公开(公告)号：WO2015189376A1

公开(公告)日：2015-12-17

申请号：PCT/EP2015/063144

申请日：2015-06-12

Applicant: SESVANDERHAVE N.V.

Inventor： WEYENS, Guy ,  LEFÈBVRE, Marc ,  THAMMANNAGOWDA, Shivegowda ,  LOMMEL, Murielle

IPC: C07K14/415 ,  C12N15/82

CPC classification number: C12N15/821 ,  A01H1/02 ,  A01H1/04 ,  C12N9/88 ,  C12N15/52 ,  C12N15/8209 ,  C12N15/8241 ,  C12N15/8279 ,  C12N15/8281 ,  C12N15/8282 ,  C12N15/8285 ,  C12N15/8286

Abstract: The present invention is related to a method for the transformation of sugar beet protoplasts comprising the steps of: – obtaining protoplasts from stomatal guard cells isolated from a sugar beet plant, - transforming the protoplasts with a nucleic acid construct comprising a nucleotide sequence of interest and a selection marker sequence, - applying to an in vitro culture of said protoplasts, one or more ALS inhibitors at a concentration that is lethal to the in vitro culture of the protoplasts and - regenerating sugar beet plants from the surviving protoplasts having integrated the nucleic acid construct comprising the sequence of interest and the selection marker sequence, wherein the selectionmarker sequence is the mutated BvALS113 sequence carrying in its sequence a mutation at amino acid 113 position from Alanine to Tyrosine.

Abstract translation: 本发明涉及一种用于转化甜菜原生质体的方法，包括以下步骤： - 从分离自甜菜植物的气孔保护细胞获得原生质体， - 用包含感兴趣的核苷酸序列的核酸构建体转化原生质体，以及 选择标记序列， - 以对原生质体的体外培养致死的浓度应用所述原生质体的一种或多种ALS抑制剂的体外培养，以及从具有整合核酸的存活的原生质体再生甜菜植物 构建体，其包含感兴趣的序列和选择标记序列，其中选择标记序列是突变的BvALS113序列，其在氨基酸113位从氨基酸到酪氨酸的突变。

公开(公告)号：WO2015189409A1

公开(公告)日：2015-12-17

申请号：PCT/EP2015/063216

申请日：2015-06-12

Applicant: SESVANDERHAVE N.V.

Inventor： WEYENS, Guy ,  LEFÈBVRE, Marc ,  THAMMANNAGOWDA, Shivegowda ,  LOMMEL, Murielle

IPC: C07K14/415 ,  C12N15/82

CPC classification number: C12N15/8213 ,  C12N9/88

Abstract: Method for the transformation of sugar beet protoplasts comprising the step of: –obtaining protoplasts from stomatal guard cells isolated from a sugar beet plant, -transforming said protoplasts with a nucleic acid construct comprising a nucleotide sequence of interest and Transcription Activator-Like Effector Nucleases (TALEN) or one or more vectors comprising sequences encoding these Transcription Activator-Like Effector Nucleases (TALEN)sequences, wherein these TALEN target and process a target sequence and replace this target sequence through homologous recombination with the nucleic acid construct comprising the nucleotide sequence of interest, -possibly applying to an in vitroculture of these protoplasts, a medium that is toxic, preferably lethal to the in vitroculture of the protoplasts, and -regenerating sugar beet plants from the cell culture, preferably from the surviving protoplasts having integrated the nucleic acid construct comprising the sequence of interest that possibly renders the transformed cell resistant to the toxic activity of the applied medium.

Abstract translation: 用于转化糖用甜菜原生质体的方法，包括以下步骤： - 从分离自甜菜植物的气孔保护细胞获得原生质体， - 用包含感兴趣的核苷酸序列和转录激活子样效应核酸酶的核酸构建体转化所述原生质体（ TALEN）或包含编码这些转录激活子样效应核酸酶（TALEN）序列的序列的一种或多种载体，其中这些TALEN靶标并通过与包含感兴趣的核苷酸序列的核酸构建体的同源重组来处理靶序列并替换该靶序列 可能地应用于这些原生质体的维生素培养物中，这种培养基是有毒的，优选对原生质体的维生素培养是致死的，并且来自细胞培养物的再生甜菜植物，优选来自已经整合了核酸构建体的存活的原生质体 包括可能的兴趣序列 使得转化细胞对所施用的培养基的毒性活性具有抗性。

公开(公告)号：WO2015188870A1

公开(公告)日：2015-12-17

申请号：PCT/EP2014/062291

申请日：2014-06-12

Applicant: SESVANDERHAVE N.V.

Inventor： WEYENS, Guy ,  LEFÈBVRE, Marc ,  THAMMANNAGOWDA, Shivegowda ,  LOMMEL, Murielle

IPC: C07K14/415 ,  C12N15/82

CPC classification number: C12N15/8201 ,  C07K14/415 ,  C12N15/8278

Abstract: The present invention is related to a method for the transformation of sugar beet protoplasts comprising the step of: – obtaining protoplasts from stomatal guard cells isolated from a sugar beet plant, - transforming said protoplasts with a nucleic acid construct comprising a nucleotide sequence of interest and a marker sequence, - applying to an in vitro culture of said protoplasts, one or more ALS inhibitors at a concentration that is lethal to the in vitro culture of the protoplasts and - regenerating sugar beet plants from the surviving protoplasts having integrated the nucleic acid construct comprising the sequence of interest and the selectable marker sequence where the selectable marker sequence is the mutated BvALS113 sequence carrying in its sequence a mutation at amino acid 113 position from Alanine to Tyrosine.

Abstract translation: 本发明涉及一种用于转化甜菜原生质体的方法，包括以下步骤： - 从分离自甜菜植物的气孔保护细胞获得原生质体， - 用包含感兴趣的核苷酸序列的核酸构建体转化所述原生质体，以及 标记序列， - 应用于所述原生质体的体外培养物，一种或多种ALS抑制剂，其浓度对于原生质体的体外培养是致命的，并且从具有整合核酸构建体的存活原生质体再生甜菜植物 包括感兴趣的序列和选择性标记序列，其中选择标记序列是突变的BvALS113序列，其携带从氨基酸113位置到丙氨酸到酪氨酸的突变。