Development of a protocol for genetic transformation of Malus spp

Federico Martinelli,Abhaya M Dandekar,Anna Perrone

doi:10.36253/caryologia-1248

Federico Martinelli, Abhaya M Dandekar + Show 1 more

Open Access

https://doi.org/10.36253/caryologia-1248

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Abstract

A protocol to produce transgenic shoots of Malus X domestica cv Greensleaves was optimized using two gene constructs previously used to create parthenocarpic tomato, Ino-IaaM and DefH9-IaaM. The aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. We investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an Agrobacterium containing “VIP” genes in the gene construct and 2) two different hormones or hormone combinations. More shoot regeneration was obtained with a combination of three hormones (BA:NAA:TDZ) during co-cultivation instead of IBA and no co-transformation was performed using the VIP gene. For the DefH9-IaaM transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “IBA treatment” and 4.42% with the Agrobacterium co-transformation treatment. More shoot regeneration occurred with the combination of three hormones (BA:NAA:TDZ) instead of with only IBA and no co-transformation was performed using VIP gene. Experiments using Ino-IaaM confirmed the results shown for the DefH9-IaaM transgene. The regenerated shoots were multiplied in selective media containing kanamycin and roots were obtained.

Highlights

Traditional genetic improvement in woody fruit species used selection and breeding, resulting in relatively few genotypes and a restricted germplasm base
Different hormone combinations were used to improve the genetic transformation protocol, using two constructs containing the IaaM gene driven by DefH9 or Ino, two ovule-specific promoters previously used to transform tomato
The number of transgenic shoots was greater with the hormone combination (1.52% vs 0.78 and 0.47%, respectively)

Summary

Introduction

Traditional genetic improvement in woody fruit species used selection and breeding, resulting in relatively few genotypes and a restricted germplasm base. This genetic uniformity has increased vulnerability of woody crops to insect pests and pathogens and caused excessive use of chemicals (Norelli et al 1994) Genetic transformation provides an alternate approach through introduction of genes encoding desirable traits (Jia et al 2019), bypassing the long periods required for genetic crosses and selection. Since production of most fruit tree species is based on a few cultivars, the impact of genetically transforming them is important.The characterization of induced overll metabolism changes using omic tools has been previously done (Tosetti et al 2010; Rizzini et al 2010)

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