Transgenic Plants as a Tool for Plant Functional Genomics

Haploid Strategies for Functional Validation of Plant Genes

“…Changes of gene expression levels may result in modified phenotypes, which can be another powerful approach for elucidating gene function [5]. Gene silencing or down-regulation induced by RNA interference (RNAi) can be achieved by expressing gene-specific double-stranded RNA (known as siRNA) or single-stranded RNA (known as microRNA) in plant cell, which in turn generates loss-of-function phenotypes [17,18].…”

“…In contrast to RNAi, overexpression or misexpression of an inactive gene or a gene coding a limiting protein will lead to gain-of-function phenotypes in transformants [21]. In this way, even the phenotypes of individual members in a gene family are observable without interference from functionally redundant genes [5].…”

“…Theoretically, we can find a EMS mutation in any given gene by screening no more than 5000 plants from the mutagenized M1 generation for the model plant Arabidopsis [3,8]. Conventional mutagenesis has been widely used in forward genetic strategies that start with a phenotype of interest and address identification of genes affecting this phenotype [9][10][11].As an alternative forward-genetic tool, insertional mutagenesis, including T-DNA (Transferred DNA) and transposon tagging, facilitate the identification of genes disrupted by these elements [5]. Currently, T-DNA-tagged lines have been generated in large numbers, becoming a popular resource for plant gene function [12,13].…”

“…Targeting Induced Local Lesions in Genomes (TILLING) is the first reverse-genetic tool, in which chemical or physical mutagenesis is followed by a high-throughput screening for point mutations [3,4,16]. TILLING is practicable for plant species with large-sized genomes and without transformation system because it is not different from traditional chemical or physical mutagenesis in creating mutations [12].Changes of gene expression levels may result in modified phenotypes, which can be another powerful approach for elucidating gene function [5]. Gene silencing or down-regulation induced by RNA interference (RNAi) can be achieved by expressing gene-specific double-stranded RNA (known as siRNA) or single-stranded RNA (known as microRNA) in plant cell, which in turn generates loss-of-function phenotypes [17,18].…”

“…In contrast to RNAi, overexpression or misexpression of an inactive gene or a gene coding a limiting protein will lead to gain-of-function phenotypes in transformants [21]. In this way, even the phenotypes of individual members in a gene family are observable without interference from functionally redundant genes [5].Targeted mutagenesis can be achieved by ZFNs, TALENs or CRISPR/Cas in which custom DNA binding motifs direct non-specific nucleases to cleave a double strand in the genome at a specific site that further stimulates error-prone nonhomologous end joining or homology-directed repair at specific genomic locations [22][23][24]6]. More applicable and easier to manipulate than ZFNs and TALENs, the CRISPR/Cas system only requires a single short RNA to generate target specificity [6] …”