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. 2015 Sep 15;14(5):1175–1182. doi: 10.1111/pbi.12466

Plant artificial chromosome technology and its potential application in genetic engineering

Weichang Yu 1,✉, Yuan‐Yeu Yau 2, James A Birchler 3
PMCID: PMC11389009  PMID: 26369910

Summary

Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first‐generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands.

Keywords: plant artificial chromosome, genetic engineering, gene stacking, food security, sustainable agriculture

Introduction

Agriculture provides food for nutrition, fibre for clothing, lumber for shelter and biofuel for increasing energy demand. The burden to produce sufficient agricultural products has been increasing due to the expanding human population, while there is a reduction of farmland and natural resources. According to United Nations' prediction (2015), world human population will reach 9.7 billion by 2050 from the current 7.3 billion (http://esa.un.org/unpd/ppp/index.htm). To maintain the current living standard will require a production of at least 23% more agricultural products. Some have estimated a demand of 50%–70% increase considering both the expanding population and the change of consumption pattern (Davies et al., 2009; Ronald, 2014). Theoretically, this increase can be achieved by scaling up farmland, breeding and cultivation of high‐yield varieties, and the reduction of losses caused by biotic and abiotic adverse conditions during the growing season and postharvest period.

However, the following constrains apply to agriculture. First, the world arable land cannot substantially increase (Zhang and Cai, 2011), fresh water is limiting, urbanization in developing countries will use more farmland for industry, and arable land will be lost to environmental deterioration such as flooding, salinification and desertification. Second, our current agriculture practices rely on heavy use of fresh water and fertilizer, which is not sustainable. The Green Revolution brought a large increase in crop yield in the last century because of the introduction of hybrid seeds and dwarf varieties that resisted lodging. However, these varieties usually consume large amount of fertilizers and require good irrigation to maximize the yield (O'Neill et al., 2004). Thus, to keep high yields, heavy applications of water and fertilizers are required, but a high percentage of these run off into the river system and have already caused serious problems in the ecosystem. Third, there is an increase in both biotic and abiotic stresses in agriculture. All these problems in our current agricultural practices suggest the need for sustainable agriculture, in which yield increase is based on reduced input of natural resources and is environmentally friendly. This will require the design of superior crop varieties based on new technologies. In this review, we will discuss the plant artificial chromosome (PAC) technology (Yu et al., 2006, 2007) and its potential application in crop genetic engineering.

Crop genetic engineering

Genetic engineering is the technology to manipulate directly the genome of an organism with recombinant DNA technology. The discovery that Agrobacterium can transfer a piece of its own DNA into a host plant genome has greatly facilitated plant genetic engineering. Reviews on plant genetic engineering and the discovery of Agrobacterium can be found elsewhere (Zambryski, 2013). Basically, by cloning into a modified Ti‐plasmid of Agrobacterium, foreign genes can be mobilized into Agrobacterium cells, which can be used subsequently to infect a plant host. Foreign genes on the Ti‐plasmid can be transferred into the host plant genome by Agrobacterium, and expressed. This process bypasses the sexual hybridization barrier in traditional plant breeding, can transfer genes across organismal kingdoms and thus allows the direct use of genes from viruses, bacteria, fungi and animals in crop improvement. In addition to the Agrobacterium‐mediated genetic transformation, another genetic transformation technology, called direct gene transformation either by the DNA uptake into plant protoplasts or through bombardment of DNA coated onto the surface of gold particles by gene gun, is also frequently used in plant genetic engineering.

Genetically engineered crops as first approved for commercialization in 1994 (James and Krattiger, 1996) have rapidly spread into the major crop producing countries in the world. Until 2014, genetically engineered crops have been grown in 29 countries with a total acreage of 181.5 million hectares (James, 2014). The introduction of both Bt toxin and herbicide resistance genes from micro‐organisms into plants has greatly changed agriculture with efficient weed control and reduced use of chemical pesticides, which are harmful to both human health and the environment (Lu et al., 2012; Pray et al., 2002). Although worldwide application of genetically engineered crops still faces some resistance (Leyser, 2014), genetic engineering technology is the most influential invention for agriculture in the last century after the Green Revolution and has huge potential in the future to meet the surging demand for agricultural products.

