Generation of Self-Incompatible Arabidopsis thaliana by Transfer of Two S Locus Genes from A. lyrata Mikhail E. Nasrallah, Pei Liu, and June B. Nasrallah

Plant transformation:

Agrobacterium-mediated transformation of A. thaliana ecotype Columbia by the floral dip method (1) used a 10-kb SmaI fragment containing SRKb (2) and a 9-kb EagI fragment containing SCRb (2) cloned into pBINPLUS (3).

Generation of SRKb/SCRb doubly transformed plants:

T2 transgenic plants with SRKb or SCRb transgene integration at a single chromosomal locus were identified by DNA gel blot analysis. Plants homozygous for a transgene locus were those which, upon selfing, produced only kanamycin-resistant progenies in ~100 seedlings tested.

Molecular analysis of transgenic plants:

DNA gel blot analysis verified the presence of the transgenes in primary transformants and their kanamycin-resistant progenies, and demonstrated the independent derivation of transgenic families. Expression of SRKb and SCRb was demonstrated by RT-PCR of pistil and anther total RNA, respectively, using primers specific for each gene. Primers were designed from intron-flanking sequences to distinguish between RNA-derived and genomic DNA-derived products. The relative amounts of SRKb transcripts in SRKb/SCRb transformants and A. lyrata SaSb plants, polyA+ RNA was isolated from pistils and subjected to RNA gel blot analysis. Blotted RNA was hybridized first with an SRKb probe and then with an actin probe. After normalization using the actin hybridization signal, transcripts were quantitated with ImageQuant (Molecular Dynamics).

Pollination assays:

Pollinations were routinely perfomed on pollen-free stigmas of stage-13 buds [flower stages according to (4)] and in young flowers (early stage 14). For developmental studies of SI in SRKb/SCRb plants, stigmas from buds at stages 12 through 14 were analyzed. Stigmas were examined for absence of contaminating pollen under a stereoscope, and appropriate pollen was manually transferred onto stigmas. For single-stigma self/cross pollination assays, unpollinated flowers were placed on 1% agar plates, and 30-40 self pollen grains were applied to one side of the stigma and the same number of cross pollen grains to the opposite side. Two hours after pollination, flowers were fixed for 30 minutes in a 3:1 mixture of ethanol and acetic acid, softened for 30 minutes in 1N NaOH at 65ºC, washed for 30 minutes in water, stained in decolorized aniline blue, and mounted for examination by epifluorescence microscopy (5). Under these conditions, an incompatible response is typically manifested by the total or near-total absence of pollen grains on the stigma surface, because pollen grains that are not anchored by a pollen tube are dislodged during fixation, softening, and washing treatments. To avoid this loss of non-germinated pollen grains (Fig. 2A), the pollinated stigma was treated in NaOH at room temperature for 12-24 hours. All pollination assays were performed using three stigmas per replicate, and were repeated on three different dates. We analyzed at least 6 transgene-expressing plants in each T2 transgenic family and 6 SRKb/SCRb plants.

Inter-species pollinations:

Inter-species pollinations were carried out using transgenic A. thaliana stigmas and A. lyrata pollen. The reciprocal pollinations are not informative because of an inter-specific pollination barrier independent of SI.

References

1. 1. S.J. Clough, A. Bent, Plant J. 16, 735 (1998).

2. M. Kusaba et al., Plant Cell 13, 627 (2001).

3. F.A. van Engelen et al., Transgenic Res. 4, 288 (1995).

4. D.R. Smyth, J.L. Bowman, E.M. Meyerowitz, Plant Cell 2, 755 (1990).

5. Y.O Kho and J. Baer, Euphytica 17, 298 (1968).