**2.1** *Agrobacterium***-mediated genetic modification method**

*Agrobacterium*-mediated genetic modification has been the method of choice for the development of genetically modified crops. The major merits of Agrobacterium-mediated genetic modification are its high frequencies of singlecopy integration, high reproducibility, transformation efficiency, stable expression of transgenes, utilization of simple equipment, ease of accessibility, ability to transfer low copies of DNA fragments carrying the desirable genes at higher efficiencies with minimal cost and the transfer of very large DNA fragments with low rearrangement [17].

The first attempt to transform cotyledonous embryos of cassava MPer183 with *Agrobacterium tumefaciens* CIAT1182 started in 1993–1994, but the transgenic

#### **Figure 1.**

*Illustration of an in vitro plant regeneration system of a typical root crop. Redrawn from Zhang [16].*

nature of regenerated plants could not be verified by Southern analysis [18]. The first successful Agrobacterium mediated cassava genetic transformation was done in Potrykus laboratory at ETH Zurich in 1996 [19]. Several Agrobacterium strains sheltering different binary vectors such as LBA4404 (pTOK233), LBA4404 (pBin9GusInt), C58C1 (pIG121Hm), and EHA105 (pBin9GusInt) were used for the transformation of cassava somatic cotyledons. Gonzalez et al. [20] successfully transformed TMS60444 with the *A. tumefaciens* strain ABI using Agrobacteriummediated FEC transformation. The transgenic nature of the two plant lines resistant to paromomycin was confirmed by glucuronidation glycosidase (GUS) assay and Southern analysis. The embryogenic suspensions of TMS60444 were transformed with *A. tumefaciens* LBA4404 using negative- and positive-selection agents [21]. A total of 12 morphologically-normal transgenic lines have been developed, of which, are five are mannose selected and seven hygromycin resistant. Moreover, polymerase chain reaction (PCR) and Southern analysis confirmed the successful integration of the transgene into the genome. The confirmation of expression of the transgene in the regenerated plants was done using reverse transcription (RT)–PCR and Northern analyses. In 2000, Sarria et al. [22] successfully transformed a herbicide (phosphinotricin, ppt)-resistance gene into the cotyledons of cassava MPer183 by an Agrobacterium-mediated method and found stable transgenic plants resistant to Basta spray (at concentrations of 200 mg/L or more). An efficiently robust and reproducible transformation protocol for cassava embryogenic suspension culture with *A. tumefaciens* has also been reported [23]. Of the 31 GUS-active plants identified, 14 were found with 100% GUS activity, whilst the remaining lines had 72% GUS activity. The transgenic nature of these plants was detected using the southern blot analysis. Zhang et al. [24] successfully introduced a synthetic artificial storage protein 1 (ASP1) gene encoding a storage protein rich in essential amino acids (80%) into embryogenic suspensions of cassava using Agrobacterium-mediated FEC transformation. The ASP1 tetramer was detected in the leaves and primary roots of transgenic cassava plants by Western analysis. Another achievement in the cassava genetic modification is the development of transgenic cassava with a lower cyanide content using MCol2215 cotyledon explants [25]. Jørgensen et al. [26] constructed several RNAi and antisense vectors to interfere with the expression of CYP79D1 and AYP79D2, and transformed the somatic cotyledons of cassava MCol22 using an Agrobacterium-mediated technique. In 2009, a multi-autotransformation (MAT) vector system of isopentenyl transferase (ipt) type was utilized for the production of marker-free transgenic cassava plants with conversion efficiency up to 19–21% via shoot organogenesis of KU50 somatic cotyledons [27]. Zhang et al. [28] developed transgenic cassava with senescence-inducible expression of the ipt using Agrobacterium-inoculated TMS60444 somatic cotyledons via shoot organogenesis. These achievements contributed to cassava transformation for the verification of a tissue-specific promoter [29, 30], the resistance to African cassava mosaic virus (ACMV) [31], increased protein content [32], and improved cassava brown streak virus resistance [33].

