17. Transgenic Breeding

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by Zachary King, Maria Ortega, and Thomas Jacobs; Institution of Plant Breeding, Genetics and Genomics, University of Georgia

Expanding Beyond the Tertiary Gene Pool: History, Challenges and Prospects of Using Transgenes in Breeding Programs

Introduction

Long since the development of agriculture, humankind has desired to tailor plants to their liking so that they may better meet their needs. Currently, plant breeders work to produce crops to feed the masses, or support the burdening need for fiber and fuel. The process of cultivar development within a crop species is possible by the element of selection on a population, which has alleles of variable worth. Sexual recombination allows for alleles to be recombined in meiosis resulting in plants which can be selected with novel traits (Tracy, 2004). However, what does a plant breeder do if desired alleles are in distantly related plants? There are several solutions to this dilemma, and most revolve around the gene pool concept first developed by Harlan and de Wet (1971). Their descriptions of gene pools define the limits to which plant breeders can exploit genetic diversity for cultivar development (Figure 1).

The primary gene pool (GP-1) allows for crosses to be easily made, whereby hybrids are fertile and chromosome pairing is normal, thus allowing Mendelian segregation of traits. The biological species is portioned into two categories, (A) subspecies composed of agronomically used lines, and (B) subspecies, which consists of weedy or wild relatives of subspecies A. The secondary gene pool (GP-2) refers to all biological species, which cross with a different combinable species. Although mating and gene transfer are possible, there is a high probability of hybrid sterility or a lack of chromosome pairing. Additionally, hybrids can be weak and thus difficult to bring to sexual maturity. The recovery of a desired phenotype from the cross may not be possible or difficult to recover even when traits are integrated back into advanced breeding lines. In the tertiary gene pool (GP-3) although crosses are possible with the crop of interest, hybrids are lethal or completely sterile. Radical means must be taken to recover hybrids including embryo rescue, grafting, the application of mutagens to break apart chromosomes or double chromosome number. A bridge cross can also be utilized to obtain partial sterility in the F1 hybrid, known as a complex hybrid. The quaternary gene pool (GP-4) does not allow any transfer of DNA between the crop of interest or other organism by mating and sexual recombination. In this case the process of transgenesis must be used to insert a DNA sequence from any biological or synthetic source known as a transgeneinto the crop plant of interest. The DNA can be chimeric where different segments e.g. the promoter, gene of interest and terminator can be taken from any source and put together in a functional manner so that the DNA cassette is expressed once transferred into the genome of the crop of interest.

Sears (1956)demonstrated how traits containing disease resistance from distantly related species could be introgressed into cultivated varieties, a process commonly referred to as a wide-cross. In this example, TriticumaestivumL.(wheat) was crossed with an intergeneric hybrid, also known as an amphiploid, formed by T. dicoccosides (wild emmer) x AegilopsumbellulataZhuk (goatgrass). The F1 progeny were then backcrossed to the recurrent parent, T. aestivum, to produce the BC2F1, which possessed an extra chromosome (isochromosome), that carried the disease resistance. However, the chromosome carrying resistance to Pucciniatriticina(wheat leaf rust) also produced an undesirable phenotype and poor pollen performance. Plants harboring the fungal resistance isochromosome were subjected to x-rays prior to meiosis to break up the chromosome, and were subsequently backcrossed to the recurrent parent, T. aestivum. In this manner, 6,091 BC3F1 plants were produced, and 132 individuals were resistant to P. triticina (2.17%). Of the resistant plants, 17 unique translocations were present, and one of the offspring showed agronomic qualities equivalent to that of T. aestivum.

Sears, thus illustrated in 1956 a way to transfer what was then termed “alien” genes to confer a fungal resistance to cultivated wheat, using wide crosses from the tertiary gene pool (Figure 1). However, using germplasm resources in the tertiary gene pool cannot solve all of the problems facing plant breeders today. With the advent of sophisticated biotechnology tools, we can now exploit the “quaternary gene pool,” which would otherwise be inaccessible with techniques involving traditional plant breeding, even those of Sears (Figure 1).

The process of exploiting the quaternary gene pool is accomplished through genetic engineering, also referred to as transgenesis, whereby a gene or genes from any gene pool whether a bacterium, virus, plant or even synthetically derived DNA sequences can be introduced to a plant’s genome in a manner where the transgenewill be inherited by the offspring. Plants that have been engineered to contain transgene(s) are known as transgenic plants and will be the focus of this chapter. For the purpose of clarity, all genetically engineered plants will be referred to as transgenic plants, as the term genetically modified organism or GMO is incorrect, as it infers that domesticated plants or plants themselves have not experienced genome modifications, which is clearly not the case as many crop plants have experienced approximately 10,000 years of “genetic modifications” controlled by the hands of humankind (Gepts, 2002)

Over 148 million hectares (365 million acres) of transgenic crops were grown in 29 countries, planted by over 15 million farmers in 2010. The 17 countries which represent the largest hectarage of transgenic crops are listed (Table 1), as reported by James(2010). Generally speaking, the most prevalenttransgenic traits of interest still remain herbicide and insect resistance, which rank first and second, respectively(James, 2010). The adoption of transgenic crops is at a record high and continues to increase, which is evident from the 87-fold increase in hectarage since the deregulation of the first transgenic crops in 1994(Krieger et al., 2008). This chapter will describe the history, challenges, benefits, and technology behind transgenic crops in breeding programs.


Fig 1: A diagram redrawn from Harlan and de Wet (1971) to include a description of gene pools as they relate to the use of transgenes. Circles are representations of the primary- (GP-1), secondary- (GP-2), tertiary- (GP-3), and quaternary-gene pools (GP-4).


Table 21 1.jpg


Cell culture and transformation

Cell Culture and Somatic Embryogenesis, a Means to Obtain Transgenic Plants

In 1839, a botanist, Matthias Jacob Scheiden and a zoologist, Theodore Schwann, observed that cells removed from a plant were able to live freely afterwards; thereafter Schwann wrote “We must therefore… ascribe an independent life to the cell as such” (Kyte and Kleyn, 1996). Scheiden and Schwann made a critical observation, that plants possess a remarkable ability to generate free-living cells from plant tissues. Plant cell and tissue cultureusing sterile technique and in vitro (within glass) conditions are key elements to obtaining transgenic crop plants.

Somatic Embryogenesis

Currently, we know a plant cell or cells are able to live independently, and also possess the ability to regenerate into a whole plant under the right environmental cues, a phenomenon referred to as totipotentcy. For this reason, cell culture is a core technology for crop plant transformation. Single cells give rise to whole plants in one of two ways. The first methodology of generating plants from cell culture is through the process of somatic embryo formation, whereby somatic cells (those not involved in sexual reproduction), produce an embryo similar to one produced by zygotic embryogenesis. The process of somatic embryo formation, and subsequently development is termed somatic embryogenesis. Somatic embryos formed with root and shoot apical meristems are termed a bipolar embryo, and germinate into whole plants (Parrott, 2000). The process of somatic embryo formation has been used to producesynthetic seed, much like seed which comes from a zygotic embryo, whereas the former is genetically identical to the tissue source in all cases(Gray and Purohit, 1991).

Organogenesis

The second methodology of regenerating single cells into whole plants is termed organogenesis, where a meristematic cell from a root or shoot primordiumis used to form organs (e.g. shoots, leaves or roots); these recovered organs can then be cultured into whole plants(Kyte and Kleyn, 1996; Parrott, 2000).