However, current plant genetic engineering uses only a few genes, such as the Bt toxin genes for insect resistance and the herbicide resistance genes for weed control. This situation is probably because of either the paucity of useful genes or the lack of technology to manage large number of genes in the current transgenesis technology. While the first generation of genetic engineering has generated great benefits for the farmers with a few genes, the next generation must rely on multiple genes to achieve benefit to both the consumers and society (Halpin, 2005). The development of the following factors will make it possible to design the next generation of genetically engineered crops with multiple transgenes.

First, high‐throughput sequencing technology and bioinformatics tools have greatly boosted the speed and efficiency of gene discovery. Instead of only a few useful genes, more and more genes or genetic networks will be discovered and made available for genetic engineering. Second, the development of synthetic biology in the last few years makes it possible to assemble genes efficiently. This will allow the construction and transformation of a group of genes rather than single or a few genes at a time. Third, with the development of a site‐specific recombination (SSR) system and the recent genome‐editing technology, one can precisely add, delete or replace transgenes in the genome. Last but not least, PAC platforms have been developed as super vectors for foreign gene organization, expression and manipulation (Yu et al., 2007). Reviews on sequencing technology and gene discovery can be found elsewhere (Salgotra et al., 2013). In this review, we will mainly discuss the development of PACs and other related technologies for gene assembly, gene targeting and chromosome delivery, and their potential applications for future PAC‐based genetic engineering.

Plant artificial chromosomes

PACs are chromosome‐based vectors (minichromosomes) for genetic engineering. Minichromosomes are engineered chromosomes with the following properties. First, minichromosomes are small and essentially have no genes of their own, and thus can be used as super vectors to express foreign genes, but have minimal interference with the host growth and development. Second, minichromosomes are stable during both mitosis and meiosis to allow the resident genes to be faithfully expressed and transmitted from cell to cell and from generation to generation. Third, minichromosomes will allow the addition, deletion and replacement of genes on them by either SSR systems or direct editing with recent genome‐editing technology.

To fulfil these requirements, minichromosomes are usually created as small in size with only essential components such as the centromeres, telomeres and origins of replication. There are generally two approaches for minichromosome construction: de novo assembly and chromosomal truncations. In the first approach, components of chromosome centromere repeats, telomeres and origins of replication are cloned, assembled in vitro and transformed into cells to direct minichromosome assembly. This approach has been successful in yeast and mammalian cells (Harrington et al., 1997; Murray and Szostak, 1983), but no definitive examples have been reported in plants (Gaeta et al., 2011; Houben et al., 2008; Mette and Houben, 2015). In one case, Carlson et al. (2007) claimed to have produced an in vitro‐assembled autonomous circular minichromosome in maize by biolistic transformation of assembled maize minichromosomes (MMCs). But, as pointed out in a previous review (Gaeta et al., 2011), the claimed behaviour of the reported MMCs is not consistent with the known behaviour of small ring chromosomes because ring chromosomes usually do not follow Mendelian inheritance (McClintock, 1932). The apparent high transmission rate and lack of evidence of active centromeres such as the CenH3 marks on the MMC centromeres could not validate the claimed MMCs as autonomous minichromosomes. In another case, Ananiev et al. (Ananiev et al., 2009) reported the production of minichromosomes in maize by biolistic transformation of constructs with cloned centromeric repeats, DNA fragments from maize 18–26S rDNA as the replication origin, and telomere repeats. As the authors pointed out, some of the minichromosomes were clearly from telomere truncations, while it might be possible that there were cases formed de novo. Even the most likely de novo minichromosomes still contain retrotransposon sequences not present in the input DNA and are much larger than the construct delivered during transformation suggesting that the centromere was actually endogenous. The difficulty in assembling autonomous minichromosomes in plants is most likely because of the strong epigenetic influence on centromere function (Birchler, 2015). In addition, de novo PACs will have a species limitation because plant centromeric repeats are species specific if they play any role in centromere function and variation exists even among closely related species.

