A short review on sugarcane: its domestication, molecular manipulations and future perspectives.

Sugarcane (Saccharum spp.) is a special crop plant that underwent anthropogenic evolution from a wild grass species to an important food, fodder, and energy crop. Unlike any other grass species which were selected for their kernels, sugarcane was selected for its high stem sucrose accumulation. Flowering in sugarcane is not favored since flowering diverts the stored sugar resources for the reproductive and developmental energy needs. Cultivars are vegetatively propagated and sugarcane breeding is still essentially focused on conventional methods, since the knowledge of sugarcane genetics has lagged that of other major crops. Cultivar improvement has been extremely challenging due to its polyploidy and aneuploidy nature derived from a few interspecific hybridizations between Saccharum officinarum and Saccharum spontaneum, revealing the coexistence of two distinct genome organization modes in the modern variety. Alongside implementation of modern agricultural techniques, generation of hybrid clones, transgenics and genome edited events will help to meet the ever-growing bioenergy needs. Additionally, there are two common biotechnological approaches to improve plant stress tolerance, which includes marker-assisted selection (MAS) and genetic transformation. During the past two decades, the use of molecular approaches has contributed greatly to a better understanding of the genetic and biochemical basis of plant stress-tolerance and in some cases, it led to the development of plants with enhanced tolerance to abiotic stress. Hence, this review mainly intends on the events that shaped the sugarcane as what it is now and what challenges ahead and measures taken to further improve its yield, production and maximize utilization to beat the growing demands.

Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice.

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged ß-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

Immunogenicity of Brugia malayi Abundant Larval Transcript-2, a potential filarial vaccine candidate expressed in tobacco.

KEY MESSAGE: Transgenic tobacco plants with Bm ALT-2, a filarial vaccine candidate, were developed. The plant-produced antigen showed immunogenicity on par with the E.coli product. Transgenic tobacco plants were developed using Brugia malayi Abundant Larval Transcript-2 (Bm ALT-2), a major antigen produced from recombinant E.coli found to be experimentally successful as potential vaccine candidate against lymphatic filariasis. Results of experiments on the transformation and expression of the Bm ALT-2 in tobacco plant to produce plant-based vaccine are presented here. We have successfully transformed the tobacco plant with Bm ALT-2 and confirmed that the plants expressed the filarial protein by PCR analysis and Western blotting. The level of expression varied from 50 to 90 ng/µg of total soluble protein for ALT-2. Immunization of mice with plant-extracted protein indicated that the plant-produced protein had immunological characteristics similar to the E.coli-produced protein. Antibody titres produced by plant-produced recombinant ALT 2-immunized mice were on par with those immunized with recombinant protein produced by E.coli. Antibody isotype assay showed that plant-produced recombinant ALT-2 induced significant IgG1, whereas E.coli-produced recombinant ALT-2 induced IgG3. This result is a step forward towards the development of a model eukaryotic system for the production of recombinant filarial proteins, which can be utilized to produce therapeutic and diagnostic molecules against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. In addition, this is the first report of the immunogenicity of a plant-derived filarial antigen.