Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

M. Arun; K. Subramanyam; T. S. Mariashibu; J. Theboral; G. Shivanandhan; M. Manickavasagam ; A. Ganapathi -

Department of Biotechnology & Genetic Engineering, School of Biotechnology, Bharathidasan University

Applied Biochemistry and biotechnology (2015)

Objetivo

•Desenvolver a transformação genética eficiente de nó cotiledonar de soja cv. PK 416, utilizando sonicação e infiltração á vácuo;

•Germinação e preparo do explante•Características da construção usada na transformação

•LBA4404, EHA101 e EHA105

•pCAMBIA 1304 - 6.128 bp

•Gene marcador

•Gene repórter

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

• Infecção por Agrobacterium e co-cultivo de explantesCMM• Métodos de transformaçãoEHA105

0 510 20 30 40 50 s

20s 0, 100, 250, 500, ou 750 (mm Hg)

0, 1, 2, ou 3 min.

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

• Meios de regeneração

Cefotaxima, vancomicina e higromicina B

SIM

SEM

RM

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

no. of GUS-GFP+ plants/total no. of infected explants×100

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

Análise histoquimica

AND ST

ST

GPANT

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean CultivarsEnsaio Visual GFP

Primes especificos gene hpt II - amplificam fragmentos de 407 bpPresença e integração do gene EcoRI

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean CultivarsAnálise molécular

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars

• Conclusões

• Desenvolvimento de um protocolo de transformação genética reprodutível e altamente eficiente mediada por Agrobacterium para nós cotiledonares de soja;

• Possibilidade de infectar mais número de explantes dentro de um curto período de tempo;

• A combinação de ultra-sons e infiltração a vácuo auxilia na transferência de T-DNA resultando em melhor eficiência de transformação

• Grande potencial para ser usada em diversas cultivares de soja para transferir e expressar traços agronomicamente e economicamente importantes

Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in

Indian Soybean Cultivars