Trait stacking in transgenic crops: Challenges and opportunities

Transcription

1 GM Crops ISSN: (Print) (Online) Journal homepage: Trait stacking in transgenic crops: Challenges and opportunities Qiudeng Que, Mary-Dell M. Chilton, Cheryl M. de Fontes, Chengkun He, Michael Nuccio, Tong Zhu, Yuexuan Wu, Jeng S. Chen & Liang Shi To cite this article: Qiudeng Que, Mary-Dell M. Chilton, Cheryl M. de Fontes, Chengkun He, Michael Nuccio, Tong Zhu, Yuexuan Wu, Jeng S. Chen & Liang Shi (2010) Trait stacking in transgenic crops: Challenges and opportunities, GM Crops, 1:4, To link to this article: Copyright 2010 Landes Bioscience Published online: 28 Jul Submit your article to this journal Article views: 1634 View related articles Citing articles: 70 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 22 December 2017, At: 08:59

2 REVIEW GM Crops 1:4, ; July/August/September/October Landes Bioscience Trait stacking in transgenic crops Challenges and opportunities Qiudeng Que,* Mary-Dell M. Chilton, Cheryl M. de Fontes, Chengkun He, Michael Nuccio, Tong Zhu, Yuexuan Wu, Jeng S. Chen and Liang Shi Syngenta Biotechnology, Inc.; Research Triangle Park, NC USA Key words: trait stacking, insect resistance, herbicide tolerance, molecular stack, large DNA transformation, mini-chromosome, targeted integration, transgene expression Downloaded by [ ] at 08:59 22 December 2017 In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e., the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression. Introduction Since the introduction of genetically modified (GM) crops in the mid-1990s, there has been a rapid adoption of the technology and the global acreage of GM crops reached a record 125 million hectares (309 million acres) in 25 countries in The country with the largest area of GM crops (half of the world s total acreage) is the United States of America (USA), followed by Argentina, Brazil, Canada, India and China. Currently, the major GM field crops are soybean, corn, cotton and canola. Minor GM crops include papaya, sugarbeet, squash, potato and alfalfa. The most widely adopted GM traits are herbicide tolerance and insect resistance. These traits provide growers with benefits of increased yield, reduced insecticide use and simplified management of weed control with fewer and more flexible herbicide applications. Herbicide (glyphosate) tolerance is the most widely used trait and has the largest acreage in soybean, corn, cotton and sugarbeet, followed by traits for insect resistance in corn and cotton. Other *Correspondence to: Qiudeng Que; qiudeng.que@syngenta.com Submitted: 06/25/10; Revised: 08/25/10; Accepted: 08/27/10 Previously published online: DOI: /gmcr transgenic traits include virus resistance, male sterility and oil quality. In 2010, 86, 93 and 93% of corn, cotton and soybean, respectively, planted in the US contained at least one biotech trait ( Until the introduction of glufosinate tolerance varieties recently, all GM soybean varieties planted in the US contained only one single trait, i.e., glyphosate tolerance. In corn and cotton, there is still significant acreage with a single herbicide tolerance or insect resistance trait. However, the percentage of products with stacked traits (herbicide tolerance and insect resistance) has increased rapidly, reaching 47% in corn and 58% in cotton in 2010 ( Current Stacked Trait Products and Trends The insect resistant trait based on Bacillus thuringiensis (Bt) toxin is becoming an indispensible tool in modern high efficiency agriculture. In order for this trait to be used sustainably, it is important to prevent the emergence and buildup of insects resistant to Bt toxins. To help manage insect resistance, the first Bt crops introduced in the USA were required to have host refuge areas without the trait ( In the USA, the Environmental Protection Agency (EPA) required that at least 20% of a grower s corn acres be planted with non-bt corn as a refuge area. In cotton-growing regions, growers can only plant up to 50% of their corn acreage with Bt corn borer resistant hybrids since maize is also an alternate host for cotton bollworm ( If the refuge area could be decreased from the standard 20 to 5%, a significant production benefit would be realized from this additional 15% Bt crop. Therefore, it makes sense to develop alternative approaches to preventing target pests from developing resistance. Recently, the industry has rapidly moved in the direction of providing two or more modes of action for the control of major insect pests, thus reducing the chance for resistance to develop and decreasing the percentage needed for insect refuges. Table 1 lists some products with stacked traits currently on the market. Additional information of these transgenic events and other transgenic events in non-stacked products not listed in the table can be found in the CERA database. 2 In the US, information on genetically engineered crop plants intended for food or feed that 220 GM Crops Volume 1 Issue 4

3 REVIEW REVIEW Downloaded by [ ] at 08:59 22 December 2017 Table 1. Transgenic products with molecularly stacked trait genes currently on the market * Trait developer(s) Crop Product name Transgenic event(s) Trait genes Trait targets Bayer CropScience Canola InVigor SeedLink MS8 (DBN ), RF3 (DBN ) bar, barnase, barstar ; Male fertility Monsanto Canola Genuity Roundup Ready GT73 (RT73) CP4 EPSPS, gox Bayer CropScience Cotton FiberMax LibertyLink Bollgar II LLCotton25, MON15985 bar, Cry1Ac, Cry2Ab Cotton WideStrike DAS , DAS Cotton Cotton WideStrike /Roundup Ready WideStrike /Roundup Ready Flex DAS , DAS , MON DAS , DAS , MON pat, Cry1Ac, Cry1Fa pat, Cry1Ac, Cry1Fa, CP4 EPSPS pat, Cry1Ac, Cry1Fa, CP4 EPSPS Monsanto Cotton Roundup Ready, Bollgard MON531, MON Cry1Ac, CP4 EPSPS Monsanto and Pioneer Hi-Bred and Pioneer Hi-Bred and Pioneer Hi-Bred and Pioneer Hi-Bred Cotton Bollgard II/Roundup Ready Flex MON , MON15985 CP4 EPSPS, Cry1Ac, Cry2Ab Maize Herculex CB TC1507 Cry 1Fa, pat Maize Herculex RW DAS Cry34Ab1/Cry35Ab1, pat Maize Herculex XTRA TC1507, DAS Maize Herculex XTRA/Roundup Ready 2 DAS , TC1507, NK603 Cry 1Fa, Cry34Ab1, Cry35Ab1, pat pat, CP4 EPSPS, Cry34Ab1, Cry35Ab1, Cry1Fa2 Lepidopteran pests (European