiling and assessment of the influence of different independent variables (mainly elicitors) on the yields of the target bioactive substances and the development of commercial production process is not visible at the time. Therefore in the near future it is necessary an integrated approach for optimizing production processes to be developed. In our opinion, this is the basis for successful achievement of the commercially important yields of bioactive substances produced by hairy roots of Salvia species.
Development of transgenics is the need of the modern era of plant breeding, as they possess the potential to incorporate those characters in crop varieties which are either difficult or impossible through conventional breeding approaches. In case of ornamental crops, the progress made in transgenic breeding is not that impressive like in cereals, pulses and vegetables, but the initiatives taken and advancements made have implicated the bright future of this technology in ornamental crops. Improved morphology, flower colour, resistance and fragrance are some of the desired novel traits in ornamental crops where transgenic approaches need to intervene. Transgenic breeding in major cut-flower crops like rose, chrysanthemum, gladiolus and carnation has provided avenues for incorporation of novel traits in other ornamental crops as well and has made such crops an ideal target for application of other advanced technologies.
For the transfer of a β-Glucuronidase (GUS) reporter gene to sugarbeet (Beta vulgaris L.) callus and leaf expiants by microprojectile bombardment, various rupture disk pressures and sample plate distances were tested. By coating the pB1221.23 DNA construct over the gold particles, bombardments were carried out by the DuPont PDS-1000/He system and the results clearly indicated the superiority of leaf expiants over the callus structures as targets. The sample plate distances affected the distribution pattern of the particles and the cells expressing the GUS reporter gene were noted to be aggregated in short distances whereas longer distance shots yielded better distribution of transformed cells. For both leaf expiants and callus structures the rupture disk pressure of 1350 psi was observed to yield the highest gene expression results and the sample plate distance of 12 cm for the leaf expiants and 9 cm for the callus were found to increase the penetration success of the gold particles.
We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.
Intimin is the primary adhesin protein of enterohemorrhagic Escherichia coli O157:H7, a pathogen carried by cattle and transmitted to humans via contaminated food and water. This work describes the rapid production of 1.3 kg dry weight of transgenic tobacco cells expressing the C-terminal 261 amino acids of intimin (Int261) for subsequent oral vaccine testing to reduce cattle colonization by E. coli O157:H7. Both a stirred-tank bioreactor (60 L) and a less capital-intensive oxygenated carboy culture were shown feasible for generating this scale of transgenic plant tissue culture biomass. Antibiotic selection pressure was not required during scale-up to maintain Int261 expression over the 13-wk culture period. Extended medium autoclave times of up to 90 min used for bioreactor sterilization had only minimal impact on nutrient uptake, culture growth, and intimin expression. Plant tissue was transformed, produced in vitro, and available for feeding studies in a fraction of the time required to develop and grow transgenic plants.
Biotechnology is broadly defined in a 1991 Office of Technology Assessment report as "any technique that uses living organisms (or parts of organisms) to make or modify products, to improve plants or animals, or to develop microorganisms for specific uses." This technology has been instrumental in the development and implementation of processes for the manufacture of antibiotics and other pharmaceuticals, industrial sugars, alcohols, amino acids and other organic acids, foods, and specialty products through the application of microbiology, fermentation, enzymes, animal cell and separation technology. Engineers, working with life scientists, often achieved scale-up to industrial production in remarkably short periods. A relatively small number helped to catalyze, over a period of 50 years, the growth of the pharmaceutical, food, agricultural-processing, and specialty-product sectors of the Indian economy to the point where sales now exceed $500 billion/year.
The past decades have witnessed an enormous development in biotechnology with regard not only to the isolation, synthesis, structure identification, and elucidation of the mode of action of molecules, but also to their application as tools within the life sciences. Biomolecules have proved to be of interest not only in biochemistry, but also in chemistry, biology, pharmacology, medicinal chemistry, biotechnology, and gene technology.
We are aware however that, despite all our efforts, it is impossible to include all aspects of biotechnology research in one book. We are not under the illusion that the text, although carefully prepared, is completely free of errors. Indeed, some colleagues and readers might feel that the choice of priorities, the treatment of different aspects of biotechnology research, or the depth of presentation may not always be as expected. In any case, comments, criticisms and suggestions are appreciated and highly welcome for further editions.
