**3.3 Co-transformation system**

The co-transformation system involves the co-transformation of plant cells with a pair of plasmid vectors, each carrying a selectable-marker gene or a target gene. The selectable marker and target gene integrate into different loci on the chromosome at the same period. Since the two genes integrate at different loci, the selectable-marker gene segregates to produce marker-free transgenic plants. This procedure works for sexually producing crops [46]. For asexually reproducing crops, conventional breeding is utilized for development of new varieties. Thus, co-transformation via sexual hybridization technique is a useful means of producing marker-free cassava, sweetpotato and yam transgenic plants as an intermediate putative parental genotype. The major demerits of this method include its labor intensiveness and the long cycle for the occurrence of genetic transformation [15].

## **3.4 The recombination system**

The site-specific recombination systems reported include Cre/loxP (Cre: causes recombination; loxP: locus of crossing X2 over in P1), R/RS (R: recombinase; RS: recognition site), Gin/gix (Gin: inversion of the G loop; gix: Gin-inversion complex sites), and FLP/FRT (FLP: flipping DNA; FRT: FLP recombination target), consist of a recombinase enzyme and corresponding specific recognition sequences [15]. The functions of the recombinase enzyme are to identify and mediate the recombination of two specific recognition sequences in the same direction leading to the simultaneous and independent formation of the cyclic DNA and the chromosome [15]. This facilitates the elimination of the selective marker gene. Of the three recombination systems, Cre/loxP is the most widely used and studied system. Dale and Ow [47] first utilized the Cre/loxP system to develop transgenic tobacco. The application of site-specific recombination system is still in its infancy stage in root and tuber crops due to its lower transformation efficiency. Saelim et al. [27] reported marker-free transgenic KU50 cassava produced using the MAT vector system (containing the yeast site-specific recombination Rint/RS system mediated excision of DNA fragments and the ipt phenotypic marker gene from recombination sites) developed by Ebinuma et al. [48]. The development of excessive and overgrowth buds in transgenic plants is caused by isopentenyl transferase gene. Genetic recombination during subculture eliminates the expression of this gene to produce marker-free transgenic plants. The conversion efficiency and the proportion of normal growing plants are 19–21% and 32–38%, respectively, indicating

feasibility of its use. Zuo et al. [49] developed the Cre-loxP-XVE system and could be worth testing in root and tuber transgenic program.

### **3.5 The transposon system**

Transposable elements (TEs) or jumping genes or transposons are sequences of DNA that move from one site in the genome to another. The transposons take advantage of their characteristic conservative cut-paste mechanism to transfer genes from one site to another in the chromosome [15]. This attribute of transposons reduces the probability of genetic linkage at the new site even after occurrence of transposition to the new site. This peculiar attribute of TEs is useful for the removal of unwanted marker gene from the transgenic plants. The removal of the unwanted marker gene is accomplished in two ways: (i) the marker gene could be placed between the TEs and the repeat sequence Dissociator (Ds) element; or (ii) the target gene could be placed between the Ds sequences. During transposition, the marker gene maybe lost or separates from the target gene. The occurrence of the transposon is achieved by progeny segregation; therefore, its application in root and tuber crops is very difficult.
