. 2020 Sep 2;11:1298. doi: 10.3389/fpls.2020.01298

Table 7.

Optimizing heat shock conditions for controlled gene excision using heat shock promoters Hsp17.7 and Hsp26 driving Cre expression.

Promoter	Treatments	Embryos transformed	T0 plants	T0 transformation (% ± SE)	Single copy events (numbers)	Quality events(numbers)	Excision frequency (%)	Usable event (%)
Hsp17.7	none	102	56	54.9 (4.4)^a	18	6	33.3	5.9
	42°C, 2 h/d, 2 d	102	39	38.2 (2.1)^b	16	9	56.2	8.8
	42°C/24 h	102	16	15.7 (1.8)^c	8	6	75	5.9
	45°C/2 h	102	50	49.0 (3.2)^a	18	14	77.8	13.7
Hsp26	none	100	53	53.0 (4.0)^a	18	1	5.6	1
	42°C, 2 h/d, 2 d	100	35	35.0 (1.2)^b	18	12	66.7	12
	42°C/24 h	100	41	41.0 (2.2)^b	15	10	66.7	10
	45°C/2 h	100	50	50.0 (2.3)^a	21	13	61.9	13

The heat treatments increased excision rates, which varied with the conditions applied. Of the two Hsp promoters tested, Hsp17.7_pro resulted in events with higher excision frequency (75% at 42°C for 24 h and 76.6% at 45°C for 2 h) compared to Hsp26_pro (66.7 and 61.9%). The treatment, 45°C for 2 h worked best for both Hsp promoters.

Four different conditions were evaluated side-by-side using split ears including no heat (control) and three heat shock treatments (42°C, 2 h/d for 2d; 42°C/24 h; and 45°C/2 h). Transformation results and qPCR detection of the number of excised quality events, frequencies of excision and usable event are presented.

Data from two independent transformers was used to determine T0 transformation frequency. The quality events (QE) were identified as single copy, backbone-free, and morphogenic gene-free (excised). The excision frequency was determined as the ratio of the number of excised single-copy events relative to the total single-copy events. The number QEs was divided by the total number of events recovered to calculate the QE frequency. The usable event (UE) frequency is a measure of the number of acceptable transgenic events per 100 embryos that was determined as the product of QE frequency and transformation frequency. Mean values followed by the same letter are not statistically different from each other at the significance level of 0.05.