For sweetpotato, Kyndt et al. [12] reported that the Agrobacterium-mediated gene delivery system was utilized for T-DNA integration, the interruption of an *F-box* gene, and the subsequent insertion of foreign T-DNA into the sweetpotato genome. This is believed to have occurred during the evolution and domestication of this crop. White et al. [34] suggested that the identification of gene sequences in IbT-DNA1 and IbT-DNA2 imply that the transforming Agrobacterium was probably Agrobacterium rhizogenes, an ancestral form of A. rhizogenes or a closely related species (perhaps extinct) of A. rhizogenes. The *Ib*T-DNA1 corresponds to TR-DNA (typically containing the auxin biosynthesis genes *iaa*M and *iaa*H), and *Ib*T-DNA2 corresponds to TL-DNA (harboring the *Rol* genes). The gene organization and DNA

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

sequences of the T-DNAs are similar to, but distinctly different from, the ORFs of the Ri and Ti-plasmids in well characterized *Agrobacterium* strains. The identified *RolB*/*RolC* region represents a new member of the *RolB* family indicating that, unlike the T-DNA found in *Nicotiana* spp. [35], the *Agrobacterium* strain (or species) that transferred its T-DNA into the sweetpotato genome is uncommon.

For yams, the Agrobacterium-mediated gene delivery system is the most preferred technique utilized for the genetic modification of the crop [14, 36]. Initial development of a transient genetic modification of *Dioscorea rotundata* using the Agrobacterium-mediated produced no transgenic plants [37]. However, the first fast, efficient and reproducible protocol for Agrobacterium-mediated transformation of *D. rotundata* resulted in the generation of stable transformations and the regeneration of complete transgenic yam plants [8]. This achievement laid the foundation for the full implementation of genetic engineering and gene editing in yam. Based on the review, it is clear that Agrobacterium-mediated transformation system is the most prominent genetic modification technique due to the availability of a large number of transgenic plants. The transformation efficiency of this technique can be improved using a protocol based on somatic cotyledons as explants for the transformation of cassava, sweetpotato and yams. Thus, it is the most widely-used method for genetic engineering in the studied root and tuber crops.

#### **2.2 Biolistic-mediated genetic modification method**

Biolistic transformation or gene gun or particle bombardment technique is often utilized for plant transformation studies. The technique involves FEC induction, subculturing, somatic embryogenesis, and plant germination. The biolistic transformation technique involves series of protocols and stages [15]. These include the subculturing in SH liquid medium, followed by supplementation with 50 μM picloram, without selection for 2 weeks. The samples are further subjected to SH liquid medium with 25 μM paromomycin for 4–5 weeks, solid SH medium with 25 μM paromomycin for 4 weeks, and Murashige and Skoog (MS) medium with 5 μM picloram for embryogenesis. The next stage involves the development of transformed cell clusters into somatic embryoids in maturation media (MS medium supplemented with 0.5% activated charcoal). The final stage is the regeneration and verification of transgenic plants exhibiting GUS-positive and paromomycin resistance using Southern analysis [15]. This technique was successfully conducted on somatic cotyledons of cassava genotypes CMC40, MPer183, MCol22, and TMS60444 suspensions [21, 38]. Twenty transgenic plants of TMS60444 and 11 transgenic lines of MCol22 were obtained. The GUS, Southern blot, and RT-PCR assays indicated the successful integration of the transgene into the plant genome. Transgenic cassava has also been produced from the plasmid constructs pHB1 and pJIT100 using FEC of TMS60444 and particle bombardment [39]. Of the dozens of transgenic plants produced using the pHB1 and pJIT100 constructs, some have been analyzed at the molecular level. Zhang and Puonti-Kaerlas [40] used particle bombardment to transfer the plasmid pHMG into TMS60444 embryogenic suspensions. Selection from the dozens of transgenic cassava plants produced in less than 15 weeks was based on either negative hygromycin or positive mannose. Zhang et al. [41] also utilized FEC and a particle bombardment technique to investigate ACMV resistance in cassava.

