About 200 mg of leaves and internode sections of young branches were sampled ... and non-transgenic eucalypts clones maintained in the screened house for.
Supplemental information Supplemental method
Expression analysis About 200 mg of leaves and internode sections of young branches were sampled by transgenic and non-transgenic eucalypts clones maintained in the screened house for >1 or >5 years. RNA extraction was performed using RNeasy Mini Kit (QIAGEN, Venlo, Netherlands). To remove some inhibitor of reverse transcription reaction and PCR, the extracted RNA samples were purified by lithium chloride precipitation. Reverse transcription reaction was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO CO., LTD, Osaka, Japan). The quantitative PCR reaction mix were prepared with THUNDERBIRD SYBR qPCR Mix (TOYOBO) and the primer pairs (5´-CAATGAAGTCGTGCTCTCCA-3´ x 5´-GAGTCCACCAGGACCTCGAT-3´, and 5´-GTGGCTTATGCGCTAAAGAG-3´
x
5´-TGGGCTAAGAAATCACTCTGG-3´)
for
amplification of codA and EgICDH, respectively. The quantitative PCR was performed and analyzed by ABI7900 HT Real Time PCR Instrument (Life Technologies, Carlsbad, California, United States) with following programs; 95ºC 2 min, 45 cycle of 95ºC 10 sec/60ºC 30 sec/72ºC 30 sec. Additional dissociation analyses were also performed and confirmed specific amplificaitons.
Supplement Figure
Transgene expression in different tissues and different growth stages The relative expression levels of codA gene were estimated by quantitative reverse-transcriptional PCR (qRT-PCT) and normalized by expression levels of the
NADP-isocitrate dehydrogenase (ICDH) gene as the control. JL: juvenile leaf, AL: adult leaf, B: internode section of young branch. i and ii indicate growth stage of sampling trees; 1 m in height grown in screened house for ca. 1 year (i), and 3-5 m in height maintained in screened house for >5 years (ii). Error bar indicates standard error (n=3).
ANOVA omitting data of no u n-transgenic lines No.1, and M001.
Degrees of freedom
Sum of squares
Standard deviation
Variance ratio against error. Significant differences at probability for F-distribution (P) < 0.01 and < 0.001 are indicated as “**” and “***”,
b
c
d
e
f
respectively.
ANOVAsing data of six lines (transgenic lines 107-1, 1-9-1, and 2-1-1 and non-transgenic lines No.1, M001, and No.18-5).
a
Analysis of variance of biomass production.
Supplemental table1
Standard deviation
c
“***”, respectively.
ratio against error. Significant differences at probability for F-distribution (P) < 0.05 and