The coupling of in vitro techniques and genetic engineering allow for the recovery of transgenic crop plants, where both use the same set of principles for success. Firstly, genetic engineering is performed on a single-cell level. Thus, totipotency and cell culture come into play, as scientists need to be able to regenerate whole plants from single cells through somatic embryogenesis or organogenesis.

Once the DNA is integrated into the cell’s genome, engineered cells need to be selected from those cells that are not engineered. This process is accomplished using selectable marker genes, or reporter genes, which allow engineered cells the ability to grow on a nutritional medium supplemented with a selection agent where cells would normally not survive (e.g. antibiotic or herbicide), or to be visualized (e.g. fluorescence from the green fluorescent protein), respectively (Hraska et al., 2006; Kaeppler et al., 2001; Kaeppler et al., 2000a; LaFayette et al., 2005; Stewart, 2001; Sundar and Sakthivel, 2008). Birch (1997) provides an excellent summary on the bottlenecks researchers face when developing transformation techniques, and how to trouble shoot general problems.

Many tools and technologies for plant transformation have been reported(Vain, 2007). However, there are two main processes for transformation:(1) particle bombardment-mediated transformation (direct transformation), and (2)Agrobacterium tumefaciens-mediated transformation (biological transformation). As of 2003, both techniques composed 25% and 57% of all transgene delivery to plants, respectively; and 82% of all plant transformations total (Vain, 2007).

Methods of Plant Transformation

Direct Transformation

There are a variety of direct DNA delivery methods to recover transgenic plants including, but not limited to, silicon-carbide whisker-mediated transformation, microinjection, protoplast transformation using polyethylene glycol and particle bombardment, see review by Altpeter et al. (2005). Direct transformation methods use physical and/or chemical methods to integrate transgenes to the plant cell nucleus, chloroplast, or mitochondria(Hunold et al., 1994; Yamashita et al., 1991).

For this review particle bombardment will be discussed as the main focus due to the prevalence of its use for the production of commercial transgenic crops, as nearly all deregulated transgenic corn and soybean varieties were engineered with the gene gun (www.aphis.usda.gov/brs/not_reg.html) (Stewart Jr., 2008; USDA, 2010). Typically the Biolistic® PDS-1000/He Particle Delivery System (PSD-1000) (Bio-Rad, Hercules, CA), also known as the gene gun (Kikkert, 1993; Sanford, 1988; Sanford, 2000), a particle inflow gun (PIG) (Finer et al., 1992; Vain et al., 1993), or the electrical discharge gun (Russell et al., 1993) are used for biolistic transformation.

Biolisitic transformation introduces DNA coated particles, known as microcarriers, into plant cells or tissues, physically using a burst of helium air within a vacuum (Kikkert, 1993). Millions of microcarriers can be introduced with a single shot, known commonly as a bombardment to the target cells or tissues, similar to a buck-shot pattern, (Kikkert, 1993). Traditionally, 1 m tungsten microcarriers were used for plant transformation. However, tungsten has a more heterogeneous size and shape distribution than gold, has been shown to catalytically degrade DNA, and can be cell-toxic (Russell et al., 1992; Sanford et al., 1993). Modern biolistic protocols typically use gold microcarriers, which are non-cell toxic, and more spherical (Kikkert, 1993). Overtime, microcarrier size has decreased; in particular, maize transformation efficiency is improved, on average over 13% when using 0.6 m gold microcarriers, as opposed to 1.0 m microcarriers (Frame et al., 2000).

Transgenic DNA is typically attached onto the microcarriers by precipitation with calcium chloride and spermidine(Kikkert, 1993; Knowle et al., 2006; Sivamani et al., 2009). Biolistic transformation offers several advantages over Agrobacterium-mediated transformations. Firstly, some cell cultures have a hypersensitive response to Agrobacterium (Kikkert et al., 2004) which will result in the death of the cell cultures. Additionally, when using Agrobacterium, it is necessary to eliminate the bacterium after transformation with antibiotics and false positives in initial PCR screening may occur due to residual Agrobacterium cells presences.

A perceived concern with biolistic transformation is the inability to produce single copy insertions; many dated gene gun methods using high quantities (micrograms) of plasmid DNA can produce high copy numbers of the transgenes, complex, or rearranged insertions and post-transcriptional gene silencing in plants. Additionally, high DNA concentrations can lead to lower transformation efficiencies (Oard, 1991) and higher amounts of transgenic lines with multiple-copy transgene insertions (Lowe et al., 2009). Efficient engineering protocols range considerably in the amount of DNA bombarded, from only a few nanograms to hundreds of nanograms per bombardment, and must be optimized for the species and project goals (Chen et al., 1998; Goldman et al., 2003; Lowe et al., 2009; Trick et al., 1997a). However, improved methods deliver low concentrations of linear DNA cassettes or plasmids (2.5 ng per bombardment), to routinely produce up to 46% single-copy transformants in maize (Lowe et al., 2009). Single copy transformants are ideal, and linear DNA cassettes allow for the “clean” insertion into the plant genome, void of superfluous vector backbone.

In summary, the cell type bombarded must be highly transformable, and in the proper physiological state to receive the microcarriers and transgene(s) with high-efficiency of transformation and low-transgene copy number, and linearized cassettes free of vector backbone are a must. Finally, the cells that receive the transgenes must be highly regenerable to create an efficacious transformation system.

Biological Delivery Methods

Besides direct transfer of DNA into plant cells, there are biological systems that can do the same thing. A. tumefaciens is a bacterium that naturally infects plants at wounded sites; typically at the crown, resulting in the creation of a crown gall. The gall produces opines which are nitrogenous compounds that the bacterium can metabolize, but the plant cannot. In this manner Agrobacterium is able to parasitize the plant(Citovsky et al., 2007).

Agrobacterium infects plants and delivers a Tumor inducing (Ti) plasmid that contains genes for opine and plant hormone synthesis. When a bacterium finds a wounded plant cell, it inserts a specific region of the Ti plasmid into the plant cell. Proteins coat the DNA strand for protection from plant endonucleases and to direct the bacterial DNA to the plant nucleus. Once in the nucleus one of the coating proteins cleaves the plant DNA and the bacterial DNA is inserted into the plant genome, using the plants DNA repair machinery (for review (Tinland, 1996)). The plant hormones encoded by the bacterial DNA stimulate the infected cells to enlarge and create the characteristic gall. Research first showed that a recombinant plasmid homologous to the bacterial Ti plasmid would also be transferred and incorporated in the host’s genome. By eliminating virulent genes from the bacterial Ti plasmid, scientists are able to generatedisarmedTi plasmids(Citovsky et al., 2007). Disarmed plasmids allow plant cell transformation using a recombinant plasmid without the production of galls. These transformed cells can then be regenerated into whole transgenic plants.

Various methods exist to transform plant cells with Agrobacterium. The simplest to date is the floral dip method used for arabidopsis transformation. Transformation of arabidopsis is crucial to research as it is usually the model of choice for identifying gene candidates.The procedure involves dipping the flowering structures into a solution of Agrobacterium(Clough and Bent, 1998). When immature ovules are transformed and pollinated the resulting seed is transgenic. Arabidopsis transformation can be done quickly, efficiently and without a tissue culture step. Unfortunately this technology has not been able to be applied in other plant systems. A similar type of tissue-culture-free transformation system has been reported in several crop species, but these reports are controversial and attempts to repeat them have failed (Shou et al., 2002; Stewart Jr., 2008)

Agrobacterium transformation remains the preferred method for many researchers and companies. This is partially due to the perception that Agrobacterium mediated transformation results in fewer single copy transgene insertions, compared to events generated with particle bombardment. However, evidence has shown just the inverse to be true (Agrawal et al., 2005; Rai et al., 2007). In terms of copy number and the complexity of the insertion, the differences may be attributed to variations in protocols between different laboratories, rather than inherent differences between the two systems.

