Description

Technical field

[0001] The invention is directed to the production of suberin, the depolymerisation process of suberin and to the use of a transcription factor as activator for the production of suberin.

Background art

[0002] Suberin is a natural aliphatic-aromatic cross-linked polyester, almost ubiquitous in the vegetable realm, albeit in very variable proportions. It is mostly found in the cell walls of normal and wounded external tissues of aerial and/or subterranean parts of plants where it plays the fundamental role of a protective barrier between the organism and its environment (see studies of Gandini A. et al, Prog. Polym. Sci., 2006, 31, 878-892).

[0003] The main components of the aliphatic domains of suberin are long chains of ω-hydroxyfatty acids, α-, ω-dicarboxylic acids and homologous mid- chain di-hydroxy or epoxy derivatives, whereas the aromatic domains are dominated by variously substituted phenolic moieties.

[0004] Suberin acts as a highly efficient barrier which limits water, solute, gas and ion exchange. Indeed, suberized cell walls could act as barriers to minimize the movement of water and nutrients, thus restricting pathogen invasion and impeding toxic gas diffusion. In addition, suberized cell walls provide a barrier to radial oxygen loss from roots to the anaerobic root substrate in wetland plants (see studies of Ranathunge K. et a/., Plant Science, 201 1 , 180, 399-413).

[0005] Suberin is particularly abundant in the oak cork, birch outer barks and potatoes. However, these resources are natural, and therefore limited.

[0006] The research of Legay S. et al. has shown that, while a strong correlation between the expression of a MYB93 transcription factor and key suberin biosynthetic genes such as GAPT5, CYP86A1 and CYP86B1 exists, the function of said MYB93 transcription factor is not known (Legay S. ef a/., Plant Mol. Biol., 2015, 88, 21 -40). [0007] The main utility of suberin and/or of its monomers is the fabrication of polyurethane and/or tackifiers, i.e. chemical compounds used in formulating adhesives to increase the stickiness of the surface of the adhesive. These tackifiers are mainly used in the tyre industry.

Summary of invention

Technical Problem

[0008] The invention has for technical problem to provide an industrial source of suberin and suberin monomers which supplements the limited natural resources.

Technical solution

[0009] The first object of the invention is directed to a process for the production of a suberin polymer, comprising the steps of (a) introducing an orthologous gene of MYB93 transcription factor into plant cells from a plant organism and/or a plant tissue leading to genetically engineered plant cells; (b) stimulating said genetically engineered plant cells; and, (c) isolating a suberin polymer that is produced as result of step (b).

[0010] According to a preferred embodiment, said step (a) is performed by /Agrobacfer/um-mediated method, by viral-mediated method, by gene gun process or by electroporation, preferably by

method.

[001 1] According to a preferred embodiment, said /Agrobacfer/um-mediated method is performed by inserting at least one vector comprising an orthologous gene of MYB93 transcription factor into a bacterium and by infecting said plant cells from a plant organism and/or a plant tissue with said bacterium, wherein said bacterium is Agrobacterium tumefaciens.

[0012] According to a preferred embodiment, said at least one vector further comprises a constitutive promotor or an inducible promotor.

[0013] According to a preferred embodiment, said constitutive promotor is CaMV 35S.

[0014] According to a preferred embodiment, said inducible promotor is induced by oestrogens, ethanol or by a heat shock. [0015] According to a preferred embodiment, said step (b) is performed with hormones, preferably oestrogens.

[0016] The second object of the invention is directed to a process for the production of monomers of suberin, comprising (a) the process of the production of suberin in accordance with the first object of the invention and the subsequent steps of (b) removing the lipids contained in the suberin so as to produce a delipidized suberin; and (c) depolymerizing said delipidized suberin so as to produce the monomers of suberin.

[0017] According to a preferred embodiment, said step (c) is performed by ester- breaking chemical reactions.

