**7. Transformation and reverse genetic in sorghum**

Methods for sorghum transformation have been available since the beginning of the 90's, initially by protoplasts [59] and cell culture [60], and subsequently *in planta* [61, 62], using *Agrobacterium* and protocols based in microprojectiles which are now available and with substantially improved efficiencies [63–69]. Sorghum is a crop hard to transform, since it is a recalcitrant genus for tissue culture and the transformation protocols reported are scarce and not very reproducible. In the particular case of sweet sorghum, [70] proposed a transformation system based on optimizing tissue culture conditions using embrionary callus with a regeneration of 90% in 12 weeks. Also, hygromycin resistance selection conferred by the Ubi-*hpt* transgene was performed, followed by particle bombardment. This method proved to be highly reproducible with an efficiency of transformation of 0.09% in every embryo. In 2012, Liu and Godwin, published a method with a better transformation efficiency in *S. bicolor*, in which using pure line embryos (IEs) Tx430, reaching an efficiency of 20.7% in the three independent experiments [71]. The protocol, which involves the use of microprojectiles and transgenes regulated under the *ubi1* constitutive promoter, improves the conditions of the media culture for embryos, as well as the parameters for transformation with microprojectiles. In this experiment, the transgenes were inherited by the T1 generation.

After, Tien-Do *et al*. [72] developed a fast and efficient system for sorghum transformation using binary vectors and the AGL1 *Agrobacterium* strain instead of microprojectiles. With the public genotype P898012 and the *bar* gene as a selective marker, callus regeneration time was reduced to 7–12 weeks, producing 18 plants per callus. This experiment achieved a frequency of 14%, where 40–50% of the transformation events possessed a single copy of integrated T-DNA with a segregation of mendelian 3: 1 estimated by Southern blot. An example of the utility of genetic transformation of sweet sorghum is presented by Zhu *et al.* [73], where using *Agrobacterium* and induce early flowering, the gene Bt cry1Ah was introduced. BT or Cry proteins are produced naturally in aggregates or crystals by *Bacillus thuringiensis.* There proteins are specific for the digestive system of different insects. Protein Cry1Ah, confers resistance to *Ostrinia furnacalis.* In this study, the generated plants, after being selected for herbicide resistance to confirm transformation, showed a high resistance at T0. Apart from the resistance tests, transgene expression was confirmed by RT-PCR and the presence of BT proteins produced by the plant by Western blot and ELISA.

One of the main arguments against the use of transgenics is the use of selection markers such as herbicide tolerance and the fact that they stay inside the genome of the transformed plant. The main issue is the possibility of the cultivar's pollen to pollinate related weeds and therefore the resistance is inherited to undesired plants. Against this problematic, there are several efforts to generate marker-free transgenics. An example is presented by Lu *et al.* [74], where at the cost of reducing the selection pressure, they manage to obtain marker-free transgenics.

In other hand, sorghum offers the opportunity to complete what has been previously described in the reverse genetics of rice and maize, providing to the genetic and familiar studies, those genes which are hard to manipulate in these crops. This allows the directed functional analysis to specify the genes in sorghum related to traits such as hydric stress and the production of certain sugars by genetic association. To accelerate the specific direct identification of genes, mutant lines using ethyl methane sulfonate (EMS) have been created. For the genotype BTx623 there are 1,600 M3 annotations, individual pedigrees, which was characterized [75]. Currently, each of the inspected M3 lines is distinguishable from the original stock and some have multiple mutant phenotypes. The additional M2 mutants are available for the scientific community for the production of thousands of M3 additional lines.

Until few years ago, even with the genome sequencing technology for the elite line BTx623, the genetic sources and sorghum germplasm where limited, making hard the functional validation of the sequenced genes. In 2016, 4,600 M4 mutant pedigrees where created by EMS mutagenizing of BTx623 seed, which were later transformed in lines by single-seed descendant method [76]. The sequencing of 256 mutant lines revealed more than 1.8 million of induced canonical mutations, affecting 95% of the sorghum genome.

### **8. Perspectives**

The studies here presented represent an introduction to the current state of sorghum genomics. Regardless these advances have contributed relevant achievements to what it is known about genetic diversity of this species, it is still necessary to develop further studies, which its aim is focused in sweet sorghum. However, the knowledge acquired in grain sorghum and other related species, constitute an important molecular base to continue developing research studies which allow to know sweet sorghum and its unreported genomic regions. In them could lie the key for the increased production of sugars, lignin and other traits of interest such as tolerance to new plague's appearances such as yellow aphids and/or diseases. It is also necessary

**121**

**Author details**

Mexico

Arturo Díaz-Franco3

Raymundo Rosas-Quijano1

2 Lund University, Lund, Skane, Sweden

provided the original work is properly cited.

