Stable transformation of Cp Genome Using Particle Gun Method
Particle gun method (also described as biolistic method) has been found to be the most successful method for transformation of the chloroplast genome. Following steps, outlined are involved; (i) A leaf is bombarded by DNA – coated tungsten particles, each carrying 20 to 50 copies of the donor plasmid vector carrying the gene of interest; only one or a few plastid genomes in an organelle are transformed and 16 to 17 cell divisions are needed to achieve homoplasmic state; (ii) selection of transformed cells is achieved in the presence of spectionmycin using spectionmycin resistance encoded by mutant 16S rRNA genes and a chimaeric aadA gene encoding aminoglycoside 3 adenyltransferase*; (iii) selected cells are regenerated into whole plants, which can be tested for the presence of desired gene in the plastid genome.

Once the vector is introduced into the chloroplast, the transforming DNA is incorporated into the chloroplast genome by two homologous recombination events in either of the following two ways (i) the cloned plastid DNA fragment replaces completely the homologous inverted repeat region of the resident genome, as done in vector pJS75; (ii) in other cases (e.g., pZS197), homologous recombination events involve the flanking DNA sequences, rbcL and ORF512.

Stable Transformation of Cp Genome using Particle gun Method

As an example of the vectors used for chloroplast transformation, pZS148 contained the following DNA sequences: (i) 16S rRNA gene cloned as a 3.7 kb SacI EcoRV fragment derived from tobacco genotype (SPC2); this fragment carried resistance to both streptomycin and spectinomycin due to mutations in 16S rDNA; and also had a PstI site. (ii) A 2.9 kb SalI fragment carrying cp ori region needed for CpDNA replication; (ii) A 3.0 kb segment of pBluescript I KS+ phagemid vector.

For transformation, following two lines of Nicotina tabacumwere used: (i) Nt(tbc) having original cytoplasm and (ii) Nt(pbg)carrying the cytoplasm of Nicotiana plumbaginifolia; this was functionally male sterile due to short filaments in stamens, but seeds could be obtained by manual pollination. Whole leaves of both, Nt(tbc)and Nt(pbg)plants were bombarded with tungsten particles coated with pZS148 DNA (carrying resistance against spectinomycin as well as streptomycin). pZS 134 carrying resistance against only spectinomycin (not against streptomycin) was used as control. Three transplastomic clones were isolated from calli obtained from leaves bombarded with pZS148 and none from those bombarded with pZS 134. Screening for Pstlsite confirmed the replacement of 16SrDNA region in leaves by that of engineered 16S rDNA using SPC2. The transplastomic plants obtained as above, were selfed and crossed to wild type plants. It was shown that in plants raised from seeds obtained due to selfing, there was no segregation, while in crosses the resistance was maternally inherited.

In higher plants, the transformation of plastids by bombardment is 100 fold less efficient despite the high number of CpDNA copies in a cell (a cell may have upto 100 plastids with ~1000 to 10,000 copies of CpDNA molecules). In Chlamydomonas(80 Cp DNA copies per cell), however, the efficiency is as high as that for nuclear genes (2 100 transplastomic clones per bombardment). Further, the selection for a non-lethal marker (e.g. neomycin, kanamycin, spectinomycin resistance) is critical for obtaining the transplastomic lines, because it allows sufficient time for the resistant plastid genome copies to increase in number to allow phenotypic expression. This explains the low efficiency of transformation of CpDNA, since eventually all transformed plastids may be derived from a solitary plastid or from a solitary DNA molecule that is transformed.