corn borer); Coleopteran pests (Corn rootworm); pests; pests; Monsanto Maize Yieldgard VT Pro MON89034 Cry1A.105, Cry2Ab2 Lepidopteran pests Monsanto Maize Yieldgard VT MON88017 CP4 EPSPS, Cry3Bb1 Monsanto Maize Yieldgard VT Triple MON810, MON88017 Cry1Ab, Cry3Bb1, CP4 EPSPS Monsanto Maize Genuity VT Triple Pro MON89034, MON88017 Cry1A.105, Cry2Ab2, Cry3Bb Coleoptera pests (corn rootworm); Lepidopteran and coleoptera pests; pests; Monsanto and Dow AgroSciences Maize Genuity SmartStax TM MON88017, DAS- MON89034, TC1507, Syngenta Maize Agrisure GT/CB/LL Bt11, GA21 PAT, CP4 EPSPS, Cry1Fa2, Cry1A.105, Cry2Ab, Cry3Bb1, Cry34Ab1, Cry35Ab1 Cry1Ab, pat, mutant maize EPSPS pests; Lepidopteran pests (European corn borer); Syngenta Maize Agrisure CB/LL/RW Bt11, MIR604 Cry1Ab, mcry3aa, pat pests; Syngenta Maize Agrisure 3000GT (GT/CB/ LL/RW) GA21, Bt11, MIR604 pat, Cry1Ab, mcry3aa, mutant maize EPSPS pests; * Information was obtained from the CERA GM Crop database ( ), the US Regulatory Agencies United Biotechnology website( and the Product and/or Services section of the following company websites: GM Crops 221

4 have completed all recommended or required reviews for planting, food or feed use can be found in the National Biological Information Infrastructure (NBII) database (usbiotechreg.nbii. gov/database_pub.asp). The NCGA database also has the latest up-to-date listing of the available maize biotech traits and their Japan and EU approval status ( In the past few years, the US EPA approved reduction of the refuge areas for cotton and corn products with dual Bt genes with different modes of action. For example, Syngenta s Agrisure Viptera TM 3111GT ( with two lepidopteran active proteins (Cry1Ab and Vip3A) and one coleopteran active protein (mcry3a) qualifies for a reduced corn borer refuge in cotton growing regions from 50 to 20%. Monsanto s Genuity TM YieldGard VT Pro TM (with Cry1A.105 and Cry2Ab) and Triple Pro (with Cry1A.105, Cry2Ab and Cry3Bb) hybrids also qualify for a reduced corn borer refuge in cotton growing regions from 50 to 20% because they have two corn borer traits ( VT-Triple-PRO.aspx). Monsanto s Genuity TM Bollgard II cotton (with Cry1Ac and Cry2Ab) and WideStrike Insect Protected cotton (with Cry1Fa and Cry1Ac) also use the natural refuge option relying on surrounding fields with non-cotton hosts in certain areas to replace the once required non-bt cotton refuge ( page/updates/2007/bollgard-cotton.htm). Also, the US EPA has approved a natural refuge option for Syngenta s VipCot TM cotton which produces both Cry1Ab and Vip3A proteins (news. agropages.com/news/newsdetail htm). In 2009, Monsanto and received EPA approval for the SmartStax TM hybrid corn product with eight different genes for herbicide tolerance and insect-protection ( SmartStax TM is created by breeding crosses bringing together several previously approved transgenic events including Herculex I (Cry1Fa) and Herculex RW (Cry34Ab and Cry35Ab) technologies, Monsanto s Genuity TM YieldGard VT Rootworm (Cry3Bb)/RR2 and Genuity TM YieldGard VT PRO (Cry1A.105 and Cry2Ab) technologies, and the two weed control technologies, Roundup Ready 2 (RR2) based on the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium strain CP4 and LibertyLink based on the phosphinothricin phosphotransferase (PAT) gene from Streptomyces. The use of dual modes of control for each pest in SmartStax TM allowed for the refuge requirement to be reduced from 20 to 5% in the US Corn Belt ( biopesticides/pips/smartstax-factsheet.pdf). Syngenta Seeds, Inc., has submitted to the US EPA an application for the registration of its Agrisure Viptera TM 3,220 trait stack, also featuring two modes of action against all major lepidopteran corn pests and requesting a reduced refuge of 5% for the US Corn Belt (agrisureviptera.com/newsdetail.aspx?newsid = 118). Another development in the marketplace for managing emergence of resistance is the Refuge in a Bag (RIB) concept, which simplifies the refuge management by incorporating non- GM seeds or seeds without the target insect traits in the Bt seed bag directly. Pioneer Hi-Bred s Optimum AcreMax 1 TM (featuring the single mode of action Herculex RW) offers a RIB option for corn rootworm resistance management but still requires growers to plant a separate 20% corn borer refuge (farmindustrynews.com/seed/0201-acremax-technology/). The emergence of glyphosate tolerant weeds in many regions of the world will likewise require the use of additional and/or alternative herbicide tolerance traits for complete control of weeds in the future, especially in dicot crops such as canola, soybean and cotton that have fewer available effective herbicide options. It has been reported that several companies are currently developing alternative non-glyphosate weed control technologies. 3 For example, in collaboration with the University of Nebraska, Monsanto is developing dicamba-tolerant soybean for control of glyphosate-resistant weeds using the dicamba mono-oxygenase (DMO) gene. DuPont is developing a dual herbicide resistance to both acetolactate synthase (ALS) inhibitors and glyphosate for use in corn and soybean. 3 is developing a soybean trait for resistance to 2,4-dichlorophenoxyacetic acid (2, 4-D) and pyridyloxyacetate herbicides based on the AAD- 12 (aryloxyalkanoate dioxygenease) gene. 3 Bayer CropScience is developing soybean traits for resistance to glufosinate (Liberty herbicide) and HPPD (4-hydroxyphenolpyruvate dioxygenase) inhibitors such as isoxaflutole. 