**3.6 Non-antibiotic selection**

In the non-antibiotic selection system, growth of transformed cells is favored by the development, promotion or additional metabolic activity, without affecting non-transformed cell growth by hormonal stimulation or inhibition by starvation from nonmetabolizable sugars, or death by antibiotics [15]. Unlike the conventional antibiotic-selection systems, the non-antibiotic techniques exhibit better acceptability by the public as they are considered to be safer than the antibiotic-selection systems. Selectable-marker genes from hormonal action Isopentenyl transferase (ipt) and indole-3-acetamide hydrolase (iaaH) are reportedly the most widely used hormone metabolism genes [15]. The Isopentenyl transferase gene was cloned from *A. tumefaciens* T-DNA and is related to cytokinin biosynthesis. The overexpression of ipt in transgenic plants has been observed to cause phenotypic variations in them [15]. This necessitates use of an inducible promoter to regulate its expression or the ipt gene should be applied in a site-specific recombination enzyme system or transposon system to build a highly-efficient marker-removal system. Saelim et al. [27] opined the usefulness of the Rint/RS system for excision of the ipt gene from transgenic cassava KU50. This technology is useful in the genetic transformation of crops with long cycle of breeding. The iaaH is also a safe selective-marker gene detected in the regulation of hormone metabolism. The overexpression of iaaH results in abnormal production of transgenic plants. However, the expressivity of iaaH can be modulated, inactivated or removed [15].

#### **3.7 Selectable-marker genes based on sugar metabolism**

The selectable genes consist of selectable markers utilized to facilitate the isolation of plasmid-containing transformants [15]. Selectable markers are useful for genetic transformation as they permit plant cells to grow under conditions that prevent the growth of untransformed tissue. Selectable-marker genes based on sugar metabolism comprise of the mannose phosphate isomerase (pmi) gene and the xylose isomerase (xylA) gene [50]. The pmi system is a positive selection system that uses d-mannose as a selection agent. The mechanism of the system is well reviewed by Liu et al. [15]. The pmi gene has been widely applied in the transformation systems of rice, corn, wheat, and sugar beet [51]. The pmi-mannose system has

#### *Genetic Modification and Application in Cassava, Sweetpotato and Yams DOI: http://dx.doi.org/10.5772/intechopen.101037*

also been used in cassava transformation system [40]. For validation of this technique, Zhang [16] constructed a pHMG binary vector that uses the visual marker GUS gene, independent expression cassettes of the pmi and hygromycin phosphotransferase genes in its T-DNA region. They observed 82.6% Agrobacteriummediated transformation of embryogenic suspensions by the mannose selection system compared to 100% hygromycin selection system. Transgenic plants were also generated from application of the biolistic transformation of somatic cotyledons as explants, and mannose as the selective agent [40].

Another selectable marker xylA gene, encodes xylA and catalyzes the conversion of d-xylose through d-xylulose tautomerism. The growth of transformed cells is promoted in the medium containing a carbon source material such as d-xylose, whereas the growth of non-transformed cells is inhibited by the lack of a suitable carbon source [15]. Haldrup et al. [52] noted the production of transgenic plants using d-xylose as the carbon source. Application of this technique is yet to be fully exploited in root and tuber crops.

### **3.8 Tissue-specific promoter**

Liu et al. [15] reported three types of promoters based on spatial and temporal expression patterns including inducible promoters, constitutive promoters, and tissue- or organ-specific promoters. For cassava genetic transformation, the CaMV 35S promoter and methyl jasmonate and salicylic acid-induced nopaline synthase gene promoter are the widely used promoters. These promoters regulate genes of interest and selective marker gene or reporter gene. The gene expression level of CaMV 35S promoter in transgenic cassava leaves was noted to be higher relative to the storage roots indicating possible influence on the function of certain genes in cassava storage roots [29].