The editors, authors and publisher are pleased to present the book on Basic Concepts of Biotechnology. After years of studying the individual components of living systems, we can now study the systems themselves in comprehensive scope and in exquisite molecular detail. We therefore face the tasks of effectively employing new technologies, of dealing with mountains of data, and, most important, of adjusting our thinking to understand complex systems as opposed to their individual components.
Transgenic forage- and turf-type tall fescue (Festuca arundinacea Schreb.) and red fescue (F. rubra L.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using chimeric hygromycin phosphotransferase (hph) gene constructs driven by CaMV 35S promoter or rice actin 1 5' regulatory sequences. Bombardment parameters of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric β-glucuronidase (gusA) gene construct. For the recovery of stably transformed clones, hygromycin selection using liquid and solidified media was tested. Initial selection in liquid culture medium allowed for a twofold — compared with continuous plate selection using solid medium — recovery efficiency of transformed hygromycin resistant clones. Plants were regenerated from 35 % and 85 % of the hygromycin resistant calli obtained in tall and red fescue, respectively. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the trans gene in transformed mature tall and red fescue plants was confirmed by northern analysis and hygromycin phosphotransferase enzyme assay.
1. INTRODUÇÃO O meloeiro (Cucumis melo L.), pertencente à família curcubitaceae, é uma planta anual, herbácea, de hastes trepadoras e folhas pecioladas grandes e aveludadas. Seu fruto é rico em sais minerais como ferro, cálcio, fósforo e vitaminas A, C e do complexo B, possuindo também propriedades estimulantes, diuréticas e laxativas (GAYET, 2003). O estado do Rio Grande do Sul é o segundo maior produtor de meloeiro do país com uma produção de 14.586 toneladas e 2.392 ha de área colhida, sendo a região sul do estado responsável por uma produção de 12.394 t e 2.126 ha de área colhida. O Brasil é um país exportador de melão, principalmente para Europa, incluindo Espanha (19.119 t) e Países Baixos (17.736 t) (AGRIANUAL, 2008). Um dos graves problemas da cultura do melão é a rápida deterioração de seus frutos (PECH et al., 1994). O melão é um fruto climatérico que apresenta baixo potencial de armazenamento, variando, dependendo da cultivar, de 5 a 10 dias em condições ambientais. Uma das principais causas da alta perecibilidade do melão é o acelerado metabolismo, envolvendo principalmente a produção do etileno, considerado o hormônio do amadurecimento (ZAREMBINSKI; THEOLOGIS, 1994). Através da biotecnologia moderna, utilizando técnicas de biologia molecular e celular, foi possível introduzir o gene da ACC oxidase em sentido anti-sense em células de meloeiro, cv. Cantaloupe, visando diminuir a produção de etileno nas plantas regeneradas e consequentemente aumentar o período de armazenamento dos frutos. A introdução de um gene através da técnica de transformação genética visa tão somente alterar a característica em que está envolvido o referido gene, sem modificar outros aspectos fisiológicos e/ou morfológicos das plantas. No entanto, estudos realizados por Pinto (2000) e Romano (2001) encontraram algumas diferenças quanto à área foliar em plantas de tabaco transformadas com o gene Lhcb1*2 de ervilha. O emprego da análise de crescimento produz conhecimento de valor prático e informações exatas, referentes ao desenvolvimento e comportamento das plantas, possibilitando assim a comparação entre cultivares e plantas transformadas e não transformadas, quando cultivadas em determinado ambiente (BORÉM, 2005). Desta forma o objetivo do presente trabalho foi analisar os parâmetros primários de crescimento e comparar, através deles, as plantas de melão Cantaloupe transformadas com o gene da ACC oxidase em sentido anti-sense e seu genótipo não transformado.