Agrobacterium is not alone in its ability to transform cells. Indeed evolutionary evidence points to several instances where genes have been transferred from bacteria to plants and vice versa. One study has shown that bacteria species closely related to Agrobacterium are capable of transferring a Ti plasmid to plant cells (Broothaerts et al., 2005). Given the diversity of bacteria species it is quite possible that other bacteria, capable of transforming plant cells with different mechanisms, are waiting to be discovered and utilized by plant scientists.

Selecting Transgenic Events

Firstly, in order for a transgenic breeding program to be successful, a large number of independent transgenic plants need to be generated. Each independent transgenic event arises from a single engineered cell, commonly referred to as an event. The first transgenic events recovered from the engineering process are referred to as the T0 generation, whereas each subsequent generation follows in a linear fashion (i.e. T0, T1, T2 etc.). There are several reasons why selecting from multiple events is often necessary. Firstly, events with single copy insertions of the DNA vector are preferred as they allow for “marker-free” transgenic lines to be selected. In this manner plants are first transformed with two cassettes, one which contains the transgene(s) coding for the trait and the other transgene selection cassette allows for the recovery of the transgenic plant from the cell-culture process (Lowe et al., 2009). The selectable marker transgene cassette can then be segregated out if integrated at separate loci from the transgene of interest. The transgene(s) of interest coding for an agronomic trait are then integrated into elite germplasm through breeding (Lowe et al., 2009; Mumm, 2007). Transgenic lines with numerous copies of the transgene(s), and (or) vector backbone sequences can cause complications such as illegitimate recombination, or instability of transgenes, and will not pass in the current regulatory climate (Agrawal et al., 2005).

Zhong(2001) summarizes three desired characteristics of transgenic plants after initial selection. Firstly, the transgenic trait needs to have high efficacy in field conditions, which is controlled in part by how well the transgene is expressed. The event may never reach the market if the desired traits of interest do not perform at a threshold level that would allow the trait to be commercially viable (e.g. superior insect resistance, herbicide tolerance etc.).

Secondly, the transgene(s) need to follow Mendelian segregation at every generation, which should be verified at the T2 generation and have high stability under various environmental conditions and in different genetic backgrounds (Visarada et al., 2009; Zhong, 2001). In this manner, much like tradition plant breeding, a given event must be selected based on high trait expression and stability in multiple locations for several years (Gepts, 2002).

Lastly, if the crop plant of interest has a known genome sequence, special care should be taken to verify that the transgene did not insert into a known gene, which could have a negative effect on the plant’s phenotype, and therefore its agronomic worth. If endogenous gene function is destroyed or altered, key biochemical pathways could be made inefficient or destroyed altogether. Additionally, transgenes may interfere with biochemical pathways based on substrate competition, or by producing interfering compounds as a product of some step within the transgene biochemical pathway (Zhong, 2001). Overexpression or silencing of transgenes above or below certain threshold limits, respectively, may prove to be deleterious to the phenotype of the plant, thus careful considerations need to taken when evaluating transgenic phenotypes (Zhong, 2001). The insertion sites of transgenes are still largely imprecise; therefore event selection based on the insertion site within the genome, also known as position effect, is of special interest (Gepts, 2002; Visarada et al., 2009).

Molecular Biology for Plant Transformation

Overexpression Vectors

The regulation of gene expression is one of the goals in the development of transgenic cultivars. Promoters, enhancers and cis-acting regulatory sequences are all DNA elements, which control gene expression. Biotechnology has developed the ability to engineer chimeric expression cassettes, by combining promoters and coding regions from different genes. Promoters have been isolated and characterized from the cauliflower mosaic virus CaMV 35S(Odell et al., 1985), maize (Zea mays)polyubiquitin 1 with intron (ZmUbi1) (Christensen et al., 1992)and rice (Oryza sativa L.) actin1 with intron (OsAct1) (McElroy et al., 1990). These promoters are widely used to construct DNA cassettes for the constitutive expression of genes in dicotyledonous and monocotyledonous plants.

The most basic design of transformation vectors permits the cloning of a gene of interest that is driven by a specific promoter, and is often arranged in parallel with a selection marker (Davey et al., 2010). The traditional design of plant transformation vectors includes the use of the cauliflower mosaic virus CaMV 35S and the A. tumefaciensnopaline synthase gene (nos) constitutive promoters. These promoters typically drive the constitutive expression of target genes and selection markers, respectively. The presence of a limited number of multiple-cloning sites (MCS) in the expression cassettes, and their absence outside of the expression cassette makes cloning of additional genes within the same vector difficult. An ideal plant-transformation vector system should then allow the user to: (1) easily clone the target gene; (2) select from a wide array of promoter, and terminator sequences; (3) express several target genes in a single plasmid simultaneously; and (4) choose from a wide array of selection and reporter genes (Chung et al., 2005). Cloning vectors that are designed to facilitate these requirements are currently available (Curtis and Grossniklaus, 2003; Earley et al., 2006; Goderis et al., 2002; Karimi et al., 2005).

Since the integration of foreign genes in the T-DNA region of the Agrobacterium Ti plasmid, transformation vectors have evolved into disarmed binary and super-binary vectors (Davey et al., 2008; Davey et al., 2010; Komori et al., 2007). For plant transformation, a binary vector system is typically used in combination with a disarmed strain of A. tumefaciens carrying a Ti plasmid,which wild-type T-DNA was replaced by an artificial T-DNA that allows the plasmid to be replicated in Escherichia coli and A. tumefaciens.These vectors are one of the most popular tools in the plant transformation community. The most recent modification of binary vectors have provided scientists with useful features such as a wide selection of cloning sites, high plasmid copy number in E. coli, high cloning capacity, improved compatibility with bacterial strains, a high diversity of plant selectable markers, and high frequency of bacterial transformation (Komori et al., 2007). Super-binary vectors were designed taking in account that Agrobacterium virulence genes have gene-dosage effects (Jin et al., 1987), and the presence of additional virulence genes (Komari, 1990)enhances the transformation efficiency of several plant species, especially those species recalcitrant to transformation, such as some cereals (Hiei et al., 1994; Ishida et al., 2004).

Promoters for the Expression of Transgenes

Venter(2007)stressed the importance of promoters in vector construction; as the choice of a promoter sequence and its optimization determines the constitutive, spatial, and/or temporal expression of transgenes. Efficient transgene expression is achieved using plant-derived promoters; or promoters known to be active in plant cells, such as those from of viruses. Constitutive transgene expression at the incorrect time may have a negative effect in plant development, therefore, tissue specific promoters have been characterized to control the spatial and, or temporal expression of a transgene. Among the currently available plant tissue-specific promoters are those belonging to fruit ripening and seed-specific genes (Zakharov et al., 2004), including the seed storage glutenin genes (Qu et al., 2008); glycoproteins in tubers and roots; and flower specific promoter (Annadana et al., 2002). Some plant promoters are induced by biotic and abiotic stresses (Pino et al., 2007); including wounding (Yevtushenko et al., 2004), iron deficiency (Kobayashi et al., 2007), and exogenously applied chemicals (Peebles et al., 2007). Synthetic promoters, designed to contain only defined regulatory elements, therefore reducing complexity of expression profiles are available. These promoters were evaluated for their potential use in pyramiding transgenes, where they may reduce homology-dependent gene silencing due to minimum sequence similarity with plant gene promoters (Oszvald et al., 2008). However, some synthetic promoters are non-suitable for plant transformation (Song, 2008). For a review of promoters used in plant transformation refer to Potenza et al. (2004).

