Carvalho M J S, Oliveira E J, Souza A S, Pereira J S, Diamantino M S A S, Oliveira S A S
Núcleo de Recursos Genéticos e Desenvolvimento de Variedades, Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil.
Núcleo de Recursos Genéticos e Desenvolvimento de Variedades, Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil
Genet Mol Res. 2017 May 31;16(2):gmr-16-02-gmr.16029556. doi: 10.4238/gmr16029556.
This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.
本研究旨在开发一种通过结合热处理和四环素从离体茎尖培养物中消除木薯蛙皮病（CFSD）的方法。根据疾病症状的目视检查，选取感染CFSD的不同种质（BGM0232、BGM0315、BGM0464、BGM584、BGM0841和BGM1342）的插条进行除菌处理。为了验证是否存在其他病害，对植株进行了木薯普通花叶病毒-CsCMV（通过酶联免疫吸附测定法）和木薯脉花叶病毒-CsVMV（通过聚合酶链反应，PCR）检测，结果证明除BGM0315和BGM0464感染CsVMV外，其他种质均未感染这些病毒。随后，将插条在不同浓度的四环素中浸泡3分钟，然后在不同温度（35°、38°、40°、45°和55°C）下进行热处理。收获2厘米长的茎段，在层流罩中对其表面进行消毒。随后，切取不同大小（0.2、0.4、0.5和1.0毫米）的茎尖，接种于含有与插条浸泡浓度相同的四环素的培养基中。培养60天后，将外植体转移至不含抗生素的增殖培养基中。转移30天后，评估再生植株的活力，然后将其在温室中驯化70天，再移栽至田间。7个月后，对有症状的根部进行目视分析，并进行PCR分析，以分别证明感染这些病毒（BGM0315和BGM0464）的种质中CFSD和CsVMV已被消除。大多数处理使感染CFSD的植株100%除菌。在同时感染CsVMV的种质中，仅有2%的植株仍被感染，这也证明了该方案对这种病害的除菌效率。