Further studies to increase the specific expression of a target gene in cassava organs and tissues led to the discovery of specific promoters in leaves or storage roots. Zhang et al. [29] detected two cassava promoters such as p15/1.5 of a cytochrome P450 protein, and p54/1.0 of the cassava glutamic acid-rich protein, Pt2L4, from a cassava storage root cDNA library. The gene expression patterns of these promoters show close association with cassava vascular tissues and storage root development, especially in the starch-rich parenchyma cells of the storage root. Their activities are also stronger than those of CaMV 35S promoter. The function of the glutamic acid-rich protein promoter has been well articulated [30, 53–55]. Application of p54/1.0 promoter regulated the dsRNA expression for interference with the granule-bound starch synthase (GBSS)I expression producing amylosefree transgenic cassava [56].

Tuber-specific class I patatin promoter was noted to regulate several gene expression. Ihemere et al. [57] reported its gene regulation activity in the expression of the *Escherichia coli* gene, glucose-1-phosphate adenylyltransferase (glgC) with insensitivity to the substrate, by site-directed mutation in cassava for increased starch content. Siritunga and Sayre [25] noted its regulation activity in CYP79D1 and CYP79D2 antisense gene expression in cassava for decreased toxicity of cyanide. Abhary et al. [32] reported the role of the promoter in the expression of a zeolin fusion protein to increase protein content. A promoter regulating protein family AAI\_LTSS of unknown function in cassava was found strongly expressed in the secondary xylem of the carrot [58].

Leaf senescence inducible promoter SAG12 was found to regulate the ipt gene that moderate extended leaf longevity and improved the drought resistance in transgenic [28]. Leaf specific cab1 promoter applied in transgenic cassava regulated transgene expression Siritunga and Sayre [25]. Based on existing information on

tissue-specific promoters, greater application of this technology is envisaged in future root and tuber breeding programs.

## **4. Applications of genetic modification technology in cassava, sweetpotato and yam improvement programs**

Application of functional genomics and genetic engineering has contributed to resolve the problems associated with the germplasm enhancement of root and tuber crops. For cassava, improved agronomic traits achieved by transgenic technology include virus resistance, improved nutritional quality, reduced cyanide content, improved biomass, and delayed post-harvest physiology deterioration in storage roots.

#### **4.1 Resistance to pests and viral diseases**

Pests and viral diseases are transmitted via infected stems, vines, tubers from generation to generation, subsequently causing yield losses. For instance, the cassava mosaic disease (CMD) accounts for about 20–95% yield reductions [59]. It is the major cassava disease in Africa and the Indian peninsula. The CMD is caused by several cassava geminiviruses and their satellite components, including the ACMV, Eastern ACMV, and Indian cassava mosaic virus [59, 60].

Transgenic technology has played an important role in obtaining virus resistant cultivars [61]. Chellappan et al. [62] utilized pILTAB9001 and pILTAB9002 harboring the wild-type and mutant AC1 genes of ACMV-Kenya. These wild-type and mutant AC1 genes regulate the cassava vein mosaic virus promoter and the pea Rubisco terminator, which enables the production of transgenic TMS60444 plants with increased resistance to mosaic disease [62]. The initial inoculation assay detected transgenic plants resistant to several cassava geminivirus diseases from Africa. However, these transgenic plants were later susceptible to CMD infection in a closed-field trial in Kenya. With the aid of improved antisense RNA technology, Zhang et al. [63] developed transgenic cassava plants with increased ACMV resistance targeting the viral mRNAs of Rep (AC1), TrAP (AC2), and REn (AC3). Several transgenic clones remained symptomless after biolistic inoculation of ACMV at infection pressure of 100 ng viral DNA plant−1. Decreased and attenuated symptom development were also detected even at higher viral DNA doses. Significant reduction in viral DNA accumulation was observed in the leaves of transgenic ACMV-resistant plants. Application of RNAi-mediated gene-silencing approaches, siRNAs, homologous to either the common region or AC1 in transgenic cassava plants suppressed the replication of African mosaic virus, leading to recovery after infection with ACMV [64] or immunity to infection by the virus [31]. Bi et al. [65] screened cassava germplasms from using Agrobacterium-mediated inoculation in combination with CMD-resistant molecular markers RME1, SSRY28, and NS158. Findings showed that the cassava germplasms lacked CMD-resistant genes, suggesting the necessity of introducing and integrating disease-resistant cassava genotypes from Africa into the current breeding program, while developing CMD-resistant cassava using different transgenic approaches.