Although a wide array of promoters have been characterized for their suitability in different plant species transformation systems, promoter development is still a young field. The recent advances in genomics, transcriptomics, and proteomics will likely contribute to the discovery, and characterization of a wider array of plant promoters. High-throughput genomic approaches have been used for the identification of promoter sequences in rice (Yu et al., 2007); and similar approaches are being developed for the identification, and characterization of regulatory sequences in other crop species (Amarasinghe et al., 2006; Hernandez-Garcia et al., 2010).

Promoters that allow the allocation of transgene products, spatially or temporally in planta, have been also used in the development of transgenic cultivars. This will be the case of promoters driving the expression of genes able to modify the profile of seed storage components in grain tissues, which are adapted for the stable accumulation of proteins, lipids or starch (Shewry et al., 2008). The promoter of the glutenin subunit 1Dx 5 drives its strong expression in the wheat endosperm. This promoter has been proposed as a candidate for the modification of protein profile of the wheat grain (Lamacchia et al., 2001). The promoter of genes such as the wheat ADP glucose phosphorylase(Thorneycroft et al., 2003), and storage albumin and globulin gene families have also been identified driving expression in the endosperm of several plants, including flax (Truksa et al., 2003), and rice (Wu et al., 1998).

RNA Interference

Many agronomically beneficial traits are caused by reduction or elimination of gene expression (Gepts, 2002). One example is the yellow seeded soybean. Soybeans are naturally rich in isoflavonoids, which results in a black seed coat found in many wild and non-adapted varieties. The yellow seed coat found in many cultivated varieties is due to the elimination of isoflavone precursors. It has been shown that dominant alleles in the I locus of soybean are responsible for the elimination of chalcone synthase (an isoflavone precursor) messenger RNA, and the resulting yellow bean phenotype (Tuteja et al., 2009). This is but one example.Being able to down regulate any plant gene would surely give scientists great control over plant phenotypes.

With improvements in gene sequencing and discovery, thousands of genes have been identified in the major crop species. Determining the function of newly discovered genes is done by the production of mutants defective in certain pathways to evaluate the effect of the gene in the plant. Individuals of plants with reduced or eliminated expression of a single gene are typically called loss of function mutants or knockout mutants. There are several ways in which to make knockout mutants. Screening large populations for mutants has been historically important, however this method is very inefficient as the natural mutation rate in plants is low. Mutagens such as ionizing radiation orethylmethanesulfonate (EMS) have been employed for the past century. However, these mutagens require the screening of very large plants populations to find the mutation mutant of interest. Koornneef et al. (1982) found the mutation rate using EMS in arabidopsis to be on average 2 X 10-4 per locustested, and even lower rates for x-rays and fast neutrons.Furthermore, these methods generally result in several mutations at once, making downstream analysis very difficult. Another difficulty is that these mutations are typically recessive, thus requiring inbreeding or several rounds of crossing for the mutations to even be detected.

Recent research in gene silencing has resulted in the production of transgene cassettes capable of silencing specific genes in a dominant fashion. This process is termed RNA interference, or RNAi, and results from the production of double-stranded RNA (dsRNA) in the plant cell. The dsRNA produced specifically prevents the production of protein from messenger RNA of the target gene [for review, (Bucher and Prins, 2006; Eamens et al., 2010)]. These technologies circumvent the problems of generating knockout mutants described above, but are still functionally identical to them.

Although the mechanism was unknown at the time, the very first transgenic plants released for crop production were the product of gene silencing events. The FlavrSavr tomato was the result of silencing a gene responsible for fruit ripening, thereby extending the shelf-life of the transgenic tomato(Krieger et al., 2008). The second deregulated transgenic plant was the virus resistant squash, Freedom II. By silencing the viral coat proteins from the zucchini yellow mosaic virus and the watermelon mosaic virus in squash, the virusescould no longer replicate itself within the cells of the transgenic squash, as the virusescould no longer produce their coat proteins. Thus,Freedom II is a variety of squash that has effective immunity to virus infection (APHIS/USDA, 1994).

Research is currently underway to produce crops that are resistant to other diseases as well. Transgenic maize expressing a silencing vector were shown to be resistant to the coleopteran western corn rootworm (Baum et al., 2007). Work is still underway for fungal (Nowara et al., 2010), and nematode (Sindhu et al., 2009) resistance.

Zinc-Finger Nucleases

Generally plant transformation technologies rely on random insertion of transgenes into plant genomes. This randomness can disrupt important genes or pathways and can be linked to deleterious traits, limiting future breeding efforts.One of the best documented examples is Monsanto’s Roundup Ready® soybean lines, which contain on average, a 5-10% yield loss compared to isogenic lines that do not contain the insertion (Elmore et al., 2001a).Transgenes may be linked with undesirable alleles making future breeding goals difficult to achieve. Given these constraints, a large number of independent events must be produced in order to find one with high expression and good field performance.

Zinc-finger nuclease technology allows for constructs to be designed to target transgene insertion into a specific locus within the plant genome, avoiding many of the problems addressed above. To employ zinc-finger nuclease technology researchers first engineer plants with zinc-finger nuclease constructs. The transformed plants produce zinc-finger nucleases that target loci specified in the construction of the zinc finger. The zinc fingers then cut both strands of DNA at the locus and then through recombination, insert in the transgene (for review (Urnov et al., 2010)). Zinc fingers can also be used to produce knockout mutations by disrupting the sequence of the target gene; i.e. causing a frame-shift mutation, elimination of the start codon or important motif, or a complete removal of the target gene(Zhang et al., 2010). Also, researchers can target a known gene with zinc fingers and replace the sequence with a selectable maker(Shukla et al., 2009). The transgene insertion destroys the function of the endogenous gene and the selectable maker allows one to select only transformed cells (Osakabe et al., 2010). The zinc finger transgene, which is most likely inserted at a separate locus, can then be segregated out from the mutation it caused; thus resulting in a modified plant, without any transgenic sequences.

The benefit of this approach is the lack of recombinant protein, which should be advantageous when petitioning for deregulation. While the advantages of site directed gene insertion are quite profound, the efficiency of the system is quite low.Osakabe et al.(2010) showed that out of the transgenic arabidopsis lines recovered, only 0-3% of the progeny had germinal mutations in the target gene. Furthermore the design of the zinc-finger nucleases is a limiting step. Of the thirty-two zinc fingers designed by Townsend et al. (2009), only three were able to bind DNA. The upfront screening assays required make zinc-finger nuclease technology unappealing for many researchers (Shukla et al., 2009).

Breeding with Transgenics

Backcross Breeding with Non-Elite Germplasm

Currently, most transformation programs still use non-elite genotypes, as most transformation procedures remain genotype specific due to the low tissue culture response and transformation frequencies observed with elite genotypes(Gepts, 2002; Mumm, 2007; Parrott et al., 1989; Visarada et al., 2009; Zhong, 2001).