Infection of actinorhizal plants roots by the actinomycete Fmnkia leads to the formation of a nitrogen-fixing root nodule (actinorhiza) consisting of multiple lobes, each of which is a modified lateral root. Actinorhiza development involves several specific steps, for example, root hair infection, prenodule formation, and initiation of lobe primordia from root pericycle. This article summarizes the latest development in the isolation and characterization of nodule-specific and -enhanced transcripts isolated from actinorhiza. The amino acid sequence derived from the nucleotide sequence of the cDNAs, in combination with localization data, showed that gene products are involved in nitrogen, carbon, and oxygen metabolism. Furthermore, some transcripts represented encoded gene products that might be part of infection and senescence mechanisms in actinorhiza. The article also reviews experiments designed to establish genetic transformation systems for actinorhizal plants. This research has led to the obtain-ment of transgenic plants of the Casuarinaceae family by using A rhizogenes and A. tumefaciens. These new findings are discussed in view of future studies on actinorhizal symbiosis. As molecular and cellular studies on Casuarinaceae and Betulaceae are more advanced than on the other six actinorhizal plant families, we concentrate primarily on species within these two families.
Embryogenic suspension cultures of Picea abies were bombarded with gene constructs containing the uidA reporter gene driven by different promoters to study how biological factors affect transient gene expression. A number of cell-lines were tested of which A66 and B17 were chosen for further specific analysis. These cell-lines were representative of a type A and type B embryogenic suspension culture, respectively, with this classification being based upon embryo morphology and regeneration capacity. In both cell-lines intermediate sized cells in the embryonic region of the somatic embryos appeared uidA positive, irrespective of which promoter was used. However, there were significant differences in transient gene expression depending on cell-line, promoters and time of bombardment after subculture. All promoters (35S, 2 x 35S, Act1-D, and Dc8) showed significantly higher expression in cell-line B 17 than A66, despite the higher growth rate of A66. The variation in transient gene expression with time after subculture was characteristic for promoter and cell-line, but it appeared not to relate to changes in mitotic index. Therefore, biological factors other than embryo morphology, growth rate or mitotic index account for the differences in transient expression.
This review focuses on the improvement of wheat (Triticum aestivum L.) via tissue and cell culture and its use in gene transfer techniques. Success of the latter critically depends on the ability to regenerate plants from cells or tissues cultured in vitro. Hence, we have devoted attention to the attempts made so far in obtaining regenerants from diverse explants. Although it is known that immature embryos are the best source for initiating morphogenic cultures, basic information related to the process of differentiation can also be gained by studying less responding tissues. The opportunity provided by anther and microspore culture in wheat improvement and the progress made is also presented.To enhance tissue culture responses, identification of chromosomes, gene loci, and genes is of cardinal importance. We have also surveyed the progress made in this regard by conventional but incisive plant-breeding techniques. Gene rearrangements in tissue culture leading to the appearance of somaclonal or gametoclonal variation are of interest in selection of useful cell lines. The last part of the review is devoted to work done on transient gene expression and transformation with emphasis on recent developments.
Referee: Dr. Ian Ray, Plant Breeding and Genetics, Department of Agronomy & Horticulture, New Mexico State University, MSC 3Q, P.O. Box 30003, Las Cruces, NM 88003-8003 Forage and turf grasses are the backbone of sustainable agriculture and contribute extensively to the world economy. They play a major role in providing high quality and economical meat, milk, and fiber products and are important in soil conservation, environmental protection, and outdoor recreation. Conventional breeding contributed substantially to the genetic improvement of forage and turf grasses in the last century. The relatively new developments in genetic manipulation of these species open up opportunities for incorporating cellular and molecular techniques into grass improvement programs. For some commonly used forage and turf species, significant advances have been achieved in the following areas: (1) establishment of a tissue culture basis for the efficient regeneration of fertile and genetically stable plants, (2) generation of transgenic plants by biolistic transformation and direct gene transfer to protoplasts, (3) recovery of intergeneric somatic grass plants by protoplast fusion, (4) development of molecular markers for marker assisted selection, and (5) sequencing of expressed sequenced tags and the development of DNA array technologies for gene discovery. Although difficulties still exist in genetic manipulation of these recalcitrant monocot species, impressive progress has been made toward the generation of value-added novel grass germplasm incorporating traits such as improved forage quality. The joint efforts of molecular biologists and plant breeders make the available biotechnological methods a useful tool for accelerating forage and turf grass improvement.