Cassava brown streak disease (CBSD) is another important viral disease of cassava in Africa. A sequence analysis of CBSD showed that the causative virus belongs to potato virus-Y of the ipomovirus family [66]. Two subspecies of CBSD are cassava brown streak virus (CBSV) and cassava brown streak Mozambique virus (CBSMV) [67]. Transgenic approach is noted to be more promising for development of CBSD clones relative to the traditional cassava-breeding method. Resistant

#### *Genetic Modification and Application in Cassava, Sweetpotato and Yams DOI: http://dx.doi.org/10.5772/intechopen.101037*

cassava lines have been developed by transferring the virus coat protein gene or through RNA (siRNA) interference [33, 68]. Cassava bacterial blight (CBB) disease (gum disease), caused by *Xanthomonas manihotis*, mainly affects cassava leaves. Transcriptomic studies have shown a rapid change in cassava genes after infection by this disease [69]. The main insects that infest cassava are whiteflies, cassava mealybugs, cassava green mites, and stemborers, while root-knot nematodes are the most widely-reported parasitic nematodes on cassava. The cultivation of insect resistant cassava genotypes increases the yield and quality of the crop. Insecticide proteins including Bt Cry proteins, protease inhibitors, α-amylase inhibitor, and plant lectins, could aid insecticides, as a high expression of these products in transgenic cassava might facilitate increased insect resistance.

Targeted genome alteration technique is a promising tool for yam breeding. Successful application of the CRISPR/Cas9 technology resulted into inactivation of the endogenous banana streak virus by editing the virus sequences to develop resistant plantain [70]. Yam viruses have also been found to be integrated into the genome of yam and for the development of yam genotypes resistant to yam mosaic virus using CRISPR/Cas9 approach [71, 72]. The major challenge of the CRISPR/ Cas9 technology is that it may recognize sequences with up to five mismatched bases suggesting high rates of off-target effects [73]. However, techniques such as DNA-RNA chimeric guides, Cpf1, a single RNA endonuclease that employs a T-rich PAM on the 5′ side of the guide, and specific point mutations have been developed to mitigate this challenge [74, 75].

The recently established gene-editing technique, the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system, resulting from the adaptive immune system of *Streptococcus pyogenes*, is a notably potent tool for targeted genome editing in many species [76]. Gene editing and genetic engineering technologies have been reported to facilitate improvement of the productivity and nutritional quality of yam. This was achieved through the Genome-Enabled Platforms for Yam Project launched in 2016 in collaboration between scientists at the International Institute of Tropical Agriculture (IITA) and Iowa State University (https://www.nsf.gov/awardsearch/showAward?AWD\_ID=1543888). Moreover, a genome-editing tool for yam using phytoene desaturase (a key enzyme in the β-carotene biosynthesis pathway, which converts the colorless phytoene to colored carotenoids) as a marker is being developed [77]. Targeted traits such as resistance to yam mosaic virus and anthracnose diseases, herbicide tolerance and nematode resistance are being investigated using yam gene editing and genetic engineering technique.

Feng et al. [76] successfully applied the CRISPR/Cas9-mediated targeted mutagenesis in *D. zingiberensis* using an *A. tumefaciens*-mediated transformation method. Their study aimed at an essential gene involved in the synthesis of secondary metabolites, known as the farnesyl pyrophosphate synthase gene (Dzfps). They detected five types of mutations among the transformed plants at the predicted double-stranded break site. Feng et al. [76] also found that the transcript levels of Dzfps and the content of squalene in isolated mutants were drastically decreased relative to those in wild-type plants and concluded that CRISPR/Cas9 is a rapid and efficient method for targeted genome modification in *D. zingiberensis*.