For these reasons the principal strategy used to incorporate transgenes into crop species is backcross breeding to elite genotypes, which are used as the recurrent parent. Firstly, backcrossing lowers mutations acquired throughout the cell culture process, known as somaclonal variation, which can have negative agronomic effects (Dale and McPartlan, 1992; Zhong, 2001). Backcrossing performed with transgenic lines follows the same regiment as traditional backcrossing methods. When introgressing transgenes into an elite background using molecular markers, it is critical to monitor the trait expression in the progeny to ensure the trait is not suppressed, and guarantee optimal functionality in different backgrounds, or generations (e.g. using insect challenge assays, or herbicide sprays, etc.).

The backcrossing process is completed in order to give an existing line, known as the recurrent parent, the transgene(s) from the non-elite donor parent (Fehr, 1987). The backcrossing process is repeated as needed to recover the phenotype of the recurrent parent while minimizing the chromatin contribution from the donor, as only the transgenes are of interest. Fortuitously, transgenes are perfect markers for marker-assisted backcrossing (MABC), as the transgene(s) per se, can be easily screened by the polymerase chain reaction (PCR). In this way the transgene(s) can be backcrossed into an elite inbred or other elite parents based on mating system of the plant (self-pollinating or outcrossing) to establish a BC2 population. For each cross, the transgenic lines are typically the pollen donors. In order to reduce linkage drag from the donor parent, recurrent parent markers can be utilized for background selection and recombination markers can be used to verify the disruption of the donor parents’ chromatin around the transgene due to crossover event(s) in meiosis (Iftekharuddaula et al., 2011; Young and Tanksley, 1989).

Iftekharuddaula et al. (2011) illustrates an excellent use of MABC, where a gene from a weedy rice (Oryza sativa L.) relative was integrated into elite varieties rapidly and efficiently using a non-transgenic approach. The authors used 116 simple sequence repeat (SSR) markers for background selection of their elite parent, and six donor markers to select BC2F2 progeny (three crosses total) with their SUB1 allele for flood tolerance from the donor parent and 99.8% of the recurrent parent’s genome, wherethe average recovery of the recurrent genome is 93.75% (Fehr, 1987). The authors credit the success in part to the 1,421 BC1F1population initially generated which captured a recombination event on both sides of the SUB1 locus, coupled with a high density of background markers to recover the recurrent parent genome. This same strategy can be used in transgenic breeding to rapidly and effectively recover a commercial line for deregulation.

Fehr (1987) states general guidelines for successful backcross breeding, which require from one to more than five backcrosses. When applied to selecting transgenic lines the number of backcrosses is (1) based on the unwanted level of chromatin linked to the transgene, (2) the importance of the recurrent parent phenotype, (3) the amount of selection imposed during recurrent backcrosses, (4) andthe level of eliteness of the donor parent.

Additional Transgenic Breeding Strategies

Pedigree method, and single seed descent can be used for forward breedingto develop inbred transgenic lines (Mumm, 2007), however, forward breeding is only favorable if the transgene donor also contains favorable allele(s) other than the transgenes per se. Due to the lack of elite, transformable genotypes, backcross breeding would likely be preferred. Additionally, the deregulation process can take many years, thus making nearly every transgenic line no longer elite by the time it reaches the current commercial market (Wayne Parrott, personal communication).

Pyramiding transgenes at separate loci would favor backcrossing, although transgenes can also be pyramided as a single DNA cassette or multiple cassettes, which allows for easy breeding schemes, less crosses and potentially less cost in the regulation aspect of generating a transgenic cultivar, which is currently estimated at≤100 million US dollars (Monsanto, 2005; Mumm, 2007; Naqvi et al., 2010).

In conclusion, when selecting a breeding strategy Mumm(2007) has outlined several considerations which have been generalized to the issue at hand (Table 2). (1) Forward breeding can be employed to derive new lines to create better hybrids [e.g. in Zea mays (maize) breeding], or breed new lines while simultaneously introgressing transgenes, as with outcrossing species. (2) If elite hybrids are already developed, backcross breeding can integrate transgenes into the recurrent parent while minimizing the chromatin of the donor parent, especially if using MABC. (3) Combinations of both backcross and forward breeding programs can be run in parallel. In this was the breeding population, or inbred lines are improved using traditional breeding methods, while the event is integrated into elite germplasm to reduce linkage drag. The transgenes are thenintrogressed from an elite parent into an elite breeding population or elite inbredsdeveloped through forward breeding.


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Effectively Breeding With Transgenes

Effects of Genetic Background on Transgene Expression

Just as different genetic backgrounds can influence the expression of a trait, so too can transgenic expression be affected. A transgene may be expressed in the transformable line, but when transferred to an elite line, expression could be reduced or highly variable. When tomato plants engineered to express miraculin, a synthetic sweetener, were crossed to different lines, there was a reduction in the total amount of protein per fruit (Kim et al., 2010). This was attributed to the amount of tissue where the protein accumulated. In the transformed line miraculin content was highest in the exocarp (the outermost layer of a fruit). This pattern of expression was maintained in the progeny; however the other parent had a smaller exocarp in the mature fruit. The size of the exocarp influenced the total amount of miraculin accumulated and therefore resulted in reduced yield of miraculin in the progeny of the cross.

Just as variation in transgene expression can occur between different genetic backgrounds, variation can occur within a single background and even within progeny of a single event. Variation between separate events of the same transgene is a common problem. This phenomenon is usually attributed to the position of the insert in the genome, where it is thought that inserts into heterochromatic locations will have a reduced expression(Butaye et al., 2005). Other possibilities include the complexity of the insert as described above. Furthermore, many transgenes can be controlled by epigenetic factors resulting in different patterns of expression. This can occur between transgenic events, or even within clones of the same event. However this variability in expression is not a dead end. A transformation program that employs some simple breeding techniques can create transgenic lines with high levels of expression.

Transgenes, just like native genes, can be selected for levels of expression. Breeding experiments on white clover using recurrent phenotypic selection have shown that transgene expression can be selected for, and enhanced over several generations (Schmidt et al., 2004). Interestingly in this study, the expression of the reporter gusA gene, which is linked to the kanamycin selection gene, did not correlate withits expression.

Quantification of Transgene Expression

The generation of transgenic plants has become efficient for a wide range of crop species, therefore the focus of transformation technologies has started to shift towards the optimization of transgene expression (Butaye et al., 2005). Significant differences have been found in the expression level of transgenes among plants produced under identical conditions, and using the same gene construct. It is also known that plant transformation may result in a high frequency of transgenic plants with undesired and unpredictable transgene expression. In the case of transgenic plants containing the gusA gene driven by the CaMV 35S promoter, a bimodal pattern of gene expression has been identified. This pattern is characterized by multiple low-expressing, and few high-expressing plants (De Bolle et al., 2003; Hobbs et al., 1993). It has also been demonstrated that transgenic plants selected for a desirable level of gene expression might lose this trait after subsequent generations (Bhat and Srinivasan, 2002; Scheid et al., 1991; Vain et al., 2002). Variation of transgene expression is undesirable, as it requires the screening of large sets of transgenic plants for the identification of individuals, whose expression level is acceptable, and phenotype is desirable. Another constraint of the variation in transgene expression is that it complicates the interpretation of transgene effects.

Variation of gene expression has become one of the major constraints of plant transformation. One of the major challenges of plant biotechnology is to develop methodologies conducive to the generation of a high proportion of transgenic plants exhibiting a desirable and stable phenotype, while minimizing variation in gene-expression (Butaye et al., 2005).