3 Syngenta has also announced the development of HPPD tolerant soybeans (native trait followed by GM) (www2.syngenta.com/en/investor_relations/ pdf/syngenta_sri_presentation.pdf). Soybean varieties tolerant to the imidazolinone class of herbicides developed by BASF and EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária) received regulatory approval for commercial cultivation in Brazil recently ( The gene stacking situation in corn is becoming complex. In the US, at least 8 genes will be needed in corn to provide for combined weed control and at least two modes of action for controlling four major pests: Ostrinia nubilalis (European corn borer, ECB), Helicoverpa zea (corn earworm, CEW), Diatraea grandiosella (Southwestern corn borer, SWCB) and Diabrotica virgifera (western corn rootworm, WCR) in the mid-west corn growing region. In addition to the above, several other insect pests, Agrotis ipsilon (black cutworm), Loxagrotis albicosta (western bean cutworm), Spodoptera frugiperda (Fall armyworm, FAW) and Diatraea saccharalis (sugarcane borer) also cause serious leaf, stock or ear damages. The complete control of these corn pests may require use of an additional insecticidal gene such as VIP3A. In addition to the herbicide tolerance and insect resistance traits, there are several agronomic and quality traits that the agricultural biotechnology industry is currently developing, including yield enhancement, drought tolerance, nitrogen utilization efficiency, disease resistance, fertility control (male sterility), grain quality (amino acid composition, protein content and oil composition) and grain processing (phytase for animal feed and amylase for corn ethanol). Thus the number of trait genes that need to be transformed into corn could easily add up to 15 or more, assuming that most traits can be achieved with only one or two genes. In addition, multiple genes will probably be needed for robust performance of complex agronomic traits such as yield enhancement and drought tolerance. In the past, transgenic events were created 222 GM Crops Volume 1 Issue 4

5 by transforming constructs carrying one or two trait genes individually into plants. Events with the required level of efficacy in the field were generated relatively easily in some cases. Products with single or double traits provide flexibility in trait combinations for each region. However, the effort required to bring a large number of trait loci into multiple cultivars even in a single crop quickly becomes unmanageable if each transgenic locus only carries one or two traits. From a scientific and breeding point of view, it is very desirable to deliver several traits simultaneously in a single locus. Delivery of multiple trait genes in one or more pieces of recombinant DNA simultaneously or consecutively into a single transgenic locus is called molecular stacking of traits. Trait molecular stacks are tightly linked and exhibit an extremely low rate of segregation, essentially behaving as a single gene, thus making trait introgression and line conversion much simpler. Current Trait Combination Tools: Breeding Stacks and Molecular Stacks Molecular stacking of two to three gene expression cassettes is straightforward. Each of these expression cassettes can contain a gene encoding a natural or mutant protein or an engineered chimeric protein or protein fusion with multiple functions such as dual Bt that can control a broad spectrum of insects. Many of the first generation trait events were generated in this fashion to contain stacks of both herbicide tolerance and insect resistance traits, such as Syngenta s maize Agrisure event Bt11 which contains a PAT gene expression cassette conferring glufosinate tolerance and a Cry1Ab gene expression cassette conferring European corn borer resistance. 2 Currently, the following maize events with double trait stacks are still on the market (see Table 1): Syngenta s Bt11 with PAT and Cry1Ab, TC1507 with PAT and Cry1Fa, Monsanto s MON with Cry1A.105 and Cry2Ab, and DAS Herculex RW event with PAT, Cry34Ab and Cry35Ab. However, as the number of traits increases, the assembly of each stack becomes more cumbersome. More importantly, the expression of each gene and efficacy of each trait may become less predictable. 4,5 Co-expression can be achieved by using a linker peptide to make a translational fusion of two different trait genes products. 6,7 In tobacco plastids, multiple open reading frames can be placed under the control of a single promoter and be transcribed as an operon, producing multiple separate proteins. Tobacco plastids have also been engineered with vectors carrying multiple genes. 8 However, the plastids of major field crops like maize, cotton, canola and soybean are not routinely transformed and no commercial plastid trait product is on the market yet. It remains to be seen whether there will be a breakthrough in transformation technology that will allow routine plastid transformation for trait development in major crops. Therefore, plastid transformation technology will not be discussed further here. All of the current multi-trait stack products on the market are derived by combining previously existing transgenic events by traditional breeding methods; thus they are called breeding stacks. Breeding stacks have the advantage of flexibility. One can utilize previously tested, approved and commercialized transgenic events. Usually, combining several loci carrying simple traits together results in products with good trait efficacy. In some countries, no additional regulatory approval is required for commercializing breeding stacks. Even when a new approval is needed, the approval of breeding stacks of existing traits is usually faster than a new trait event since all traits in the breeding stack are previously well characterized. Breeding stacks are also flexible: the trait offering can be tailored to specific markets where the insect pest spectrum might be different, thus requiring different trait stacks. However, combination of several trait loci is very time consuming and expensive because of the number of back-crosses that are needed to convert hundreds of elite lines for commercial launch in each crop. When breeding for trait stacks containing two or three loci, it is convenient to use a linked herbicide tolerance trait to identify desired progeny containing the intended trait combinations. However, since the number of herbicide tolerance traits is limited, introgression of multiple loci must be achieved in part by use of more expensive molecular (either DNA or protein) analysis tools. The problem becomes more complicated when multiple third party traits are combined together, because the genetic background could be very different among the donor lines. Seed production of varieties with multiple trait loci is also hard to track from the perspective of quality management and product stewardship. Potential erroneous