Applied Plant Genomics and Biotechnology

Palmiro Poltronieri, Yiguo Hong

• Publisher: Woodhead Publishing
• Year: 2015
• ISBN:
  9780081000717
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

Applied plant genomics and biotechnology reviews the recent advancements in the post-genomic era, discussing how different varieties respond to abiotic and biotic stresses, investigating epigenetic modifications and epigenetic memory through analysis of DNA methylation states, applicative uses of RNA silencing and RNA interference in plant physiology and in experimental transgenics, and plants modified to produce high-value pharmaceutical proteins. The book provides an overview of research advances in application of RNA silencing and RNA interference, through Virus-based transient gene expression systems, Virus induced gene complementation (VIGC), Virus induced gene silencing (Sir VIGS, Mr VIGS) Virus-based microRNA silencing (VbMS) and Virus-based RNA mobility assays (VRMA); RNA based vaccines and expression of virus proteins or RNA, and virus-like particles in plants, the potential of virus vaccines and therapeutics, and exploring plants as factories for useful products and pharmaceuticals are topics wholly deepened. The book reviews and discuss Plant Functional Genomic studies discussing the technologies supporting the genetic improvement of plants and the production of plant varieties more resistant to biotic and abiotic stresses. Several important crops are analysed providing a glimpse on the most up-to-date methods and topics of investigation. The book presents a review on current state of GMO, the cisgenesis-derived plants and novel plant products devoid of transgene elements, discuss their regulation and the production of desired traits such as resistance to viruses and disease also in fruit trees and wood trees with long vegetative periods. Several chapters cover aspects of plant physiology related to plant improvement: cytokinin metabolism and hormone signaling pathways are discussed in barley; PARP-domain proteins involved in Stress-Induced Morphogenetic Response, regulation of NAD signaling and ROS dependent synthesis of anthocyanins. Apple allergen isoforms and the various content in different varieties are discussed and approaches to reduce their presence. Euphorbiaceae, castor bean, cassava and Jathropa are discussed at genomic structure, their diseases and viruses, and methods of transformation. Rice genomics and agricultural traits are discussed, and biotechnology for engineering and improve rice varieties. Mango topics are presented with an overview of molecular methods for variety differentiation, and aspects of fruit improvement by traditional and biotechnology methods. Oilseed rape is presented, discussing the genetic diversity, quality traits, genetic maps, genomic selection and comparative genomics for improvement of varieties. Tomato studies are presented, with an overview on the knowledge of the regulatory networks involved in flowering, methods applied to study the tomato genome-wide DNA methylation, its regulation by small RNAs, microRNA-dependent control of transcription factors expression, the development and ripening processes in tomato, genomic studies and fruit modelling to establish fleshy fruit traits of interest; the gene reprogramming during fruit ripening, and the ethylene dependent and independent DNA methylation changes.

• provides an overview on the ongoing projects and activities in the field of applied biotechnology
• includes examples of different crops and applications to be exploited
• reviews and discusses Plant Functional Genomic studies and the future developments in the field
• explores the new technologies supporting the genetic improvement of plants

1

Transgenic, cisgenic and novel plant products

Challenges in regulation and safety assessment

Palmiro Poltronieri and Ida Barbara Reca, CNR-ISPA, Department of Food, Agriculture, Fisheries and Biotechnology, National Research Council, Italy

Plant science has made considerable progress in developing new biotechnology-based plant breeding techniques to alter genetic and epigenetic factors. In this chapter, we will discuss novel plant products (NPPs) obtained by cisgenesis, intragenesis and genome engineering using site-specific nucleases and gene-targeting oligonucleotides. Among these, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeats-associated Cas9 nucleases. Reverse breeding methods and backcrossing of the engineered plants with natural varieties has provided improved plants and fruit trees devoid of transgenes and cisgenes. There is an increasing higher public acceptance of the NPPs devoid of virus sequences and antibiotic genes, and containing only genetic material derived from the species itself or from closely related species. An overview is presented on the differences between regulation and regulatory bodies in various countries, with a need for harmonization in the ruling and in definition differences between modified and non-modified plant genomes.

Keywords

Genetically modified plants; novel plant products; cisgenesis; intragenesis; homologous recombination; non-homologous end joining; site-directed nucleases; zinc finger nucleases; transcription activator-like effector nucleases; clustered regularly interspaced short palindromic repeats; antisense technology; RNA interference; safety assessment; harmonization

