scholarly journals Devising an Innovative, Genotype-free Transformation Protocol for Recalcitrant Indica Rice Genotypes: Samba Mahsuri and Sundarbans’ Salt-tolerant Indica Rice Cultivars White Getu and Hamilton to Establish an Ingenious Platform for CRISPR/Cas9-mediated Genome Editing

2021 ◽  
Author(s):  
Sonia Khan Sony ◽  
Khaled Fathy Abdel Motelb ◽  
Tanushri Kaul ◽  
Mamta Nehra ◽  
Jyotsna Bharti ◽  
...  
Keyword(s):  
Genome Editing ◽  
Callus Induction ◽  
Indica Rice ◽  
Rice Cultivars ◽  
Wild Type ◽  
Salt Tolerant ◽  
Transformation Protocol ◽  
Rice Genotypes ◽  
Agronomical Traits

Abstract BackgroundPlant genetic transformation involves in vitro callus induction & regeneration strategies that are quintessential for introduction of novel agronomical traits employing CRISPR/Cas9-based genome editing. However, lack of effective regeneration and transformation techniques for indica rice cultivars pose as the foremost hurdle towards genetic improvement in rice crop. We devised an astounding road-map to genotype-independent and efficacious in vitro callus induction, transformation and shoot regeneration protocol that emerges as an optimal therapy, universally adaptable to invariably any rice cultivar, in order to establish an ingenious CRISPR/Cas9-based genome editing platform in this crop.ResultsWe developed a genotype-independent regeneration and transformation protocol employing mature seed-derived calli for indica rice (one mega variety- Samba Mahsuri and two salt tolerant wild genotypes- White Getu & Hamilton) genotypes to introduce important agronomical traits via CRISPR/Cas9-based genome editing system. MS- and N6-salt based media reinforced with 2,4-D (2.5 mg/L); dicamba (1.5 mg/L); TDZ (0.1 mg/L), proline (1000 mg/L), and glutamine (2.5 mg/L) exhibited highest percentage (95-98%) of embryogenic calli initiation and development. Employing this novel protocol, we achieved unparalleled regeneration efficiencies within untransformed calli (90-94%) and transformed calli (81-86%) in these recalcitrant indica genotypes and significantly enhanced number of shoots (18-20) on MS medium containing BAP (1.5 mg/L), NAA (0.5 mg/L), TDZ (1.0 mg/L), zeatin (0.2 mg/L) and proline (500 mg/L). We successfully transformed rice calli with pCAMBIA1300-based marker- free NICTK-1_pCRISPR-Cas9 vector harbouring the cassette of plant codon optimized Cas9 via biolistic approach that exhibited notably enhanced transformation efficiencies (67-69%). The integration of Cas9 gene into rice genome was validated by PCR, Southern blotting and Sanger sequencing analyses. The transgenic lines were phenotypically indistinguishable from the wild type as no significant differences in phenotypic performances were revealed between transgenic and wild type lines. ConclusionWe devised a promising, time-efficient, universally adaptable, optimal hormonal-media therapy for triggering enhanced embryogenic callus formation, regeneration and transformation efficiencies, across recalcitrant indica rice genotypes.

scholarly journals In vitro Callus Induction and Regeneration of Popular Indica Rice Genotypes

2020 ◽  
Vol 13 (4) ◽  
Author(s):  
Aananthi. N
Keyword(s):  
Callus Induction ◽  
Callus Formation ◽  
Indica Rice ◽  
Immature Embryo ◽  
Coconut Milk ◽  
Rice Genotypes ◽  
Mist Chamber ◽  
Regenerated Plant ◽  
Better Than

Five rice cultivars viz., ASD 16, White Ponni, Pusa Basmati 1, Pusa Sugandh 4 and Pusa Sugandh 5 belonging to subspecies indica were compared for its ability in callus formation and regeneration. In this experiment, the different parameters viz., the effect of hormones (2,4-D and kinetin), organic supplement (coconut milk O1-CM 100 mll-1, O2-CM 75 mll-1, O3-CM 50 mll-1), explants (seed and immature embryo), media (MS and N6), carbon source (sucrose and maltose) using five genotypes on callus response was studied. The effect of hardening methods was also assessed. Results showed that for enhanced callus induction was with MS medium supplemented with 2.0 mgl-1 2, 4-D + 0.5 mgl-1 kinetin + 30 gl-1 maltose irrespective of explants used. Addition of 100 ml l-1 coconut milk was found have improvement in callus response. The performance of immature embryo was better than seed for callus induction, emrbyogenic callus formation, rhizogenic callus formation and regeneration. MS media provided superiority over N6. Among the genotypes Pusa Basmati 1 rendered outstanding performance in callus behavior. The treatment combination MS + 2.5 mgl-1 BAP + 0.5 mgl-1 NAA + 1.0 mgl-1 KN gave the highest organogenesis response and regeneration of plantlets. Hardening in mist chamber was recognized as the best method to give the highest per cent of regenerated plant lets.