In contrast, minichromosome construction by telomere‐mediated chromosomal truncation (TMCT) has been very successful. TMCT is based on the discovery that transformation of telomere‐containing sequences into the genome will seed new telomeres at the site of integration (Farr et al., 1992; Yu et al., 2006). We have previously shown that a 2.6‐kb telomere repeat assembled from the Arabidopsis telomere clone (Richards and Ausubel, 1988) is efficient in breaking maize chromosomes after transformation (Yu et al., 2006). Although longer telomere repeat is required in maize TMCT, the length of transforming telomeric DNA can be as short as 100 bp in chromosomal truncations of Arabidopsis (Nelson et al., 2011), 392 bp in barley (Kapusi et al., 2011) and 1.3 kb in rice (Xu et al., 2012). The efficiencies of TMCT vary from around 9% in maize (Yu et al., 2006), up to 32% in rice (Xu et al., 2012), 56% in Arabidopsis (Nelson et al., 2011) and 40%–100% in tetraploid barley depending on the transformed telomere repeat size (Kapusi et al., 2011). Because telomeric repeat sequence is conserved in the plant kingdom (Richards and Ausubel, 1988), TMCT should work in most plant species with the typical Arabidopsis type of telomeres. Indeed, since the first reported TMCT in plants (Yu et al., 2006), minichromosomes have been constructed in many plant species including maize (Gaeta et al., 2013; Yu et al., 2007), rice (Xu et al., 2012), Arabidopsis (Nelson et al., 2011; Teo et al., 2011) and barley (Kapusi et al., 2011).

In addition, minichromosomes must have functional centromeres to keep them stable during cell division. Minichromosomes produced by TMCT have natural centromeres with little or no modifications because the TMCT only allows the transmission of functional centromeres while those without function due to large deletions will be lost. The stability of minichromosomes created by TMCT has been confirmed. In maize, TMCT‐produced minichromosomes from both A and B chromosomes are stable during both mitosis and meiosis (Yu et al., 2007). In rice, minichromosomes can be maintained in both callus culture and suspension culture for over 2 years (Yang et al., 2015). Besides, we have also shown that minichromosomes do not pair with their progenitor chromosomes and thus avoid recombination with the genome (Yu et al., 2007). This property is important for the application of minichromosomes because they will be stable during meiosis and remain as independent vectors in the genome so that their activity will not interfere with or be interfered by the genome.

To allow the manipulation of genes, minichromosomes are usually designed to have site‐specific recombination (SSR) systems, such as the Cre‐lox (Abremski et al., 1983), FLP‐frt (Golic and Lindquist, 1989) or the phiC31‐att integrase systems (Thorpe and Smith, 1998). Strategies to manipulate minichromosomes with SSR systems will be discussed below.

Genetic engineering with PACs

Genetic engineering with PACs has many advantages over the traditional genetic transformation. With the PAC system, an almost unlimited number of genes can be integrated and expressed. These genes could be simple stacking of multiple genes for plant disease resistance, the prevention of insect infestation, weed control, or the tolerance to harmful environmental conditions such as heat, cold, drought and salinity. It is also possible to engineer compound gene complexes to enable plants with new traits such as more efficient utilization of water (Borland et al., 2014) and fertilizer, metabolic engineering to produce valuable metabolic products for crop nutritional improvement or medicine, and the adaptation of plants to produce high biomass for biofuel. Another benefit of genetic engineering with PACs is the avoidance of linkage drag when transgenes need to be stacked or transferred to other germplasm in breeding. Linkage drag is a condition when a desired gene is linked to deleterious genes which, when they exist, will have a negative effect on the fitness of the plant. Some linkage drag cannot be broken by repeat backcrossing, which is a big challenge in plant breeding (Young and Tanksley, 1989). Because PACs act as independent chromosomes, no other genes from the genome are linked; thus, the whole package of genes on the PAC acts as one linkage group and can be transferred in a single cross without introducing other genes from the genome. In addition, genetic engineering with a PAC can prevent the disruption of endogenous gene function, which frequently occurs during a randomized genetic integration event by traditional genetic engineering, and the expression of transgenes on a PAC is less likely influenced by genes in the genome due to position effects (Yu et al., 2007).