1.1 Genetically modified plant products in the United States

The growing area of genetically modified (GM) crops has substantially expanded since they were first commercialized in 1996. Correspondingly, the adoption of GM crops has brought huge economic and environmental benefits (processed proteins and carbohydrates, soy sauce, proteins for feeds). All these achievements have been primarily supported by two simple traits of herbicide tolerance and insect resistance in the past years (Chen and Lin, 2013). The populations of at least nine pest species have evolved resistance to Bacillus thuringiensis (Bt) toxins in the field. It has been reported that widespread control failures of Bt cotton associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac have happened in the state of Gujarat in western India. Moreover, the wide adoption of HR crops in the United States also appeared to accelerate the evolution of resistance weeds to glyphosate compared with areas not growing GM crops (Tabashnik et al., 2012; Carriere et al., 2010; Dhurua and Gujar, 2011). Twenty-four glyphosate-resistant weed species have been identified since Roundup-tolerant crops were introduced in 1996. However, studies on Palmer amaranth (Amaranthus palmeri) showed that the plant can easily grow in transgenic cotton fields since 2008. Palmer amaranth is a weed especially in the south-eastern United States. It outcompetes cotton for moisture, light and soil nutrients and can quickly take over fields. Farmers had historically used multiple herbicides, which slowed the development of resistance, and controlled weeds through ploughing and tilling, practices that deplete topsoil and release carbon dioxide but do not encourage resistance. Monsanto has changed its recommendations on glyphosate use, so that farmers change and use a mix of chemical products and ploughing.

For GM plants (GMPs), it takes almost 6 years and US$ 35 million to generate the data for a regulatory dossier, limiting the use of this technology to the major agrobiotechnology companies and to high-value crops and traits (Podevin et al., 2013).

1.2 GMP products in Europe

The purpose of European Union (EU) legislation on GM food and feed products is to protect not only the environment but also the public by ensuring food safety and to uphold consumers’ rights to choose between GM and non-GM through food labelling (EC No 1829/2003, 1830/2003). The enforcement of EU legislation requires reliable qualitative and quantitative analytical methods. There are several different aspects in risk assessment of GM plants and derived products that must be discussed with EU authorities in the accomplishment of a regulatory dossier: a thorough analysis of the environmental risk assessment (persistence and invasiveness assessment including agronomic and phenotypic performance, interactions with target and non-target organisms, horizontal gene transfer, impacts on biogeochemical processes, impacts on agricultural management practices); food/feed safety assessment (composition, toxicology, allergenicity, nutritional assessment, dietary exposure) and molecular characterization of genetic modifications (transformation process, molecular analysis and expression of inserted DNA, inheritance and stability of inserted DNA, bioinformatic analysis).

Public attention is mainly focussed on the introduction and marketing of GM crops, food and seeds. Since 1980, the regulation of health safety and environmental risks are generally much stricter in EU than in the United States. The EU introduced the Cartagena protocol on biosafety (http://bch.cbd.int/protocol), which extends the United Nations Convention on Biological Diversity (CBD) and implemented it in its legislation. While the US Food and Drug Administration (FDA) delegates the determination of safety of production of modified plants to the company filing the submission, it regulates the environmental release of GM organisms (GMOs) that could deliver plant pests or genes from plant pests under the Plant Protection Act. Depending on the nature and intended use of the plant it may still be subject to other regulatory authorities such as the Environmental Protection Agency (USEPA) and FDA.

The precautionary principle and recently the social and economic aspects that were included in the decision process of GM risk assessment are the main reasons that delay GM plants approval in Europe, while in other advanced countries these constraints are less tight.

At a subsequent step, most regulatory authorities require that GMO be devoid of unnecessary DNA, especially vector backbone sequences (VBSs), containing bacterial resistance genes and origins of replication (OR).

In 2013, only one transgenic crop (Bt maize) is commercially planted in EU. Bt maize was planted in six countries (Spain, Czech Republic, Slovakia, Portugal, Romania and Poland) in about 300 ha.

Considering field trials in EU countries, both for intended commercialization and research purposes, many projects were performed recently (e.g. potatoes with modified sugar content, changed composition of starch polysaccharides or resistance against infection, virus-resistant plum trees, flax with changed linseed oil properties, trees for bioremediation, resistance against illnesses, growth acceleration or changed technological properties of wood).

New field trials have been stopped since the year 2008, according to EU directive 2001/18/EC (http://www.efsa.eu.int/science/gmo/gmoopinions/384en.html) (EPEC, 2011).

Further studies with different purpose are realized in the category of the ‘contained use’ (laboratory scale). The most promising are the experiments testing the application of transgenic plants for the production of various recombinant proteins (molecular farming, e.g. antibodies).

Under the EU legislation for authorization of release of new GMOs, detection methods are also required to be submitted to the EU reference laboratory on GMOs for assessment and validation. The European Network of GMO Laboratories (ENGL) serves the analytical laboratory community with up-to-date information on the latest developments in GMO testing.

Several previous EU projects have sought to develop such methods to satisfy a wide range of requirements, e.g. high quantification accuracy, on-site detection, high throughput and wide target range. These projects included QPCRGMOFOOD (FP5), SIGMEA (FP6), COEXTRA (FP6) and GMOSeek (SAFEFOOD ERA-NET).