#### **4.2 Improved stress resistance**

Cassava is susceptible to cold stress, resulting into decrease root yield and cumbersome maintenance of cassava stems. In southern China, winter, rain, snow, and freezing temperatures cause severe frostbite to cassava stems [15]. In 2008, damage of cassava stems caused by cold stress in Guangxi amounts to several billion RMB. Thus, development of low temperature resistant cassava clones aids easier stem storage and increasing cultivation in temperate countries, thereby meeting the demand for raw cassava materials for the industries. High expression of the C-repeat/dehydration-responsive element-binding factor 3 gene regulated by a low temperature inducible or CaMV 35S promoter has been noted to significantly improve cold resistance in transgenic cassava [15]. The growth and yield of cassava are influenced by intermittent drought in tropical and subtropical regions. Application of leaf senescence induced promoter, SAG12, for expression of the ipt gene, transgenic cassava revealed prolonged leaf life, and improved resistance to drought stress [28]. This study demonstrates a useful strategy for the improvement of drought resistant and high yield cassava clones. Several authors have also noted a large number of genes associated with stress resistance using high-throughput genomic and transcriptomic analyses of cassava [78–80]. Functional verification of these genes is needed for better understanding of the molecular mechanisms of cassava resistance to different stresses and establishment of the theoretical basis for cassava genetic improvements.

#### **4.3 Enhanced nutritional quality**

Nutritional quality enhancement in roots and tubers can improve the dietary and nutritional balance of people who utilize them, particularly in central and western Africa. Zhang et al. [24] noted the production of transgenic cassava exhibiting an artificial storage protein enriched with essential amino acids, under the control of the CaMV 35S promoter. The transgenic plants were detected expressing ASP1 at both the RNA and protein levels. The leaves of transgenic plants had increased proline and serine contents, while the amounts of aspartic acid, alanine, and methionine were decreased compared to non-transgenic plants. Altered composition of amino acids and reduced cyanide content were also observed in transgenic plants [32]. Thus, cassava protein enhancement using transgenic approach is practically feasible and is a useful means of reducing protein deficiency in poverty-stricken regions [81].

The BioCassava Plus project team has developed transgenic cassava clones with value-added traits including virus resistance, improved protein content, and increased vitamin A, iron, and zinc contents [10]. The HarvestPlus project team has also developed β-carotene-rich cassava clones [82].

### **4.4 Reduced cyanide content**

Siritunga and Sayra [25] utilized an Arabidopsis leaf-specific promoter to drive the antisense expression of cytochrome P450 genes (CYP79D1 and CYP79D2). In vitro tests of the linamarin content of the transgenic leaves revealed a decrease of 60–94% compared to the control, while a 99% decrease was observed in the storage roots of cassava, suggesting the transport of linamarins from leaves to storage roots. White et al. [83] noted that at transcript level, the hydroxynitrile lyase content in cassava roots is only 6% of that in the leaves. The overexpression of hydroxynitrile lyase reduces the acetone cyanohydrin content of roots, thereby accelerating the detoxification process. The overexpression of hydroxynitrile lyase was achieved through the cloning of the cDNA of the gene encoding hydroxynitrile lyase between the CaMV 35S promoter and the pea ribulose bisphosphate carboxylase terminal sequence, and transformed into MCol2215 [84]. The authors found a 40–135% increase in the hydroxynitrile lyase activity in transgenic plants, compared to 800–1300% found in the storage roots of cassava. However, no changes were detected in the total amount of linamarin and lotaustralin detected in the whole

plant [84]. After harvesting, the detoxification capacity of the root was greatly enhanced. Jørgensen et al. [26] conducted similar trial by RNAi and found a 92% decrease in the cyanogenic glucoside contents of cassava storage roots.