Several factors responsible for variable transgene expression have been identified. One such source of variation is associated with the transgene copy number of individual plants. Multiple transgene copies tend to integrate into one, or only a few genomic regions (De Buck et al., 1999; Pawlowski and Somers, 1998). Increased transgene copy number can result in higher expression level[review by Altpeter et al. (2005)]; however, multiple-copy integration patterns are often associated with low levels of expression, this is particularly noticeable in the case of tandem repeats (Jorgensen et al., 1996; Sijen et al., 1996; Wang and Waterhouse, 2000), and inverted repeats (Depicker and Van Montagu, 1997; Hobbs et al., 1993; Meyer and Saedler, 1996; Stam et al., 1997). In previous cases, reduced transgene expression has been associated with the interaction of the transgene with homologous sequences. However, low levels of expression have also been observed when the multiple transgene copies are present at unlinked sites. The reduction in expression of multiple copies may occur at the transcriptional or translational level, and is believed to be one of the major factors affecting transgene expression in plants. Another factor associated with variations in gene expression is the transgene integration position within the genome, as integration may happen in regions with high or low transcriptional activity, or close to transcriptional regulators (Matzke and Matzke, 1998). Besides transgene copy number, silencing, and position effects; there are other factors such as somaclonal variation, which add variation of transgene expression (Duncan, 1996; Kaeppler et al., 2000b; Phillips et al., 1994). Additionally, the regulatory elements chosen for the construction of the transgene expression cassette, such as the promoter and terminator play a critical role in transgene expression (Beyene et al., 2011; De Bolle et al., 2003; Schledzewski and Mendel, 1994).

Methodologies for the Analysis of Transgenic Plants

Kohli et al., (2010)reviewed the currently available techniques for the identification of transgenic plants. The majority of transformed tissue is identified on the basis of the phenotype (i.e. antibiotic selection during tissue culture or herbicide resistance) conferred by the transgene. This is strong evidence that the transgene has integrated in the targeted genome, and is functional. The most definitive proof of the transgene integration is a Southern blot, which serves as a genetic signature of a particular transformed plant and its descendants, and allows the estimation of transgene copy numbers. PCR is also used to confirm the integration of transgenes, however false positives may appear from the amplification of non-integrated plasmid DNA from Agrobacterium, hence it is usually used as an indicative technique. DNA sequencing provides the best resolution for the analysis of transgenic plants, permitting the characterization of the structural organization of transgene loci, and their possible rearrangements. The availability of high-throughput sequence technologies and reference genomes from crop species provides an adequate tool for the analysis of entire genomes from transformed plants; this has been the case of the sequencing of the SunUp transgenic papaya (Ming et al., 2008).

Gene Flow

A major concern of releasing transgenic plants into the environment is the possibility of resistance genes (e.g. herbicide or insect resistance) spreading to wild, or weedy relatives of the transformed crop. Such an occurrence could seriously impact a farmer’s ability to control weed problems, and lead to shifts in the genetic compositions of wild species (Chapman and Burke, 2006). The potential of gene flow from transgenic crops to their weedy relative(s), is limited to environments where both are grown in the same location. For most transgenic crops such as corn and soybean this is not a problem unless they are grown in their respective Centers of Origin; Central America or eastern Asia, respectively. Besides gene flow from transgenic cultivars to wild relatives, gene flow from a transgenic cultivar to non-transgenic cultivars could create problems for farmers when they go to sell their seed. If their target market wants transgenic-free products, and is aware that the crop contains some transgenic material, they may not purchase the seed from the farmer.

To date much attention has been paid to gene flow via cross-pollination, but additional pathways exist. Seed dispersal is another mode by which transgenes can flow. Seeds may spread by(1) improperly cleaned equipment, (2)movement of seed, (3)persistence in a field, and(4) human error during planting. For some crops vegetative cuttings may also serve as a source of transgene flow (Mallory-Smith and Zapiola, 2008); however this is limited since most crops are annuals. Indeed investigations into transgene flow in cotton, a primarily self-pollinated crop, haverevealedgeneflow via seeds is a more serious concern than pollen flow (Heuberger et al., 2010).

In corn, which sheds relatively large amounts of pollen and is wind dispersed, gene flow was shown to be limited to 5-15 meters of the pollen source, and most pollen was found within 5 meters of the plant(Robson et al., 2011). The greater distance of spread was correlated with the direction of wind flow. These data show that while gene flow can occur, it is generally limited to the immediate surrounding area. Given proper buffering distances, prevention of gene flow can be limited to a reasonable extent.

Transgenic Breeding Case Studies

Breeding with a Self-Pollinated Crop – Herbicide Resistant Soybean

Soybean [Glycine max (L.) Merr.] is grown in more than 50 countries and is the leading oilseed crop for worldwide production and consumption(Wilcox, 2004). The scale of soybean production, and the crop’s economic importance have driven substantial private and public research efforts for the crop’s improvement. Although conventional breeding is the mainstay improvement strategy of soybean (Sleper and Shannon, 2003), genetic transformation has been an important component for its improvement. A remarkable example is the development of Roundup Ready® soybean, which became the most extensively planted transgenic crop worldwide, and is the product of some of the first successful transformations events in soybean (Padgette et al., 1995). In 2008, 62,47 million hectares of herbicide tolerant soybeans were planted worldwide (Brookes and Barfoot, 2010).

Soybean was once considered to be recalcitrant to tissue culture, regeneration, and transformation. Today, soybean transformation is possible with an array of transformation methods including Agrobacterium-mediated transformation, and microprojectile bombardment-mediated transformation using somatic embryos as the target tissue; these methodologies are extensively reviewed(Clemente and Klein, 2004; Olhoft and Somers, 2007; Trick et al., 1997b; Widholm, 2004; Widholm et al., 2010). After transformation, plants are regenerated via either somatic embryogenesis or shoot organogenesis (Olhoft and Somers, 2007; Trick et al., 1997b). Soybean transformation technologies have been employed to confer economically important traits including herbicide resistance, modified seed oil and protein composition, nematode resistance, isoflavone content, insect resistance, disease resistance, and low phytate content (Widholm et al., 2010). Herbicide resistance is the most commercially important trait that has been introduced to soybean (Table 3).

In 1970, Glyphosate, a broad-spectrum and highly translocated herbicide was discovered (Franz et al., 1997). This herbicide inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the plant shikimate biosynthetic pathway leading to the production of essential aromatic amino acids required for the synthesis of phenolics, lignins, tanins, and other phenylpropanoid products(Senseman et al., 2007). In 1983 Monsanto, in collaboration with Washington University, isolated the A. tumefaciens strain CP4, which is highly tolerant to glyphosate because its ESPSP enzyme is less sensitive to inhibition by the herbicide(Watrud et al., 2004). In 1986, researchers inserted the CP4 epsps gene into the soybean genome; and 10 years later, glyphosate tolerant soybean was commercialized as the Roundup Ready® cultivar. With the rapid adoption of glyphosate tolerant crops, the use of this herbicide became a simple, effective, and relatively inexpensive method to control weeds compared to traditional methods (Green, 2009). Roundup Ready® soybean was rapidly adopted; ten years after its introduction composed nearly 60% of the world’s soybean acreage (Konduru et al., 2008), and currently represents more than 90% of soybean acreage in the US (http://www.ers.usda.gov/Data/ BiotechCrops).