[0018] According to a preferred embodiment, said plant organisms are tobacco plant and/or apple plant, and/or wherein said plant tissues are leaves, fruits, roots and/or stems.

[0019] According to a preferred embodiment, the coding sequence of orthologous gene of MYB93 transcription factor is the genes MDP0000320772 (SEQ- ID NO: 1 ) and/or MDP0000228252 (SEQ-ID NO:2).

[0020] The third object of the invention concerns the use of orthologous gene of MYB93 transcription factor as activator for regulating with an inducible promotor the expression of SMT1 , KCS1 , KCS4, 4CL1 , 4CL3, AT2G39350, WBC1 1 , AT3G55090, AT2G37360, AT5G19410, PDR4, AGL21 , AT4G24140, AT4G36610, AT4G27450, ANNAT8, AT2G03200, AT4G32480, AT4G 17800, CT-BMY, BETA-TIP, AT2G44300, AT3G22620, GPAT4, GPAT6, BT4, CSLC12, AT5G38200, AT4G15610, AT5G44550, AT5G07475, CYP94C1 , CYP96A10, CYP86A8, CYP86A1 , CYP86B1 , AT5G1 1880, IRX12, ENODL17, AT2G18360, WRI 1 , CER4, DIN10, AT1 G74460, AT5G03610, AT5G22810, AT5G37690, AT1 G75900, AT2G23540, AT5G23370, ASN1 , GPAT5, AT5G49350, GH9C1 , AT1 G80160, AT5G24080, AT4G27300, Hsp70b, AT5G24580, AT1 G29000, AT1 G71050, HHP1 , AT1 G32910, AT5G09520, LAC 12, AT5G48480, AT1 G54540, AT3G22800, AT5G13900, LACS4, TAA1 , AT2G23790, SKS1 , AT4G15700, MYB67, MYB93, MIOX2, NAC038, NAC058, NAC073, NAC075, AT1 G09380, LP1 , LTP4, NAT7, AT3G49190, OLEO3, AT4G35160, AT3G59850, PE1 1 , AT5G19640, AT3G52790, AT1 G68850, AT5G05340, AT5G14130, PAL2, AT3G09925, AT5G47635, IRX9, XTR6, AT3G22600, AT1 G05450, BST1 , CER3, EDA4, EXO, OXS3, AT5G09530, RPD1 , TMN7, TBL19, TBL33, AT2G14830, AT5G23750, AT5G41400, ATL8, RIC4, AT2G33205, AT5G18840, SULTR1 , AT5G1 1620, MYB84, UCC3, PGSIP3, AT5G27020, AT4G10265 and/or AT1 G68360 gene in plant organisms and/or in plant tissues.

[0021] According to a preferred embodiment, the expression of SMT1 , KCS1 , KCS4, 4CL1 , 4CL3, AT2G39350, WBC1 1 , AT3G55090, AT2G37360, AT5G19410, PDR4, AGL21 , AT4G24140, AT4G36610, AT4G27450, ANNAT8, AT2G03200, AT4G32480, AT4G 17800, CT-BMY, BETA-TIP, AT2G44300, AT3G22620, GPAT4, GPAT6, BT4, CSLC12, AT5G38200, AT4G 15610, AT5G44550, AT5G07475, CYP94C1 , CYP96A10, CYP86A8, CYP86A1 , CYP86B 1 , AT5G 1 1880, I RX12, ENODL17, AT2G18360, WR11 , CER4, DIN10, AT1 G74460, AT5G03610, AT5G22810, AT5G37690, AT1 G75900, AT2G23540, AT5G23370, ASN1 , GPAT5, AT5G49350, GH9C1 , AT1 G80160, AT5G24080, AT4G27300, Hsp70b, AT5G24580, AT1 G29000, AT1 G71050, HHP1 , AT1 G32910, AT5G09520, LAC 12, AT5G48480, AT1 G54540, AT3G22800, AT5G13900, LACS4, TAA1 , AT2G23790, SKS1 , AT4G15700, MYB67, MYB93, MIOX2, NAC038, NAC058, NAC073, NAC075, AT1 G09380, LP1 , LTP4, NAT7, AT3G49190, OLEO3, AT4G35160, AT3G59850, PE1 1 , AT5G19640, AT3G52790, AT1 G68850, AT5G05340, AT5G14130, PAL2, AT3G09925, AT5G47635, IRX9, XTR6, AT3G22600, AT1 G05450, BST1 , CER3, EDA4, EXO, OXS3, AT5G09530, RPD1 , TMN7, TBL19, TBL33, AT2G14830, AT5G23750, AT5G41400, ATL8, RIC4, AT2G33205, AT5G18840, SULTR1 , AT5G1 1620, MYB84, UCC3, PGSIP3, AT5G27020, AT4G10265 and/or AT1 G68360 gene in plants organisms and/or in plant tissues leads to the production of suberin in plants organisms and/or in plant tissues.

