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Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

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Abstract

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

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Abbreviations

ASAL:

Allium sativum leaf agglutinin

BBMV:

Brush border membrane vesicle

BPH:

Brown planthopper

cv.:

cultivar

C-PRINS:

Cycling-primed in situ labelling

ELISA:

Enzyme-linked immunosorbent assay

GLH:

Green leafhopper

GUS:

β-glucuronidase

hpt :

Hygromycin phosphotransferase coding gene

MS:

Murashige and Skoog

PRINS:

Primed in situ labelling

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Acknowledgements

The authors are grateful to Council of Scientific and Industrial Research, Government of India for providing fellowships to PS, PM and ID. TR acknowledges the Department of Biotechnology, Government of India for providing financial support at CAS, Department of Botany, Calcutta University. We are thankful to Regional Rice Research Station, Chinsurah, West Bengal, India for providing seed stock of IR64 rice cultivar. We thank Dr. A. Chatterjee for reading the manuscript. The backup service of Mr. Arup Kumar Dey is sincerely acknowledged.

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Saha, P., Majumder, P., Dutta, I. et al. Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests. Planta 223, 1329–1343 (2006). https://doi.org/10.1007/s00425-005-0182-z

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