Delivery of Transforming DNA to the Chloroplast

Delivery of foreign DNA to the chloroplast requires its transport through several physical barriers: the cell wall, the cytoplasma membrane, and the chloroplast double-membrane system. Since no bacterial or viral pathogen is known which could be utilized for DNA delivery, transgene transmission needs to employ rather rigid physical methods. The most effective and widely used system utilizes microprojectile bombardment with plasmid-coated gold or tungsten particles, the so-called biolistic method, which was first used to transiently transform onion epidermal cells (Klein et al. 1987). Subsequent refinement of the technique eventually enabled the transformation of smaller cell types as well as subcellular targets, like plastids in the unicellular algae Chlamydomonas (Boynton et al. 1988) or in tobacco (Svab et al. 1990). Other sophisticated methods have also been developed,

Table 2.1 Plant species for which plastid transformation has been achieved. Common and scientific names are given, with the transgenes integrated so far, as well as the efficiency of the transformation process.

Species

Transgenes integrated

Explants per bombardment

(efficiency)

Reference

Tobacco (Nicotiana tabacum

aadA +

1/1 (100%)

Zoubenko et al. (1994)a

Petit Havana SM1)

uidAa

Potato (Solanum tuberosum

aadA + gfp

3/104 (2.8%)

Sidorov et al. (1999)

FL1607; S. tuberosum cv

aadA +

14/435 (3.2%)

Nguyen et al. (2005)

Desiree)

gfp

Tomato (S. lycopersicum var.

aadA

1-3/20 (5-15%)

Ruf et al. (2001)

IAC-Santa Clara)

Petunia (Petunia hybrida var.

aadA + gusA

3/31 (9.6%)

Zubko et al. (2004)

Pink Wave)

Soybean (Glycine max L. cv

aadA

11/80(13.7%)

Dufourmantel et al.

"Jack")

(2004)

Lettuce (Lactuca sativa L. cv

aadA/gfp

5/85 (5.8%)

Kanamoto et al. (2006)

Cisco)

Lesquerella fendleri

aadA + gfp

2/51 (3.9%)

Skarjinskaia et al. (2003)

Carrot (Daucus carota L. cv

aadA + badh

1/7 (14%)

Kumar et al. (2004a)

Half Long)

Cotton (Gossypium hirsutum)

aphA-6 + nptII

1/2.4 (41.6%)

Kumar et al. (2004b)

Poplar (Populus alba)

aadA + gfp

44/120 (36.6%)

Okumura et al. (2006)

Sugar beet (Beta vulgaris ssp.

aadA + gfp

3/40 (7.5%)

De Marchis et al. (2008)

vulgaris)

Rice (Oryza sativa japonica)

aadA + gfp

2/100 (2%)

Lee et al. (2006)

Cabbage (Brassica oleracea L.

aadA + uidA

3-5/150 (2-3%)

Liu et al. (2007)

var. capita L.)

Canola (B. napus)

aadA +

4/1000 (0.4%)

Hou et al. (2003)

cry1Aa10

1/5b

Cauliflower (B. oleracea var.

aadA

Nugent et al. (2006)

botrytis)

Arabidopsis thaliana ecotype

aadA

2/201 (0.9%)

Sikdar et al. (1998)

aFor tobacco, numerous more transformations have been reported bTransformation was achieved by PEG-mediated transformation like polyethylene glycol (PEG)-mediated transformation of protoplasts (Golds et al. 1993) or even the direct injection of DNA into the organelle via a femtoliter syringe (Knoblauch et al. 1999). Although plastid transformation with PEG requires some experience in the enzymatic digestion of the cell wall and the treatment of protoplasts as well as the regeneration of plants, it can be basically performed with standard laboratory equipment. Micromanipulation of cells on the other hand requires specialized equipment, which is limiting for its use; and so far no reports have shown the successful regeneration of transplastomic plants from this particular gene transfer method. However, the most widely and successfully used method is the biolistic transfer of DNA, depicted by the successful transformation of the plastids of numerous plant species (Table 2.1).

Chloroplast Transformation Methods

0 wild type plastid genome

Dna Chloroplast
  1. 2.1 Schematic drawing of the plastid transformation and regeneration process. An explant, usually a leaf, is bombarded with DNA-coated tungsten or gold particles. When the DNA is delivered to one chloroplast, integration of the transgene takes place, generating a heteroplastomic cell in which a small number of plastid genomes are transgenic (open circles). Subsequent differentiation and shoot regeneration from this cell results in a heteroplastomic plantlet. To obtain a homoplastomic transgenic plant requires several cycles of regeneration under selection
  2. 2.1 Schematic drawing of the plastid transformation and regeneration process. An explant, usually a leaf, is bombarded with DNA-coated tungsten or gold particles. When the DNA is delivered to one chloroplast, integration of the transgene takes place, generating a heteroplastomic cell in which a small number of plastid genomes are transgenic (open circles). Subsequent differentiation and shoot regeneration from this cell results in a heteroplastomic plantlet. To obtain a homoplastomic transgenic plant requires several cycles of regeneration under selection

Once the transgene DNA has been delivered to the chloroplast (Fig. 2.1), stable integration via homologous recombination has to take place (see below) to generate a stable transgenic trait which will be passed on after plastid division to its descendants. Every chloroplast harbors up to a hundred copies of its genome, grouped in nucleoids representing aggregates of 7-10 copies. Since cells can contain up to

100 chloroplasts, a single integration event creates a transplastomic cell in which only a minority of genomes is altered, the so-called heteroplastomic state. For the generation of a stable transplastomic plant, wild-type plastid genomes have to be winnowed. As the sorting of plastid DNA during cell division in shoot-regeneration is a stochastic process a small percentage of altered homoplastomic plants can be generated in the absence of selection pressure (5.6%; Lutz and Maliga 2008). To increase the efficiency of transformation routinely, a homoplastomic state of the engineered plants is reached by successive regeneration under strong selective pressure with an appropriate antibiotic. It is estimated that it takes between 20 and 30 cell divisions to deplete the undesired wild-type chloroplast genomes (Maliga 2004; Verma and Daniell 2007). Since this number could not be reached in a single plant regeneration process, explants have to go through several cycles of regeneration under selection (Fig. 2.1). So far a given plants ability to regenerate from fully differentiated tissue is the biggest obstacle for applying the plastid transformation to a large number of plant species. Tobacco is by far the best analyzed system regarding plastid transformation, and therefore most experiments referred to in this section are made in tobacco.

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    How to transform glycine max transiently?
    5 years ago

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