[0022] According to a preferred embodiment, said plant organisms are tobacco plant and/or apple plant, and/or wherein said plant tissues are leaves, fruits, roots and/or stems.

[0023] According to a preferred embodiment, the coding sequence of said orthologous gene of MYB93 transcription factor is the genes MDP0000320772 (SEQ-ID NO: 1 ) and/or MDP0000228252 (SEQ-ID NO:2). Advantages of the invention

[0024] The invention is particularly interesting in that it provides a reliable source of suberin which then can be depolymerized and transformed in very useful material. The process of the present invention represents an important source of biopolymers that can be used in the industry.

Description of an embodiment

[0025] The invention relates to the controlled production process of the suberin polymer (i.e. a copolyester) by the use of DNA sequence genes, named MDP0000320772 (SEQ-ID NO: 1 ), MDP0000228252 (SEQ-ID NO:2) isolated from Malus x domestica cv. Cox Orange pippin and/or orthologous genes of MYB93 transcription factor and coding a MYB-family transcription factor, named MYB93 and similar to AtMYB93 (Arabidopsis thaliana model plant), which is able to finely regulate the expression of the suberin downstream synthesis dedicated enzymes, such as those described in table 1 below.

[0026] These suberin synthesis genes are found in plant organisms, for example tobacco plant (Nicotiana benthamiana) and/or apple plant, or plant tissues, for example plant leaves, fruits, roots and/or stems.

[0027] Suberin productive transformed plants, tissues or cells are obtained via the use of the bacterium Agrobacterium tumefaciens carrying a plasmid in which the DNA sequence genes coding for the MYB93 transcription factor is inserted. The plasmid, or the vector, comprises an inducible promotor or a constitutive promotor. An example of constitutive promotor is CaMV 35S. An example of inducible promotor is XVE, OLexA (induced by oestrogens), Ale, AlcA (induced by ethanol) or Gmhsp17.3 (induced by heat shock).

Table 1 : MYB93-like activated genes coded according to Arabidopsis thaliana orthologous genes (the corresponding gene code in Nicotiana benthamiana is also indicated). Gene symbol

Gene code In (Arabidopsis

Nicotiana Annotation description thaliana nr benthamiana (Arabidopsis thaliana nr database) database)

Nbv5tr6228038 24-sterol c-methyltransferase SMT1

Nbv5tr6328789 3-ketoacyl- synthase 1 KCS1

Nbv5tr6222668 3-ketoacyl- synthase 4 KCS4

Nbv5tr6215803 4-coumarate- ligase 1 4CL1

Nbv5tr6243580 4-coumarate- ligase 3 4CL3

Nbv5tr6236478 abc transporter g family member 1 AT2G39350

Nbv5tr6217020 abc transporter g family member 1 1 WBC1 1

Nbv5tr6206538 abc transporter g family member 16 AT3G55090

Nbv5tr6220564 abc transporter g family member 2 AT2G37360

Nbv5tr6391581 abc transporter g family member 23 AT5G19410

Nbv5tr6236569 abc transporter g family member 32 PDR4

Nbv5tr6244229 agamous-like mads-box protein agl21 AGL21

Nbv5tr6389537 alpha beta fold family protein AT4G24140

Nbv5tr631401 1 alpha beta fold family protein AT4G36610 aluminum induced protein with ygl and Irdr