Soybean Event Selection

Three strategies where evaluated to introduce glyphosate resistance into crop species: (1) over-expression of sensitive target enzymes, (2) detoxification of glyphosate molecules, and (3) expression of an insensitive form of the target enzyme. The method that was used to generate the glyphosate resistant soybean was the introduction of an insensitive form of the target enzyme in the soybean genome. The first glyphosate resistant soybean was the GST 40-3-2 event, which was field tested in 1991, and was first released in 1996 as Roundup Ready® soybean. The adoption of the technology was very rapid, with glyphosate tolerance now present in over 1,000 soybean varieties (Green, 2009). GST 40-3-2 was produced using particle bombardment-mediated transformation of the cultivar A5403 with the cp4 epsps gene from A. tumefaciens(Padgette et al., 1995). The plasmid used for transformation was PV-GMGT04, carrying the cassettes for the expression of the cp4 epsps gene, and the β-glucuronidase (gusA) gene that was used as selectable marker. In the cp4 epsps cassette, the production of the EPSPS enzyme was regulated by the CaMV 35S promoter, the nosterminator from A. tumefaciens, and the CTP4 chloroplast transit peptide from Petunia hybrida; this resulted in the expression of cp4 epsps gene throughout the plant life cycle, and the accumulation of CP4 EPSPS in the chloroplast where the shikimate pathway is localized. It was determined that the GTS 40-3-2 event did not have a significant effect in the morphology, agronomic characteristics, and weediness of the soybean plant. Many growers found that glyphosate tolerant soybean yielded less that conventional varieties, and university research also indicated that they yielded from 5 to 7% less than conventional varieties. It was never determined if the yield loss was due to linkage drag from A5403, the glyphosate tolerance trait per se, or the insertion point of cp4 epsps in the soybean genome (Elmore et al., 2001b).

The event MON89788 ushered in the new generation of glyphosate tolerant soybean currently marketed as the Roundup Ready 2 Yield® cultivar, and the first new transgenic trait in soybeans for more than a decade; it was approved by regulatory agencies in the USA since 2007. It is expected that Roundup Ready 2 Yield® will have higher yields than Roundup Ready soybean. Roundup Ready 2 Yield® soybean contains the same epsps gene used in Roundup Ready soybean, with a different insertion site in the soybean genome, and stronger promoters and regulatory elements that enhance the expression of the ESPS gene in male reproductive, and vegetative tissues (Watrud et al., 2004). In contrast with GTS 40-30-2, MON89788 was produced by Agrobacterium transformation of tissue from the elite cultivar A3244. The plasmid GMGOX20 contained the cp4 esps gene regulated by the Figwort Mosaic Virus 35S promoter enhancer, and the A. thaliana tsf1 leader and intron. The transit peptide CTP4 from the epspsgene from P. hybridawas replaced by CTP2 from the epsps gene from A. thaliana. This event did not have a significant effect in the morphology, agronomic characteristics, or weediness of soybean. Although MON89788 exhibited significantly reduced height when compared to A3244.


Fig 2: Monsanto’s breeding scheme for the production of MON89788. All generations are self-pollinated . R1 generation was used for selection of homozygous. R5 seeds were used for commercial development, or regulated field trials. Generations R5, R6, R7, andR8 were used form molecular stability analysis. Figure redrawn from (USDA, 2010).


Table 21 3.jpg


Breeding with the Model Crop – Insect Resistant Maize

The following is based on information made available from the Petition for Determination of Nonregulated Status of Ciba Seeds’ Corn Genetically Engineered to Express the CryIA(b) Protein from Bacillus thuringiensis subspecies kurstaki prepared for the USDA (http://www.cera-gmc.org).

One of the first deregulated transgenic plants was B. thuringiensis(Bt) Corn, engineered to be resistant to lepidopteran insects, specifically the European corn borer (ECB) Ostrinianubialis. The Bt gene is derived from the soil bacterium B. thuringiensis subspecies kurstaki, which produces an endotoxin crystal protein [Cry1A(b)]. When ingested,[Cry1A(b)] creates pores in the midgutof lepidopteran insects, which results in death. The crystal protein is highly selective as it is only active in alkaline environments and binds to specific receptors present in the midgut of lepidopteran insects. Prior to the development of Bt Corn, Bt had been produced and used effectively for decades as an insecticide sprayed directly on crops.

The justification for the production of Bt Corn was the extensive damage ECB causes to corn on a yearly basis. Thiscatepillarfeeds by tunneling through the leaves and stems of corn, which can increase lodging, and allows bacteria and fungi to infect the plants. At the time of writing the petition (1994), it was estimated that the cost of ECB damage was $50 million annually in the state of Illinois. In highly infested fields, yield loss could be as high as 30%. Insecticide application is costly, hazardous, and could also kill non-targetand beneficial insects. With these concerns in mind Ciba Seeds (a division of Ciba-Geigy Corporation) developed a corn event that would produce the Bt toxin in the plant tissue.

The proprietary elite inbred line CG00562 was used to generate the transgenic event through biolistic transformation. The explants were co-transformed with two plasmids. One plasmid contained two truncated Bt genes, one under the control of a green-tissue specific promoter, and the other under a pollen specific promoter. These allow the expression of Bt in tissues where ECB is known to feed. The second plasmid contains the selectable marker bar, which confers resistance to the herbicide phosphinothricin (glufosinate).

Clonal plants from event 176 were used in an initial insect-feeding assay to determine the effectiveness of the trait. The event 176 was crossed to non-transgenic inbred line CG00562, as well as 19 other elite inbred lines that represent a wide range of heterotic groups. Segregation analysis in the F2, BC1, and BC2, showed that Bt and bargenes were dominant traits inherited in a Mendelianfashion. Southern hybridizations were performed using the Bt gene as a probe and the same banding pattern was observed in each of the plants regardless of the cross. Restriction fragment length polymorphism analysis determined that both,Bt and bar genes,were inserted within chromosome 1. These two genes are tightly linked; in one assay, out of 3,240 hybrid plants, only 5 (0.15%) plants were tolerant to herbicide, but susceptible to ECB.

The agronomic performances of the lines derived from event 176 were evaluated in 1992, 1993, and 1994. Isogenic lines homozygous for Bt were created and compared to crosses from non-transformed CG00526 and the other elite lines. After infestation with ECB, transgenics containing the Bt gene had significantly less damage in comparison to the non-transgenic control plants, which had broken midribs and lesions, signs associated to ECB damage. Throughout the season, those lines containing the Bt gene stayed green and healthy, while the controls were killed.

Multi-location testing in 1993 (5 locations) and 1994 (8 locations) measured the effect of ECB damage on yield. There was a significant reduction in the amount of damage on Bt lines compared to control lines. However, there was not always a significant difference in yield. In all cases, the Bt lines either yielded just as well, or significantly better than the controls.

Large scale yield trials at 42 locations in naturally infested fields demonstrated the effectiveness ofBt corn compared to the non-transgenic variety. On average, Bt corn yielded 13.78 more bushels per acre more than control lines even though ECB was not a major problem throughout the United States in 1994. At each of the locations trained agronomists and plant breeders observed that other than resistance to ECB and glufosinate, the Bt plants behaved similarto non-transgenic corn plants.

Currently, 63% of the corn planted in the US contains a Bt gene either by itself, or as a stacked trait (www.ers.usda.gov). It has been calculated that Bt corn has saved growers approximately $6.9 billion from 1995-2009 (Hutchison et al., 2010).

