Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment

1. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 1.

   Transmission of paternal ptDNA in alloplasmic crosses. (A) Flower morphology of the male sterile N. tabacum CMS92 mother line and fertile Nt-pMSK56 transplastomic father line. Below are listed nuclear, plastid, and mitochondrial markers. (B) PSpc32 line has petaloid flowers and is male sterile, as the CMS92 mother line. (C) The PSpc4 (RM protocol) and PSpc2, PSpc3, and PSpc66 (RMOP protocol) paternal pollen transmission events. Arrows point to green resistant sectors. (D) Classification of spectinomycin-resistant lines. The aadA gene in paternal ptDNA confers streptomycin resistance to PSpc4 cells. Spontaneous spectinomycin-resistant mutant Spc93 is sensitive to streptomycin. Mother line N. tabacum CMS92 (sensitive to both drugs) and father line Nt-pMSK56 (resistant to both drugs) were controls. Leaf sections were cultured on RMOP medium containing 500 mg/liter spectinomycin or 500 mg/liter of both drugs.

   
   
2. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 2.

   Transmission of paternal ptDNA between parents with normal cytoplasm. (A) Nuclear (N), plastid (Pt), and mitochondrial (Mt) genotypes of parental lines and exceptional hybrid with paternal ptDNA. (B) Identification of PSpc70 paternal ptDNA transmission event and Spc69 spontaneous spectinomycin-resistant mutant in RMOP/RMOP protocol. (C) Classification of spectinomycin-resistant lines. Paternal aadA gene confers streptomycin resistance to PSpc70. Spc69 spontaneous spectinomycin-resistant mutant is sensitive to streptomycin. Both lines are gentamycin-resistant. Nt-pHC19 and Nt-pMSK56 are parental lines. (D) Germination of seedlings on spectinomycin medium indicates that the PSpc70 paternal ptDNA transmission line is homoplastomic for the aadA transgene and segregates for the nuclear gentamycin resistance marker.

   
   
3. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 3.

   RFLP mapping of paternal ptDNA. DNA gel blot to identify RFLP markers in the CMS92 ptDNA. Lanes contain total cellular DNA of N. tabacum cv. Petit Havana (t), the CMS92 line (u), and transplastomic Nt-pMSK56 line (TP). The genes covered by the probe and restriction enzymes used for DNA digestion are listed on top. Fragment sizes are shown for N. tabacum.

   
   
4. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 4.

   Plants with paternal ptDNA have mtDNA from both parents. Note N. tabacum- (t) and N. undulata- (u) specific mtDNA fragments in parental lines and in plants obtained by spectinomycin (PSpc2–66) and streptomycin (PSt1, PSt6) selection. Total cellular DNA was probed with the 6.9-kb atp6 probe.