Infection of actinorhizal plants roots by the actinomycete Fmnkia leads to the formation of a nitrogen-fixing root nodule (actinorhiza) consisting of multiple lobes, each of which is a modified lateral root. Actinorhiza development involves several specific steps, for example, root hair infection, prenodule formation, and initiation of lobe primordia from root pericycle. This article summarizes the latest development in the isolation and characterization of nodule-specific and -enhanced transcripts isolated from actinorhiza. The amino acid sequence derived from the nucleotide sequence of the cDNAs, in combination with localization data, showed that gene products are involved in nitrogen, carbon, and oxygen metabolism. Furthermore, some transcripts represented encoded gene products that might be part of infection and senescence mechanisms in actinorhiza. The article also reviews experiments designed to establish genetic transformation systems for actinorhizal plants. This research has led to the obtain-men...
Covalently coupling toluidine blue O-tiopronin to a gold nanoparticle forms an enhanced, exceptionally potent antimicrobial agent when activated by white light or 632 nm laser light. Aqueous solutions of tiopronin-gold nanoparticles had no antimicrobial effect and, when added to solutions of toluidine blue O, did not enhance the antimicrobial effect of the latter. The minimum bactericidal concentration of the covalently coupled toluidine blue O-tiopronin gold conjugate for Staphylococcus aureus was at least four times lower than that of free toluidine blue O.
Cells from a suspension culture of Sorghum vulgare (sorghum) have been transformed to either hygromycin or kanamycin resistance following uptake of pBC1 or pNGI plasmids, respectively, introduced on DNA-coated high velocity microprojectiles. Hygromycin- and kanamycin-resistant transformants contained hygromycin B phosphotransferase- and neomycin phosphotransferasehybridizing restriction fragments of the expected size, respectively. A second introduced, but unselected for, reporter uidA gene which encodes ß-glucuronidase activity was also detected by DNA gel blot analysis in these transformants and shown to be expressed at low levels in two of the ten transformants analyzed. Transcripts from the introduced foreign genes accumulated to detectable levels in only these two transformants, both of which had a high copy number of genes integrated into their genome. This report further establishes the biolistic method as a useful route for delivery of DNA into the difficult-to-transform monocotyledonous plant species and represents the first stable transformation of this agronomically-important cereal grain.
Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata 'Crandon' and P. nigra 'Betulifolia' x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.
We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.
A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.
We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.
Transgenic soybean (Glycine max L.) plants derived from electric discharge particle acceleration experiments exhibited varying degrees of chimerism which was followed by the expression of the introduced β-glucuronidase (gus) gene. Degrees of chimerism in transgenic plants were established by determining expression of the gus gene observed as blue spots, streaks or sectors in stem and leaf tissues in in vitro grown plantlets and greenhouse plants. Clonal plants were also obtained. Presence of the gene was confirmed by Southern blot analysis. These studies permitted the reconstruction of a partial picture for the development of the soybean plant.
In the following review, I present an assessment of the realities and possibilities of effecting gene transfer to cereal crops. I discuss why Agrobacterium has been unsuccessful with cereals, what alternatives have been tested, the extent to which they have yielded transgenic plants, and their potential agronomic utility. The discussion, necessarily subjective, is framed within a rigid definition of what constitutes proof of gene integration, and the biological factors affecting transformation competence.
Electric discharge particle acceleration was used to introduce three foreign genes into the American cranberry (Vaccinium macrocarpon Ait.). These genes were NPTII (conferring resistance to the antibiotic, kanamycin), GUS (allowing for visual verification), and B.t. (conferring resistance to lepidopteran insects). Adventitious buds were induced on stem sections prior to bombardment with DNA-coated gold pellets. Bombarded stem sections were then transferred to a selection medium containing kanamycin. The surface of the medium was overlaid with a thin layer of kanamycin solution. Approximately 35 days after blasting, proliferating cell masses and elongating shoots were observed amidst the mass of kanamycin-inhibited tissue. Seven weeks after blasting, a histochemical assay verified GUS expression in these tissues, and polymerase chain reaction was used to confirm the presence of the introduced genes.
Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.
Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1(-1) 2,4-D, 400 mg 1(-1) glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1(-1) kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS.
Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,β-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%-27%. Similar rates of cotransformation were observed among various marker pairs.