Nbv5tr6313510 motifs AT4G27450

Nbv5tr62351 12 annexin d8 ANNAT8

Nbv5tr6235088 aspartyl protease family protein AT2G03200

Nbv5tr6220091 at4g32480 f8b4_180 AT4G32480

Nbv5tr6252744 at-hook dna-binding family protein AT4G 17800 bam3_arath ame: full=beta-amylase

Nbv5tr6268817 chloroplastic CT-BMY

Nbv5tr6217187 beta-tonoplast intrinsic protein BETA-TIP bifunctional inhibitor lipid-transfer protein

Nbv5tr6200335 seed storage 2s albumin superfamily protein AT2G44300 bifunctional inhibitor lipid-transfer protein

Nbv5tr6223644 seed storage 2s albumin-like protein AT3G22620 bifunctional sn-glycerol-3-phosphate 2-o-

Nbv5tr6395628 acyltransferase phosphatase GPAT4

bifunctional sn-glycerol-3-phosphate 2-o-

Nbv5tr6318400 acyltransferase phosphatase GPAT6

Nbv5tr6215068 btb and taz domain protein 4 BT4 Nbv5tr6227571 cellulose-synthase-like c12 CSLC12 class i glutamine amidotransferase-like

Nbv5tr6237322 protein AT5G38200 csplm_arath ame: full=casp-like protein 1 d 1

Nbv5tr6232726 short= 1 d1 AT4G 15610 csplw_arath ame: full=casp-like protein 1 b1

Nbv5tr6223406 short= 1 b1 AT5G44550

Nbv5tr6199034 cupredoxin superfamily protein AT5G07475

Nbv5tr6220824 cytochrome family subfamily polypeptide 1 CYP94C1

Nbv5tr6340302 cytochrome family subfamily polypeptide 10 CYP96A10

Nbv5tr6346089 cytochrome family subfamily polypeptide 8 CYP86A8

Nbv5tr6408715 cytochrome p450 86a 1 CYP86A1

Nbv5tr6237440 cytochrome p450 86b1 CYP86B1

Nbv5tr6233583 diaminopimelate decarboxylase 2 AT5G1 1880

Nbv5tr6203380 diphenol oxidase IRX12

Nbv5tr6200724 early nodulin-like protein 17 ENODL17

Nbv5tr6216023 esterase lipase domain-containing protein AT2G 18360

Nbv5tr6237694 ethylene-responsive transcription factor wril WRI 1

Nbv5tr6203422 fatty acyl- reductase cer4 CER4

Nbv5tr6396810 galactinol-sucrose galactosyltransferase 6 DIN10

Nbv5tr6222730 gdsl esterase lipase AT1 G74460

Nbv5tr6202105 gdsl esterase lipase AT5G03610

Nbv5tr6216569 gdsl esterase lipase AT5G22810

Nbv5tr6291 143 gdsl esterase lipase AT5G37690

Nbv5tr6207792 gdsl esterase lipase exl3 AT1 G75900

Nbv5tr6222428 gdsl-motif lipase hydrolase AT2G23540

Nbv5tr6234393 gem-like protein 8 AT5G23370

Nbv5tr6336275 glutamine-dependent asparagine synthetase ASN1

Nbv5tr6232718 glycerol-3-phosphate acyltransferase 5 GPAT5

Nbv5tr6232901 glycine-rich protein AT5G49350

Nbv5tr6391909 glycosyl hydrolase 9c1 GH9C1

Nbv5tr6236952 glyoxylase i 7 AT1 G80160 g-type lectin s-receptor-like serine threonine