, Abraham Ontiveros-Cisneros2

to develop genetic maps which allow the localization of genetic codifying regions to certain traits of agronomic interest. Regarding molecular studies in Mexico, there are no reports of genetic maps or genomics performed in sweet sorghum. This represents an opportunity to develop research lines which allow to generate the country's own sweet sorghum genotypes carrying tolerances to adverse biotic and abiotic conditions predominant in the country. This would allow the growth on its production and of its sub products, focusing in alternative environment friendly energy sources.

1 Bioscience Institute, Autonomous University of Chiapas, Tapachula, Chiapas,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Alfredo Vázquez-Ovando1

3 INIFAP, Río Bravo Experimental Field, Río Bravo, Tamaulipas, Mexico

\*Address all correspondence to: didiana.galvez@unach.mx

, Noé Montes-García<sup>3</sup>

and Didiana Gálvez-López1

,

\*

*A General Overview of Sweet Sorghum Genomics DOI: http://dx.doi.org/10.5772/intechopen.98539*

#### *A General Overview of Sweet Sorghum Genomics DOI: http://dx.doi.org/10.5772/intechopen.98539*

*Biotechnological Applications of Biomass*

the plant by Western blot and ELISA.

microprojectiles. With the public genotype P898012 and the *bar* gene as a selective marker, callus regeneration time was reduced to 7–12 weeks, producing 18 plants per callus. This experiment achieved a frequency of 14%, where 40–50% of the transformation events possessed a single copy of integrated T-DNA with a segregation of mendelian 3: 1 estimated by Southern blot. An example of the utility of genetic transformation of sweet sorghum is presented by Zhu *et al.* [73], where using *Agrobacterium* and induce early flowering, the gene Bt cry1Ah was introduced. BT or Cry proteins are produced naturally in aggregates or crystals by *Bacillus thuringiensis.* There proteins are specific for the digestive system of different insects. Protein Cry1Ah, confers resistance to *Ostrinia furnacalis.* In this study, the generated plants, after being selected for herbicide resistance to confirm transformation, showed a high resistance at T0. Apart from the resistance tests, transgene expression was confirmed by RT-PCR and the presence of BT proteins produced by

One of the main arguments against the use of transgenics is the use of selection markers such as herbicide tolerance and the fact that they stay inside the genome of the transformed plant. The main issue is the possibility of the cultivar's pollen to pollinate related weeds and therefore the resistance is inherited to undesired plants. Against this problematic, there are several efforts to generate marker-free transgenics. An example is presented by Lu *et al.* [74], where at the cost of reducing the

In other hand, sorghum offers the opportunity to complete what has been previously described in the reverse genetics of rice and maize, providing to the genetic and familiar studies, those genes which are hard to manipulate in these crops. This allows the directed functional analysis to specify the genes in sorghum related to traits such as hydric stress and the production of certain sugars by genetic association. To accelerate the specific direct identification of genes, mutant lines using ethyl methane sulfonate (EMS) have been created. For the genotype BTx623 there are 1,600 M3 annotations, individual pedigrees, which was characterized [75]. Currently, each of the inspected M3 lines is distinguishable from the original stock and some have multiple mutant phenotypes. The additional M2 mutants are available for the scientific community for the production of thousands of M3

Until few years ago, even with the genome sequencing technology for the elite line BTx623, the genetic sources and sorghum germplasm where limited, making hard the functional validation of the sequenced genes. In 2016, 4,600 M4 mutant pedigrees where created by EMS mutagenizing of BTx623 seed, which were later transformed in lines by single-seed descendant method [76]. The sequencing of 256 mutant lines revealed more than 1.8 million of induced canonical mutations,

The studies here presented represent an introduction to the current state of sorghum genomics. Regardless these advances have contributed relevant achievements to what it is known about genetic diversity of this species, it is still necessary to develop further studies, which its aim is focused in sweet sorghum. However, the knowledge acquired in grain sorghum and other related species, constitute an important molecular base to continue developing research studies which allow to know sweet sorghum and its unreported genomic regions. In them could lie the key for the increased production of sugars, lignin and other traits of interest such as tolerance to new plague's appearances such as yellow aphids and/or diseases. It is also necessary

selection pressure, they manage to obtain marker-free transgenics.

**120**

additional lines.

**8. Perspectives**

affecting 95% of the sorghum genome.

to develop genetic maps which allow the localization of genetic codifying regions to certain traits of agronomic interest. Regarding molecular studies in Mexico, there are no reports of genetic maps or genomics performed in sweet sorghum. This represents an opportunity to develop research lines which allow to generate the country's own sweet sorghum genotypes carrying tolerances to adverse biotic and abiotic conditions predominant in the country. This would allow the growth on its production and of its sub products, focusing in alternative environment friendly energy sources.
