Plant transformation by particle bombardment of embryogenic pollen

Abstract

Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10(4) pollen grains were GUS(+). Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS(+) pollen, 1-5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.

Improved efficiency of microspore culture of Brassica campestris ssp. pekinensis (Chinese cabbage)

“…In recent years, isolated microspore culture has attracted a lot of interest because of its great potential for DH production. A microspore-derived embryo is also a suitable system for gene mapping (Graner, 1996;Zhang et al 2005;Geng et al, 2007;Yu et al, 2008), genetic transformation (Stǒger et al, 1995) and selection of dominant and recessive traits (Polsoni et al, 1988;Swanson, 1989;Liu et al, 2003;Wang et al, 2008;Zou et al, 2009). Since Lichter (1982) first reported the embryogenesis in microspore cultures of Brassica napus, embryogenesis has been induced in microspore cultures of different Brassica species (Babbar et al, 2004).…”

The Role of Abscisic Acid in Induction of Androgenesis: A Comparative Study Between Hordeum vulgare L. Cvs. Igri and Digger

“…Advantages include accelerated and simplified breeding procedures (Morrison and Evans 1988). Microspores can be used as target cells for stable transformation, for example, by co-cultivation with Agrobacterium or particle bombardment (Creissen et al 1990, Pechan 1989, Stöger et al 1995, Twell et al 1989). Successful use of microspores for such applications requires optimized culture procedures to achieve high regeneration frequencies.…”

Genetic transformation technology in the Triticeae

“…Provided that transformed pollen cells can be made to proliferate in vitro and subsequently be converted into haploid plants, then chromosome doubling (whether spontaneous or chemically induced) will result in instant homozygosity for the transgene. However, the cell wall of the immature pollen grain (consisting of the intine and the exine) represents a significant physical barrier to transgene introduction (Stoeger et al 1995). In addition, the gene transfer process imposes a substantial level of stress (either through wounding where biolistic transformation is attempted, or response to pathogen infection in methods employing Agrobacterium), while the need to interrupt the normal pollen maturation process and to confer regenerability by the imposition of temperature and/or nutritional stress can also be injurious to the host cell.…”