TY - JOUR

T1 - Optimization of Rhizobium rhizogenes-mediated transformation, regeneration and characterization of Malus domesticaBorkh. Ri lines

AU - Rüter, Philipp

AU - Eeckhaut, Tom

AU - Dhooghe, Emmy

AU - De Keyser, Ellen

AU - Bartels, Jens

AU - Winkelmann, Traud

AU - Hung Doan, Manh

N1 - Funding Information: Open Access funding enabled and organized by Projekt DEAL. This research was funded by the DFG (Deutsche Forschungsgemeinschaft, WI 2002/6\u20131) and VLAIO as part of the RootsPlus project. RootsPlus was carried out under the second call of the ERA-NET Cofund SusCrop, being part of the Joint Programming Initiative on Agriculture, Food Security and Climate Change (Facce-JPI). SusCrop has received funding from the European Union\u2019s Horizon 2020 research and innovation program under grant agreement No 771134.

PY - 2024/4/20

Y1 - 2024/4/20

N2 - This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.

AB - This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.

KW - Adventitious shoot regeneration

KW - Agrobacterium rhizogenes

KW - Apple

KW - Digital PCR

KW - Hairy roots

KW - Ri phenotype

UR - http://www.scopus.com/inward/record.url?scp=85190815786&partnerID=8YFLogxK

U2 - 10.1007/s11240-024-02742-4

DO - 10.1007/s11240-024-02742-4

M3 - Article

AN - SCOPUS:85190815786

VL - 157

JO - Plant Cell, Tissue and Organ Culture

JF - Plant Cell, Tissue and Organ Culture

SN - 0167-6857

IS - 2

M1 - 32

ER -