Breeding an Obligate Outcrossing Polyploid – Herbicide Resistant Alfalfa

The following is based on information made available by the USDA (USDA, 2010) and (agbioworld.org). Alfalfa (M. sativa ssp. sativa) is a tetraploid forage grass (2n = 4x = 32) and is currently grown as a high quality forage grass, primarily for dairy cattle and horses, on 10 million hectares (25 million acres) of U.S. land, and ranks third in crop value at 8 billion dollars.

Roundup Ready® Alfalfa (Medicago sativa L.) events J101 and J163 were filed for non-regulated status by Monsanto and Forest Genetics Incorporated and were approved January 2011. The T0 events, J101 and J163, acted as the donor of the epspsgene, and were generated using a conventional, transformable genotype known as R2336. The events have the 5-enolpyruvylshikimate-3-phosphatesynthase (epsps) gene that confers resistance to the broad-spectrum herbicide glyphosate, the active ingredient in the Roundup® herbicide. The transgene was delivered by A. tumefaciens-mediated transformation (strain CP4). Both events were transformed and contain a single intact insertion of the epspsgene, and none of the vector backbone is present outside of the T-DNA borders.

When in field conditions, it was noted that both events were phenotypically equivalent to commercial populations and contained no additive weediness. Additionally, events were equally tolerant of diseases and pests as conventional alfalfa. Furthermore, forage equality was substantially equivalent to conventional varieties, and there are no native relatives of alfalfa in the United States, which limits the potential of gene flow to non-transgenic varieties, or feral alfalfa. Over two breeding seasons, events J101 and J163 showed transgene stability as verified by Southern blot hybridization, and Mendelian segregation, as well as trait quantification expression, which monitored EPSPS protein production in fresh tissue of the events.

The companies promoted Roundup Ready alfalfa to have many advantages over standard commercial varieties including; easy stand establishment, ease of cropping on marginal lands, increased seed purity in stands, a low-environmental risk for weed control, and flexibility of weed control based on an as needed basis.

Separately, each event was used in a modified backcross method to the elite cultivar Fall Dormancy 3 (FD3) to generate the BC3F1, where each genotype was a heterozygote for the epspstrait. For each cross, the transgenic lines were the pollen donors. The populations were crossed (J101 BC3F1 x J163 BC3F1) to generate a synthetic cultivar with 95% resistance to glyphosate herbicide (Figure 3).


Fig 3: Monsanto’s breeding scheme for the production of Roundup Ready alfalfa using backcross breeding with two events, J101 and J163, to release a synthetic cultivar (USDA, 2010).


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Glossary

Agrobacterium tumefaciens:A rod shaped, gram-negative soil dwelling bacterium used to transfer transgenes to plant cells for the generation of transgenic plants.

Backcross breeding: A breeding method for the introgression of transgenes into an elite genotype from a transgenic event. This is routinely done because the transformable genotype is typically a poor agronomic performer.

Cassette: The transcriptional unit of a gene of interest within a vector, including everything from the start of the promoter sequence to the end of the terminator sequence.

Cis-acting sequence: Regulatory sequences that are recognized by DNA binding proteins and affect the expression of adjacent genes on the same DNA molecule.

Coding region: The portion of a gene composed of exons that encodes a protein.

Constitutive expression: Refers to a gene that is transcribed at a constant level.

Disarmed plasmids: Plasmids that have lost their T-DNA, but still retain virulence genes,which allows the transference of transgenic plasmids containing left and right borders into plant cells. Disarmed Agrobacterium strains are able to transform plant cells without creating a gall.

Elite genotypes: Genotypes, which have high agronomic qualities and yields, allowing for the commercial production of crops or use as desirable parents in breeding programs.

Enhancer: DNA sequences that promote gene transcription.

Forward breeding:A breeding approach aimed to generate new lines while simultaneously introgressing transgenes.

Gene pool concept: First proposed by Harlan and de Wet (1971) to characterize crop plants and their related species in regards to their cross compatibility and ability to recombine gene pools.

Knockout mutants:Mutant lines that lack the expression of certain genes. These are useful for genetic analyses.

Microcarriers: The microscopic metal particles coated with DNA that deliver transgene(s) to the nucleus, chloroplast, or mitochondria of a plant cell.

Multiple cloning site: Small region of DNA that contains many restriction sites that is used for gene cloning.

Non-elite genotypes: Genotypes which have poor agronomic qualities, and yields that are below average which would not allow for their commercial production or use as parents in breeding programs.

Opines: Low molecular weight compounds encoded by Ti plasmids in Agrobacterium that serve as a nitrogen and energy source for the bacterium, but cannot be metabolized by plants.

Organogenesis: The production of shoot or root meristems, without somatic embryogenesis, which can be cultured to recover whole plants.

Particle bombardment: Process where DNA is introduced to the nucleus, chloroplast, or mitochondria of a plant cell. The DNA is delivered mechanically on microscopic metal particles (often gold) coated with the DNA.

Plant cell and tissue culture: The in vitro manipulation of plant cells and organs to generate transformable cells or tissues, which can be genetically engineered and subsequently regenerated into whole plants. Additionally, cell culture can be used to rapidly and effectively propagate elite genotypes.

Promoter: The DNA region, usually upstream to the coding sequence of a gene, which functions as a regulator of gene expression, and determines the start site of transcription.

Pyramiding transgenes:Stacking more than one transgene into a plant.

Recalcitrant to transformation:A genotype which has poor tissue culture response or transformability.

RNAi: RNA interference is the term coined when RNA negatively regulates the expression of a gene.

Selection marker: A gene engineered into a vector that allows the selection of transformed tissue.

Single copy transformant:A transgenic plant with a singletransgene integration site inits genome. Single copy transgenics are desired for their ease in breeding due to Mendelian segregation of the transgenes and for deregulation purposes.

Somaclonal variation: Genotypic and phenotypic variation generated from mutations or chromosomal rearrangements induced by plant cell culture.

Somaclonal mutations are almost never desirable and can be minimized by keeping cells in culture for a relatively short period of time.

Somatic embryo: A bipolar embryo formed in vitro from cultured plant cells. Somatic embryos are genetically identical to the plant that acted as the tissue donor. They can be propagated in vast numbers quickly, are transformable and can be regenerated into whole plants under the right conditions.

Synthetic seed: Mass propagation of elite genotypes using in vitro derived somatic embryos, which have been encapsulated to mimic seed.

T0:The initial transgenic plant generated from the plant transformation process.

Terminator sequence: The DNA sequence that marks the end of transcription for a gene.

Ti plasmid: A plasmid that induces the characteristic crown gall, or tumor, when transformed into plant cells. Ti plasmids contain phytohormone and opine synthesis genes to establish a gall where Agrobacterium can proliferate.

Totipotency: A term used to define the ability of a plant cell to regenerate into a whole plant under the right environmental cues.

Transfer-DNA (T-DNA): The DNA that Agrobacterium transfers into a plant cell to ultimately transform it.

Transgene: DNA that has been generated or obtained from a biological source, virus or is synthetically produced. The transgene is stably introduced into the genome of a plant to generate a transgenic plant. Transgenes may be chimeric; meaning each part of a functional gene (e.g. promoter, gene and terminator) is from a different source.

Transgenic plants: Plants containing transgenes or DNA introduced purposely by scientific methods conceived by humans.

Vector: The total part of transgenic DNA that will be transferred into a plant cell. Generally vectors contain a selectable marker cassette and one to several gene-of-interest cassettes.