**6.** *In vitro* **regeneration of transgenic shoots**

Transgenic shoot proliferation and multiplication are not easily achieved from the meristematic regions of the *Agrobacterium* infected double cotyledonary-node explants. This is the case, even if shoot development may be stimulated by the removal of the epicotyls during explant preparation and incisions on pre-determined axillary meristematic cells found on the cotyledonary junctions. In this study, tissue browning caused by oxidation and accumulation of phenolic compounds affected shoot initiation (**Figures 1d** and **4c** and **d**). More than 50%


**Table 5.** The response of soybeans to modified MS culture media and infection with *Agrobacterium tumefaciens* constituting a pTF101.1 vector.

support to shoots and buds initiated on the cotyledonary junction and exhibit minimal deficiency symptoms on their cotyledons. Even though, these explants show little sensitivity to culture conditions compared to the hypocotyl segments or immature cotyledons as indicated by Zhang et al. [41], their response was much better, especially subsequent *Agrobacterium* infection. The shoots and buds appeared to be well and effectively supported by these explants. Double cotyledonarynode explants produced better responses than the use of single coty-node explants. These single coty-nodes are prepared by longitudinally splitting the double coty-nodes at the cotyledonary junction into two single nodes [38]. Even if high proliferation of shoots can be achieved on other types of explants by directly stimulating pre-existing axillary meristems and induce rapid cell division because of severe wounding, tissue senescence due to bacterium overgrowth and callus formation instead of shoots still make double coty-nodes preferable than single coty-nodes.

initiated with on explants cultured on medium containing antibiotics without *Agrobacterium* infection.

**Figure 4.** Shoot induction from double-cotyledonary node explants on MS culture medium supplemented with 2.0 mgL−1 6-BA. (a, b) Shoot multiplication on un-infected coty-node explants cultured on MS medium used as control without antibiotics. (c) Callus formation and senescence from cotyledonary-nodes cultured on MS medium containing cefotaxime and vancomycin antibiotics. (d) Shoot buds initiated on explants infected with *Agrobacterium*. (e) Shoots

Transgenic shoot proliferation and multiplication are not easily achieved from the meristematic regions of the *Agrobacterium* infected double cotyledonary-node explants. This is the case, even if shoot development may be stimulated by the removal of the epicotyls during explant preparation and incisions on pre-determined axillary meristematic cells found on the cotyledonary junctions. In this study, tissue browning caused by oxidation and accumulation of phenolic compounds affected shoot initiation (**Figures 1d** and **4c** and **d**). More than 50%

**6.** *In vitro* **regeneration of transgenic shoots**

30 Transgenic Crops - Emerging Trends and Future Perspectives

of explants exhibiting shoot growth inhibition due to oxidative browning induced compact callus. This had negative effects on the rate of shoot and thus, the regeneration of transformed microshoots was dramatically decreased in all cultures tested for *Agrobacterium*-mediated genetic transformation. All soybean genotypes used in this study had been affected by oxidative browning, despite modifying the medium according prescriptions made in the literature. Several approaches have been reported, such as; the addition of dithiothreitol, polyvinylpyrrolidone (PVP), activated charcoal and other antioxidant mixtures like ascorbic acid. Jones and Saxena [48] report a novel approach of introducing PAL inhibitor, inhibiting the activity of phenyl-alanine ammonia lysate (PAL) enzyme. This enzyme catalyses the formation of phenolic compounds through phenylpropanoid metabolic pathway. However, shoot induction frequency of all cultivars ranged between 0.0 and 20% as indicated in **Table 5**. Only 0.31% regeneration frequency was obtained in LS 677 (**Table 5**). The detrimental effect of oxidative stress on the number of shoots induced per explants, induced shoots growth and transformation efficiency was also reported by Li et al. [49]. This problem has been correspondingly reported to have affected the efficiency of many cultures in plant tissue organogenesis and embryogenesis. For example; Hartmann et al. [50] reported failure to achieve *in vitro* plantlets regeneration during mass propagation of non-transgenic plants.
