Stable and efficient transformation of apple.

Apple is one of precious fruit crop grown in temperate zone. In the post genomic era, the analysis of gene functions in horticultural crops such as apple is required for agricultural utilization. For analysis of such crops, the protocol establishment of tissue culture and transformation is essential. Although transformation efficiency in family Rosaceae is generally very low, some cultivars of Malus species have high transformation ability. Apple cultivars are usually clonally propagated by grafting on rootstocks, which can affect fruit quality and maturity and scion productivity. Apple rootstock cultivar Japan Morioka 2 (JM2) was produced at the Division of Apple Research, Institute of Fruit and Tea Science, NARO, in Japan. JM2, which was developed for dwarfing scions and improving disease resistance, is easily propagated by hardwood cutting. Furthermore, JM2 can be stably transformed at a high efficiency, which is better than other JM series rootstocks derived from the same parent. Leaflets of cultured shoots of JM2 have been transformed using Agrobacterium (Rhizobium) with a transducing gene. In this article, the JM2 transformation protocol is introduced in detail. Various genes and promoters have been confirmed to function as expected, with the resultant transformants exhibiting specific staining and fluorescent signals, and modified floral organ shapes, precious blooming and other characteristics. JM2 is thus a useful rootstock material for the enhancement of genetic research on apple and its relatives.

Characterization of FLOWERING LOCUS C Homologs in Apple as a Model for Fruit Trees.

To elucidate the molecular mechanism of juvenility and annual flowering of fruit trees, FLOWERING LOCUS C (FLC), an integrator of flowering signals, was investigated in apple as a model. We performed sequence and expression analyses and transgenic experiments related to juvenility with annual flowering to characterize the apple FLC homologs MdFLC. The phylogenetic tree analysis, which included other MADS-box genes, showed that both MdFLC1 and MdFLC3 belong to the same FLC group. MdFLC1c from one of the MdFLC1 splice variants and MdFLC3 contain the four conserved motives of an MIKC-type MADS protein. The mRNA of variants MdFLC1a and MdFLC1b contain intron sequences, and their deduced amino acid sequences lack K- and C-domains. The expression levels of MdFLC1a, MdFLC1b, and MdFLC1c decreased during the flowering induction period in a seasonal expression pattern in the adult trees, whereas the expression level of MdFLC3 did not decrease during that period. This suggests that MdFLC1 is involved in flowering induction in the annual growth cycle of adult trees. In apple seedlings, because phase change can be observed in individuals, seedlings can be used for analysis of expression during phase transition. The expression levels of MdFLC1b, MdFLC1c, and MdFLC3 were high during the juvenile phase and low during the transitional and adult phases. Because the expression pattern of MdFLC3 suggests that it plays a specific role in juvenility, MdFLC3 was subjected to functional analysis by transformation of Arabidopsis. The results revealed the function of MdFLC3 as a floral repressor. In addition, MdFT had CArG box-like sequences, putative targets for the suppression of flowering by MdFLC binding, in the introns and promoter regions. These results indicate that apple homologs of FLC, which might play a role upstream of the flowering signals, could be involved in juvenility as well as in annual flowering. Apples with sufficient genome-related information are useful as a model for studying phenomena unique to woody plants such as juvenility and annual flowering.

The use of a fertile doubled haploid apple line for QTL analysis of fruit traits.

Apple is an economically important crop, and various approaches to genetic analysis in breeding programs have been attempted, including the production of doubled haploid (DH) lines, which are genetically homozygous. In this study, we used a DH line for QTL analyses, for the first time in a fruit tree, expecting it to simplify the analysis of the inheritance of quantitative traits and thus to enhance QTL detection power. Using an F1 population from 'Prima' × 'Apple Chukanbohon 95P6' (DH), we constructed a genetic map of 'Prima', and identified 19 QTLs for 13 traits. These QTLs had comparatively high LOD scores and explained a large part of the variation of the phenotypes. In particular, acidity, juice browning, and skin splitting clearly segregated at a 1:1 ratio, consistent with the segregation of the alleles at the detected QTLs in linkage group 16; these traits appeared to be regulated by single genes, despite general consideration that they are quantitative traits. Using this simple genetic composition of the F1 population, we concluded that the skin splitting of apple fruit has recessive inheritance, and that the allele for splitting is tightly linked with those for high acidity and low juice browning in 'Prima'.

CRISPR-Cas9-mediated genome editing in apple and grapevine.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

Expression and functional analysis of apple MdMADS13 on flower and fruit formation.

Apple MdMADS13 has a transcription factor with MADS domain. Moreover, it is expressed specifically at petals and carpels. The product forms a dimer with MdPISTILLATA (MdPI) protein as a class B gene for floral organ formation. Reportedly, in parthenocarpic cultivars of apple (Spencer Seedless, Wellington Bloomless, Wickson and Noblow) the MdPI function is lost by genome insertion of retrotransposon, which cultivars show a homeotic mutation of floral organs, petals to sepals and stamens to carpels. Apple fruit is pome from receptacle tissue, and MdSEPALLATA (MdMADS8/9) and AGAMOUS homologues MdMADS15/22 involved in the fruit development, the transgenic apple suppressed these gene showed poor fruit development and abnormal flower formation. This article describes that the MdMADS13 retained expression after blossom and small fruits of parthenocarpic cultivars. Yeast two-hybrid experiment showed specific binding between MdPI and MdMADS13 proteins. Furthermore, transgenic Arabidopsis with 35S::MdMADS13 have malformed stamens and carpels. These results suggest strongly that MdMADS13 is related to flower organ formation as a class B gene with MdPI.

Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.

Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.

The analysis of transgenic apples with down-regulated expression of MdPISTILLATA.

In parthenocarpic cultivars of apple (Malus×domestica Borkh.), MdPISTILLATA (MdPI) expression has been suppressed by retrotransposon insertion into the MdPI genome. In this study, transgenic apple lines were produced that exhibited the same level of MdPI depression. The 1P-2 promoter from the MdPI genome, which specifies its expression in the petals and stamens, was used for antisense-MdPI expression, and rolC:AtFT was included to accelerate flowering. The transgenic apple with rolC:AtFT/1P-2:antisense-MdPI showed homeotic changes in the floral organs, whereby petals and stamens were replaced with sepals and pistils, respectively. Line 9-2 of this transgenic apple also showed strong suppression of MdPI. Some individuals from this line had deformed floral organs, suggesting that the homeotic changes were incomplete. Other transformants of line 9-2 that had double sepals in the first and second whorls, and many pistils in the third and fourth whorls, as seen in apple cultivars with class B mutations, which demonstrated MdPI functioned for floral organs formation same as Arabidopsis PISTILLATA gene. The transgenic apples set parthenocarpic fruits (15.7%). However, precocious transgenic apples with rolC:AtFT exhibited more parthenocarpy (14-27%). This indicates that MdPI depression cannot explain fruit formation in parthenocarpic cultivars of apple, and so some other as yet unidentified genes must be responsible.

Expression patterns of several floral genes during flower initiation in the apical buds of apple (Malus × domestica Borkh.) revealed by in situ hybridization.

The apple (Malus × domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4 years) and a long reproductive period between the flower initiation and the mature fruit (14-16 months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.

Promotion of flowering and reduction of a generation time in apple seedlings by ectopical expression of the Arabidopsis thaliana FT gene using the Apple latent spherical virus vector.

Tree crops have a long juvenile period which is a serious constraint for genetic improvement and experimental research. For example, apple remains in a juvenile phase for more than five years after seed germination. Here, we report about induction of rapid flowering in apple seedlings using the Apple latent spherical virus (ALSV) vector expressing a FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana. Apple seedlings could be flowered at 1.5-2 months after inoculation to cotyledons of seeds just after germination with ALSV expressing the FT gene. A half of precocious flowers was normal in appearance with sepals, petals, stamens, and pistils. Pollen from a precocious flower successfully pollinated flowers of 'Fuji' apple from which fruits developed normally and next-generation seeds were produced. Our system using the ALSV vector promoted flowering time of apple seedlings within two months after germination and shortened the generation time from seed germination to next-generation seed maturation to within 7 months when pollen from precocious flowers was used for pollination.

An efficient method for sonication assisted Agrobacterium-mediated transformation of coat protein (CP) coding genes into papaya (Carica papaya L.).

An efficient method for the production of transgenic papaya was developed via Sonication Assisted Agrobacterium-mediated Transformation (SAAT) of somatic embryos. The plasmid pGA482G was modified to contain gene PTi-Epj-TL-PLDMV with CP coding sequence of PLDMV Japan strain and chimeric gene PTi-NP-YKT with multiple CP coding sequences from PRSV Taiwan strain, PRSV Hawaii strain and PRSV Thailand strain, respectively. Disarmed Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pGA482G with the CP genes and nptII gene was used to transform embryo calli of papaya variety Sunset to produce transgenic papaya plants. The experiment was focused on the screening of effective transformation method. The engineered Agrobacterium grown overnight was diluted with an infection media of high osmotic pressure (1/2 MS medium contain 6% sucrose and 1% glucose, pH 5.7) and adjusted to optical density OD600nm = 0.15-0.20, embryonic calli were immerged in it for 30 min and treated with 5 s, 15 s, and 20 s sonication respectively during the infection. Results indicated that 15 s sonication treatment improved the transformation efficiency dramatically. After 15 s sonication treatment on embryo calli loaded in 15 ml sterile plastic tubes, 21 putative transgenic lines were produced from 80 pieces embryonic calli (26.3%) transformed by Agrobacterium [pGA482G/CPG] and 8 putative transgenic lines was produced from 48 pieces embryonic calli (16.7%) transferred by Agrobacterium [pGA482G/CPB], while only a single line came out of 64 pieces embryonic calli (1.6%) transformed by Agrobacterium [pGA482G/CPG] and none from 25 pieces embryonic calli transformed by Agrobacterium [pGA482G/CPB] in the non-treatment control. Results also showed that the best concentration of selection antibiotic was 120 mg/L kanamycin. A total of 42 resistant shoots were produced from 421 pieces of original embryonic calli in 9 months. The presence of the CP genes in the transgenic plants and their integration into the papaya genome were confirmed by PCR and Southern hybridization respectively.