Toolkit for PACs

The application of PACs in crop genetic engineering requires the development of a full kit of tools for chromosome engineering and gene manipulation. Fortunately, recent development in molecular biology has made huge progress in such technologies such as multiple gene assembly and transformation technologies, gene targeting, genome editing and chromosome delivery (Figure 1).

Figure 1.

Figure 1

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PAC platforms and the toolkit for manipulation. TMCT, telomere‐mediated chromosomal truncation; PAC, plant artificial chromosome; BIBAC, binary bacterial artificial chromosome (Hamilton, 1997; Hamilton et al., 1996); TAC, transformation‐competent artificial chromosome (Lin et al., 2003; Liu et al., 1999); Gateway‐based platform, Gateway® technology‐based gene assembly technology (Chen et al., 2006); GoldenBraid, an iterative cloning system for standardized assembly of reusable genetic modules (Sarrion‐Perdigones et al., 2011, 2013); Modular cloning, a modular cloning system for standardized assembly of multigene constructs (Weber et al., 2011; Werner et al., 2012); RMDAP, recombination‐assisted multifunctional DNA assembly platform (Ma et al., 2011); MISSA, multiple‐round in vivo site‐specific assembly system (Chen et al., 2010).

Gene assembly is a synthetic biology technology to ligate multiple genetic elements into functional gene expression cassettes in vitro. Traditional gene cloning technology relies on step‐by‐step ligation of DNA fragments in a plasmid vector. However, most plasmid vectors are small and have limited capacity to accommodate large DNA inserts. To clone large DNA pieces, bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs) have been used. In plant genetic engineering, BAC vectors can be modified to include T‐DNA border sequences to allow the transfer of cloned DNA in a BAC to the plant genome by Agrobacterium‐mediated transformation. The modified vectors are called binary BAC (BIBAC) (Hamilton, 1997; Hamilton et al., 1996) or transformation‐competent artificial chromosome (TAC) (Lin et al., 2003; Liu et al., 1999). The development of BIBAC and TAC has made it possible to transform plant cells with large DNA fragments. In addition to the enlarged capacity of cloning vectors, progress has also been made to increase the cloning efficiency using an SSR system, Gateway® cloning technology or the recent utilization of the type IIS restriction enzymes (Golden Gate cloning). Many gene assembly systems have been developed in recent years including the Gateway® technology‐based gene assembly technology (Chen et al., 2006), GoldenBraid system (Sarrion‐Perdigones et al., 2011, 2013), modular cloning (MoClo) system (Weber et al., 2011; Werner et al., 2012), recombination‐assisted multifunctional DNA assembly platform (RMDAP) (Ma et al., 2011), and multiple‐round in vivo site‐specific assembly (MISSA) system (Chen et al., 2010). Interested readers can examine these references for the details of these technologies. In one example, 17 transcription units (~50 Kb) have been assembled from 68 DNA fragments using Golden Gate cloning and the MoClo systems into a binary vector (Werner et al., 2012). The assembled constructs can be used directly in genetic transformation, or used to target PACs.

