EFFICIENT IN PLANTA GENETIC TRANSFORMATION THROUGH Agrobacterium-MEDIATED FLORAL-DIP TECHNIQUE IN INDIAN FLAX

Main Article Content

SOURAV DUTTA
PRANIT KUMAR MUKHERJEE
ABHIJIT MITRA
SUFIA ZAMAN
ASIT B. MANDAL

Abstract

An efficient in planta genetic transformation technique through Agrobacterium-mediated floral-dipping involving an Indian flax (Linum usitatissimum L.) var. Tiara has been developed. The technique is very simple, reproducible and found to be efficient in development of transgenic lines. Floral buds of Tiara were infected with Agrobacterium strain LBA 4404 harbouring cry1A(b) in a binary vector pCAMBIA1301. Agrobacterium cells at an O.D.600 of 0.5 in infiltration medium (IM) containing 8.0% sucrose and 0.1% Tween 20 were used for floral-dip transformation. Transgenic status of the transformants were established by assessing transgene expression at transient level through GUS histochemical assay and PCR at T1 generation and stable integration of the intruded transgene at genomic level was confirmed through PCR and semi-quantitative RT-PCR analysis at T2 generation. Successful stable integration of cry1A(b) transgene was observed in 19 lines at T2 generation. Progenies of those lines deemed to impart tolerance to combat attack of serious lepidopteran insect pests in Indian flax. This technique prospects amply in augmenting the quality and productivity of Indian flax, especially at the interface of discernible global climate changes and increase of resilient insect pests damage to this crop of economic importance.

Keywords:
Indian flax (Linum usitatissimum L.), Agrobacterium-mediated genetic transformation, Lepidopteran insect pest management, floral-dipping, Gus histochemical assay, semi-quantitative RT- PCR

Article Details

How to Cite
DUTTA, S., MUKHERJEE, P. K., MITRA, A., ZAMAN, S., & MANDAL, A. B. (2021). EFFICIENT IN PLANTA GENETIC TRANSFORMATION THROUGH Agrobacterium-MEDIATED FLORAL-DIP TECHNIQUE IN INDIAN FLAX. PLANT CELL BIOTECHNOLOGY AND MOLECULAR BIOLOGY, 22(53-54), 21-36. Retrieved from https://www.ikprress.org/index.php/PCBMB/article/view/7011
Section
Original Research Article

References

Balakrishnan NP, Rao RR, Hajra PK. Flora of India. B. D. Sharma (Ed.). Calcutta: Botanical Survey of India. 1993;1:1-45.

Goyal A, Sharma V, Upadhyay N, Gill S, Sihag M. Flax and flaxseed oil: An ancient medicine & modern functional food. Journal of Food Science and Technology. 2014;51(9):1633-1653.

Reddy PS, Pati D. Indian oilseeds: present status and future needs. Indian Journal of Agricultural Science. 1998;68(8):453-459.

Mackiewicz-Talarczyk M, Barriga-Bedoya J, Mankowski J, Pniewska I. Global flax market situation. In ID# 97 International Conferences on Flax and Other Bast Plants. 2008;408-412. Gill KS. Linseed. Linseed; 1987.

Khush GS. Green revolution: the way forward. Nature Reviews Genetics. 2001; 2(10):815-822.

Vijayachandra K, Palanichelvam K, Veluthambi K. Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation. Plant Molecular Biology. 1995;29(1):125-133.

Reddy SSS, Singh B, Peter AJ, Rao TV. Production of transgenic local rice cultivars (Oryza sativa L.) for improved drought tolerance using Agrobacterium mediated transformation. Saudi Journal of Biological Sciences; 2018,2016.

Habben Jeffrey. Improving Drought Tolerance in Maize: Transgenic Approaches to Improving Grain Yield Under Water Limiting Conditions. Plant and Animal Genome XXIV Conference. Plant and Animal Genome; 2016.

Nishizawa T, Nakajima N, Tamaoki M, Aono M, Kubo A, Saji H. Fixed-route monitoring and a comparative study of the occurrence of herbicide-resistant oilseed rape (Brassica napus L.) along a Japanese roadside. GM Crops & Food. 2016;7(1):20-37.