We have developed a method for reproducibly obtaining transgenic rice at a high frequency (10(-4)): electroporation with a buffer in which chloride ions are replaced with organic acids. Co-transformation frequencies of the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes located on two separate plasmids were higher than 50%. Transgenic rice plants contained multiple copies of introduced genes integrated into their genomes in a complex manner. GUS enzyme activity was not proportional to gene copy number. Introduced HPT genes were detected and expressed in the progeny of transformants.
Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii. The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii.
Biologists commonly wish to introduce a wide range of substances into living cells. Such substances include biological stains, proteins (antibodies or enzymes), and genetic material (either RNA or DNA). The cell membrane and, in the case of plant ceils, the cell wall pose formidable barriers which exclude many macromolecules. The concept of particle bombardment has been put forward as a universal mechanism for transporting substances into any living cell. An acceleration device has been designed and constructed which can accelerate small tungsten particles (1 to 4 urn in diameter) to velocities of about 1,000 to 2,000 ft/sec. We have found that these particles can penetrate cell walls and membranes and enter cells in a nonlethal manner. Thousands of cells can be penetrated simultaneously, in situ, as they occur in tissues. Particle bombardment has been shown to be effective in delivering foreign substances into a variety of plant species, including onion, tobacco, corn, and rice. This new method of delivering macromolecules into living cells and tissues should have diverse scientific applications.
DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration
to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants
were followed in the first (R1) and second (R2) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for β-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for β-glucuronidase.
Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R1 generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover,
all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R2 generation. R1 plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R2 generation, demonstrating recovery of both homozygous and heterozygous R1 plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can
be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers
are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from
single transformed cells by a de novo organogenic pathway.
Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration
method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had
been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then
transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were
confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines
gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable
to plant species that can be infected by Agrobacterium and regenerated from leaf explants.
We have regenerated whole plants of rice (Oryza sativa L.) derived from protoplasts, which had been electroporated with plasmid DNA possessing a chimeric gene encoding aminoglycoside phosphotransferase II (APH(3')II). Transformed calli were selected on the basis of tolerance to the antibiotic, G418. Several plants were regenerated possessing functional APH(3')II activity due to the integration of intact foreign DNA into their genome.
Genetically engineered plants of commercial cotton varieties were obtained by Agrobacterium–mediated transformation. Inoculated tissues selected on kanamycin–containing medium gave rise to transformed calli that were resistant to the antibiotic and expressed the neomycin phosphotransferase enzyme. Amplified tissues were plated onto hormone–free medium to promote embryogenesis. Somatic embryos germinated and whole plants also expressed the marker enzyme.
We have recovered transgenic rice plants from a number of commercially important cultivars, including until now recalcitrant Indica varieties, using electric discharge particle acceleration. Immature embryos from greenhouse–grown plants were bombarded with gold particles carrying DNA, and transgenic plants were recovered following a simple culture protocol. Mendelian segregation of foreign genes was observed in R1 progeny and stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from progeny plants. Alternative transformation protocols that are dependent on the development of protoplast and suspension culture systems are no longer necessary as we have shown that a wide variety of diverse cultivars can be transformed. Transgenic plants expressing agronomically useful traits such as herbicide resistance have been obtained and are currently undergoing further evaluation. This report also demonstrates that it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos.
We have established an efficient protocol for plant regeneration from haploid Indica-type rice protoplasts. Incubation of these protoplasts with the selectable hygromycin phosphotransferase (hph) gene expressed under control of the 35S promoter of cauliflower mosaic virus (CaMV) and polyethyleneglycol (PEG), and subsequent culture in the presence of hygromycin B, led to the recovery of numerous resistant clones from which 77 plants were regenerated. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed, and that it was inherited in offspring.
THE generation of transgenic plants using gene transfer techniques is important to both the investigation of gene regulation and the genetic engineering of crops1. The Ti plasmid of Agrobacterium tumefaciens is now routinely used to transform dicotyledonous plants2, and the transfer of foreign genes to unorganized tissue3–6 and plants7,8 has been accomplished using direct DNA transfer methods9–11. A protocol for the easy and reproducible production of fertile transgenic cereals, however, has not yet been described. We report here the production of fertile transgenic rice plants obtained by introducing the bacterial hph gene, encoding hygromycin B resistance12 (Hmr), into protoplasts of Oryza sativa (L.) by electroporation. The non-selectable gene encoding β-glucuronidase was also transferred with the hph gene and its expression was detected in the progeny of the stable transformant.
