scholarly journals 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

Agronomy ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 179
Author(s):  
Tanika Thakur ◽  
Kshitija Sinha ◽  
Tushpinder Kaur ◽  
Ritu Kapoor ◽  
Gulshan Kumar ◽  
...  

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.

Forests ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 564
Author(s):  
Gaetano Distefano

The main challenges for tree crop improvement are linked to the sustainable development of agro-ecological habitats, improving the adaptability to limiting environmental factors and resistance to biotic stresses or promoting novel genotypes with improved agronomic traits [...]


2021 ◽  
Vol 12 ◽  
Author(s):  
Andrés M. Bellido ◽  
Eduado D. Souza Canadá ◽  
Hugo R. Permingeat ◽  
Viviana Echenique

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.


2017 ◽  
Vol 2 (6) ◽  
pp. 599 ◽  
Author(s):  
Tifa R. Kusumastuti ◽  
Rizkita R. Esyantia ◽  
Fenny M. Dwivany

Banana is one of the major fruit crops, though its conventional breeding has limitations, such as sterility and high polyploidy  levels.  Biotechnological  approach  using genetic  transformation  crop for improvement  offers  an alternative  solution.  In  this  study  a  protocol  was developed  for  establishing genetic  transformation  from embryogenic callus and somatic embryos of the banana cv Ambon Lumut . Embryogenic callus was obtained in ID4 medium (MS-based medium) supplemented with 1 mg L-1 IAA, 4 mg L-1 2,4D, and 0.03 g L-1 active charcoal. Embryogenic callus was transferred into liquid mediu m to establish somatic embryos. Embryogenic callus and somatic embryos were used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain A GL1, containing pART-TEST7 p lasmid with gfp gene as a reporter and CaM V35S as a promoter, was used for transformations. The embryogenic callus and somatic embryos were transformed using heat-shock method followed by centrifugation  (2000 rpm) and co-cult ivation in liquid medium containing acetosyringone (100 M) for 3 days. Results of the GFP analysis showed transient expression from gfp gene reporter in transformed embryogenic callus and somatic embryos. Transformation efficiency in somatic embryos (85,9%) was higher than  that in embryogenic callus (32.09%). PCR analysis using CaMV primer showed bands that compatible with CaMV35S promoter at 507 bp. This is a report showing establisment of embryogenic callus and somatic embryo culture transformation by using A. tumefaciens-mediated transformation protocol of the local banana cv Ambon Lumut. This study proved  the huge potential for genetic transformation of banana cv Ambon Lumut for crop improvement, such as pest or disease  resistance and abiotic factor stress tolerance. Keywords: banana; embryogenic callus; somatic embryos.


2011 ◽  
Vol 9 (01) ◽  
pp. 97-108 ◽  
Author(s):  
H. D. Upadhyaya ◽  
K. N. Reddy ◽  
Shivali Sharma ◽  
R. K. Varshney ◽  
R. Bhattacharjee ◽  
...  

Pigeonpea (Cajanus cajan(L.) Millsp. is one of the most important legume crops as major source for proteins, minerals and vitamins, in addition to its multiple uses as food, feed, fuel, soil enricher, or soil binder, and in fencing, roofing and basket making. ICRISAT's genebank conserves 13,632 accessions of pigeonpea. The extensive use of few parents in crop improvement is contrary to the purpose of collecting a large number of germplasm accessions and has resulted in a narrow base of cultivars. ICRISAT, in collaboration with the Generation Challenge Program, has developed a composite collection of pigeonpea consisting of 1000 accessions representing the diversity of the entire germplasm collection. This included 146 accessions of mini core collection and other materials. Genotyping of the composite collection using 20 microsatellite or simple sequence repeat (SSR) markers separated wild and cultivated types in two broad groups. A reference set comprising 300 most diverse accessions has been selected based on SSR genotyping data. Phenotyping of the composite collection for 16 quantitative and 16 qualitative traits resulted in the identification of promising diverse accessions for the four important agronomic traits: early flowering (96 accessions), high number of pods (28), high 100-seed weight (88) and high seed yield/plant (49). These accessions hold potential for their utilization in pigeonpea breeding programmes to develop improved cultivars with a broad genetic base. Pigeonpea germplasm has provided sources of resistance to abiotic and biotic stresses and cytoplasmic-male sterility for utilization in breeding programmes.


2018 ◽  
Vol 150 ◽  
pp. 9-17 ◽  
Author(s):  
Claudia D. Norzagaray-Valenzuela ◽  
Lourdes J. Germán-Báez ◽  
Marco A. Valdez-Flores ◽  
Sergio Hernández-Verdugo ◽  
Luke M. Shelton ◽  
...  

Author(s):  
Mehi Lal ◽  
Saurabh Yadav ◽  
Rajendra Prasad Pant ◽  
Vijay Kumar Dua ◽  
B. P. Singh ◽  
...  

The quality and quantity of the potatoes produced is directly affected by the climatic factors that prevailed during the crop season. It is also well established that abiotic and biotic stresses cause tremendous losses to the crop. Host plants and their pathogens are prone to various climatic factors like temperature, relative humidity, rainfall and CO2 which are behaving in erratic manner. Phytophthora infestans has adapted itself at higher temperature so there are chances to spread at a larger area. The other potato diseases like early blight, bacterial wilt, soft rot and viral diseases may also behave differently at elevated temperature and high rainfall. Viral diseases of potato are serious threat to potato industry as most of the viruses are transmitted by vectors and vector populations are bound to increase with these changed climatic conditions. Therefore, potato researchers need to simulate these conditions and devise mitigation strategies for sustained potato production.


2019 ◽  
Vol 17 (11) ◽  
pp. 2143-2152 ◽  
Author(s):  
Yu Liu ◽  
Yu Wang ◽  
Shuqing Xu ◽  
Xianfeng Tang ◽  
Jinshan Zhao ◽  
...  

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