Development of a facile genetic transformation system for the Spanish elite rice paella genotype Bomba

Abstract

We report the development of an efficient and reproducible genetic transformation system for the recalcitrant Spanish elite rice paella genotype, Bomba. Preconditioned embryos derived from dry seeds were bombarded with gold particles carrying a plasmid containing a screenable and a selectable marker. We confirmed integration and expression of hpt and gusA in the rice genome. Transformation frequency was ca: 10% in several independent experiments. We show Mendelian inheritance of the input transgenes and zygosity determination of the transgenic lines in the T1 generation. A unique and critical step for the regeneration of plants from transformed tissue was shading during the early stages of regeneration, combined with a specific cytokinin:auxin ration at the onset of shifting callus to regeneration media.

Keywords: Direct DNA transfer; Genetic transformation; Oryza sativa; Zygosity determination.

Fig. 1

Bomba rice transformation system using mature seeds as explants. A Scutellum tissue 6 days after geminating seeds on proliferation medium; B seed-derived embryos (6 days after germination) immediately prior to bombardment; C hygromycin-resistant embryogenic callus on selection medium, 2 weeks after bombardment (prior to removal from the scutellum); D hygromycin-resistant embryogenic callus on selection medium 6 weeks after bombardment (after two successive subcultures of 2 weeks each); E regenerating (green) tissue derived from embryogenic callus after 2 weeks on regeneration medium under shade; F regenerating plantlet under selection; G plants on rooting medium; H established plants in soil

Fig. 2

GUS expression in early transformation events on scutellum tissue. Transient GUS expression 48 h after bombardment: A 6 day-old and B 7 day-old embryos. C, D GUS scores based on the number of blue foci on scutellum tissue (from images A and B); E transient GUS expression measured 6 or 7 day-old embryos after bombardment (no significant difference at P ≥ 0.05); F plantlet regeneration after 3 subcultures on regeneration medium (with selection) and GUS expression in leaf; G, H impact of selection pressure (hygromycin at 30 mg L⁻¹) on phenotype of regenerating embryogenic callus. Three week-old callus tissue on selection media (bombarded with pGUS-HPT); G inset: homogeneous embryogenic tissue; H inset: non embryogenic tissue (bombarded only with gold particles-no transgenic DNA)

Fig. 3

DNA and GUS analyses. A PCR from genomic DNA of callus lines transformed with pGUS-HPT. Lane C +  contains the PCR product from pGUS-HPT used for bombardment, as a positive control. Lane L contains the 1 kb DNA Ladder (ThermoFisher Scientific SM0313). Lane wt contains the PCR product from a wilt type (wt) plant. Lane C- contains the master mix used in the PCR reaction without DNA. Lanes 13 to 24 contain the PCR products amplified from independent callus lines. Samples 14 and 16 were negative. The expected PCR product for the hpt gene is 1271 bp; B Gus expression analysis of different callus lines and wt

Fig. 4

Gel blot analyses of XhoI-digested genomic DNA (20 μg) from independent lines. A, B Ethidium bromide-stained gel showing complete digestion of genomic DNA; C, D blot probed with the 466-bp-DIG-labelled PCR product from hpt; E, F blot probed with the 676-bp-DIG-labelled PCR product from gusA; G, H copy number of each line calculated from comparisons with pGUS-HPT genome-equivalent amounts. L, 1 kb DNA Ladder (ThermoFisher Scientific SM0313) in kilobases (kb). Numbers indicated independent transformed lines; wt, wild type; 1, 3 and 6 copies of XhoI digested pGUS-HPT

Fig. 5

Gel blot analysis of XhoI-KpnI-digested genomic DNA (20 μg) from independent lines and gene expression analyses. A Blot probed with the 466-bp-DIG-labelled PCR product from hpt; B blot probed with the 676-bp-DIG-labelled PCR product from gusA. L, 1 kb DNA Ladder (ThermoFisher Scientific SM0313) in kilobases (kb). Numbers indicate independent transgenic lines; wt, wild type; 1, and 3 copies of XhoI-KpnI digested pGUS-HPT. C Normalised relative mRNA expression for the hpt and gusA genes

Fig. 6

Gel blot analysis of XhoI-digested genomic DNA (20 μg) (progeny plants). Blot probed with the 466-bp-DIG-labelled PCR product from hpt; L, 1 kb DNA Ladder (ThermoFisher Scientific SM0313) in kilobases (kb). wt, wild type; T0 plant #27; numbers indicate T1 progeny plants; 1, 3 and 6 copies of XhoI digested pGUS-HPT