We have used particle acceleration by electric discharge to introduce DNA-coated gold particles into meristems of immature soybean seeds. Approximately 2 percent of shoots derived from these meristems via organogenesis were chimeric for expression of the introduced gene. Experiments using this protocol have resulted in mature RO and R1 plants expressing the introduced gene(s). We regard this work as a major step in the practical application of genetic engineering to the useful modification of food-crop species.
Transgenic sugarcane plants were produced by bombardment of embryogenic callus with high-velocity DNA-coated microprojectiles, followed by a selection and regeneration procedure designed for this target tissue. Optimal bombardment conditions for embryogenic callus required microprojectile velocities higher than those previously found effective for sugarcane suspension culture cells. Bombardment of target tissues twice increased the number of transiently expressing cells in regenerable callus regions, to more than 300 per treated plate. Stable transformants were obtained following bombardment with the neomycin phosphotransferase (npt-II) gene under the control of the Emu strong monocot promoter. Stepped increases in antibiotic concentration during selection and regeneration allowed recovery of actively growing callus and plants on media containing geneticin concentrations completely inhibitory to untransformed controls. NPT-II levels in transformed plants were 20–50 times the background levels in control plants in ELISA assays, and Southern analysis revealed integration of one to three copies of the introduced gene in the sugarcane genome. The procedures described yield one to three transgenic plants per treated plate within 16 weeks of bombardment and provide a simple, efficient and broadly applicable system for genetic transformation of sugarcane. A similar approach should be applicable to other members of the Poaceae able to form embryogenic callus.
Chimeric soybean plants derived from electric discharge particle acceleration experiments were used to develop relationships between tissue-specific expression patterns of a marker gene and germ-line transformation events in Ro plants. Using the GUS histochemical assay the likelihood of germ-line transformation was correlated to the localization of enzyme activity in specific tissues of the parent plant. The expression patterns of the 35S-GUS marker gene in specific tissue types in Ro plants and correlation with transmission of the introduced gene to progeny allowed the production of a clear picture of the development of these plants and their germ-line cells. Advances reported in this article resulted in the development of a commercial process for the genetic improvement of this important staple crop which until recently was very recalcitrant to conventional transformation methods.
Successful transient expression of genes (luciferase and -glucuronidase) in cultured tobacco cells and adventitious shoots of egg-plant hypocotyls can be obtained by use of a simple biolistic device. This device is driven by controlled gas pressure from a cylinder of nitrogen gas. It has the advantages of being free of explosive heat and of avoiding cell damage caused by expanding gas, due to the self-sealing effect of the projectile.
The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal
cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors
of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular
salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation
procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor
synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional
collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic
analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of
granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature
of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor
as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen
matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in
the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from
collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein
with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor
was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation
of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and
its receptor and signal transduction pathway.
We have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed
genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato
(LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated
onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression
between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit
regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this
transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile
bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve
stable transformation of pollen.
The biolistic technique transforms cells by bombardment with DNA-coated microprojectiles. It has been used to transform plants,
microbes, and organelles. We adapted a standard Biolistic PDS-1000 device for use with animals and have successfully transformed
tissues in live mice. The firefly luciferase gene was introduced into mouse skin and ear tissue. One day after transformation
344±74 and 1648±254 pg of luciferase were detected in skin and ear samples, respectively. Expression of the gene product was
transient but detectable up to 7 days after bombardment. A further modification of the device allowed transient transformation
of liver tissue in vivo. Liver contained 293±122 pg of luciferase 1 day postransformation. Expression of the gene in liver
tissue was unchanged at Day 3 but declined to low levels by Day 5. This new device allowed a fourfold increase in gene expression
in ear tissue extending a minimum of 14 days. This technology is applicable to a broad range of tissues and organs in situ
and makes it possible to test numerous reporters and the tissue specificity of promoters. It may also be useful in protocols
for somatic cell therapy.