Nbv5tr6228455 protein kinase AT5G24080 g-type lectin s-receptor-like serine threonine-

Nbv5tr6247978 protein kinase sd1 -1 AT4G27300 Nbv5tr6217644 heat shock protein 70b Hsp70b heavy metal transport detoxification domain-

Nbv5tr6225223 containing protein AT5G24580 heavy-metal-associated domain-containing

Nbv5tr6217989 protein AT1 G29000 heavy-metal-associated domain-containing

Nbv5tr6199828 protein AT1 G71050

Nbv5tr6363705 heptahelical transmembrane proteinl HHP1

Nbv5tr6203661 hxxxd-type acyl-transferase-like protein AT1 G32910 hydroxyproline-rich glycoprotein family

Nbv5tr6206592 protein AT5G09520

Nbv5tr6223276 laccase 12 LAC 12 lactoylglutathione lyase glyoxalase i-like

Nbv5tr6199430 protein AT5G48480 late embryogenesis abundant hydroxyproline-

Nbv5tr6223877 rich glycoprotein AT1 G54540

Nbv5tr6235965 leucine-rich repeat extensin-like protein 6 AT3G22800

Nbv5tr6199910 lipid transfer-like protein vas AT5G 13900

Nbv5tr6246568 long chain acyl- synthetase 4 LACS4

Nbv5tr6204044 l-tryptophan-pyruvate aminotransferase 1 TAA1

mcu2_arath ame: full=calcium uniporter

Nbv5tr6214962 protein mitochondrial flags: precursor AT2G23790

Nbv5tr6200242 monocopper oxidase-like protein sksl SKS1

Nbv5tr6224169 monothiol glutaredoxin-s3 AT4G 15700

Nbv5tr6217099 myb domain protein 67 MYB67

Nbv5tr6229127 myb domain protein 93 MYB93

Nbv5tr6235613 myo-inositol oxygenase 2 Ml OX2

Nbv5tr6226272 nac domain containing protein 38 NAC038

Nbv5tr6224556 nac domain containing protein 58 NAC058

Nbv5tr6205412 nac domain containing protein 73 NAC073

Nbv5tr6222834 nac domain containing protein 75 NAC075

Nbv5tr6221699 nodulin 21 -like transporter family protein AT1 G09380

Nbv5tr6200284 non-specific lipid-transfer protein 1 LP1

Nbv5tr6201754 non-specific lipid-transfer protein 4 LTP4

Nbv5tr6290585 nucleobase-ascorbate transporter 7 NAT7

Nbv5tr6314641 o-acyltransf erase (wsd1 -like) family protein AT3G49190 Nbv5tr6307449 oleo3_arath ame: full=oleosin kda OLE03

Nbv5tr6214000 o-methyltransferase-like protein AT4G35160

Nbv5tr6369799 pectin lyase-like superfamily protein AT3G59850

Nbv5tr6216642 pectinesterase 1 1 PE1 1

Nbv5tr6206160 peptide nitrate transporter AT5G 19640 peptidoglycan-binding domain-containing

Nbv5tr6217608 protein AT3G52790

Nbv5tr6227940 peroxidase 1 1 AT1 G68850

Nbv5tr6222399 peroxidase 52 AT5G05340

Nbv5tr6235366 peroxidase 55 AT5G14130

Nbv5tr6236806 phenylalanine ammonia-lyase 2 PAL2

pollen ole e 1 allergen and extensin family

Nbv5tr6243328 protein AT3G09925 pollen ole e 1 allergen and extensin family

Nbv5tr6231268 protein AT5G47635

Nbv5tr6394602 probable beta- -xylosyltransferase irx9 IRX9

probable xyloglucan endotransglucosylase

Nbv5tr6200854 hydrolase protein 23 XTR6

protease inhibitor seed storage lipid transfer

Nbv5tr6217812 protein family protein AT3G22600 protease inhibitor seed storage Itp family

