T1 seeds

Nomenclature used for identifying seeds stocks collected on primary transformants and progeny plants

The T0 plants generated in Montpellier are individually identified by a code consisting of 4 letters et 2 numbers (AAA A01). This code corresponds to the DNA isolation plate number followed by the coordinates of the well in 96-well extraction plate and is printed as a barcode which allow a reliable tracking of the information and seed stocks related to an individual line.� All the information related to the generation and phenotypic characterization of the primary transformant �including observations on T1 seeds borne by panicles of the primary transformant – are gathered in the Oryza Tag Line phenotype database.

Nomenclature for first generation progeny plants (T1)

As mentioned above, the library is systematically seed increased and evaluated under field conditions. 20 T1 seeds harvested on the T0 plant are generally sown and generate a variable number of plants (ideally 20), depending on the seed germination ability. Whether no obvious mutant phenotype is observed, panicles bearing the T2 seeds are harvested in bulk and the corresponding seed stock is labelled with the T0 code number followed by /0 (e.g. AAAA01/ 0).

Whether segregation of phenotypes is observed on the field, T1 plants are individually harvested and T2 seed stocks identified with the T0 code number followed by /n� (AAAA01/n).� T2 seeds collected on individual T1 plant are therefore labelled from AAAA01/ 1 to AAAA01 /99. �The use of /0 to /99 labels are restricted to the field evaluation and seed increase.

Whether seeds of specific lines are increased under greenhouse conditions, panicles are individually harvested and are identified by numbers resuming from 101 (AAAA01/101) to 199 (AAAA01/199) for individual harvest or AAAA01/100 for a bulk harvest.

Whether a first user exceptionally ask for T1 seeds while accepting to return part of T2 seeds to the stock centre, seeds collected on these plants will be attributed a number starting from 201 (AAAA01/201 to AAAA01/299) for individual harvest or AAAA01/200 for a bulk harvest.

For a second user the numbering will be from 301 to 399 (individual harvest) or 300 (bulk harvest).

All the information related to the generation and phenotypic characterization of the T1 progeny plants �including observations on T2 seeds borne by panicles of the primary transformant – are gathered in the O ryza T ag L ine phenotype database.

Nomenclature for second generation progeny plants (T2)

Whether a second seed increase on the field is necessary due to a poor harvest of T2 seeds on T1 plants (low germination ability, low fertility�), bulk harvest of T3 seeds collected on propagated T2 plants are labelled AAAA01/0/0. Please note that seed samples may be prone to biased segregations due to the possible reduction of plant numbers at the T1 generation.��

T3 seeds resulting from specific multiplications in greenhouse of T2 plants are identified the same way as T2 seeds ( i .e. for bulked T2 seeds from AAAA01/0/101 to AAAA01/0/199 for individual harvest or AAAA01/0/100 for bulk harvest).�

[Genetic analysis of T1 progeny of transgenic tobacco with metallothionein gene and metallothionein domain mutant alpha alpha gene]

The chimeric gene containing a cloned mouse metallothionein processed gene or a cloned mouse metallothionein domain mutant alpha alpha gene was respectively introduced into tobacco (Nicotiana tobacum L. cv. NC89) on a disarmed Ti-plasmid of Agrobacterium tumefaciens. T1 seeds from self-fertilized transgenic tobacco were germinated on media containing cadmium or herbicide PPT. The PPT tolerance trait followed Mendelian inheritance and co-segregated with heavy metal tolerance. Meanwhile Southern blot and Western blot verified the existence of the MT gene and alpha alpha mutant gene in the T1 generation plants which keep tolerance to heavy metal. All the results demonstrated the stable integration and inheritance of exotic genes. In addition, assay of the root length and fresh weight of T1 seedlings indicate that transgenic tobacco plants with alpha alpha mutant gene still have a little higher tolerance than that with matural MT gene.

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