Tesi etd-11162020-111127
Link copiato negli appunti
Tipo di tesi
Corso Ordinario Secondo Livello
Autore
DELLA MAGGIORA, LORENZO
URN
etd-11162020-111127
Titolo
CRISPR/Cas9 – technology in poplar: example of an approach for the production of uniform knockout mutants for the metal transporter MTP1
Struttura
Cl. Sc. Sperimentali - Agraria
Corso di studi
SCIENZE AGRARIE E BIOTECNOLOGIE - SCIENZE AGRARIE E BIOTECNOLOGIE
Commissione
Presidente Prof. PE', MARIO ENRICO
Relatore Prof. SEBASTIANI, LUCA
Membro Dott. DELL'ACQUA, MATTEO
Membro Prof.ssa MENSUALI, ANNA
Membro Prof.ssa PUCCIARIELLO, CHIARA
Membro Dott.ssa MOONEN, ANNA CAMILLA
Membro Dott.ssa BARTOLINI, SUSANNA
Relatore Prof. SEBASTIANI, LUCA
Membro Dott. DELL'ACQUA, MATTEO
Membro Prof.ssa MENSUALI, ANNA
Membro Prof.ssa PUCCIARIELLO, CHIARA
Membro Dott.ssa MOONEN, ANNA CAMILLA
Membro Dott.ssa BARTOLINI, SUSANNA
Parole chiave
- CRISPR/Cas9
- homozygous editing
- MTP1
- Populus
Data inizio appello
15/12/2020;
Disponibilità
completa
Riassunto analitico
CRISPR/Cas9 – technology has been successfully applied to edit the genome of different plants species, including poplar trees. However, chimerism and heterozygosity for the desired mutation in primary transgenic plants are drawbacks often present. Uniform mutants cannot be obtained through sexual reproduction because of the long life – cycle of woody perennial plants. For this reason, different approaches have been used in poplar to solve this problem.
Several Populus species are tolerant to zinc (Zn) excess. The study of their mechanisms of response to Zn excess and other heavy metals has identified promising candidates for soil decontamination through phytoremediation. Populus alba ‘Villafranca’ clone is a poplar species highly tolerant to Zn excess and the poplar tonoplast metal transporter MTP1 is involved in Zn sequestration inside the vacuole.
In this work, lines of Populus alba ‘Villafranca’ clone previously mutagenized for the inactivation of MTP1 using CRISPR/Cas9 – technology were used. These lines showed an incomplete inactivation of the gene. In order to obtain uniform knockout mutants, three in vitro lines were subjected to a second round of shoot regeneration through an indirect morphogenesis protocol and using leaf explants. Seventeen new lines were produced. The 11 survived lines were then propagated in vitro. In future, all lines will be characterized for the inactivation of MTP1 and the uniformity of the mutation will be assessed.
Several Populus species are tolerant to zinc (Zn) excess. The study of their mechanisms of response to Zn excess and other heavy metals has identified promising candidates for soil decontamination through phytoremediation. Populus alba ‘Villafranca’ clone is a poplar species highly tolerant to Zn excess and the poplar tonoplast metal transporter MTP1 is involved in Zn sequestration inside the vacuole.
In this work, lines of Populus alba ‘Villafranca’ clone previously mutagenized for the inactivation of MTP1 using CRISPR/Cas9 – technology were used. These lines showed an incomplete inactivation of the gene. In order to obtain uniform knockout mutants, three in vitro lines were subjected to a second round of shoot regeneration through an indirect morphogenesis protocol and using leaf explants. Seventeen new lines were produced. The 11 survived lines were then propagated in vitro. In future, all lines will be characterized for the inactivation of MTP1 and the uniformity of the mutation will be assessed.
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