Metry EA, EL MOGHAZY GM, Masoud MS, Youssef MH, Omar TA, Yehya T, EL-ITRIBY HA. Nutritional and biological evaluation of transgenic potato lines resistant to potato tuber moth. Egyptian Journal of Genetics and Cytology. 2008; 37(1).

Hinchee MA, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Horsch RB. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technology. 1988;6(8):915-922.

Burkitbayeva S, Qaim M, Swinnen J. A black (white) hole in the global spread of GM cotton. Trends in Biotechnology. 2016;34(4):260-263.

Zhang HX, Blumwald E. Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotechnology. 2001;19(8):765-768.

McHughen A, Holm FA. Transgenic flax with environmentally and agronomically sustainable attributes. Transgenic Research. 1995;4(1):3-11.

Czemplik M, Szopa J. Optimizing biomedical and industrial products development based on flax. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 2009;4(062):1-10. Available:http://www.isaaa.org

Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: Beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal. 1987; 6(13):3901-3907.

Dong JZ, McHughen A. Transgenic flax plants from Agrobacterium mediated transformation: incidence of chimeric regenerants and inheritance of transgenic plants. Plant science. 1993;91(2):139-148.

Islam AS, Miah SA. Transgenic plants: Risks, concerns and effects on ecosystem and human health. Plant Tissue Culture and Biotechnology. 2006;16(2):139-164.

Castagnola AS, Jurat-Fuentes JL. Bt crops: Past and future. In Bacillus thuringiensis biotechnology. Springer, Dordrecht. 2012; 283-304.

Lereclus D, Delecluse A, Lecadet MM. Diversity of Bacillus thuringiensis Toxins and Genes; 1993.

Haider MZ, Ellar DJ. Mechanism of action of Bacillus thuringiensis insecticidal δ-endotoxin: interaction with phospholipid vesicles. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1989;978(2):216-222.

Adang MJ, Crickmore N, Jurat-Fuentes JL. Diversity of Bacillus thuringiensis crystal toxins and mechanism of action. Advances in Insect Physiology. 2014;47:39-87.

Kuo WS, Chak KF. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Applied and Environmental Microbiology. 1996;62(4):1369-1377.

Avisar D, Eilenberg H, Keller M, Reznik N, Segal M, Sneh B, Zilberstein A. The Bacillus thuringiensis delta-endotoxin Cry1C as a potential bioinsecticide in plants. Plant Science. 2009;176(3):315-324.

Chaudhary B, Tripathi MK, Pandey SK, Bhandari HR, Meena DR, Prajapati SP. Variety TIARA (JRF-2); 2019. Available:http://www.cambia.org Available:https://www.snapgene.com

Saha P, Datta K, Majumder S, Sarkar C, China SP, Sarkar SN, Datta SK. Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants. Plant Cell, Tissue and Organ Culture (PCTOC). 2014;118(2):313-326.

Bastaki NK, Cullis CA. Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate. JoVE (Journal of Visualized Experiments). 2014;(94):e52189.

Clarke JD. Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harbor Protocols. 2009;2009(3):pdb-prot5177.

Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22(3):474-481.

Mullis KB. The unusual origin of the polymerase chain reaction. Scientific American. 1990;262(4):56-65.

Simms D, Cizdziel PE, Chomczynski P. TRIzol: A new reagent for optimal single-step isolation of RNA. Focus. 1993; 15(4):532-535.

Huis R, Hawkins S, Neutelings G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biology. 2010;10(1):1-14.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402-408.

Whelan JA, Russell NB, Whelan MA. A method for the absolute quantification of cDNA using real-time PCR. Journal of Immunological Methods. 2003;278(1-2): 261-269.

Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW. Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnology Letters. 2006;28(19):1601-1613.

Mangeot-Peter L, Legay S, Hausman JF, Esposito S, Guerriero G. Identification of reference genes for RT-qPCR data normalization in Cannabis sativa stem tissues. International Journal of Molecular Sciences. 2016;17(9):1556.

Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW. mRNA and microRNA quality control for RT-qPCR analysis. Methods. 2010;50(4):237-243.

Saha D, Mukherjee P, Dutta S, Meena K, Sarkar SK, Mandal AB, Mitra J. Genomic insights into HSFs as candidate genes for high-temperature stress adaptation and gene editing with minimal off-target effects in flax. Scientific Reports. 2019;9(1): 1-18.