scholarly journals Establishment of Morphogenic and Regeneration Ability in two Indica Rice Cell Suspension Cultures after Exposure to NaCl

1970 ◽  
Vol 19 (2) ◽  
pp. 185-197
Author(s):  
T.L. Aditya
Keyword(s):  
Cell Suspension ◽  
Callus Induction ◽  
Somatic Embryos ◽  
Indica Rice ◽  
Cell Suspension Cultures ◽  
Nacl Stress ◽  
Callus Cultures ◽  
Regeneration Ability ◽  
Rice Varieties

An efficient protocol was developed for in vitro morphogenic ability along with plantlet regeneration of two Bangladeshi indica rice varieties (BR24 and BR26) via somatic embryogenesis by applying 50 mM NaCl stress in callus induction and suspension initiation media. Osmotic stress was induced by NaCl (50, 100, 150, 200 and 250 mM) on the cell growth in suspension maintenance media. In viability test stress adapted cells showed 85 - 95% viability up to 200 mM NaCl compared with stress shocked (MS1-50) and control (MS1-0) treatments. Higher stress adapted cells showed growth retardation and the induction of plasmolysis. For both genotypes somatic embryos were obtained in both MS based liquid and semisolid media with or without 50 and 100 mM NaCl. Cell suspension-derived micro-calli were partially desiccated (6 - 12 hr) and subsequently maintained in MS1 callus induction media supplemented with proline (12 mM), ABA (2 mg/l) and 0.6% phytagel in the presence or absence of 50 and 100 mM NaCl. Subsequently, desiccated somatic embryos were transferred in MS based regeneration media with or without 50 and 100 mM NaCl. Proline mediated callus was found to be more effective in embryo differentiation than ABA. Partial desiccation dramatically enhanced callus growth and partially increased regeneration percentage. BR24 showed a better regeneration response producing plantlets in presence of proline in control media while BR26 restored regeneration potential in the presence of ABA and 100 mM NaCl. Plantlets regenerated from salt stressed callus cultures were then grown in compost in a glasshouse and produced normal, fertile plants.  Key words: Indica rice, Cell suspension, Morphogenic, Regeneration D.O.I. 10.3329/ptcb.v19i2.5436 Plant Tissue Cult. & Biotech. 19(2): 185-197, 2009 (December)

scholarly journals Studies on In Vitro Response to Callus Induction and Regeneration of Five High Yielding Indica rice Varieties

2019 ◽  
Vol 7 ◽  
pp. 97-104
Author(s):  
Md. Niuz Morshed Khan ◽  
Md. Monirul Islam ◽  
Dr. Md. Shahidul Islam
Keyword(s):  
Callus Induction ◽  
Callus Formation ◽  
Indica Rice ◽  
Induction Medium ◽  
Embryogenic Calli ◽  
Rice Varieties ◽  
New Variety ◽  
Increasing Demand ◽  
Embryogenic Callus Formation

Due to growing population, there is an increasing demand of rice production but the productivity of rice is lessened day by day. To overcome this problem various biotechnological tools can be used for developing various rice varieties. However, the lack of a simple and efficient protocol for callus induction, embryogenic callus formation and quick plant regeneration in this cereal crop. In this study embryogenic calli from mature seeds of five indica rice varieties viz. Binadhan-5, Binadhan-6, BRRI dhan-48, BRRI dhan-58 and IR-64 were observed that is done in four different types of media composition. The highest callus induction were observed in media containing Sucrose as a carbon source. Among those varieties Binadhan-6 and BRRI dhan-48 showed highest rate of callus induction respectively. This study will be useful for selecting suitable callus induction medium for callus induction in future that will be useful for not only national but also international plant breeders for producing new variety and so on.

scholarly journals Mature Embryo-Based in vitro Regeneration of Indica Rice Cultivars for High Frequency Plantlets Production

2017 ◽  
Vol 20 (2) ◽  
pp. 81-87
Author(s):  
HN Barman ◽  
ME Hoque ◽  
RK Roy ◽  
PL Biswas ◽  
MAI Khan ◽  
...  
Keyword(s):  
Callus Induction ◽  
High Frequency ◽  
Indica Rice ◽  
In Vitro Regeneration ◽  
Mature Embryo ◽  
Ms Medium ◽  
Rice Varieties ◽  
Growing Medium ◽  
Regeneration Efficiency