Nbv5tr6235917 protein AT1 G05450

Nbv5tr6203465 protein deformed root hairs 4 BST1

Nbv5tr6246004 protein eceriferum 3 CER3

Nbv5tr6207869 protein embryo sac development arrest 4 EDA4

Nbv5tr6236780 protein exordium EXO

Nbv5tr6228779 protein oxidative stress 3 OXS3

Nbv5tr6232591 protein pro-glu-leu AT5G09530

Nbv5tr6212895 protein root primordium defective 1 RPD1

Nbv5tr6215888 protein transmembrane nine 7 TMN7

Nbv5tr6223226 protein trichome birefringence-like 19 TBL19

Nbv5tr6202866 protein trichome birefringence-like 33 TBL33 regulator of vps4 activity in the mvb pathway

Nbv5tr6216437 protein AT2G 14830

Nbv5tr6203790 remorin family protein AT5G23750

Nbv5tr6217669 ring u-box superfamily protein AT5G41400 Nbv5tr6397663 ring-h2 finger protein atl8 ATL8

Nbv5tr6237939 rop-interactive crib motif-containing protein 4 RIC4

serinc-domain containing serine and

Nbv5tr6226886 sphingolipid biosynthesis protein AT2G33205

Nbv5tr6202392 sugar transporter erd6-like 16 AT5G 18840

Nbv5tr6268371 sulfate transporter SULTR1

swim zinc finger family protein mitogen- activated protein kinase kinase kinase -

Nbv5tr6232134 related AT5G1 1620

Nbv5tr6218132 transcription factor rax3 MYB84

Nbv5tr6232053 uclacyanin 3 UCC3

udp-glucuronate:xylan alpha-

Nbv5tr6223206 glucuronosyltransferase 2 PGSIP3

weak similarity to elegans transposase

Nbv5tr6232339 (tr:g 1 125840) AT5G27020

Nbv5tr6264015 wound-responsive protein AT4G 10265

Nbv5tr6222913 zinc finger-related protein AT1 G68360

[0028] The suberin production was assessed using delipidized extracts, which were then depolymerized using any type of with ester-breaking chemical reactions, such as:

[0029] Methanolysis with NaOMe catalyst (Graca J., Pereira H., Phytochem.

Anal., 2000, 11, 45-51 ) can be employed.

[0030] Otherwise, alkaline hydrolysis with KOH in water/ethanol (Sousa A. F., et a/., ChemSusChem, 2008, 1, 1020-1025) can be used.

[0031] Alternatively, a solution of trifluoroboron in methanol can be employed (Franke R. et al., Phytochem., 2005, 66, 2643-2658).

[0032] Alternatively yet, sulfuric acid in methanol can be used (Vishwanath S. J. ef a/., Plant Physiol., 2013, 163, 1 1 18-1 132).

[0033] Less aggressive techniques than alkaline hydrolysis like base and acid- catalyzed methanolysis, and hydrogenolysis, are preferred for analytical convenience (Kolattukudy, Advances in Biochemical Engineering/Biotechno., 2001 , 71, 1 -49).

[0034] The composition of the resulting depolymerized fraction is then analysed using a GC-MS analysis. [0035] Example 1 :

[0036] The stable expression of a MYB93-like transcription factor or an

orthologous gene of MYB93 transcription factor in plant cells was also performed in accordance with the following experimental protocol:

a. The MDP0000320772 contig (SEQ-ID NO: 1 ) is isolated from a cDNA library made from RNA extracted from Malus x domestica cv. "Cox Orange Pippin".

b. The following primers are used: Forward(F) 5'- CACCATGGGAAGATCTCCTT-3',

Reverse(R) 5'-AGCAATCTCATTGGAAAAAGG-3' using the Q5 High Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instructions (Tm=60°C).