labeling or handling of parent lines may result in cross-contaminated seed lots. Currently, from a practical point of view, at least two loci are needed for regulatory or technical reasons. For example, if the regulatory agency requires refuge planting for corn hybrids with insect resistance traits, the refuge plants will probably still require herbicide tolerance for weed management. Thus, it would be convenient to separate traits into an herbicide tolerance trait stack on one inbred and an insect resistance trait stack on a separate inbred. Inbreds containing each of these separate stacks can be generated individually. Improving Tools to Deliver Large Multi-Gene Stack Vectors Generation of molecular stack of multiple traits presents several challenges, including cassette design, vector assembly, transformation and gene expression analysis. Many trait genes in the current approved products are under the control of a few frequently used promoters such as Cauliflower Mosaic Virus (CaMV) 35S, maize ubiquitin-1 and rice actin-1. For successful molecular stacking it will be necessary to increase the number of available regulatory elements for trait development. Despite a reasonable number of available promoters, there are very few studies describing how they work when combined at a single locus. 9 It is also important to understand how other regulatory elements such as enhancers and insulators interact when placed close to each other, and to analyze the impact of the overall chromatin environment on their activity. Routine recombinant DNA technology depends on restriction enzymes for DNA manipulation. Currently, the main workhorse for most laboratories is recombinant DNA technology based on restriction endonucleases and DNA ligation, as well as recombinase-mediated assembly. However, as the GM Crops 223

6 number of components and size of DNA constructs increase, it becomes more difficult to find unique restriction sites for tailoring DNA manipulation. Even though assembly of 8 10 gene expression cassettes onto one T-DNA can be done with careful vector design, detailed planning and use of rare cutting endonucleases, application of new tools would be very helpful. Novel restriction endonucleases capable of recognizing and cleaving long sequences are extremely useful for assembly of complex DNA structures. Recently, it has been demonstrated that novel mega-endonucleases and zinc finger nucleases can be customdesigned to allow digestion of specific sequences. 10,11 Dafny-Yelin and Tzfira have reviewed the different approaches for assembling multigene vectors, including traditional cloning methods, rare cutting endonucleases, artificial restriction enzymes and recombinases. 12 Encouragingly, recent advances in DNA synthesis technologies may greatly facilitate vector assembly by providing genes with unwanted restriction sites removed and avoiding sub-cloning steps. Advances in DNA synthesis may make it costeffective to synthesize an entire gene vector and replace routine vector construction and assembly. 13 Enzymatic assembly of DNA molecules up to several hundred kilobase (kb) pairs has been demonstrated. 14 This kind of large DNA assembly method will be needed for efficient construction of transformation vectors containing large native genomic regions, metabolic pathways and whole redesigned organelle genomes and mini-chromosomes. For further reading on the different aspects of synthetic biology, the reader is encouraged to explore other articles in the same focus issue as for the above ref. 13. Large multi-gene constructs have been transformed into plants using both Agrobacterium-mediated and physical delivery methods. The upper size limit (if any) for biolistic delivery is unknown. Biolistic bombardment of plant cells has been used to deliver large DNA molecules such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and minichromosomes Micro-projectile bombardmentmediated transformation often results in insertion of truncated and/or concatenated plasmid DNA. However, biolistic delivery of a linear construct with no vector backbone has been shown to produce a high percentage of events with intact single copy insertions. 19 Use of lower DNA concentrations along with careful handling of donor DNA have led to successful biolistic transformation and regeneration of maize plants containing a circular minichromosome. 17 For Agrobacterium-mediated gene delivery, special binary vectors with low copy bacterial origins of replication have been designed for introducing large T-DNA inserts, especially for genomic DNA containing repetitive sequences. The Binary Bacterial Artificial Chromosome (BIBAC) vector system has been used successfully for transformation of intact high molecular weight DNA into plant chromosomes in several species including Arabidopsis, tobacco, tomato and rice The BIBAC vector has been reported to deliver at least 150 kb of intact T-DNA to the tobacco nuclear genome. The efficiency of that process was found to be enhanced by the presence of additional Agrobacterium tumefaciens virulence genes. 24 In fact, a helper plasmid containing extra copies of virg was found to be an absolute requirement for obtaining tomato transformants with the BIBAC system. 20 Similar to BIBAC, Liu et al. 25 reported construction of TAC (transformation-ready artificial chromosome) vectors based on P1 phage origin of replication. T-DNA regions of TAC vectors with 40 to 80 kb genomic DNA fragments have been transferred back into Arabidopsis with high efficiency and the transgenes were shown to be inherited faithfully by the progeny. Both BAC and TAC vectors contain very low copy bacterial origins of replication (f1, P1 and Ri) which allow the insert to be more stable in both E. coli and Agrobacterium. However, BAC and TAC vectors may not be essential for large T-DNA construction and delivery as long as the inserts can be stably maintained in the E. coli and Agrobacterium cells. It has been reported that conventional binary vectors such as one based on the RK2 origin of replication can also be used for construction of large T-DNA vectors. 26 Launching of T-DNA from the Agrobacterium chromosome has also been tested for delivery of transgene. 