Telomere‐mediated chromosomal truncation

Telomeres are the natural ends of chromosomes, which protect the chromosome from deterioration or fusion with other chromosomes. Most higher plants have the Arabidopsis‐type of telomeres with long arrays of telomere repeat (TTTAGGG)n except some plant species in the order of Asparagales and the Solanaceae family where the telomere repeat sequences are replaced with (TTAGGG)n and (TTTTTTAGGG)n, respectively (Peska et al., 2015). The minisatellite telomere repeats are lost in Allium and are probably replaced with an unknown mechanism (Peska et al., 2015; Sykorova et al., 2006). The discovery that telomere sequences can make chromosome breakage during genetic transformation has made telomeres as valuable tools in chromosome engineering (Farr et al., 1992; Yu et al., 2006). TMCT was originally designed to construct PACs by placing telomere repeats, selection markers and SSR systems on one plasmid (Yu et al., 2006, 2007). After genetic transformation with either Agrobacterium‐ or biolistic‐mediated transformations, transgenic cell lines or plants were selected by their resistance to herbicide or antibiotics conferred by the selection marker gene, and then subsequently screened by fluorescence in situ hybridization (FISH) for chromosomal truncations. Minichromosomes can be identified if TMCT removes both chromosome arms from a metacentric or submetacentric chromosome, or removes one arm from an acrocentric or a telocentric chromosome. However, this strategy has recently been shown not to be essential. By direct transformation of a mixture of DNA elements containing telomere repeats, a selection marker and an SSR system, minichromosomes have been produced in both rice (Xu et al., 2012) and maize (Gaeta et al., 2013). This modified TMCT is based on the fact that DNAs cotransformed by bombardment can integrate into the same location (Makarevitch et al., 2003). This practice will greatly reduce the work in PAC construction and the freedom to choose assembled gene packages, selection markers and SSR systems. In combination with the gene assembly technology, it will be possible to construct PACs with packages of gene complexes.

Site‐specific recombination (SSR) systems derived from prokaryotic or lower eukaryotic cells (such as yeast) are powerful tools for precise gene manipulations, including gene deletion and gene targeting. It is also an essential component in PAC technology to allow the retrofitting of minichromosomes with useful genes. We have demonstrated in principle that SSR systems can be used in gene replacement and gene deletion (Gaeta et al., 2013; Yu et al., 2007). To test the feasibility of using SSR systems on minichromosomes, we used the Cre‐lox as the test system (Yu et al., 2007). Two transgenic maize lines were crossed: one harboured the construct 35S‐lox66‐Cre, and the other harboured the promoterless construct lox72‐DsRed on a minichromosome. After crossing, the expression of Cre protein would promote the recombination between lox66 and lox72 sites rendering the exchange between Cre and DsRed genes, resulting in the generation of the recombinant 35S‐lox‐DsRed. The 35S promoter would then drive the DsRed gene and produce red fluorescence in cells. The results demonstrated that the SSR system can be used for site‐specific recombination in a minichromosome. In another demonstration, Gaeta et al. (2013) successfully removed a selection marker gene from a minichromosome by the Cre‐lox system.

