The Peculiarities Of Sri Lankan Crop Cultivation

Introduction

Being an agricultural based country, Sri Lankan crop cultivation faces several challenges due to its specific climatic conditions. Prolonged seasonal lack of rainfall is one of such major abiotic stresses affecting plant growth and reducing crop productivity. The development of molecular cloning techniques has opened up pathways to determine and alter the genes responsible for development of drought resistance in commercial crop products. The in vivo protein overexpression to induce drought resistance is frequently observed globally in developing agro-economy. However, the selection of a most suitable vector for the gene transfection has been a challenge in several crop plants as the subsequent selection of most suitable host-vector complex is a key factor in overexpression of recombinant proteins at high yield.

Methods

This study aims to review the possible vectors that can be used for in vivo protein overexpression of genes responsible for drought resistance, through a thorough published literature survey at ‘Google scholar’ search engine and the ‘NCBI’ database with the search terms of ‘drought resist*’, ‘drought toleran*’, ‘vector’, ‘overexpression’.

Results and Discussion

The literature search indicated the wide use of in vivo protein overexpression to stimulate drought resistance in many crop products at global scale. A late embryogenesis abundant (LEA) protein gene OsLEA3-1 has been recognised as a gene, to test drought tolerance and it was overexpressed in rice to test drought resistance. In the study, three binary expression constructs (OsLEA3-S, OsLEA3-A, and OsLEA3-H) were generated by inserting the full-length cDNA (released from the cDNA vector pSPORT1 by BamHI and KpnI) into backbone vectors pCAMBIA1301S, pCAMBIA1301A, and pCAMBIA1301H, respectively. (Xiao et al., 2007). Insertion of a double CaMV 35S, rice Actin1 and HVA1-like promoter, respectively, into the multiple cloning sites (HindIII and SacI) of pCAMBIA1301 that has developed backbone vectors. All the backbone expression vectors were introduced into drought-sensitive Japonica rice Zhonghua 11 by Agrobacterium mediated transformation (Hiei, et al., 1994; Lin and Zhang, 2005).

Another recent study overexpressed of three NAC genes in Arabidopsis (ANAC019, ANAC055, and ANAC072) in order to enhance tolerance to drought stress. (Zheng et al., 2018). In transformation the full-length cDNA of ONAC045 was amplified using a cDNA clone (GenBank Accession No.CT829509) from a cDNA library of Guangluai 4 (Oryza sativa L.ssp. indica) as template. Ligation of PCR fragment into the overexpression vector pCAMBIA1300S was confirmed by the sequencing. The resultant construct, pCAMBIA1300S-ONAC045, was transformed into Nipponbare (Oryza sativa L. ssp. japonica) by Agrobacterium-mediated transformation method to generate transgenic rice plants. (Hiei et al., 1994).

The protein coding region of OsbZIP72 was amplified from seedling of Oryza sativa cv. Nipponbare by reverse transcriptase polymerase chain reaction (RT-PCR). Ligation of PCR fragment into the overexpression vector pCAMBIA1300S to construct pCAMBIA1300S-OsbZIP72 was confirmed by the sequencing. This construct was transformed into rice (Oryza sativa cv. Nipponbare) by Agrobacterium-mediated transformation method to generate transgenic rice plants (Lu, et al. 2009).

Another study amplified the native promoter from genomic DNA of upland rice IRAT109 and placed to pCAMBIA1381xb-GFP to control GFP expression (Hu, et al., 2006). The SNAC1 native promoter fragment with the first 270 bp of SNAC1-coding sequence was introduced to pCAMBIA1381xb-GFP for GFP fusion expression. The full-length cDNA of SNAC1was amplified and inserted into pCAMBIA1301 binary vector, under the control of the CaMV 35S promoter for overexpression (Hu, et al., 2006). All of the constructs were transformed into the japonica rice by the Agrobacterium-mediated transformation method.

According to M. Dalal et al., (2009) findings for the overexpression of BnLEA4-1 in Arabidopsis BnLEA4-1 was PCR-amplified from pGEMT-BnLEA4-1 vector with the BnLEA4-forward and BnLEA4-reverse primers as described above, restricted with BamHI and SacI, and cloned into modified binary vectors pCAMBIA1200-P35S::GUS::NOS and pCAMBIA1200- PRD29A::GUS::NOS, by replacing GUS with BnLEA4-1. Thus, BnLEA4-1 was cloned under constitutive CaMV35S promoter and stress-inducible Arabidopsis RD29A promoter in pCAMBIA1200-P35S::GUS::NOS and pCAMBIA1200-PRD29A::GUS::NOS, respectively. These binary vectors were transformed into Agrobacterium strain LBA4404 and were used for transformation into Arabidopsis thaliana ecotype Columbia by floral dip transformation method (Clough and Bent, 1998).

Conclusion and Recommendation

A recent study provided evidence for the availability of infrastructure and skilled labour for protein overexpression in Sri Lanka, yet the industrial practice of these methods is still lacking (Cooray, et al., 2018). The traditional plant breeding methods hold several disadvantages such as time consuming to observe productivity, and introduction of disadvantageous to the new breed. Therefore, introduction of the drought tolerant gene to the crop plant using molecular cloning techniques followed by induction of in vivo protein overexpression to stimulate the drought tolerance can be suggested as a promising biotechnological advancement.

Aforementioned literature studies showed vectors that can be modified to fit to the individual crop product and can be utilised for in vivo overexpression of drought tolerant genes. Majority of the plasmid vectors are compatible with the common host Agrobacterium. To be used as a suitable vector, the plasmid should hold several characteristics such as, a promoter to fit to the fully expression of the recombinant gene, availability of compatible linker region, and screening genes. Therefore, it is recommended as the next step of this study to extend the review to analyse these characteristics of the vector to develop into a guide for selection of vector in in vivo drought resistant protein overexpression.