Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce
Background Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importanc...
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todo:paper_07173458_v31_n_p1_Darqui2023-10-03T15:36:23Z Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce Darqui, F.S. Radonic, L.M. López, N. Hopp, H.E. López Bilbao, M. Homozygosis Kanamycin Lactuca sativa L. Lines nptII Plants Root Seed Seedling Selection Transgenesis Nucleic acids Seed Tissue culture Homozygosis Kanamycins Lactuca sativa L Lines nptII Plants Root Seedling Selection Transgenesis Plants (botany) kanamycin Article biological monitoring biological trait concentration (parameters) controlled study crop production homozygote lettuce methodology nonhuman nptII gene plant gene plant leaf plant parameters plant structures polymerase chain reaction progeny root development root length seedling separation technique shoot transgenic plant weight Background Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays. © 2017 JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_07173458_v31_n_p1_Darqui |
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Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Homozygosis Kanamycin Lactuca sativa L. Lines nptII Plants Root Seed Seedling Selection Transgenesis Nucleic acids Seed Tissue culture Homozygosis Kanamycins Lactuca sativa L Lines nptII Plants Root Seedling Selection Transgenesis Plants (botany) kanamycin Article biological monitoring biological trait concentration (parameters) controlled study crop production homozygote lettuce methodology nonhuman nptII gene plant gene plant leaf plant parameters plant structures polymerase chain reaction progeny root development root length seedling separation technique shoot transgenic plant weight |
| spellingShingle |
Homozygosis Kanamycin Lactuca sativa L. Lines nptII Plants Root Seed Seedling Selection Transgenesis Nucleic acids Seed Tissue culture Homozygosis Kanamycins Lactuca sativa L Lines nptII Plants Root Seedling Selection Transgenesis Plants (botany) kanamycin Article biological monitoring biological trait concentration (parameters) controlled study crop production homozygote lettuce methodology nonhuman nptII gene plant gene plant leaf plant parameters plant structures polymerase chain reaction progeny root development root length seedling separation technique shoot transgenic plant weight Darqui, F.S. Radonic, L.M. López, N. Hopp, H.E. López Bilbao, M. Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| topic_facet |
Homozygosis Kanamycin Lactuca sativa L. Lines nptII Plants Root Seed Seedling Selection Transgenesis Nucleic acids Seed Tissue culture Homozygosis Kanamycins Lactuca sativa L Lines nptII Plants Root Seedling Selection Transgenesis Plants (botany) kanamycin Article biological monitoring biological trait concentration (parameters) controlled study crop production homozygote lettuce methodology nonhuman nptII gene plant gene plant leaf plant parameters plant structures polymerase chain reaction progeny root development root length seedling separation technique shoot transgenic plant weight |
| description |
Background Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays. © 2017 |
| format |
JOUR |
| author |
Darqui, F.S. Radonic, L.M. López, N. Hopp, H.E. López Bilbao, M. |
| author_facet |
Darqui, F.S. Radonic, L.M. López, N. Hopp, H.E. López Bilbao, M. |
| author_sort |
Darqui, F.S. |
| title |
Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| title_short |
Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| title_full |
Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| title_fullStr |
Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| title_full_unstemmed |
Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| title_sort |
simplified methodology for large scale isolation of homozygous transgenic lines of lettuce |
| url |
http://hdl.handle.net/20.500.12110/paper_07173458_v31_n_p1_Darqui |
| work_keys_str_mv |
AT darquifs simplifiedmethodologyforlargescaleisolationofhomozygoustransgeniclinesoflettuce AT radoniclm simplifiedmethodologyforlargescaleisolationofhomozygoustransgeniclinesoflettuce AT lopezn simplifiedmethodologyforlargescaleisolationofhomozygoustransgeniclinesoflettuce AT hopphe simplifiedmethodologyforlargescaleisolationofhomozygoustransgeniclinesoflettuce AT lopezbilbaom simplifiedmethodologyforlargescaleisolationofhomozygoustransgeniclinesoflettuce |
| _version_ |
1782023979575279616 |