Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

Overexpression of Sugarcane Sucrose Transporter 1 (SoSUT1) Gene Increases Rice Yield

The growth and development of plants are determined by photosynthesis, which ultimately results in sucrose. Sucrose is synthesized in the source then translocated to all parts of the plant (sink). The translocation process of sucrose from source to sink is controlled by sucrose protein called sucrose transporter. SoSUT1 is a gene that encodes a sucrose transporter 1 (SUT1) protein in sugarcane. Rice transformation with the SoSUT1 causes overexpression SUT, which is expected to increase the translocation of sucrose into the seed of rice plants. This research was conducted by introducing SoSUT1 in rice plants Inpari 14 SS. Transformation using Agrobacterium tumefaciens vector in apical bud explant Indica rice cv. Inpari 14 SS results in 26 events positive rice contains genes SoSUT1. This study aims to elucidate the inheritance of transgene in the next generation and to characterize its effect on the morphology and the yields of a subsequent generation. The study is conducted by planting the seeds of T1 and T2 plants in media containing Hygromycin and using PCR analysis for further analysis. As the results, from 26 events on the T1 plant, only 3 events of T3 plants were confirmed on the T3 plant. The overexpression of the SoSUT1 gene could increase the number of tillers, the number of productive tillers, panicle length, and panicle exit length, also increasing the number of spherical grains, reducing the number of empty grains and increasing the weight of 1000 grains.

Pengembangan Populasi Mutan Penanda Aktivasi: I. Transformasi Padi Japonica Tropis Lokal Sulawesi cv. Asemandi dengan bantuan Agrobacterium tumefaciens

<p>The rice transformation technology<br />is not only provides valuable methods for the introduction<br />of useful genes into rice plant to improve important<br />agronomic traits, but also helps in studying gene function<br />and regulation based on rice genome sequence information.<br />Knockout of genes by insertional mutagenesis is a straightforward<br />method to identify gene functions. One of the<br />methods to develop rice mutants is through genetic transformation<br />mediated by Agrobacterium using activation<br />tagging by Ac-Ds system. A study was done with an objective<br />to obtain mutant rice of local tropical japonica cv. Asemandi<br />through genetic trans-formation mediated by Agrobacterium<br />tumefaciens. The transformation was conducted using<br />Agrobacterium vector with the strain of Agl-1 containing<br />activation tag construct. The result of experiment showed<br />that it has been obtained 17 independent line (304 plants)<br />transgenic Asemandi containing activation tag construct.<br />These starter lines will be used as materials to develop<br />several generations of stabil rice mutant through selfing.</p>

Constitutive Overexpression of the Plasma Membrane Na+/H+ Antiporter for Conferring Salinity Tolerance in Rice

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3?SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 - which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture-independent Agrobacteriummediated in planta transformation method that circumvents the problems associated with tissue culture-based indica rice transformation methods. Integration of the foreign genes (OsSOS1) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SOS1) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice.Plant Tissue Cult. & Biotech. 25(2): 257-272, 2015 (December)