c. The PCR product is purified using the QIAquick PCR purification Kit (QIAGEN, Leusden, The Netherlands), cloned into the pGEM-T easy vector with a 3: 1 (insert/vector ratio) and transformed into the E. coli JM109 competent cells following the manufacturer's guidelines (PROMEGA, Fitchburg, Wl, USA).

d. Transformed cells are selected on Xgal/IPTG plate. Ten positive colonies are validated by PCR (Tm=55°C) using the M13 primers (F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3',

R: 5'-TCACACAGGAAACAGCTATGAC-3'), sequence using a 3500 Genetic Analyzer using the BigDye terminator v3.1 kit (Thermo Fisher, Waltham, MA, USA) and blast against the Malus x domestica genome v1.

e. Plasmids are extracted using the QIAprep Spin Miniprep Kit (QIAGEN, Leusden, The Netherlands).

f. The MDP00003207772 fragment (SEQ-ID NO: 1) was then inserted in the pENTR/D TOPO vector. The recombination steps are performed following the LR clonase cloning protocol (Invitrogen) using the pENTR/D TOPO::MDP0000320772 and PMDC7 vectors (Curtis and Grossniklauss, 2003).

g. Plant cells are cultured in Murashige and Skoog medium (MS medium). Cells in suspension are subcultured every seventh day (1 ml of cells into 30 ml of liquid media). h. Agrobacterium tumefaciens GV3101 -pMP90 strain (PMDC7::MDP0000320772) is grown in a 50 ml_ LB liquid medium supplemented with gentamycin (30 mg/L), rifampicin (10 mg/L), kanamycin (50 mg/L) and acetosyringone (30 mg/L).

i. The 100 mL baffled base Erlenmeyer flask is agitated at 130 rpm/28°C for 48 h. Culture is then centrifuged at 4000 rpm for 10 min, re-suspended into a 50 mM MES (pH 5.6), 10mM MgSO ₄ infiltration buffer supplemented with 150 mg/L acetosyringone.

j. After a 2 hours of incubation, PMDC7::MDP0000320772 A. tumefaciens culture is adjusted at OD600=2

k. 5 mL plant cell culture were applied in a 100 mm Petri dish

I. Add 5 [iL acetosyringone to plant cell culture (stock is 150 mg/mL in

DMSO), swirl gently to mix

m. Add the Agrobacterium tumefaciens strain to the plate. Use different amount of bacteria i.e. 25 μί and 100 μί

n. Cover plate with parafilm and incubate two days at 25°C

o. Transfer cells in 15 mL centrifuge tube

p. Let the cells settle in the centrifuge tube and remove the supernatant. Add 10ml MS medium to the cell and mix gently.

q. Repeat step p two more times using MS medium

r. Repeat step p with MS medium with 500 μg/mL carbenicilin s. Put between 1 -5 ml of washed cells onto a petri dish with MS supplemented with BASTA (20 μg/mL) and carbenicilin (500 μg/mL) t. After 14 days BASTA under light exposure, resistant transformant calli should appear green whereas non transformant calli should appear bleached or yellow. Transfer micro-calli on a new selective plate (MS supplemented with BASTA (20 μg/mL) and carbenicilin (500 Mg/mL))

u. Allow calli to grow for 7 to 10 days

v. Repeat steps t-u at least 3 times

w. Transfer one callus in a 1 L baffled Flask filled with culture medium:

4.3 g Murashige and Skoog salt (M0221 , Duchefa), 1 mg thiamine hydrochloride, 100mg myo-inositol, 181 μί 2,4- Dichlorophenoxyacetic acid (5mM), 30g sucrose adjusted at pH=5.8.