The study was conducted at Biotechnology Division of Bangladesh Rice Research Institute (BRRI) to investigate the effects of plant growing medium and plant growth regulator (PGR) for the callus induction and high frequency plantlets regeneration of indica rice. Ten indica rice varieties viz. BR5, BR11, BRRI dhan28, BRRI dhan29, BRRI dhan33, BRRI dhan41, BRRI dhan47, BRRI dhan48, BRRI dhan49 and BRRI dhan50 were cultured on MS, N6 and LS media. The MS medium was found better for callus induction as compared to N6 and LS media. Among the tested varieties BRRI dhan48 induced the highest percent and best quality callus. Interaction effects of BRRI dhan48 to MS medium yielded 71.55% callus induction. The regeneration efficiency of BRRI dhan48 was tested on MS medium supplemented with different combination of NAA plus BAP and NAA plus kinetin. MS medium supplemented with 2.0 mg L-1 NAA and 2.0 mg L-1 Kn was found the best in respect of percent regenerated (76.67%) plantlet as well as for the growth of plantlets in vitro.Bangladesh Rice j. 2016, 20(2): 81-87

scholarly journals Optimization of in vitro regeneration protocol for a popular Indica rice (Oryza sativa L. cv Swarna)

2016 ◽  
Vol 5 (08) ◽  
pp. 1395 ◽  
Author(s):  
Vijaya Naresh Juturu ◽  
Gopala Krishna Mekala ◽  
Mallikarjuna Garladinne ◽  
Puli Chandra Obul Reddy ◽  
Akila Chandra Sekhar*
Keyword(s):  
Callus Induction ◽  
Oryza Sativa L ◽  
Indica Rice ◽  
In Vitro Regeneration ◽  
Regeneration System ◽  
Embryogenic Calli ◽  
Rice Varieties ◽  
Regenerated Plants ◽  
Rooting Medium

Though regeneration system in rice has been very well established compare to other crop plants, the fact remains that, most of the indica rice varieties are still recalcitrant for regeneration and genetic transformation. Therefore, refinement of tissue culture protocol for generation of embryogenic calli and regeneration of the fertile plants from a single cell should be a pre requisite event for development of transgenic plants. Here, in this study we reported high frequency robust regeneration protocols for a popular Indica cultivar Swarna.Mature seeds were used as initial material as explants. Highest callus induction % was observed in MSCIMP medium containing 2.0 mg-1 2,4, D + 0.5 mg-1 Kn as phytohormonal combinations. In addition, maximum regeneration was observed in 2.0 mg-l KN + 0.5 mg-l NAA. Regenerated plants were shifted to rooting medium followed by polyhouse for hardening. The callus induction and regeneration reported in this study were well suited for transformation agronomical important genes or functional genomics studies.

scholarly journals High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

2020 ◽  
Author(s):  
Karthik Murugan ◽  
Arun S. Seetharam ◽  
Andrew J. Severin ◽  
Dipali G. Sashital
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Double Strand Breaks ◽  
Target Sequence ◽  
Wild Type ◽  
Strand Breaks ◽  
Cleavage Assay ◽  
Target Sequences

AbstractCas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

scholarly journals In vitro regeneration protocol for indica rice genotypes

2017 ◽  
Vol 16 (1) ◽  
pp. 1314-1223
Author(s):  
K. Miah ◽  
B. Hossen ◽  
M. S. Haque ◽  
M. Z. Tareq ◽  
S. N. Begum
Keyword(s):  
Indica Rice ◽  
In Vitro Regeneration ◽  
Rice Genotypes

scholarly journals Embryogenic Callus Induction and Regeneration of Elite Bangladeshi Indica Rice Cultivars

1970 ◽  
Vol 17 (1) ◽  
pp. 65-70 ◽  
Author(s):  
ME Hoque ◽  
MS Ali ◽  
NH Karim
Keyword(s):  
Plant Regeneration ◽  
Callus Induction ◽  
Embryogenic Callus ◽  
Callus Formation ◽  
Indica Rice ◽  
Rice Cultivars ◽  
Embryogenic Calli ◽  
Rice Callus ◽  
Embryogenic Callus Induction ◽  
Embryogenic Callus Formation

Significant variations were observed among six elite Bangladeshi Indica rice cultivars tested in relation to total callus induction frequency (p = 0.017), embryogenic callus formation frequency (p = 0.001) and subsequent plant regeneration responses (p = 0.005). In all the cases, embryogenic callus formation frequency was much more less than the total callus (embryogenic + non-embryonegic) formation frequency. The embryogenic calli derived from mature seed embryos produced green plants, successfully established in soil and produced fertile seeds.Key words: Indica rice, Callus induction, Plant regeneration, Genotypic variationsDOI = 10.3329/ptcb.v17i1.1122Plant Tissue Cult. & Biotech. 17(1): 65-70, 2007 (June)

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