27 This design, which minimizes copy number in Agrobacterium, results in a high percentage of single copy plant transformants with intact T-DNA, although the overall transformation frequency is lower compared to binary vectors. 27 Delivering one long T-DNA from the Agrobacterium chromosome might be a good alternative to the use of several smaller binary vectors. However, the additional step of inserting T-DNA into the Agrobacterium chromosome in addition to the reduced transformation efficiency make the method more cumbersome in practice. The molecular stacking approach has been studied with an increasing number of genes assembled into a single plant transformation construct. 9 Downstream breeding effort will be greatly simplified if multiple traits with different modes of action can be carried in a single transformation vector, for example, an ECB and CRW stack with Cry1Ab, Cry1Fa, VIP3A, mcry3aa, Cry34Ab and Cry35Ab along with EPSPS conferring glyphosate tolerance. However, this kind of multi-cassette vector would require coordinated expression of all 7 trait genes in either constitutive and tissue-specific fashions. In molecular stacking, trait genes controlled by promoters with different tissue specificities must be placed in close proximity, raising the risk that there might be interaction between different promoter regulatory sequences. This is especially of concern when long-range-active viral enhancer elements such as those from the CaMV 35S promoter are used, because they may affect expression of several genes in the array. Encouragingly, the few examples in the literature suggest that molecular stacking of trait genes may not be so formidable. Cao et al. 28 have transformed rice plants by T-DNA delivery and expressed several trait genes including herbicide tolerance and yield enhancement from a ~20 kb 5 gene array under the control of different promoters. Fujisawa et al. 29 have described coordinate expression of seven trait genes to enhance carotenoid synthesis in Brassica napus. In this study trait genes were introduced on a single ~17 kb T-DNA. Recently it was shown that when a large 32 kb T-DNA vector with 10 genes was introduced into an elite maize line, most trait genes could be expressed well based on ELISA data and lepidopteran and coleopteran insect bioassay results. 30 In this study, it was shown that even though transformation frequency of the large 32 kb T-DNA vector by 224 GM Crops Volume 1 Issue 4

7 Table 2. Developers of novel trait gene stacking technologies Developer Technology AgBiotech partnership and licensees * Web site Cellectis Precision Biosciences Sangamo BioSciences Chromatin, Inc., Meganuclease-mediated site-specific integration mediated by homologous recombination Protein engineering to create novel custom meganuclease for mediating site-specific integration of transgenes BASF Plant Science, Monsanto, Limagrain, Bayer CropScience, DuPont (Pioneer Hi-Bred) DuPont (Pioneer Hi-Bred), Bayer CropScience com Zinc finger nucleases with custom specificity DowAgrosciences Mini-chromosome * From the announcements found in individual developer company s website. DowAgrosciences, Syngenta, Monsanto, Bayer CropScience Downloaded by [ ] at 08:59 22 December 2017 Agrobacterium was much lower, many events containing intact T-DNA could be recovered. Because the delivery of large T-DNA molecules has resulted in decreased transformation efficiency, it may be more desirable to generate several smaller molecular stacks from a subset of similarly expressed traits and deliver them in combinations, rather than putting all traits together. Smaller stacks of a subset of traits provide flexibility when combining different traits for product packages to fit the needs of different regions and customers and in response to the changing regulatory and business environment. These separate T-DNAs can also be delivered at the same time during transformation using either Agrobacterium or biolistic delivery to generate transgenic loci with co-integrated DNA. Such co-integration of transformed DNA molecules has been reported in many co-transformation studies. Komari et al. 31 generated co-integrated T-DNA events and also segregating T-DNA events with two different T-DNAs harbored in one or two Agrobacterium strains. McCormac et al. 32 also showed that by using a binary plasmid with three selectable T-DNA regions, the frequency of co-integration could be as high as a third within a T 0 subpopulation of co-transformants. Chen et al. 33 also codelivered 14 different plasmid DNAs into rice embryogenic tissues using biolistic bombardment, and found that many transgenic plants contained co-integrated DNA molecules. Zhu et al. 34 described similar work in maize but individual trait genes were co-bombarded to create a library of transgenic events. Their screen identified several independent events containing functional copies of all five trait genes. The reader is also encouraged to read a recent review by Naqvi et al. 35 on different options for multigene transfer, especially concerning their application for metabolic pathway engineering. New Technologies for Delivering Molecular Stacks In the last decade there has been substantial progress in the development of technologies allowing creation of transgenic crop plants with multiple gene stacks, especially in large DNA delivery, site-specific integration and mini-chromosome technologies. Table 2 lists some of the technology providers that are working on the trait stacking technologies. Site-specific integration can be mediated by site-specific recombinases or double stranded (ds)-dna breaks created by rare-cutting endonucleases such as homing endonucleases or custom-designed zinc-finger nucleases. Site-specific recombinases such as Cre, Flp and λ integrase have been demonstrated to mediate recombination and integration of transgene sequences in important crops like tobacco, rice, soybean and maize Homing endonucleases are found in nature and are involved in intron homing, a gene conversion process initiated by nuclease-mediated homing site cleavage. More than a dozen well-characterized homing endonucleases have been isolated from different organisms, including bacteria, algae and fungi. Their specificity can also be changed by mutagenesis of the amino acid sequences recognizing the DNA target sequence. 