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Agrobacterium rhizogenes-mediated DNA transfer inPinus halepensis Mill.

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Agrobacterium rhizogenes successfully transformed Aleppo pine (Pinus halepensis) embryos, seedlings, and shoots. This research demonstrates efficient genetic transformation for root and callus regeneration in this important tree species.

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Area of Science:

  • Plant Biotechnology
  • Forest Genetics
  • Molecular Biology

Background:

  • Agrobacterium rhizogenes is a soil bacterium known for its ability to genetically transform plants, inducing hairy root disease.
  • Pinus halepensis (Aleppo pine) is a significant coniferous tree species with economic and ecological importance, but efficient transformation methods are limited.
  • Understanding and optimizing plant transformation protocols are crucial for genetic improvement and research in woody species.

Purpose of the Study:

  • To investigate the efficacy of Agrobacterium rhizogenes-mediated transformation in various explants of Pinus halepensis.
  • To assess the potential for stable genetic transformation and regeneration of transgenic tissues (roots, calli) in Aleppo pine.
  • To evaluate the role of Agrobacterium rhizogenes in inducing adventitious root formation and its potential for plant regeneration.

Main Methods:

  • Transformation of mature embryos, seedlings, and shoots of Pinus halepensis using Agrobacterium rhizogenes strain LBA9402.
  • Monitoring transformation efficiency via β-glucuronidase (GUS) expression assays in different explants.
  • Induction of adventitious roots and calli through various inoculation methods, including hypocotyl injection and dipping seedling cuttings.
  • Regeneration of adventitious shoots followed by Agrobacterium infection and subsequent root induction.
  • Confirmation of transgene integration using Polymerase Chain Reaction (PCR) and Southern blot analysis.

Main Results:

  • Mature embryos showed high transient GUS expression (85%) in the radicle and lower expression in cotyledons (24%).
  • Stable transformation was confirmed by the regeneration of GUS-expressing roots and calli from infected seedlings.
  • Inoculum injection into hypocotyls resulted in callus and root formation in 64% of seedlings.
  • Dipping seedling cuttings yielded adventitious roots in 71% of cases, all exhibiting GUS activity.
  • Infection of induced adventitious shoots led to regeneration of adventitious roots (74%) and root primordia (40%) in older shoots.
  • PCR and Southern blot analyses confirmed the presence of the uidA gene and rolC/rolB genes in transgenic tissues.

Conclusions:

  • Agrobacterium rhizogenes is an effective tool for the genetic transformation of Pinus halepensis across different developmental stages.
  • The study highlights the potential for regenerating transgenic roots and calli, crucial for further genetic manipulation and research in Aleppo pine.
  • The findings provide a foundation for developing improved transformation and regeneration protocols for Pinus halepensis, contributing to forest genetics and biotechnology.