Recent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. Abiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. The present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

中文翻译：

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通过农杆菌介导的转化方法开发部分非生物胁迫耐受性柑橘和柑橘


最近的研究表明，由于疾病、干旱、寒冷和土壤盐分等生物和非生物胁迫，导致柑橘产量下降，农民面临着一些挑战。因此，有必要改良这些能够抵抗生物和非生物胁迫以及高产的品种。柑橘属物种中非生物胁迫耐受基因的转化对于利用受非生物胁迫影响的地区至关重要。本研究旨在通过农杆菌介导的转化转移 PsCBL 和 PsCIPK 基因，提高柑橘和柑橘对非生物胁迫的抗性。通过农杆菌介导的转化方法，将从豌豆中分离的耐非生物胁迫的 PsCBL 和 PsCIPK 基因转化到两种柑橘物种：Citrus reticulata Blanco 和 Citrus sinensis (L.) Osbeck。两个物种的成熟种子来源的愈伤组织用含有 PsCBL 和 PsCIPK 基因的根癌农杆菌 LBA4404 感染。将受感染的愈伤组织在黑暗条件下共培养，然后用抗生素溶液洗涤并转移至选择培养基。回收初步抗性愈伤组织并再生为植株。最大再生率分别为61.11±1.35%和55.55±1.03%。通过进行 β 葡萄糖醛酸酶 (GUS) 测定和随后的 GUS 基因 PCR 扩增证实了遗传转化。两种栽培品种的转化率均高于之前的报道。当细菌 OD600 为 0.5、乙酰丁香酮浓度为 150 μM 时，发现最大转化频率。 对转基因植株进行了耐旱、耐盐的体外评价，转基因植株表现出比对照植株更好的性能。本研究表明，用 PsCBL 和 PsCIPK 基因转化柑橘植物可提高非生物胁迫耐受性。