10,40 There have been several reports on the use of homing endonucleases I-SceI and I-CeuI for directing targeted insertion in different plants including tobacco, rice and maize via homologous recombination or non-homologous end-joining. 44 One potential drawback of using the site-specific recombinases and homing endonucleases for site-specific insertion is the need to first introduce a target recognition sequence into the genome before targeted insertion can be carried out efficiently. In contrast, zinc-finger nucleases (ZFNs) can be designed to cut any desired region of the natural or GM genome of the target plant. ZFNs are engineered chimeric nucleases created by fusing the zinc-finger DNA binding domain with the nuclease domain of the type IIs restriction endonuclease FokI. 45 The DNA sequence specificity can be altered by changing the zincfinger protein sequences. ZFNs recognizing specific transgenic and endogenous sequences have also been used to make ds-dna breaks to direct gene targeting in several plant species, including Arabidopsis, tobacco and maize, and by using different transformation techniques including electroporation, Agrobacterium and silicon carbide whiskers Since the binding specificity of the ZFN can be altered to recognize the sequence of interest directly, this technology has the potential to create insertion into any desired location in the genome. For trait development, a remaining question is how to determine the best chromosomal location for inserting potential genes of interest. We must also investigate whether stacked trait genes will be expressed as expected to give the desired trait efficacy. One straightforward approach would be to insert new genes into previously characterized loci such as sites of transgene insertion in existing commercial products. Such a strategy has been proposed as an example for converting a commercial variety for transgene stacking. 51 Since the tools of GM Crops 225

8 site-specific insertion are now becoming a reality, these questions can be answered in the near future. In addition, rapid development in crop genome sequencing and gene expression profiling will also be very useful in helping us to understand and predict suitable chromosomal locations for a particular group of trait genes. Recently, it has been shown that engineered mini-chromosomes can be formed either by deletion of non-essential portions of the supernumerary maize B-chromosomes 52 or by in vitro assembly from isolated centromere-like sequences. 17,18 Carson et al. 17 reported the in vitro assembly of artificial mini-chromosomes from cloned centromere-like sequences and transgene cassettes, and delivered them into maize cells to form autonomous functional chromosomal units de novo. It was estimated that native maize chromosomes contain from ~300 kb to >2,800 kb of CentC and CRM repeat sequences in different centromeres. 53 The smallest centromere of the artificial mini-chromosomes used by Carson et al. 17 is less than 20 kb, which is at least 15 times smaller than that of the smallest native maize chromosome centromere of ~300 kb. Genetic mapping shows that the minimal size for centromere function of B-chromosomes is around 150 kb, 54 which is still more than seven-fold larger than the centromere of the minichromosome reported by Carson et al. 17 In spite of the small size of engineered artificial mini-chromosomes, they were shown to be stably transmitted through mitosis and meiosis and were inherited through several generations. 17 These authors also found that the reporter gene carried on their artificial mini-chromosomes was expressed normally. 17 As gene vectors, mini-chromosomes offer some advantages over integration of the transgene into the host chromosomes. Transgenes on a mini-chromosome will not interrupt important endogenous genes or cause undesirable protein fusions. Likewise they avoid the problem of transgene insertion into an undesirable region of a normal chromosome resulting in negative phenotypes, such as low transgene expression or yield drag. Because all trait genes on the mini-chromosome are linked and behave genetically as a single locus, breeding to introgress all the traits into new breeding lines should be facile, in contrast to the case with integrated transgenes, which require many backcrosses. However, it still remains to be seen how the transgenes will interact when they are carried on a minichromosome, since it is not known whether the mini-chromosome has the usual chromatin structure or whether the genes will be subjected to similar epigenetic regulations as those integrated into normal chromosomes. It will be interesting to see how the mini-chromosomes are transmitted mitotically and meiotically over time under field conditions and whether trait genes will be expressed properly when the plants are subjected to various environmental stresses. Expression of Transgenes in Molecular Stacks One of the major uncertainties of molecular stacking concerns the expression of stacked genes over generations, and whether trait efficacy can be maintained in diverse genetic backgrounds under field conditions. This kind of multi-cassette vector would require coordinated expression of all genes in a tissue-specific fashion while the selectable marker and herbicide tolerance genes are usually expressed constitutively. Even though it has been shown possible to use the same promoter(s) to control several genes in pathway engineering studies, 29,34,55 it is not known how stable the expression is over generations and whether gene silencing will occur. Since repeated sequences in a transgene array may increase the chance of homology-dependent gene silencing, it would be desirable to avoid using the same promoter repeatedly in a transformation vector. However, the availability of suitable promoters is limited for practical reasons. 