Supplementary material

Article title: Characterization, regeneration and efficient transformation of Chirita pumila (Gesneriaceae), a potential model organism for functional evo-devo

Journal name: Plant Cell, Tissue and Organ Culture

Author’s names: Bo-Ling Liu1† · Xia Yang† · Jing Liu · Yang Dong · Yin-Zheng Wang

Affiliation of corresponding author:

State Key Laboratory of Systematic and Evolutionary Botany,

Institute of Botany,

Chinese Academy of Sciences,

20 Nanxincun, Xiangshan, Beijing 100093, China

E-mail address of corresponding author:

Supplementary Fig. 1 The result of artificial pollination. Seven flowers about 1.5 cm long were artificially emasculated before the stamens mature. About 48–72 h later, six flowers were artificial pollinated using pollen collected from other open flowers, while one flower as the negative control was not artificial pollinated. The photos were taken two weeks later. The left one is a fertile capsule due to successful artificial pollination, and the right one shows the negative control with undeveloped ovary. Bar, 1 cm.

Supplementary Fig. 2 Histograms of PI fluorescence intensities of nuclei isolated from internal reference standard (a) and C. pumila (b). a The C-value measurement of rice. b The C-value measurement of C. pumila (one representative plant).

Supplementary Fig. 3 Effect of BA and NAA on the adventitious shoot induction of C. pumila. a No growth regulator was applied in MS medium. b 0.5 mg l-1 BA and 0.1 mg l-1 NAA were applied. c 0.1 mg l-1 BA and 1 mg l-1 NAA were applied. The plastic Petri dish we used is 12 cm in diameter.

Supplementary Fig. 4 The plant overexpressing the GUS gene is morphological normal. a A wild-type plant. Bar, 1 cm. b A representative transgenic plant transforming the GUS gene. Bars, 1 cm.

Supplementary Table 1 The seed setting percentage of C. pumila flowers obtained in the bagging experiment.
Replicate / Analyzed flowers / Flowers setting seeds / The seed setting percentage (%)
1 / 18 / 17 / 94.4
2 / 18 / 18 / 100
3 / 24 / 23 / 95.8
Total / 60 / 58 / 96.7
Supplementary Table 2 Parameters of C. pumila chromosomes.
Chromosome number / Relative length / Arm ratio / Type*
1 / 8.7+7.3 / 1.2 / m
2 / 8.2+7.6 / 1.1 / m
3 / 8.4+3.6 / 2.3 / sm (1sat)
4 / 7.8+3.3 / 2.4 / sm (1sat)
5 / 6.3+5.3 / 1.2 / m
6 / 6.3+5.1 / 1.2 / m
7 / 6.3+5.1 / 1.2 / m
8 / 5.8+5.0 / 1.2 / m
* m, metacentric chromosome; sm, submetacentric chromosome; sat, satellite.
Supplementary Table 3 Results of the 2C values for C. pumila.
Plant / 2C Nuclear DNA content (pg DNA) / Haploid genome size (Mb) / CV (%)
1 / 1.6 / 782.4 / 4.9
2 / 1.7 / 831.3 / 6.3
3 / 1.6 / 782.4 / 4.4
Mean / 1.6 / 798.7
Supplementary Table 4 Effects of temperature on shoot induction rate of C. pumila leaf explants.
Temperature (°C) / Shoot induction rate (%)*
22 / 55.5±0b
24 / 87.8±6.7a
26 / 97.8±11.6a
28 / 86.7±3.9a
Data are presented as mean ± SD calculated from three independent experiments with about 40 leaf explants each.
*Statistical differences were evaluated using the Fisher's LSD test (P < 0.05) with the same letter indicative of no significant difference.
Supplementary Table 5 Hyg is an effective selection marker for C. pumila.
Hyg (mg L-1) / Survival rate (%) / Shoot induction rate (%)
0 / 100±0 / 97.8±3.9
5 / 93.3±6.7 / 91.1±3.9
10 / 60±11.6 / 48.9±10.2
15 / 37.8±3.9 / 33.3±6.7
20 / 0 / 0
30 / 0 / 0
Data are presented as mean ± SD calculated from three independent experiments with about 40 leaf explants each.