Abstract
Hybrid seeds are used for stimulated crop production, as they harness heterosis. The achievement of complete male-sterility in the female-parent and the restored-fertility in F1-hybrids are the major bottlenecks in the commercial hybrid seed production. Here, we report a male sterility–fertility restoration system by engineering the inmost nutritive anther wall layer tapetum of female and male parents. In the female parent, high–level, and stringent expression of Arabidopsis autophagy–related gene BECLIN1 was achieved in the tapetum, which altered the tapetal degeneration program, leading to male sterility. This works on our previously demonstrated expression cassette based on functional complementation of TATA-box mutant (TGTA) promoter and TATA-binding protein mutant3 (TBPm3), with modification by conjugating Long Hypocotyle in Far-Red1 fragment (HFR1NT131) with TBPm3 (HFR1NT131-TBPm3) to exercise regulatory control over it. In the male parent, tapetum–specific Constitutive photo-morphogenesis1 (COP1) was expressed. The F1 obtained by crossing these engineered parents showed decreased BECLIN1 expression, which was further completely abolished when COP1-mutant (COP1L105A) was used as a male parent, leading to normal tapetal development and restored fertility. The system works on COP1-HFR1 interaction and COP1–mediated degradation of TBPm3 pool (HFR1NT131-TBPm3). The system can be deployed for hybrid seed production in agricultural crops.
Highlights
Number of biotechnological strategies have been deployed to restrict self–fertilization in plants[7]
Petit Havana (NTPH) harboring this expression cassette 1370 were normal during growth and development with a similar range of pollen viability, germination, and seed setting when compared with Nicotiana tabacum cv. Petit Havana (NTPH) plants (Supplementary Fig. S1-b and c)
The gusA expression in the stage 2 anther ranges from 11.45 nmoles mg−1min−1 to 47.57 nmoles m−1min−1 (Supplementary Fig. S2-a), with an average expression of 22.00 ± 5.15 nmoles mg−1min−1 (Fig. 2b); at stage 3, the expression ranges from 10.45 nmoles mg−1min−1 to 45.65 nmoles mg−1min−1(Supplementary Fig. S2-b), with average expression being 20.43 ± 5.74 nmoles mg−1min−1(Fig. 2b) in the anthers of transgenic lines evaluated
Summary
Number of biotechnological strategies have been deployed to restrict self–fertilization in plants[7]. Several transgenic approaches have been developed to generate male sterile plants either through early tapetum degeneration by expressing BARNASE18, RNase -119, DIPTHERIA TOXIN-A32, RIBOSOME INACTIVATING PROTEIN33, and BAX28, or by delaying tapetal PCD through BAX INHIBITOR28, ethylene receptor gene Cm-ETR1/H69A34, cystein protease BoCysP1 and BoCP335. The female expression cassette has the potential to attain high-level, stringent expression of a desired gene limited to tapetal cells, but the expression switches to abolition in F1 when regulated by the male component. We successfully deployed this system to express the desired gene (we earlier demonstrated for male sterility) Arabidopsis BECLIN115, which generates the complete male sterile parent by altering the tapetal degeneration program. Nicotiana tabacum has been used for the proof of principle presented here, but the essential elements of the technology are generic and possibly will work in other crops
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