Reducing cadmium accumulation in field grown tobacco plants

A method for reducing the level of cadmium in the leaf of a field grown plant comprising the steps of:

• (a) reducing the expression or activity of two heavy metal ATPases (HMAs), said two HMAs comprising, consisting or consisting essentially of:
• (i) polypeptides having at least 65% sequence identity to SEQ ID NO:1 and SEQ ID NO:2;
• (ii) polynucleotides encoding the polypeptides set forth in (i);
• or
• (iii) polynucleotides having at least 65% sequence identity to SEQ ID NO:3 and SEQ ID NO:4 encoding HMAs
• suitably, wherein the expression or activity of the HMAs is reduced by mutagenesis or genome editing;
• (b) growing the plant in the field;
• (c) optionally, measuring the cadmium content in the plant obtained in step (b);
• and
• (d) identifying a plant in which the cadmium content therein is reduced in comparison to a control plant in which the expression or activity of the HMAs has not been reduced, suitably, wherein the plant or part thereof exhibits at least a 27% reduction, as compared to the control plant, in the accumulation of cadmium in leaf when the plant is field grown in the presence of naturally or non-naturally occurring cadmium;
• suitably, wherein the phenotype of the mutant plant or part thereof at harvest time is the same as the control plant at the same harvest time, suitably, wherein the mutant plant or part thereof does not show a biomass reduction at harvest time as compared to the control plant at the same harvest time.

A method for identifying one or more genetic alterations in a field grown plant that correlates with reduced levels of cadmium in leaf as compared to a field grown control plant that does not comprise the one or more genetic alterations, said method comprising the steps of:

• (a) identifying a plant with reduced levels of cadmium in the leaves when grown in the field as compared to a control plant grown in the field, optionally wherein the phenotype of the plant or part thereof at harvest time is the same as the control plant at the same harvest time, suitably, wherein the plant or part thereof does not show a biomass (for example, leaf weight) reduction at harvest time as compared to the control plant at the same harvest time;
• (b) providing a nucleic acid sample from the plant identified in step (a);
• and
• (c) identifying in the nucleic acid sample from step (b) one or more genetic alterations in the polynucleotide sequences encoding HMAs having at least 65% sequence identity to the non-mutated sequences set forth in SEQ ID NO:1 and SEQ ID NO:2 or the polynucleotide sequence having at least 65% sequence identity to SEQ ID NO:3 and SEQ ID NO:4.