9 For example, if it is desirable that certain Bt genes not be expressed in corn pollen, one hurdle is that there is a limited selection of strong promoters that have this attribute. Also, it is not well understood what mechanisms contribute to the expression variation between different transgenic plants that contain the same gene cassettes, even though it is generally assumed that both the position effect from the insertion site s neighboring chromosome environment and the transgene s epigenetic modification such as methylation can be factors. For example, it has been reported that the intratransformant variability of reporter gene expression could be as high as the inter-transformant variation in comparisons between single copy transformants. 56 It has also been shown that sitespecific integration of a reporter gene into the same locus can produce alleles that express at a predictable level or alleles that are differentially silenced. 57 This effect is depending on the target site of the insertion, but correlates with the amount of DNA methylation of the introduced DNA, presumably by the host cell during the transformation process. 57 Thus, there is still much to be learned about the epigenetic control of gene expression in transgene stacking. In her earlier gene stacking review Halpin 4 pointed out the challenges associated with multi-gene expression and manipulation in plants. Five years later, many of these same challenges on transgene expression remain despite considerable progress in assembling and delivering transgenes. 5 Even for transformation vectors containing only one or two traits, it is wellknown that many transformants must be generated to identify a few lead events for commercial development. The probability of getting a commercially acceptable event will decrease as the number of trait gene cassettes increases, especially if the stacked traits require very different regulatory sequences and have different expression patterns, for example a molecular stack with a lepidopteran insect resistance trait and a rootworm control trait. For trait efficacy, it is probably easier to generate a commercially viable event with a stack containing only a subset of trait genes such as a broad lepidopteran insect control stack of Cry1Ab, Cry1Fa and Vip3Aa that requires similar expression pattern, versus a large combination stack with multiple herbicide tolerance genes and several kinds of insect resistance traits under the control of promoters with different tissue specificities. In plants, enhancers present in promoters are capable of changing the activity and specificity of nearby promoters. 58,59 For example, the CaMV 35S enhancer can affect expression of genes located over 78 kb away. 60 This kind of long distance effect of regulatory elements on gene expression can potentially result in problematic expression of neighboring genes in a molecular stack event because strong promoters are frequently used to drive constitutive trait gene expression to ensure insect resistance and 226 GM Crops Volume 1 Issue 4

9 herbicide tolerance trait efficacy throughout a large part of the crop s life cycle. Another factor that may affect stacked transgene expression is the nature of the insertion site itself, i.e., the neighboring genomic context including activity of the cis-regulatory elements and chromatin environment. Variation ascribed to the locus is generally referred to as position effect. One strategy for reducing variability of transgene expression variation and position effect is to flank the transgene with chromatin boundary elements such as insulators or matrix attachment regions. Insulators are DNA sequence elements that protect genes from the inappropriate influences of nearby promoter elements (enhancer blocker) or the spreading of heterochromatin effects into the transgene from the surrounding regions (chromatin barrier). Various elements such as insulators and MARs have been studied in several laboratories to evaluate their effect on transgene expression but results have been mixed. Nagaya et al. 61 found that a sea urchin Ars (arylsulfatase) insulator can suppress variation of transgene expression in tobacco BY2 cells. Matrix-attachment regions (MARs), also known as scaffold attachment regions (SARs), are sequence elements with nuclear matrix (scaffold) binding properties and are postulated to be the boundary elements organizing nuclear DNA into discrete chromatin structural domains. One study found that in Arabidopsis a chimeric transgene flanked by tomato HSC80 MARs behaved no differently than a control without MARs. 62 These authors also showed that the use of a different MARs, the ARS1 MAR from yeast, significantly decreased expression. However, in two different tobacco studies, yeast and tobacco MARs were shown to increase transgene expression in tobacco cells. 63,64 Since these studies utilized biolistic delivery, the dramatic improvement observed in the resulting multi-copy lines may be a protection against gene silencing, which may also explain why no such protection was seen at a very high transgene copy number. 63 In maize, three MAR elements, including two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1 MAR), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. 65 In this study, it was shown that the Adh1 5' MAR and yeast ARS1 MAR reduced transgene silencing but had no effect on the variability of expression. In stable transgenic maize plants, GUS expression was lower in all tissues of the plants with Adh1 MARs flanking the GUS gene cassette compared to control plants without Adh1 MARs flanking the GUS gene cassette. However, GUS expression was found to be localized to the lateral root initiation sites in transgenic plants with Adh1 MARs flanking the GUS gene cassette. 65 To eliminate the effect of transgene position and copy number on MAR activity, Cre-lox mediated recombination was used to generate allelic loci. 66 In this study, it was found that GUS gene expression in plant populations could be shielded from RNA silencing by the presence of the chicken lysozyme A element, but expression stabilization was only observed in two of three independent tobacco transformants. 66 Hily et al. 58 showed that out of the three plant MARs tested, only the petunia TBS MAR element, but not the maize ADH1 or tobacco Rb7 MARs, was found to block interactions between the 35S enhancer and AGIP promoter without affecting the function of either 35S or AGIP promoters in transgenic Arabidopsis plants. 