The success of this method depends on the high efficiency of the particle bombardment and shoot organogenesis. The long time utilized for the FEC induction, subculturing, somatic embryogenesis, and plant germination causes a low efficiency of plant regeneration and a high rate of somaclonal variation. This indicates low probability of success due to its complicated operation and its susceptibility to

many factors. Thus, the technique is infrequently used for genetic transformation in roots and tubers. This necessitates the establishment of a robust standard protocol for FEC-based transformation for each cultivar.

#### **2.3 The friable embryogenic callus genetic modification method**

The embryogenic callus tissue utilized in this genetic modification technique are friable as they tear, slough and bleed more easily when touched. For the cassava embryogenic callus genetic modification, the induction of primary somatic embryos on the embryogenesis induction medium of the immature young leaves and apical or axillary meristems are useful for the establishment of cyclic secondary somatic embryogenesis using the subculture of the induction medium [15]. However, continual subculturing of the secondary somatic embryos on Gresshoff and Doy (GD) medium, supplementation with 12 mg/L picloram results into production of FEC, formation of non-embryogenic calli and secondary somatic embryos [15]. Establishment of an embryogenic suspension for rapid multiplication in liquid Schenk and Hildebrandt (SH) medium containing 10–12 mg/L picloram requires appropriate selection of FEC and subculturing on the GD solid medium in the longterm. Culturing of embryogenic suspension cells on MSN solid media with 1 mg/L naphthaleneacetic acid, produce somatic embryos and subsequently cotyledon-stage somatic embryos that germinate to plantlets [42, 43]. The FEC and embryogenic suspension cultures are susceptible to Agrobacterium infection, and favorable to particle bombardment, making the delivery of foreign genes easy. Cassava transformation is usually done using FEC and/or embryogenic suspensions by *A. tumefaciens* or particle bombardment.

#### **2.4 Shoot organogenesis-based genetic modification method**

This technique involves the induction of somatic embryogenesis from immature leaves and apical or axillary meristems using green cotyledons as explants and plant regeneration via shoot organogenesis to overcome genotype restrictions [44]. However, an efficient shoot organogenesis using mature green cotyledons has been developed from secondary somatic embryos in AgCOM medium supplemented with N6-benzylaminopurine and AgNO3 (an ethylene action inhibitor) [45]. The shoot organogenesis-based genetic modification method is most applicable and suitable for the biolistic or Agrobacterium-mediated genetic transformation since these techniques accommodate the initiation of shoot primordia from cut ends and epidermal cells as well as the regeneration of transgenic plants in a short cycle of 3–4 months. Zhang et al. [21] established that subjection of transgenic shoots to rooting sensitivity tests could quickly eliminates false-positive transgenic plants, thereby preventing difficult and expensive molecular analyses. This is also a reliable method for screening transgenic cassava plants.

#### **3. Novel genetic transformation technologies**

#### **3.1 Genetic transformation**

Advances in transgenic technology has led to the development of different schemes for the delivery of target genes into plant cells [15]. Selectable-marker genes including herbicide- and antibiotic-resistance genes, and the reporter genes such as GUS, luc, chloramphenicol acetyltransferase, and green fluorescent protein. The selectable-marker and reporter genes are used for screening and monitoring

of putative transgenic plants. However, there are lots of public concern about the biosafety of using these marker genes are widely used for genetic transformation. This necessitates use of suitable alternatives as selectable markers. Roots and tubers are conventionally vegetatively propagated crops with the merit of reduced risk of horizontal gene transfer to relevant organisms and pathogens through pollen. This indicates the necessity of developing safe marker genes or marker-free technology in root and tuber crops.