Stacked traits have become the trend of genetically engineered crop production. Genetically engineered crops with two or more stacked traits accounted for ~28% of over 181 million hectares of cultivated transgenic crops in 2014 (James, 2014). Several strategies have been developed for trait stacking in planta, including conventional breeding and molecular methods. Stacking genes of interest (GOIs) at the same locus can ease the management of transgenes. For example, they can be removed as a unit if needed to suit some applications, such as genetic use restriction technology (GURT) (Sang et al., 2013). Gene assembly technology can be used in combination with SSR systems to add genes to the PAC at the same locus. In addition to assembling a group of available genes in vitro, and then transferring them to minichromosomes, it will be useful if transgenes can be supplemented and stacked onto minichromosomes in vivo at different times. In plants, two SSR‐mediated methods have been employed to stack transgenes to the same locus in vivo. One is the recombinase‐mediated cassette exchange (RMCE) method (Turan et al., 2012). There are different strategies to use RMCE for genome manipulation. In principle, SSR systems in RMCE are used to facilitate the exchange of, for example, SMG1‐GOI1 and SMG2‐GOI2 between cassettes attP ϕC31‐[SMG1‐GOI1]‐attP Bxb1 and attB ϕC31‐[SMG2‐GOI2]‐attB Bxb1, where attP and attB are the ‘recognition sites’ for ϕC31‐att and Bxb1‐att SSR systems, and SMG and GOI are selectable marker gene and gene‐of‐interest, respectively (Figure 2) (Zhu et al., 2013). One of the cassettes needs to be integrated into a random locus, or precisely integrated into a desirable locus through genome editing technology in the genome (Zhu et al., 2013). Genome editing technology will be discussed in the next paragraph. In Figure 2, the attP ϕC31‐[SMG1‐GOI1]‐attP Bxb1 cassette is pre‐embedded in the genome. The other cassette and recombinase‐expressing vectors are then co‐delivered into cells for cassette exchange. The recombination between attP x attB of the two systems renders cassette exchange upon the coexpression of both ϕC31 and Bxb1 recombinases (Figure 2). By introducing multiple recognition sites, different cassettes can be integrated subsequently into the locus. RMCE has been successfully used for transgene stacking in soybean (Li et al., 2009). The other SSR‐mediated method is the use of unidirectional SSR to facilitate the sequential site‐specific integration of individual plasmids carrying different transgenes into a pre‐determined chromosomal locus through attP x attB recombination. The scheme is described in Figure 3. A proof‐of‐concept study has demonstrated the successful use of a unidirectional Bxb1‐att SSR system to stack three transgenes (gus, luciferase and gfp) into a model plant at the same locus (Hou et al., 2014). The three stacked transgenes were transmitted and inherited as a unit into the subsequent generations with predictable gene expression. The SSR systems will be valuable tools for gene stacking and deletion in PAC technology.

Figure 2.

Figure 2

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Recombinase‐mediated cassette exchange (RMCE). Coexpression of both ϕC31 and Bxb1 recombinases promotes recombination between attP x attB of the two systems, rendering the swap of SMG 1‐GOI 1 and SMG 2‐GOI 2 between cassettes attP ϕC31‐[SMG 1‐GOI 1]‐att P B xb1 and attB ϕC31‐[SMG 2‐GOI 2]‐att B B xb1. SMG: selectable marker gene; GOI: gene‐of‐interest; attP ϕC31 and attB ϕC31: attP and attB recognition sites of ϕC31‐att system; att P B xb1 and att B B xb1: attP and attB recognition sites of Bxb1‐att system. attR ϕC31 and att L B xb1 are hybrid sites derived from attP x attB recombination.

Figure 3.

Figure 3

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An example of using unidirectional Bxb1 SSR system for in vivo transgene stacking. Target construct (a) with gene‐of‐interest (GOI 1) and attP site (attP 0 ) of Bxb1‐att system was transformed and integrated into plant genome to produce target lines (b). Site‐specific recombination occurs between the attP 0 site of a target line and the attB 1 site (or attB 2 site, but attB 1 is desirable) of an integration vector (c), which contains GOI 2 and two identical attB sites (attB 1 and attB 2 ), upon the supply of Bxb1 recombinase. The resulting recombinant contains both GOI 1 and GOI 2, and a bring‐in new site (attB 2 ) which can be used for next‐run gene integration (d). Two hybrid sites (attL and attR) were also generated through attP x attB recombination (d). SMG1 flanked by loxP sites can be removed by Cre recombinase (e). Another integration vector (f) containing GOI 3 and two identical attP sites (attP 1 and attP 2 ) was then provided for next‐run gene stacking through recombination between genomic attB 2 site and plasmid attP 1 site to bring in GOI 3.

