**1. Introduction**

Transgenic plants have been around for some time and have become of age. Their strict regulation and public approval processes remain controversial with some people opposed to transgenic plants for reasons that can broadly be categorized into real, perceived or imagined based on established scientific facts. During evaluations before releasing into the environment, the transgenic plants are evaluated to confirm if they deliver the benefits they claim, and whether they are safe to human health and the environment [1]. Continued engagement with these concerns has led to the exploration of possible solutions that make the technology more efficient, safer, and more widely accepted.

Many of the concerns associated with genetically modified plants emanate from the use of reporter genes and selectable marker genes such as antibiotic or herbicide resistance genes in the process of genetic engineering [2, 3]. These genes, together with the transgene of interest, are usually part of a relatively large plasmid that has sequences that are not required in the genetically modified plant but become transferred to the plant and get integrated into the plant genome because of the general and non-specific nature of plant transformation methods. The reporter and selectable marker genes together with vector backbone sequences once present in the transgenic plant are of ecological concern because they may be passed on to other species in the environment, resulting in such characteristics as weediness and

invasiveness [1, 2]. The spread of antibiotic resistance genes is also of great concern to human health. The integration sites of these genes are random and may result in some 'unintended effects' such as inactivation of important genes and production of new toxins or allergens. All these factors are considered during evaluations for the release of genetically modified plants.

Over the years, the concept of cisgenic plants has emerged and is contrasted with transgenic plants by using DNA sequences from naturally crossable species and possibly avoiding the use of reporter and selectable marker genes as well as vector sequences [4, 5]. This chapter will explain how that is achieved, and why cisgenic plants might be more widely acceptable to regulators and consumers.

### **2. History and controversies of transgenics/GMOs**

In 1994, the genetically modified Flavr Savr™ tomato was commercialized [6]. This was a great stride for both science and commerce, and the society's response to this new type of product has helped determine how such new technologies are regulated. While some proponents of genetic engineering would have wished for no labelling and minimum statutory regulation of the development and environmental release of the GMOs, an antagonistic anti-GMO movement arose and advocated for a 'ban' on GMOs. This created a healthy, restrained environment in which real, potential and imagined dangers of the new technology could be objectively evaluated. Processes for approval and release of GMOs were established, enabling society to have a say, whatever the nature of their reservations might be.

Singh et al. [1] lists five potential risks associated with the cultivation of transgenic crops. These are: (1) Introduction of allergenic or harmful proteins into the foods; (2) Detrimental effects on non-target species and the environment; (3) Increased invasiveness and weediness of crop plants; (4) Increase pest and disease resistance in response to intense selection pressure; and (5) Fear of biodiversity loss. These potential risks must be addressed before regulatory approval for the release of a transgenic plant is granted. Scientific research has therefore continued to look for ways to eliminate the sources of these concerns, where possible.

Many of the potential risks related to the presence of a reporter and selectable marker genes in the GMOs. Vector backbone sequences often get integrated into the plant genome as well [2]. The sites of integration of these DNA sequences are often random, possibly disrupting some essential gene functions, giving rise to toxic or allergenic products and some other non-intended effects [5]. Alternative methods of genetically engineering plants have been developed to better address some of these concerns.

Two main methods are used for plant transformation: *Agrobacterium*-mediated transformation and biolistics (bombardment) [2, 3]. The processes have been studied for a long time and there is some understanding of how transgenes enter the cell cytoplasm and nucleus in both cases, the mechanisms for transgene integration into the genome cannot be easily manipulated. The mechanisms of integration involve homologous or non-homologous recombination and are reviewed by Mundembe and Hwang et al. [7, 8].

It became apparent that the methods of plant transformation and mechanism of DNA integration were intricately linked to the concerns raised against transgenic plants [2]. The selectable marker such as herbicide resistance or antibiotic resistance gene is required as a mechanism to positively select for transformed plants over untransformed plants; untransformed plants will not survive in the presence of the herbicide or antibiotic [2]. The reporter gene gives a visual marker such as colour or fluorescence that enables the experimenter to tell the transformed nature of any

tissue easily. These marker genes are usually on the same piece of DNA as the gene of interest so that the presence of the marker genes can be taken as an indication of the presence of the gene of interest as well. The presence of these genes in the environment is a major concern. In addition, the site of integration of these genes is random.

Vector backbone sequences also often become integrated into the plant genome. Applicants for approval are required to demonstrate that vector backbone sequences are absent for approval to be granted. Plant transformation experiments are designed on the assumption that only the sequences between the left and right borders of a T-DNA will be transferred to the plant genome. Widespread reports of integration of vector sequences were cited by opponents of genetic engineering as evidence that the genetic engineering of plants was not sufficiently understood to be released into the environment. The perception of 'randomness' of transfer and integration made the public uneasy about GMOs.

Site-specific recombination promised to circumvent the concerns about the randomness of the site of integration. Site-specific recombination systems have been studied since the 1980s. These include Cre-lox P ('*c*auses/*c*yclization *r*ecombination/*lo*cus of crossing over, *x*, in P1'), FLP/FRP (flippase/flippase recognition target) and integrase [9]. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats- CRISPR-associated gene 9 (CRISPR-Cas9) is another group of nucleases that has been adapted for manipulation of DNA at specific sites [10].
