**6. Genetic transformation of cane, a very powerful biotechnological tool to generate tolerant plants to water stress**

According to the International Service for the Acquisition of Agri-biotech Applications (ISAAA), the worldwide distribution of genetically modified crops involves a total of 26 developing countries and 7 industrialized countries, headed by USA, Brazil, Argentina, Canada, India, China and South Africa. There is a current approval on the use of two commercial varieties of genetically modified cane in Brazil and Indonesia. On the former, plants containing the Cry1Ab gene, which produces an insecticidal toxin capable of killing the *Diatraea* caterpillar, are being cultivated. In Indonesia plants transformed with the EcBetA gene are resistant to drought.

Scientific research in genetic transformation have focused on resistance to biotic and abiotic factors such as weed control, production of renewable primary products, energy crops and production of pharmaceutically active substances.

Some of the methods in genetic transformation of plants are by Agrobacterium or biolistic which are time consuming, laborious and have low transformation efficiency. Thus we have attempted different options to optimize genetic transformation in sugar cane. An option for efficient transformation is by using different types of vectors, for example Anderson & Birch [61] used Binary super vectors in addition of different types of promoters (constitutive and inducible). Niu et al. [62] is other case who used the SoCINI inducible promoters and the ScMybRI constitutive promoters respectively [62, 63].

On the other hand, different *in vitro* culture protocols have been tried for decades to optimize the efficiency (time and management of the explant) as well as the number of transgenic plants. Yogesh and collaborators transformed cane leaves by Biolistic [64], regenerating seedlings via direct (ED) and indirect (EI) embryogenesis [65]. Arencibia and Carmona [66] reported genetic transformation by *Agrobacterium tumefaciens* and via indirect morphogenesis resulting in regenerated seedlings. Manickavasagam et al. reported regenerated seedlings after *A. tumefaciens* transformation via axillary shoots [67]. These latter two protocols require a time lapse between 3 and 6 months to generate seedlings.

reported to be up-regulated in response to H2

100 Plant, Abiotic Stress and Responses to Climate Change

climate change.

resistant to drought.

O2

its heterologous expression in *Escherichia coli* increases the bacterial host's tolerance to NaCl and PEG [59]. The homolog of this gene in 'Mex 69-290' was slightly up-regulated in leaves, but down-regulated in roots (**Figure 6**, cluster 5). All of these mentioned DIR genes from sugarcane 'MEX69290' are interesting because they show differential expression patterns in leaves and roots in response to osmotic stress. However, their functional roles in osmotic stress tolerance and biomass accumulation still need to be experimentally analyzed. In summary, in the absence of a complete sequenced genome for sugarcane, high-throughput sequencing technologies applied to the elucidation of elite cultivars' transcriptome profile are one of the most valuable resources for the identification of genes involved in both stress tolerance and biomass accumulation, which are important agronomic traits to face global

**6. Genetic transformation of cane, a very powerful biotechnological** 

According to the International Service for the Acquisition of Agri-biotech Applications (ISAAA), the worldwide distribution of genetically modified crops involves a total of 26 developing countries and 7 industrialized countries, headed by USA, Brazil, Argentina, Canada, India, China and South Africa. There is a current approval on the use of two commercial varieties of genetically modified cane in Brazil and Indonesia. On the former, plants containing the Cry1Ab gene, which produces an insecticidal toxin capable of killing the *Diatraea* caterpillar, are being cultivated. In Indonesia plants transformed with the EcBetA gene are

Scientific research in genetic transformation have focused on resistance to biotic and abiotic factors such as weed control, production of renewable primary products, energy crops and

Some of the methods in genetic transformation of plants are by Agrobacterium or biolistic which are time consuming, laborious and have low transformation efficiency. Thus we have attempted different options to optimize genetic transformation in sugar cane. An option for efficient transformation is by using different types of vectors, for example Anderson & Birch [61] used Binary super vectors in addition of different types of promoters (constitutive and inducible). Niu et al. [62] is other case who used the SoCINI inducible promoters and the

On the other hand, different *in vitro* culture protocols have been tried for decades to optimize the efficiency (time and management of the explant) as well as the number of transgenic plants. Yogesh and collaborators transformed cane leaves by Biolistic [64], regenerating seedlings via direct (ED) and indirect (EI) embryogenesis [65]. Arencibia and Carmona [66] reported genetic transformation by *Agrobacterium tumefaciens* and via indirect morphogenesis resulting in regenerated seedlings. Manickavasagam et al. reported regenerated seedlings

**tool to generate tolerant plants to water stress**

production of pharmaceutically active substances.

ScMybRI constitutive promoters respectively [62, 63].

, NaCl, and PEG treatment [59]. Furthermore,

In contrast, a genetic transformation protocol using *A. tumefaciens* has been developed in our laboratory (in the process of obtaining patent) where *in vitro* basal micro-shoots of MEX69290 cultivars underwent the insertion of the CpRap2.4b gene from the AP2/ERF transcription factor family, and out of cDNA of papaya stressed at 40°C. This genetic transformation protocol requires only 20 minutes and has a contamination rate of 0%, as well as a 21-day seedling regeneration rate. Our results showed a 70% survival in the first subculture and 100% in the second subculture with Kanamycin; similar results were reported by Manickavasagam regenerating transgenic seedlings using micro axillary outbreaks out of field plants [67], with a very laborious genetic transformation system and with 50% survival in the first crop. In addition, this work would be the second in sugarcane to report a gene of the AP2/ERF family of transcription factors inserted in sugar cane, the other work is the one reported by Reis et al. where they over expressed AtDREB2A CA (constitutive activity) in sugar cane [68]. In the transformed sugarcane seedlings generated by the genetic transformation protocol that was developed in our laboratory, the presence of the GFP was observed at the fluorescent emission of 395–475 nm, which indicates that the seedlings are transformed (**Figure 7**).

It should be clarified that the functionality of the CpRap2.4b gene belonging to the (AP2/ ERF) transcription factors family was tested in tobacco plants, which were segregated to obtain F2 plants and were then subjected to water stress (drought) conditions to evaluate their function.

**Figure 7.** GFP fluorescence of different plant leaves of sugar cane var. MEX69290. (A) Segment of wild leaf in visible light. (B) Wild leaf segment with emission at 509 nm. (C, E and G) Transgenic plants 1, 2 and 3 in visible light. (D, F and H) Transgenic plants 1, 2 and 3 with emission at 509 nm.