Genome editing technologies are newly developed techniques for highly efficient and precise manipulation of genes in the genome. These technologies use engineered nucleases to cut genomic DNA to make double‐stranded breaks (DSBs) at the sites of recognition. The DSBs will be repaired by genome‐encoded recombination machinery such as nonhomologous end‐joining (NHEJ) or homologous recombination (HR). Commonly used genome editing tools are the zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system (Gupta and Musunuru, 2014; Puchta and Fauser, 2013). Each of these systems uses a unique mechanism to recognize genomic loci in a DNA sequence‐specific manner and makes precise cleavage of the genomic DNAs to generate knockout mutations. Detailed information about these systems is reviewed elsewhere (Gupta and Musunuru, 2014; Puchta and Fauser, 2013).

Genome editing technology can be used in PACs by the following approaches. First, precise chromosome deletions can be made at pre‐designed locations based on the specific recognition of DNA sequences of the genome editing techniques. TMCT occurs randomly because transformed DNAs are integrated randomly in the genome by both Agrobacterium‐mediated and biolistic transformations. To produce desirable minichromosomes, usually a sufficient number of transgenic plants are generated and screened. With the help of genome editing tools, it may be possible to target the telomere repeat DNAs to the DSB sites to seed telomeres at preselected locations to make targeted chromosome truncations. Alternatively, it may be possible to delete large pieces of chromosome arms by genome editing techniques. Genome editing usually generates small deletions, insertions or substitutions at the target site due to the repair of the DSBs by the NHEJ machinery. However, a recent report has demonstrated that 115–245 kb genomic DNA can be deleted by placing two targets of Cas9/sgRNAs apart on the same chromosome (Zhou et al., 2014).

In addition, genome editing can be employed for in vivo gene stacking on minichromosomes. This includes the use of engineered sequence‐specific nucleases. Among them, ZFN has been successfully used to stack two herbicide resistant genes in maize (Ainley et al., 2013). The addition of new transgenes to a pre‐determined locus on a minichromosome at different times further increases the application of PAC platforms in genetically engineered crop production. SSR and current powerful genome editing tools should be able to serve this purpose and carry out precise gene stacking on PACs.

Genome editing has also been demonstrated to be highly efficient in creating gene knockout mutations. This can be used to remove marker genes or any unwanted sequences in PACs. It is also possible to design homologous recombination by TALEN and CRISPR/Cas9 systems to replace genes in PACs. But there are still no reports on the success of any such attempts.

Delivery of PACs

PACs are frequently misunderstood as analogues of BACs or YACs, which can be cloned and introduced into cells easily. In fact, PACs are generated in vivo by TMCT and cannot be cloned in vitro. Thus, the transfer of PACs will be restricted to the same species in which the PACs are produced. In theory, PACs can be introduced into any varieties that are compatible in sexual hybridization. It may also be possible to introduce the PACs into other plant species provided that sexual hybridization between them can be performed to produce viable progeny. For example, individual maize chromosomes as well as the supernumerary B chromosomes can be introduced into oat as chromosome addition lines by genetic crosses and embryo rescue (Koo et al., 2011; Rines et al., 2009). Because maize minichromosomes from both A and B chromosomes are available, similar approaches can be performed to make oat x maize addition lines with maize minichromosomes. In practice, however, this approach introduces not only the PACs but also other chromosomes in the genome to the recipient, and it is tedious to get rid of the other chromosomes by traditional genetic crossing unless, as in the case of oat:maize crosses, there is uniparental chromosome loss. A PAC delivery system based on the combination of PACs with haploid breeding has recently been proposed (Birchler, 2014) based on a recent discovery that a maize haploid inducer line can transmit the supernumerary B chromosomes during a genetic cross to a recipient maize line (Zhao et al., 2013). Because of the elimination of paternal haploids inducer chromosomes in the zygotes, the resulting haploids only contain reduced maternal chromosomes and the B chromosomes. The haploid genome can be doubled to make doubled haploids by colchicine treatment. The utility of this proposed PAC delivery method needs to be experimentally tested.