Today, therefore, there is a very wide choice of GMO analytical methods and advice for most situations (van de Bulcke et al., 2010). However, as GMO technology has advanced in both volume and application, and as new knowledge on the nature of GMOs has become available, there are several problematic areas of GMO testing that now require new research and the development of new methods in order to ensure continued consumer safety and choice. The problematic areas are as follows:

• Increased GMO volume. As the number of GMOs released increases, so does the need to screen them simultaneously and identify them in a cost-effective and accurate way.

• Unauthorized GMO proliferation. As the number of authorized GMOs increases, so too does the number in development in non-EU countries, and the risk of unauthorized GMOs entering the EU. Superficially, many of these GMOs many appear very similar to authorized ones (same marker genes) and may therefore evade current screening methods.

1.2.1 Novel GMP producing methods and risk mitigation

Plant products produced by conventional breeding are more familiar to consumers. Reverse breeding is a technology that transiently produces parental lines that are hybridized with non-transformed plants to obtain segregants devoid of the bacterial vector, and of the gene variants inducing the desired traits. Reverse breeding is applied to reconstitute parental lines starting with an elite F1 hybrid whose genetic material is unknown. Reverse breeding combines several other techniques such as RNA interference (RNAi) to suppress meiotic recombination, tissue culture to regenerate plants from cells and the double haploidization technique to create double haploid plants, which are used as the respective parental lines to produce new elite F1 hybrids.

These novel plant products (NPPs) include GMPs that do not use traditional methods of genetic transformation and may not contain stable transgenes, so that in most cases standard molecular methods cannot detect any non-self gene or mutation. Transient or secondary genetic alterations caused by the novel transformation process may still be detected (through transcriptomic analyses and next-generation sequencing [NGS]).

Another concern about transgenic crops relates to the mixing of genetic materials between species that cannot hybridize by natural means.

1.2.2 Targeted genome modification using site-specific nucleases

In the case of higher plants, an efficient RNA delivery system has not been established. Agrobacterium-mediated transformation of DNA or direct injection of DNA using a particle gun are commonly used to express proteins of interest. In Agrobacterium-mediated transformation, a single-stranded transfer DNA (T-DNA) protected by coat proteins is delivered from Agrobacterium to the plant cell nucleus, where naked single-stranded molecules are converted to double strands. These double-stranded T-DNAs are used as templates for homologous recombination (HR) (Chen and Gao, 2014; Figure 1.1). The critical step is the introduction of DNA double-strand breaks (DSBs) at given genomic sites. Engineered nucleases generate DSBs and consequently activate DNA repair to seal the breaks along with any modifications such as mutations, insertions, replacements and chromosomal rearrangements.

Figure 1.1 Schematic representation of targeted genome modifications using site-specific nucleases.

Because transcription and translation of engineered nucleases needs some time, it is not easy to harmonize the timing of expression of engineered nuclease tightly with the existence of the HR template. In addition, Agrobacterium-mediated gene targeting (GT) is only applicable to plant species in which an efficient transformation system is established. A novel GT method called in planta GT (Fauser et al., 2012; Ayar et al., 2013) promises to solve both problems. In this system, a GT donor vector flanked by two engineered nuclease recognition sites is first stably integrated into the plant genome. Site-specific nuclease (SSN) or site-directed nuclease (SDN) exploit the presence of an oligonucleotide sequence complementary to the gene region to be targeted for modification (i.e. meganuclease, endonuclease). Once the site-specific endonuclease is expressed or introduced inside the cell, it cuts within the target and also excises the chromosomal transgenic donor if the recognition sequence of an engineered nuclease present at both ends of the GT donor is the same as that in the target gene. HR between the excised GT donor vector and target locus results in in planta GT. Conventional GT approaches rely on the generation of a very large number of transformation events, while the in ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of figures
• List of tables
• About the editors
• About the contributors
• List of abbreviations
• Introduction
• 1. Transgenic, cisgenic and novel plant products: Challenges in regulation and safety assessment
• 2. What turns on and off the cytokinin metabolisms and beyond
• 3. Apple allergens genomics and biotechnology: Unravelling the determinants of apple allergenicity
• 4. Non-food interventions: Exploring plant biotechnology applications to therapeutic protein production
• 5. In planta produced virus-like particles as candidate vaccines
• 6. Biotechnology of Euphorbiaceae (Jatropha curcas, Manihot esculenta, Ricinus communis)
• 7. Regulation framework for flowering
• 8. Epigenetic regulation during fleshy fruit development and ripening
• 9. Tomato fruit quality improvement facing the functional genomics revolution
• 10. Rice genomics and biotechnology
• 11. Genome-wide DNA methylation in tomato
• 12. Recent application of biotechniques for the improvement of mango research
• 13. Cotton genomics and biotechnology
• 14. Virus technology for functional genomics in plants
• 15. PARP proteins, NAD, epigenetics, antioxidative response to abiotic stress
• 16. Applied oilseed rape marker technology and genomics
• Index