Despite considerable efforts in the genetic engineering of plants, and notable achievements in some species, the world's major cereal crops are proving remarkably recalcitrant to genetic transformation. This paper reviews the various approaches that have been taken and assesses which of them may provide routine transformation methods for cereals. The biological peculiarities of cereals may hold the key to future success.
The biolistic process is a new process which employs high velocity microprojectiles to deliver substances into cells and tissues. It has been described in different ways and has been referred to as the particle gun method, the microprojectile method, the gene gun method, the particle acceleration method, the bio-blaster method. The inventors of the process have coined the term ‘biolistic’ (biological ballistics) to describe both the process and any associated apparatus used to shoot biological materials into living targets.
Until recently, the improvement of soybean (Glycine max) by genetic engineering has been limited due to the lack of an efficient transformation system for introducing foreign genes into regenerable tissues. We report the production of transgenic plants using an electric discharge to inject DNA-coated gold particles into soybean meristems; the transformed phenotype segregated in a mendelian fashion and the introduced genes were stable for up to six generations. The technique has been developed into a commercially viable process for introducing any foreign gene into any variety of soybean.
We have obtained stably transformed callus lines by direct delivery of DNA into plated suspension culture cells of wheat (Triticum aestivum L.) using high velocity microprojectile bombardment. Three different reporter or selectable marker genes, jointly present or on separate plasmids, were introduced: neomycin phospho-transferase (NPTII), -glucuronidase (GUS) and 5-enolpyruvylshikimate phosphate (EPSP) synthase. Kanamycin was used for the selection of resistant calli, which were screened for GUS expression by a histochemical stain. Southern analysis confirmed that the NPTII, GUS and EPSP synthase genes had been stably integrated in all of the kanamycin resistant and GUS positive lines, and NPTII and EPSP synthase activities were demonstrated in the transformed calli.
We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.
We have constructed a novel micro-projectile accelerating system for efficient gene transfer into cells in situ that avoids binding DNA to micro-projectiles and keeps the DNA in solution. Further, instead of a macro-projectile (or the equivalent), it accelerates the particles in a Bernoulli air stream. The micro-targeting approach directs highly dispersed particles to sites with diameters as little as 0.15 mm, allowing precise aiming to restricted tissues. The system is physically flexible and should therefore be adaptable to different tissues and species. Transient expression of the Escherichia coli beta-glucuronidase gene in immature wheat embryo scutella was obtained at a frequency of up to 3% of the treated cells in the surface layer. In tobacco SR1, we achieved many transgenic plants, and the efficiency of stable transformation with the neomycin phosphotransferase (NPTII) gene was approximately 10(-3) per exposed cell.
We report here a novel phenomenon, namely that nucleic acids can be
delivered into plant cells using high-velocity microprojec-tiles. This
research was conducted in the hope of circumventing some of the inherent
limitations of existing methods for delivering DNA into plant
cells1-6. After being accelerated, small tungsten particles
(microprojectiles) pierce cell walls and membranes and enter intact
plant cells without killing them. Microprojectiles were used to carry
RNA or DNA into epidermal tissue of onion and these molecules were
subsequently expressed genetically. This approach can therefore be used
to study the transient expression of foreign genes in an intact tissue.
It remains to be shown that smaller cell types, as are found in
regenerable plant tissues, can be stably transformed by this method. If
this proves possible, it would appear to provide a broadly applicable
transformation mechanism capable of circumventing the host-range
restrictions of Agrobacterium tumefaciens1, and the
regeneration problems of protoplast transformation2-5.
Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnosperm Picea glauca (white spruce). Promoter expression was assayed in three different tissues capable of in vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducible Arabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter-beta-glucuronidase-nopaline synthase 3' fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the beta-glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.
We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.
Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.
