**3. The transformation process**

As already indicated, *Agrobacterium*-mediated genetic transformation relies on the natural genetic transfer process causing crown gall disease in plants. This biological method had led to the modification of genomes or addition of genes in various crop plants like maize, cowpea, sunflower, canola and rice [12–16]. The manipulation of *Agrobacterium* by scientists allowed for the transfer of T-DNA without causing tumours in transformed plants. This was achieved by silencing the tumour-inducing genes found on the extrachromosomal plasmid [17]. The transgenes introduced are specifically defined and precisely transformed in the laboratory before being delivered in targeted host plant tissues.

The delivery protocols differ according to species and the purpose of transformation. Thus, the methods for DNA transfer and expression in various plant species are quite varied and their applications in different genotypes always require optimization. This implies that, a tremendous effort still need to be placed on developing more efficient and reproducible transformation procedures. According to Finer and Dhillon [18], *Agrobacterium* is one of the main methods routinely used by many laboratories for plant transformation. This method is considered rapid, most efficient and cheaper for the transformation of many crop plants compared to other techniques. Other methods used for genetic transformation, their advantages and setbacks are discussed below.

### **3.1. Other methods of genetic manipulation**

One of the most interesting techniques for DNA transfer is Agroinfiltration. In agroinfiltration, *Agrobacterium* is infiltrated or injected into plant cells (leaves) of a suitable host. This method induces transient expression of genes in a plant by forcing *Agrobacterium* suspension into the internal leaf tissues using a syringe [19]. Zhao et al. [20] reported transformation of *Nicotiana benthamiana* using this method with *Agrobacterium* strain harbouring pCAMBIA1301. The β-glucuronidase (GUS) expression and GUS activity showed increased transgene expression more than 6-fold for agroinfiltration suspension containing 20 μM 5-azacytidine, 0.56 mM ascorbic acid and 0.03% Tween-20. The floral dip method of *Agrobacterium*-mediated transformation also have been developed. The flowers of plants to be transformed are immersed in a suspension of *Agrobacterium* containing wetting agent, for example; Tween-20 or Silwet to allow bacterial access to pores or cracks on the flower. This method was reported by Finer and Dhillon [18] to be designed specifically for *Arabidopsis*, and there is no other plant that, currently respond positively like *Arabidopsis*. In contrast, Verma et al. [21] reported more than 1% transformation efficiency in *Brassica napus* cv. Elect and *Brassica carinata* cv. Pusa Gaurav using this method. However, various attempts in many plant species to develop floral dip transformation protocols have been met with very limited transformation efficiencies [21]. Another method is particle bombardment invented by John Sanford [22]. This microprojectile or biolistic bombardment employs particle acceleration coated DNA into the target plant tissues. Once in the cells, the DNA becomes permanently integrated into the chromosomes of the host plant genome [20]. Although, there are numerous techniques used for transformation, all methods face the same challenges of inefficiency, lack of a routinely used protocol and the genotype specificity problem.

### **3.2. Challenges faced during genetic transformation**

and assembly of proteins that get remobilised or degraded [10]. Proteases are well known for their key role in biochemical processes, implicated for the development and continuation of several diseases. Their role in disease formation, especially during programmed cell death (PCD) involves dismantling of organelles and the different macro molecules required for plant growth and development. They are largely involved in translation and folding of storage proteins, protein remobilisation, signalling controls and at lesser extent for morphogenesis [11].

As already indicated, *Agrobacterium*-mediated genetic transformation relies on the natural genetic transfer process causing crown gall disease in plants. This biological method had led to the modification of genomes or addition of genes in various crop plants like maize, cowpea, sunflower, canola and rice [12–16]. The manipulation of *Agrobacterium* by scientists allowed for the transfer of T-DNA without causing tumours in transformed plants. This was achieved by silencing the tumour-inducing genes found on the extrachromosomal plasmid [17]. The transgenes introduced are specifically defined and precisely transformed in the laboratory

The delivery protocols differ according to species and the purpose of transformation. Thus, the methods for DNA transfer and expression in various plant species are quite varied and their applications in different genotypes always require optimization. This implies that, a tremendous effort still need to be placed on developing more efficient and reproducible transformation procedures. According to Finer and Dhillon [18], *Agrobacterium* is one of the main methods routinely used by many laboratories for plant transformation. This method is considered rapid, most efficient and cheaper for the transformation of many crop plants compared to other techniques. Other methods used for genetic transformation, their advantages

One of the most interesting techniques for DNA transfer is Agroinfiltration. In agroinfiltration, *Agrobacterium* is infiltrated or injected into plant cells (leaves) of a suitable host. This method induces transient expression of genes in a plant by forcing *Agrobacterium* suspension into the internal leaf tissues using a syringe [19]. Zhao et al. [20] reported transformation of *Nicotiana benthamiana* using this method with *Agrobacterium* strain harbouring pCAMBIA1301. The β-glucuronidase (GUS) expression and GUS activity showed increased transgene expression more than 6-fold for agroinfiltration suspension containing 20 μM 5-azacytidine, 0.56 mM ascorbic acid and 0.03% Tween-20. The floral dip method of *Agrobacterium*-mediated transformation also have been developed. The flowers of plants to be transformed are immersed in a suspension of *Agrobacterium* containing wetting agent, for example; Tween-20 or Silwet to allow bacterial access to pores or cracks on the flower. This method was reported by Finer and Dhillon [18] to be designed specifically for *Arabidopsis*, and there is no other plant that, currently respond positively like *Arabidopsis*. In contrast, Verma et al. [21] reported more than 1% transformation efficiency in *Brassica napus* cv. Elect and *Brassica carinata* cv. Pusa Gaurav using this method. However, various attempts in many plant species to develop floral dip transformation protocols

**3. The transformation process**

20 Transgenic Crops - Emerging Trends and Future Perspectives

and setbacks are discussed below.

**3.1. Other methods of genetic manipulation**

before being delivered in targeted host plant tissues.

There are several challenges faced during the process of delivering segments of oncogenic DNA to susceptible plant cells. The limitations are mostly associated with *in vitro* culture conditions than the genetic transfer and expression. Plant regeneration *in vitro* can be efficiently and rapidly achieved for plantlets micropropagation. Soybean has been successfully regenerated through adventitious/axillary/meristem shoot organogenesis and direct or indirect somatic embryogenesis using different types of mature and immature explants. But, coupling *in vitro* plant tissue culture with transformation to improve production of transgenic plants presents its own challenges. To produce transformants, especially in soybeans, *in vitro* culturing strategies that are highly efficient are required. Soybean is still considered a recalcitrant crop, and the nature of culture media and susceptibility of selected explants to *Agrobacterium* influences transgenic plant regeneration efficiency.

Constraining factors such as; genotype specificity, antibiotics toxicity, selection pressure, explant type and age, *Agrobacterium* overgrowth and contaminations are still being neglected. Zia [23] indicated that, these abovementioned factors play a key developmental role in *in vitro* manipulation of plants. Failure of many tissue culture based *in vitro* transformations is mainly due to these factors. In addition, other forms of transformation like electroporation, particle bombardment and protoplast-mediated transformation pose more challenges than *Agrobacterium*-mediated genetic transformation under *in vitro* culture conditions. These techniques are expensive to carry-out, are labour intensive with prolonged steps of transformation, cause unstable transgene expression particularly due to gene silencing [24], produce multiple transgene copy number [12] and cause gene rearrangement within inserts and instability over the generation of transgenic plants [25].
