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

Abstract

Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10(-4)-10(-5)) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions.

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.

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.

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.

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.