A mammalian embryonic cell surface glycoprotein (ESGp), whose expression and biochemical structure seem to be developmentally regulated, has been isolated and characterized. The molecule expressed in two cell through morula stage mouse embryos has a molecular weight, by electrophoretic analyses, of 90 kDa. At the blastocyst stage, however, the molecule migrates as a broad, heterogeneous band ranging from 90 to 110 kDa. Evidence obtained from studies of embryonal carcinoma (EC) cells indicates that this band is actually a composite of three distinct molecules (molecular weight 90, 95, and 105 to 110 kDa), each of which is synthesized uniquely by one of the different cell types of the blastocyst: the embryonic ectoderm and visceral and parietal endoderms, respectively. A survey of various mouse tissues and cell lines has revealed that undifferentiated cells express the low molecular weight form (90 kDa) characteristic of embryonic ectoderm, whereas differentiated cells and adult tissues express the high molecular weight form (110 kDa) characteristic of parietal endoderm. Only the EC visceral endoderm cell analogues have been shown to express the intermediate molecule (95 kDa). In embryos, the antigen is uniformly distributed over the cell surface during early cleavage stages (two to eight cell); just before compaction, however, it seems to redistribute and becomes polarized at the outside exposed edges of blastomeres. In cultured EC cells, ESGp is found only in areas of cell-to-cell contact; free-standing surfaces of cells are negative for expression. It is possible, therefore, that ESGp may be involved in the intercellular adhesion of both EC cells and compacting embryos.
Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.
The genetic transformation of mitochondria and chloroplasts has been an intractable problem. The newly developed "biolistic"
(biological ballistic) process was used to deliver DNA into yeast cells to stably transform their mitochondria. A nonreverting
strain, which is respiratory deficient because of a deletion in the mitochondrial oxi3 gene, was bombarded with tungsten microprojectiles
coated with DNA bearing sequences that could correct the oxi3 deletion. Respiratory-competent transformants were obtained
in which the introduced oxi3 DNA is integrated at the homologous site in the mitochondrial genome. Organelle genomes can now
be manipulated by molecular genetic techniques in the same way as nuclear genomes.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.
Soybean protoplasts from a number of commercially important cultivars have been genetically engineered by way of electroporation using chimeric genes coding for resistance to the aminoglycoside antibiotics kanamycin and G418. Effective electroporation conditions were determined by monitoring transient expression from aminoglycoside 3'-phosphotransferase II (APHII) expression plasmids. Electroporation of protoplasts with a chimeric APHII gene and subsequent selection on media supplemented with kanamycin resulted in the recovery of calli resistant to the antibiotic. Enzyme assays for APHII activity and Southern blot hybridization confirmed the expression of the foreign DNA and its stable integration into the soybean genome. Root formation was induced from transformed calli, and these roots maintained expression of the APHII gene.
Immature soybean (Glycine max L.) embryos from commercially important cultivars were the targets of rapidly accelerated, DNA-coated, gold particles. Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene. Kanamycin-resistant calli were obtained at a rate of approximately 10(-5). Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. Our results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and indicate the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues.
A transient assay to test heterologous promoter activity in Picea glauca‐white spruce using electric discharge particle acceleration
- Ellis D.D.
Ellis, D.D., McCown, B.H., Russell, D.R. and McCabe, D.E.
(1990) A transient assay to test heterologous promoter activity
in Picea glauca-white spruce using electric discharge particle
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Asgrow's genetically engineered soybean has farmers excited
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Cutler, K. (1 991) Asgrow's genetically engineered soybean has
farmers excited. Ag. Biotech. News, Sept./Oct., 12.
Functional analysis of maize anthocyanin biosynthetic pathway gene expression by microprojectile bombardment
- Roth B.
Roth, B., Klein, T., Goff, S., Fromm, M. and Bowen, B. (1990)
Functional analysis of maize anthocyanin biosynthetic pathway
gene expression by microprojectile bombardment. In Vitro Cell.
Dev. Biol. 26, 33.
Gus messenger RNA delivery and expression in plant cells via particle bombardment
- Walker L.
Walker, L., Aoyagi, K., Ulrich, T. and Wong, J. (1990) Gus
messenger RNA delivery and expression in plant cells via
particle bombardment. In Vitro Cell. Dev. Biol. 26, 70.
Fertile transgenic rice plants regenerated from transformed protoplasts
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Shimamoto, K., Teda, R., Izawa, T. and Fujimoto, H. (1989)
Fertile transgenic rice plants regenerated from transformed
protoplasts. Nature, 338, 274-277.