Comparison of in vitro regeneration and transformation efficiency among five vietnamese indica rice cultivars

L.Q. Cao¹†, P.D. Nguyen¹, X.H. Pham¹, V.H. La², X.V. Nguyen², T.D. Nguyen²*

¹Department of Molecular Pathology, Institute of Agricultural Genetics, Vietnam Academy of Agricultural Sciences, Hanoi 11956, Vietnam
²Institute of Biotechnology and Food technology, Thai Nguyen University of Agriculture and Forestry, Thai Nguyen 24153, Vietnam
*Correspondence: dungnt@tuaf.edu.vn

Abstract:

This study optimized the in vitro regeneration and transformation processes for five popular indica rice cultivars in Vietnam named as BC15, Bac Thom 7 (BT7), Khang Dan 18 (KD18), OM5451, and TBR225. The highest callus induction rates (83-93.67%) were achieved on a medium containing 2.5 mgL^-l 2,4-D, supplemented with 500 mg L^-l each of L-proline, L-glutamine, and casein. Higher or lower concentrations of 2,4-D resulted in reduced callus induction. Among tested cultivars, BT7 exhibited the highest callus induction rate of 93.67%. Shoot regeneration was optimized on a medium with a combination of 0.5 mg L^-l kinetin, 2.0 mg L^-l BAP, and 0.5 mg L^-l NAA, resulting in regeneration rates ranging from 80.23% to 90.77% across cultivars, with BT7 exhibiting the highest regeneration rate (90.77%). Hygromycin at 20 mg L^-l was optimal for selecting transgenic calli, with BT7 showing the highest transformation efficiency opdetermined to be optimal for selecting transgenic calli, with BT7 achieving the highest transformation efficiency (44% hygromycin-resistant callus and 29% GUS expression). PCR analysis confirmed a high proportion (87.5%) of transgenic plants in the BT7 cultivar. These findings indicate that the BT7 is the most suitable cultivar for genetic transformation and gene editing applications.

Key words:

Optimizing tissue culture media for efficient transformation of different indica rice genotypes

M. A. Zaidi¹, M. Narayanan¹⋅², R. Sardana¹, I. Taga¹,³, S. Postel¹, R. Johns¹,M. McNulty¹, Y. Mottiar¹, J. Mao¹, E. Loit¹, I. Altosaar¹,*

¹ Agricultural Biotechnology Laboratories, Department of Biochemistry, Microbiology andImmunology, University of Ottawa, 451 Smyth Rd. Ottawa, ON., K1H 8M5, Canada
² Present address: Department of Agricultural Entomology, Agricultural College and ResearchInstitute, Tamil Nadu Agricultural University, Madurai-625 104, India
³ Clinical Biochemistry and Nutrition Unit, Biochemistry Department, Faculty of Sciences, P OBox 24157, University of Douala, Douala, Cameroon
* Corresponding author: altosaar@uottawa.ca Tel: +1(613) 562-5846, Fax: +1 (613) 562-5440

Abstract:

This article is dedicated to the rye breeder Count Friedrich Georg Magnus von Berg, celebratingthe 160th year of his birth. An efficient system was established for high frequency embryogenesis and regeneration of indica rice, Oryza sativa L. cv. MDU 5. Basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins were evaluated in callus induction and regeneration media to establish the most efficient regeneration protocol for MDU 5 indica rice. The optimized callus induction and regeneration media consisted of the basic MS salt mixtures and vitamin solutions supplemented with 4% maltose, 1g/L casein hydrolysate and 50 mg/L tryptophan, solidified with 1% agar. The callus induction medium was supplemented with 2,4−D 2 mg/L, kinetin 0.5 mg/L, indole acetic acid 1 mg/L, and 6−benzyl aminopurine 0.5 mg/L whereas the media for regeneration phase comprised kinetin 2 mg/L, indole acetic acid 1 mg/L and 6−benzyl aminopurine 2 mg/L. The media optimized for MDU 5 was analyzed for response of 73 other indica genotypes. Of these indica varieties 64 genotypes yielded 98.5% callus induction within eight days, 59% embryogenic calli formation, initiation of multiple green buds within eight days and a regeneration rate of 90%. The embryogenic calli derived were used for transformation with Agrobacterium tumifaciens (LBA 4404 or EHA 101 strains). Two binary vectors (pKHG4 and pIG121Hm) containing hph and GUS genes were used in these transformation studies. Fifty-one genotypes responded to the optimized media by producing hygromycin−resistant calli. The -histochemical test for ß−glucuronidase activity was positive from 32 genotypes with the transformation efficiencies ranging between 7.0% and 8.3%.

Key words:

4−D: 2, 4−Dichlorophenoxyacetic acid; NAA: Naphthaleneacetic acid, GUS Abbreviations: BAP: 6−Benzylaminopurine; 2, indica rice, regeneration, transformation