Orbović Vladimir, Ravanfar Seyed Ali, Acanda Yosvanis, Narvaez Javier, Merritt Benjamin A, Levy Amit, Lovatt Carol J
Citrus Research and Education Center, University of Florida/IFAS, Lake Alfred, FL, 33850, USA.
Department of Botany and Plant Sciences, University of California, Riverside, CA, 92521, USA.
Transgenic Res. 2021 Oct;30(5):687-699. doi: 10.1007/s11248-021-00260-z. Epub 2021 May 30.
Transgenic 'Duncan' grapefruit (Citrus paradisi Macf.) and 'Valencia' sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, 'Duncan' grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5 years old and the T1 siblings from this line flowered and fruited when they were just over 18 months old. In contrast, T1 seedlings from three lines of 'Duncan' grapefruit expressing the CsLFY gene flowered within 3 months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three 'Carrizo' citrange (C. sinensis × Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.
转基因“邓肯”葡萄柚（Citrus paradisi Macf.）和“伏令夏橙”甜橙（Citrus sinensis [L.] Osbeck）植株在拟南芥胁迫诱导型启动子AtRD29A的控制下异位表达中华柑橘（品种为华盛顿脐橙）的APETALA1（CsAP1）或LEAFY（CsLFY）基因，在非诱导性（温暖温度、水分充足）的温室条件下开花，而它们的野生型（WT）对照则不开花。开花的转基因植株除了没有野生型幼龄植株特有的刺外，形态特征没有改变。最早熟的T0代品系，即表达CsAP1基因的“邓肯”葡萄柚（Dun134 - 3），在4.5岁时开花结果，该品系的T1代子代在刚过18个月大时就开花结果。相比之下，三个表达CsLFY基因的“邓肯”葡萄柚品系的T1代幼苗在萌发后3个月内开花，但无法支持果实发育。叶片中相应转基因的转录水平与开花早熟性无关。为了进一步研究AtRD29A的活性，对在AtRD29A启动子调控下用绿色荧光蛋白（GFP）基因转化的三株“卡里佐”枳橙（C. sinensis × Poncirus trifoliata）砧木幼苗的叶片进行干旱胁迫或水分充足处理。GFP的表达不依赖于胁迫，这与在非诱导条件下CsAP1和CsLFY转基因植株开花的观察结果一致。综合来看，结果表明AtRD29A在柑橘背景中组成型表达。尽管失去了对开花时间的控制，但在拟南芥RD29A启动子控制下异位表达中华柑橘AP1或LFY基因的转基因柑橘品系表现出早熟开花、果实发育和可育转基因种子形成。这些转化品系可作为减少世代间隔以加速育种的有用工具。
