Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules

Abstract

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.

Keywords: VvHB63; VvTAU; grapevine; mRNA in situ hybridisation (ISH); ovule development; seedless.

Figure 1

The in situ hybridisation with different digestion times of protease K: 1 µg/mL of protease K digested at 37 °C for 20 min (a,c,e) and 30 min (b,d,f). (a,b) antisense probe, 10 DAF in ‘Red Globe’; (c,d) antisense probe, 10 DAF in ‘Thompson Seedless’; (e,f) sense probe. DAF: days after flowering. The ISH signal is evidenced by purple staining and indicated by a black arrow.

Figure 2

The in situ hybridisation with different probe lengths. Hybridised with the hydrolysed probe (150 bp; a,c,e) and unhydrolysed probe (402 bp; b,d,f) to produce a hybridised signal. (a,b) antisense probe, 10 DAF in ‘Red Globe’; (c,d) antisense probe, 10 DAF in ‘Thompson Seedless’; (e,f) sense probe. DAF: days after flowering. The ISH signal is evidenced by purple staining and indicated by a black arrow.

Figure 3

In situ hybridisation with different probe concentrations. The concentrations of the probe used were 50 ng/mL (a,d,g), 100 ng/mL (b,e,h) and 150 ng/mL (c,f,i). (a–c) antisense probe, 10 DAF in ‘Red Globe’; (d,f) antisense probe, 10 DAF in ‘Thompson Seedless’; (g–i) sense probe. DAF: days after flowering.

Figure 4

The in situ hybridisation with different hybridisation temperatures. The hybridisation temperatures were used included 50 °C (a,c,e) and 55 °C (b,d,f). (a,b) antisense probe, 10 DAF in ‘Red Globe’; (c,d) antisense probe, 10 DAF in ‘Thompson Seedless’; (e,f) sense probe. DAF: days after flowering.

Figure 5

Optimisation of solution washing conditions after hybridisation. Three different washing conditions were improved by in situ hybridization, including five serial washes with 0.1× SSC solution and blocking with 1% blocking solution for 45 min (a,d,g), three washes with 0.2× SSC and sealing with 1% blocking solution for 45 min (b,e,h) and three washes with 0.2× SSC and sealing with 1% blocking solution for 45 min (c,f,i). (a–c) antisense probe, 10 DAF in ‘Red Globe’; (d,f) antisense probe, 10 DAF in ‘Thompson Seedless’; (g–i) sense probe. DAF: days after flowering. The ISH signal is evidenced by purple staining and indicated by a black arrow.

Figure 6

VvHB63 expression analysis in grapevine ovules by in situ hybridisation. (a–d) 15 DAF; (e–l) 25 DAF; (i: integument; triangular label (ch): chalaza; en: endosperm; ii: inner integument; mi: medium integument; oi: outer integument; t: testa; ep: epidermis); DAF: days after flowering. At 15 DAF, VvHB63 was expressed mainly in the epidermal cells, middle integument and inner integument (the ISH signal is evidenced by purple staining and indicated by a black arrow).

Figure 7

VvTAU expression analysis in grape ovules by in situ hybridisation. (a–d) 15 DAF; (e–l) 25 DAF; (i: integument; triangular label (ch): chalaza; en: endosperm; ii: inner integument; mi: medium integument; oi: outer integument; t: testa; ep: epidermis); DAF: days after flowering. At 15 DAF, VvTAU was expressed mainly in the epidermal cells and integument of the ovule of the seeded cultivars (the ISH signal is evidenced by purple staining and indicated by a black arrow).