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. 2022 Jan 18:9:uhab014.
doi: 10.1093/hr/uhab014. Online ahead of print.

Significant improvement of apple (Malus domestica Borkh.) transgenic plant production by pre-transformation with a Baby boom transcription factor

Jiajing Chen  1   2 , Sumathi Tomes  1 , Andrew P Gleave  1 , Wendy Hall  1 , Zhiwei Luo  1 , Juan Xu  2 , Jia-Long Yao  1   3
Affiliations

Significant improvement of apple (Malus domestica Borkh.) transgenic plant production by pre-transformation with a Baby boom transcription factor

Jiajing Chen et al. Hortic Res. .

Abstract

BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1 gene. "Royal Gala" apple plants were first transformed with a CaMV35S-MdBBM1 construct (MBM) under kanamycin selection. These MBM transgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV35S-GUS transgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBM plants produced more cells than the GUS plants, indicating that ectopic-expression of MdBBM1 enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBM plants compared with the GUS plants. Plants of eight independent MBM lines were compared with the GUS plants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0-3% for the GUS plants, 3-8% for five MBM lines, and 20-30% for three MBM lines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

Keywords: Apple (M. domestica); Baby Boom (BBM); Plant hormone response; Shoot regeneration; Transformation.

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Figures

Figure 1
Figure 1
Analysis of tobacco and apple plants transformed with a 35S-MdBBM1-GR construct. a Relative transcript levels of MdBBM1 in wild-type (WT) and eight 35S-MdBBM1-GR transgenic lines (NBGs) of tobacco by ddPCR analysis. b Regeneration rates (number of independent shoots regenerated per 100 explants) for the WT and NBG8 line of tobacco on MS medium supplemented with 0.1 mg/L BA, without or with (+) DEX (5 μM). c Images show shoot regeneration from leaf explants of the WT and NBG8 on MS medium supplemented with 0.1 mg/L BA, with or without DEX (5 μM). d Relative transcript levels of MdBBM1 in WT and eight 35S-MdBBM1-GR transgenic lines (MBGs) of apple. e Regeneration rates for the WT, MBG3 and MBG8 line of apple on shoot maintenance medium with 0.1 mg/L IBA and 1 mg/L BA, without or with (+) DEX (5 mM). f Images show shoot regeneration from leaf explants of the WT, MBG3 and MBG8 line of apple on shoot regeneration medium with 1 mg/L NAA, 5 mg/L BA and 1 mg/L TDZ, without DEX. The values in a, b, d and e are mean ± SD (n = 3). The asterisks in a, b, d and e indicate significant differences to the WT according to a t-test (P < 0.01).
Figure 2
Figure 2
In vitro phenotype analyses of apple transgenic lines overexpressing MdBBM1. a–c Shoots multiplied from a single shoot of a 35S-GUS line and two types of 35S-MdBBM1 lines (types-1 MBM29 and type-2 MBM8) respectively, photographed after a single shoot was cultured on shoot maintenance medium for three weeks. d Number of shoots multiplied from each single shoot of 35S-GUS and 35S-MdBBM1 lines presented as violin-type plots. The data were collected from three independent experiments each used six to eight shoots per line. A one-way ANOVA test revealed the significant differences in shoot number between the GUS and MBM lines (**** representing p < 0.0001). e–g Root growth of the three lines at three weeks after six single shoots were cultured on rooting medium. h–j Shoot regeneration of the three lines at six weeks after leaf explants were culture on a shoot regeneration medium. k–m Shoot regeneration at six weeks after leaf explants were cultured on shoot maintenance medium. For shoot regeneration (h–m), the leaf explants were cultured for four weeks in the dark and then two weeks under a 16-h photoperiod.
Figure 3
Figure 3
Phenotype analyses of greenhouse-grown apple transgenic lines overexpressing MdBBM1. a–c Plants of a GUS line (a), and two MBM lines carrying 35S-MdBBM1 (b, c) 3 months after being transplanted and grown in a greenhouse. d–f Left panel shows the gross morphology of a fully expanded leaf from a, b and c with the cell density of the leaves shown through cross sections (middle panel) and parallel sections (right panel) through the palisade tissues.
Figure 4
Figure 4
Overexpression of MdBBM1 enhanced apple transformation efficiency. a, b Leaf explants of a GUS line (a) and a 35S-MdBBM1 line (MBM29) (b) after infection with Agrobacterium tumefaciens containing the 35S-ALS construct for herbicide resistance and cultured on shoot regeneration medium supplemented with the Glean herbicide (2 μg/L) for three months in dark. Callus tissues and shoot-regenerating foci on these leaf explants are indicated by the red arrows. c, d Shoot-regenerating foci isolated from MBM29 explants without infection or selection (c) or with infection and selection (d) four weeks after transfer to shoot maintenance medium supplemented with 4 μg/L Glean under and a 16-h photoperiod. e Three single shoots of the GUS line, MBM29 and three independent re-transformed lines of MBM29 (MBM29-ALS1, 2, 3) cultured on shoot maintenance medium supplemented with 100 μg/L Glean under a 16-h photoperiod for 4 weeks. f Expression analysis of MdALS in the GUS, MBM29 and three re-transformed MBM29 lines. The values are mean ± SD (n = 3). The asterisks indicate significant differences (P < 0.01) to the GUS line according to a t-test. g, h Plants of the GUS line (g) and MBM29-ALS (h) lines rooted in vitro, established in a greenhouse, then sprayed with 60 mg/L Glean and photographed at 4 weeks post spray.
Figure 5
Figure 5
Overexpression of MdBBM1 markedly changed the expression levels of genes in plant hormone signalling pathways. Differentially expressed genes (DEGs) identified by comparison of transcription data of a GUS line and two 35S-MdMBM1 lines (MBM8 and MBM29). Graphical representation of FKPM values from RNA-seq data of various classes of genes are shown, as are the identities of these genes (MD numbers) in the apple reference genome. a Small auxin up-regulated RNA (SAUR) genes of the auxin signalling pathway leading to cell enlargement and plant growth (upper panel). Fourteen SAUR genes significantly down-regulated in the MBM8 and MBM29 lines compared with the GUS line are shown. b Type-A ARR (A-ARR) and type-B ARR (B-ARR) genes encoding suppressors and activators, respectively, in the cytokinin signalling pathway leading to cell division and shoot regeneration (upper panel). Four A-ARR and three B-ARR genes significantly down-regulated and up-regulated, respectively, in MBM8 and MBM29 lines compared with the GUS line are shown. c D-class cyclin (CYCD) responds to brassinosteroid signalling for promoting cell division (upper panel). Six CYCD genes up-regulated in MBM8 and MBM29 lines compared with the GUS line are shown. FPKM values are means ± SD of three RNA-seq libraries. Red bars represent up-regulation of genes and blue bars represent down-regulation of genes in the MBM8 and MBM29 lines compared with the GUS line. The values in a, b and c are mean ± SD (n = 3). The asterisks in a, b and c indicate significant differences (P < 0.01) to the GUS line according to a t-test.
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