*4.2. Evaluation of Bacterial Blight Resistance*

The parental varieties (IRBB66 and TNG82), susceptible varieties (Taichung Native 1 (TN1), Taichung sen 10 (TCS10)), BC2F2 and BC2F3 generation genotypes were pyramided with the five BB-resistant genes, with IR24 as control. Different combinations were evaluated for BB resistance under greenhouse and field conditions with the pathogen, *X. oryzae* pv. *oryzae*. Pathogen inoculation was performed at the maximum tillering stage in the field through the modified leaf clipping method, as previously described [41]. A strong Taiwanese epidemic pathogen isolate, XF89-b, was used in this study. The isolate was grown in 523 medium [42] with agitation at room temperature for two days. After adjusting the optical bacterial density to 10<sup>9</sup> CFU/mL with distilled water, the cultures were used to screen the rice plants for BB resistance. Approximately six leaves from one plant were clipped from the top 2–3 cm simultaneously. All inoculation was completed within 1 h following the preparation of bacterial suspensions. Lesion length for BB was scored after inoculation when the lesion of the susceptible variety, TN1, reached approximately 3/4 of overall leaf length (approximately 21–28 days). The resistance reaction was classified as highly resistant (HR), resistant (R), moderately resistant (MR), moderately susceptible (MS), and susceptible (S) when the values of lesion length were recorded as 0–1 cm, 1.1–3 cm, 3.1–6 cm, 6.1–10 cm, and more than 10 cm, respectively [43,44].

#### *4.3. Evaluation of Agronomic Traits*

During the second and first crop season of 2017 and 2018, respectively, the 30-day-old seedlings of the BC3F3 and BC3F4 pyramided lines and both the parents were transplanted into three rows, with 27 plants per row, per entry, at 15 × 25 cm spacing, under a randomized complete-block design, with two replications at the Taiwan Agricultural Research Institute's Chiayi Agricultural Experiment Station Farm. Ten plants from each entry were recorded as one data replication. Single plant yield

was recorded for the 16 BC3F3 genotypes as a basis for selection. In BC3F4, variables for agronomic traits were recorded for nine pyramided lines, including: plant height (cm), days to 50% flowering, panicle length (cm), panicles/plant, panicle weight (g), number of grains/panicle and 1000-seed weight (g), while single plant yield (g) was recorded on a whole-plot basis. In addition, the grain quality, including palatability, protein, amylose, brown rice ratio, head rice ratio and total milled rice ratio, was investigated and analyzed. For palatability analyses, the rice grains were hulled and ground into a fine flour. Approximately 33 g of rice flour was used for the palatability evaluation, which was performed by using a palatability analyzer system (Toyo Taste Meter, Model MA-30), in accordance with the manufacturer's operation manual (TRCM Co., Toyo Rice Polishing Machine Factory, Japan), as previously described [45]. Protein and amylose contents were measured with a near-infrared spectrometer (AN820, Kett Electric Laboratory Co. Ltd., Tokyo, Japan) (Near Infrared Spectrometer, Foss Japan Co. Ltd., Tokyo, Japan). Statistical analysis was performed with independent samples using least significant difference (LSD).