58 The fact that only some MARs exhibit true insulating activity may explain their mixed effects on gene expression in plants. Challenges and Uncertainties As mentioned above, there have been many technical developments for creating and delivering multiple gene stacks in the last decade. However, the adoption of these technologies will probably depend on several factors, including technical performance, development cost, grower needs, regulatory environment and fit with the individual biotech company s breeding program. One of the considerations is the trait needs of growers in different regions, since pest and weed distributions are unique for each geographical location. Another consideration is the flexibility of breeding with another company s germplasm and traits, since many GM traits are also cross-licensed to third party seed companies. In this case, a molecular stack event with all of the company s traits might not be desirable since some traits might not be compatible with another company s existing traits or are not covered by the license. For insect resistance trait programs, it is important to consider resistance management requirements while designing the trait or molecular stacks. Also, for hybrid seed crops like maize, trait stack design should consider whether some traits should be on the male or the female side to make the intended hybrid seeds for future breeding efforts. A further consideration of whether or not to adopt a large DNA stack vs. breeding stack is the sheer cost and timeline of registering a new transgenic event. In some countries such as the US and Canada, breeding stacks from previously registered events do not require new safety assessment studies. Therefore, it is much cheaper and faster to develop and commercialize a stack product via combination of existing events. However, the US EPA does require separate review of the safety of the trait stacks if a specific hazard can be identified, for example when the parental events carry traits encoding biotoxins ( plant incorporated protectants ) such as insecticides, since combinations of biotoxins may result in synergetic or adjuvant effects. In Japan and EU countries, stacked events are considered as new GMOs and they need regulatory approval, including risk assessment of their safety, similar to single event. However, even in EU countries the safety assessments of the parental events covering all uses of the stacked events will provide a good basis for the evaluation of stacked trait events, though additional information proving the validity of the studies carried out on the GM parental lines are needed to complete the risk assessment of the GM stacked event. 67 Since the cost of performing regulatory studies and registering a transgenic event often exceeds multimillions of dollars for an individual country and may take several years for regulatory agencies to approve newly generated events, there is little incentive for biotech companies to spend additional millions on regulatory studies for a different transgenic event of a previously approved trait if the new event does not provide clear additional benefits. However, there are situations where a molecular stack might be preferred in order to avoid the cost of regulatory GM Crops 227

10 approvals of several independent events, for example in a new crop where a number of traits are needed or in a crop for which breeding is extremely difficult, for example in vegetatively propagated crops such as potato, cassava and sugarcane. Transgene stacking presents many interesting challenges, both scientific and tactical, that will require input from molecular and genome biologists, breeders, growers and regulatory agencies in order to choose the most cost-effective solution. It will be very interesting to see whether one or several means of trait stacking will ultimately predominate in the market. Will the dark horse of the field, the mini-chromosome, which is providing so much interesting science today, turn out to be a sound and useful technology that supplants the current methods of inserting transgenes into the normal chromosomes of the plant? Will the frustrating and yet tantalizing results with insulator technology develop into a valuable tool for stabilizing gene expression? In this post-structural genomic era, more crop genomes are being fully sequenced and characterized; and more technologies with higher throughput, improved accuracy and broader coverage are being developed. Various methods have been developed to predict various regulatory features, transcription factor binding sites, tandem repeats, CpG/CpNpG islands and combinatory transcription factor binding sites, to name a few, by taking advantage of the vast amount of genomic sequence information, methylation or chromatin immunoprecipitation (ChIP) profiles and transcriptome profiling data from diverse species. These predicted features can be used to develop artificial regulators to drive various expression patterns. The artificial regulators, such References 1. James C. A global overview of biotech (GM) Crops: Adoption, impact and future prospects. 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Transgenic Res 2001; 10: as minimum promoters with well characterized cis-regulatory elements, enhancers and insulators, can be used in combination to precisely control the spatial and temporal expression of each transgene or different transgenes in concert at the desired strength for trait stacks. In addition, the knowledge is rapidly accumulating about epigenetic features in a genome, such as histone modification, DNA methylation and micrornas. 68,69 Genetic features such as homologous recombination hotspots discovered by high density genotyping technologies, as well as information about functional regulation of host gene expression at the chromosomal level, could be used to select and develop the optimal landing sites for site-specific integration. Furthermore, the learning about the chromosomal structure and organization and their expression control from the host genomes could be applied to improve the design and function of mini-chromosomes or artificial chromosomes. 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