Recent Developments and Strategies for the Application of Agrobacterium-Mediated Transformation of Apple Malus × domestica Borkh

Abstract

Genetic transformation has become an important tool in plant genome research over the last three decades. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. Further, we highlight targeted gene silencing applications. Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Success has also been achieved in establishing methods for targeted genome editing (GE). For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. In particular, the current state of the rapid crop cycle breeding system and its applications will be presented.

Keywords: Agrobacterium tumefaciens; Malus; apple; genome editing; rapid cycle breeding; transformation; virus-induced gene silencing.

Figure 1

Development of the harvest volumes of selected apple cultivars in the EU in comparison with the years 2008 and 2020 (www.prognosfruit.eu).

Figure 2

GE strategies using CRISPR/Cas9. A DSB in the target locus is introduced by CRISPR/Cas9-mediated DNA cleavage. The DSB repair by NHEJ leads to random mutations at the site of the DNA break including insertions and deletions (indels). By the addition of a homologous donor DNA, the DSB can be repaired by HDR. This strategy can be used to introduce specific GE events such as insertions, substitutions or gene replacements.

Figure 3

Base editing by CRISPR-dependent cytosine base editor (CBE). (A) The CBE binds specifically to the target DNA, which is defined by the guide RNA sequence. (B) The fused cytosine deaminase catalyzes the deamination of the cytosine base, which results in the formation of uracil. Because of the changed base-pairing properties, the G-U pair forms a mismatch that is recognized by the DNA repair machinery of the cell. Repair events can result in the replacement of the original G to A. The C-to-T conversion becomes stable after replication.

Figure 4

Schematic representation of the idea behind the rapid cycle breeding program. One parent with excellent traits in terms of fruit quality (green) containing a transgenic T-DNA insertion for early flowering is crossed with another parent (red) containing a trait of interest, e.g., disease resistance. Many resistances are unfortunately only found in Malus wild species, which usually have insufficient fruit quality. The high proportion of adverse alleles in these genotypes is symbolized by the red color. Repeated backcrossing with high-quality apple varieties (green) gradually reduces the proportion of negative alleles. The flower symbolizes the transgene for early flowering. GI is the abbreviation for “gene of interest,” which confers the desired trait as, e.g., disease resistance. If both traits (early flowering and resistance) are inherited monogenically, a quarter of offspring will be produced with both traits. These genotypes can then be used in further crosses to shorten breeding cycles. Another quarter of offspring will contain only the gene of interest. Such non-transgenic null-segregants can be released from the breeding program as advanced selections.

Figure 5

Netted BpMADS4 transgenic apple trees used for crosses in the experimental orchard at Cornell University. Nets around the trees prevent pollinators from spreading the transgenic pollen outside the orchard. Source: Dr. Awais Khan who leads the rapid cycle breeding program at Cornell University in Geneva (New York, United States) kindly provided the photograph.