To deliver PACs across species where a hybridization barrier exists, it should be possible to use somatic hybridization or microprotoplast fusion techniques. Somatic hybridization is the direct fusion of protoplasts of a donor plant with those of a recipient. It plays an important role in bridging the hybridization of alien species to cultivated crops in wild hybridization, or introducing desirable genes from alien species to recipient crops for genetic improvement (Waara and Glimelius, 1995). It overcomes genetic cross barriers that prevent sexual hybridization of genetically distantly related species. Somatic hybridization can be performed as the fusion of two complete genomes (symmetric fusion) or the fusion of one complete genome (as recipient) with a donor whose genome is disrupted by pretreatment with chemicals or irradiation (asymmetric fusion). While symmetric hybridization allows the transfer of the whole genome, asymmetric hybridization only transfers a partial genome to the recipient. An extreme case is the microprotoplast fusion (also called microprotoplast‐mediated chromosome transfer, or MMCT), in which the donor protoplasts are treated to generate microprotoplasts containing a single chromosome or a few chromosomes. MMCT allows the transfer of single intact chromosomes (Ramulu et al., 1993), and thus is the ideal technology for the delivery of PACs when sexual hybridization is not possible.

Commercialization, biosafety and regulatory issues

Although technologies such as genome editing used in PACs may result in genetically engineered crops without transgenes, the PAC vector itself is a transgenic product carrying marker genes and SSR systems. The stacking of more GOIs to the PAC will add more foreign genes to produce numerous transgenic products. Thus, extensive assessment on environmental and health impact must be performed before any market release for commercialization. As genetically modified organisms (GMOs) with multiple genes, the impact of such arrays in PAC‐engineered crops can be assessed concurrently. Thus, the increased number of genes might not necessarily prolong deregulation. With the ease to transfer PACs from one cultivar to another, potentially a package of genes on a PAC can be commercially used without repeated assessment. In addition, PAC technology may have industrial applications to produce pharmaceutical products in a contained environment. With the capacity to express a large number of genes in PACs, it will be possible to engineer plants with biological pathways to produce metabolic products with biomedical activities (Wilson and Roberts, 2014).

However, the potential application of PACs might be influenced by a few factors. First, genetic engineering needs PAC technology in managing multiple genes. The value of genetic engineering with multiple genes has been realized in both public and industrial sectors. For example, to stack multiple genes, technologies have been developed for automatic handling of large numbers of samples for genotyping and marker‐assisted selection in gene stacking (Peng et al., 2014). While the current technology might be able to handle a few genes, the increase in the number of genes will need more efficient technology such as PACs in the future. Second, the time and cost for deregulation might discourage the development of high‐throughput technologies especially in noncommodity crops (Miller and Bradford, 2010; Ricroch and Henard‐Damave, 2015). It has been estimated that $1–15 million extra cost per insertion event was spent for regulatory approval before commercialization (Miller and Bradford, 2010). The gradual awareness of the nature and benefit of genetic engineering in the public might change its acceptance and reconsideration of its regulatory policy, which will greatly reduce the cost for deregulation and commercialization of GMOs, and facilitate the application of PAC technology in genetic engineering.

Conclusions

The development of PACs has opened a new avenue for the next generation of genetic engineering (Halpin, 2005). Next‐generation genetic engineering should make use of many genes rather than one or a few genes as in current practice. The increase in number of genes requires a PAC platform for efficient organization and manipulation. Recently developed technologies are useful tools for efficient manipulation of PACs, but the utility of these technologies needs to be experimentally tested on PACs. With these technology advances, improved PAC technology should play an important role in genetic engineering to produce more and higher quality agricultural and industrial products to meet future demands.

Acknowledgement

We thank Dr. Zaifeng Fan from China Agricultural University for his critic reading of the manuscript. This work was supported in part by National Natural Science Foundation of China (Project No. 31271422), a Shenzhen Commission of Science and Technology Innovation Project (CXZZ20120619150627261) and a Shenzhen Peacock Innovation Team Project (No. KQTD201101) for WY. Research on this topic in the Birchler Lab is supported by National Science Foundation Plant Genome Program grant IOS‐1339198. The authors have no conflict of interest to declare.

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