**11. Conclusions**

**10. Recommended strategies for improvements**

34 Transgenic Crops - Emerging Trends and Future Perspectives

soybean varieties, this tool requires the following amendments:

antibiotics at a range between 50 and 500 mgL−1.

There is adequate evidence gathered in this study indicating that, the soybean genotypes used will variably express and transmit the transgenes if any event of transformation is to take place. This was clearly demonstrated by the varied responses formed in plant cell initiation and microshoots formation during callus induction and shoot multiplication. Moreover, the varied intensities in oxidative browning of tissues, chlorosis and subsequent wounded tissue necrosis exhibited by explant from different genotypes, indicated the extend of the problems associated with *Agrobacterium*-mediated genetic transformation of legumes. For the successful optimisation of protocol routinely used for transformation of a wide range of

**1.** Thorough screening and selection of genotypes such as Peking that showed moderate level

**2.** Re-evaluation of culture condition, particularly the amount and type of antibiotics used in the culture medium. Mangena [29] reported successful induction of multiple shoots from cotyledonary-node explants infected with *Agrobacterium tumefaciens* containing ΩPKY vector construct on MS medium containing aminoglycoside antibiotics. This study reported effective *Agrobacterium* overgrowth control, low explant toxicity, lower levels of explant decay and better shoot proliferation under aminoglycosides compared to the β-lactam

**3.** Continued and effective use of additives. Culture agents that inhibit tissue senescence which include, but not limited to cysteine, dithiothreitol, ascorbate and sodium thiosulfate are highly recommended. The effectively optimised use of these antioxidants may have

**4.** *Agrobacterium tumefaciens* density must be thoroughly adjusted and optimised to avoid tissue senescence. Explant infection by this bacterium on the cotyledonary junctions and bases of explants causes tissue decay in culture. This effect occurs even though contamina-

Other affordable method such as *in-planta Agro*-injection must be considered by laboratories to generate new genetically improved soybean plants. This technique was introduced by Chee et al. [65] in genetic transformation of soybean and kidney bean by *Agro*-injecting seeds with a suspension of *Agrobacterium* strain EHA101 with pIG121 vector plasmid containing genes for neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT) and β-glucuronidase (GUS). The soybean and kidney bean seeds were surface sterilised using 0.6% sodium hypochlorite and germinated on moistened sterile paper towels at 25°C for 24 h in darkness. *Agro*-infection yielded 12% transgenic soybeans and 24% of transgenic kidney beans identified using NPTII amplified by polymerase chain reaction (PCR). Generally, there are difficulties in the *in vitro* regeneration and selection of transgenic plants during *Agrobacterium*-mediated genetic transformation. But, this technique is undoubtedly the best

given positive results in cultivars like Peking than in any of the cultivars used.

tion as a result of *Agrobacterium* overgrowth is efficiently controlled.

tool available for the transfer and expression of transfer DNA in host plant cells.

of resistance to production of phenolics and subsequent oxidation of tissues.

The use of double cotyledonary-node explants still remain superior for establishment of soybean cultures–callus and shoot proliferation. This is so, because the efficiency of the cultures established relied primarily on the explant type, as one of the culture factors. Explant amenability to *Agrobacterium* infection, antibiotics and growth regulator regimes impacted highly on the culture successes observed. A successful shoot and callus induction in the control media was illustrated. The negative impacts of *Agrobacterium* infection and effect of antibiotics on the culture was also observed. However, these findings clearly demonstrate that, more work still need to done, focusing on the optimisation of tissue culture conditions and bacterial cultures. For the main purpose of developing a high frequency, genotype independent and efficient protocol for use in *Agrobacterium*-mediated genetic transformation of soybean.