Shake vigorously for 2 min.

x. Incubate at 22°C for 10 days with 140 rpm agitation

y. Add 17- -oestradiol at 10μΜ final

z. Incubate at 22°C for 10 days with 140 rpm agitation

aa. Centrifuge the culture at 13000g/10min

bb. Collect both pellet and supernatant

cc. Extract the lipid fraction and suberin fraction

[0037] Example 2

[0038] The stable expression of a MYB93-like transcription factor or an orthologous gene of MYB93 transcription factor in plant cells was performed in accordance with the following experimental protocol:

a. The MDP0000320772 contig (SEQ-ID NO: 1 ) is isolated from a cDNA library made from RNA extracted from Malus x domestica cv. "Cox Orange Pippin".

b. The following primers are used: Forward(F) 5'- CACCATGGGAAGATCTCCTT-3',

Reverse(R) 5'-AGCAATCTCATTGGAAAAAGG-3' using the Q5 High Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instructions (Tm=60°C).

c. The PCR product is purified using the QIAquick PCR purification Kit (QIAGEN, Leusden, The Netherlands), cloned into the pGEM-T easy vector with a 3:1 (insert/vector ratio) and transformed into the E. coli JM109 competent cells following the manufacturer's guidelines (PROMEGA, Fitchburg, Wl, USA).

d. Transformed cells are selected on Xgal/IPTG plate. Ten positive colonies are validated by PCR (Tm=55°C) using the M13 primers (F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3',

R: 5'-TCACACAGGAAACAGCTATGAC-3'), sequence using a 3500 Genetic Analyzer using the BigDye terminator v3.1 kit (Thermo Fisher, Waltham, MA, USA) and blast against the Malus x domestica genome v1.

e. Plasmids are extracted using the QIAprep Spin Miniprep Kit (QIAGEN, Leusden, The Netherlands). f. The following steps are performed following the LR clonase cloning protocol (Invitrogen) with the pENTR/D TOPO and pEarleyGate103 (p103) vectors (Earley et al. 2006).

g. Plant cells are cultured in Murashige and Skoog medium (MS medium). Cells in suspension are subcultured every seventh day (1 ml of cells into 30 ml of liquid media).

h. Agrobacterium tumefaciens GV3101 -pMP90 strain (p103::MDP0000320772) is grown in a 50 ml_ LB liquid medium supplemented with gentamycin (30 mg/L), rifampicin (10 mg/L), kanamycin (50 mg/L) and acetosyringone (30 mg/L).

i. The 100 mL baffled base Erlenmeyer flask is agitated at 130 rpm/28°C for 48 h. Culture is then centrifuged at 4000 rpm for 10 min, re-suspended into a 50 mM MES (pH 5.6), 10mM MgSO ₄ infiltration buffer supplemented with 150 mg/L acetosyringone.

j. After a 2 hours of incubation, p103::MDP0000320772 A. tumefaciens culture is adjusted at OD600=2

k. 5 mL plant cell culture were applied in a 100 mm Petri dish

I. Add 5 [iL acetosyringone to plant cell culture (stock is 150 mg/mL in

DMSO), swirl gently to mix

m. Add the Agrobacterium tumefaciens strain to the plate. Use different amount of bacteria i.e. 25 μί and 100 μί

n. Cover plate with parafilm and incubate two days at 25°C

o. Transfer cells in 15 mL centrifuge tube

p. Let the cells settle in the centrifuge tube and remove the supernatant q. Add 10ml MS medium to the cell and mix gently.

r. Repeat steps p-q two more times using MS medium

s. Repeat steps p-q with MS medium with 500 μg/mL carbenicilin t. Put between 1 -5 ml of washed cells onto a petri dish with MS supplemented with BASTA (20 μg/mL) and carbenicilin (500 μg/mL) u. After 14 days BASTA under light exposure, resistant transformant calli should appear green whereas non transformant calli should appear bleached or yellow

v. Transfer micro-calli on a new selective plate (MS supplemented with

BASTA (20 μg/mL) and carbenicilin (500 Mg/mL)) Allow calli to grow for 7 to 10 days Repeat steps v-w at least 3 times