Melon (Cucumis melo)

Process for the production of transgenic plantlets having diploid phenotype from genetically transformed explants, said plantlets belonging to the species Cucumis melo and containing at least one gene introduced by the intermediary of Agrobacterium tumefaciens , comprising the following steps: A) inducing genetically transformed shoot buds from cotyledons of Cucumis melo in a shoot bud induction medium without forming calli, wherein the cotyledons are obtained from embryos which have germinated from 0 to 4 days before being contacted with A. tumefaciens, wherein the induction medium comprises about 440 to about 2,200 mg/L of calcium chloride calculated as CaCl₂R₂H₂ O, and about 0.3 to about 1.13 mg/L 6-benzyl aminopurine (BAP); and
B) forming genetically transformed plantlets from genetically transformed shoot buds, wherein the step of forming comprises: (i) culturing the genetically transformed shoot buds in a medium having 6-benzyl aminopurine (BAP) until the shoot buds have reached a height of at least 3 mm; and
(ii) transferring and incubating the shoot buds in a suitable macro-element plant cell culture medium sufficiently to form the genetically transformed plantlets.

Process for production of transgenic plantlets with a diploid phenotype, from genetically transformed explants, said plantlets belonging to the species Cucumis melo and containing at least one gene introduced by the intermediary of A. tumefaciens comprising the following steps: A) inducing genetically transformed shoot buds from cotyledons of Cucumis melo in a shoot bud induction medium without forming calli, wherein the cotyledons are obtained from embryos which have germinated from 0 to 4 days before being contacted with A. tumefaciens, and wherein the induction medium comprises about 440 to about 2,200 mg/L of calcium chloride calculated as CaCl₂R₂H₂ O, about 0.3 to about 1.13 mg/L of 6-benzyl aminopurine (BAP), and about 0 to about 1.3 mg/L indole-3-acetic acid (IAA); and
B) forming genetically transformed plantlets from the genetically transformed shoot buds, wherein the step of forming comprises: (i) culturing the shoot buds in a medium having 6-benzyl aminopurine (BAP) until the shoot buds have reached a height of at least 3 mm; and
(ii) transferring and incubating the shoot buds in a suitable macro-elements plant cell culture medium comprising:

• KH₂PO₄ from about 50 to about 100 mg/L;
• MgSO₄from about 75 to about 300 mg/L;
• CaCl₂R₂H₂O from about 500 to about 2500 mg/L;
• KNO₃ from about 750 to about 1200 mg/L; and
• NH₄NO₃ from about 150 to about 200 mg/L sufficiently to form the genetically transformed plantlets.

Transgenic plants having a diploid phenotype belonging to the species Cucumis melo comprising at least one DNA sequence introduced by the intermediary of Agrobacterium tumefaciens.

Transgenic plant tissue having a diploid phenotype belonging to the species Cucumis melo comprising at least one DNA sequence that confers resistance to cucumber mosaic virus.

Transgenic plants having a diploid phenotype and belonging to the species Cucumis melo comprising at least one DNA sequence that confers resistance to Cucumber mosaic virus.

Transgenic plants having diploid phenotype, belonging to the species Cucumis melo, and containing at least one gene introduced by the intermediary of Agrobacterium tumefaciens, wherein the plant tissue is produced from genetically transformed explants by a process comprising the following step: A) inducing genetically transformed shoot buds from cotyledons of Cucumis melo in a shoot bud induction medium without forming calli, wherein the cotyledons are obtained from embryos which have germinated from 0 to 4 days before being contacted with A. tumefaciens, wherein the induction medium comprises about 440 to about 2,200 mg/L of calcium chloride calculated as CaCl₂R₂H₂ O, and about 0.3 to about 1.3 mg/L 6-benzyl aminopurine (BAP); and
B) forming genetically transformed plantlets from genetically transformed shoot buds, wherein the step of forming comprises: (i) culturing the genetically transformed shoot buds in a medium having 6-benzyl aminopurine (BAP) until the shoot buds have reached a height of at least 3 mm; and
(ii) transferring and incubating the shoot buds in a suitable macro-element plant cell culture medium sufficiently to form the genetically transformed plantlets.

A process for the production of transgenic plants resistant to cucumber mosaic virus, said plants belonging to the species Cucumis melo, said process comprising the following steps:

i) introduction, via Agrobacterium tumefaciens, of a gene coding for the capsid protein of the cucumber mosaic virus, into explants of plants belonging to the species Cucumis melo, said explants being cotyledons of embryos isolated from seeds, the said cotyledons having germinated for 0 to 4 days;
ii) induction of genetically transformed shoot buds from transformed explants obtained in step (i);
iii) development of transgenic plantlets from genetically transformed shoot buds obtained in step (ii);
iv) development of transgenic plants from the transgenic plantlets obtained in step (iii).

An isolated nucleotide sequence coding for the capsid protein of the cucumber mosaic virus, comprising the coding region of the following sequence (SEQ ID:1):

gttattgtct actgactata tagagagtgt ttgtgctgtg ttttctcttt tgtgtcgtag
 60
aattgagtcg agtc atg gac aaa tct gaa tca acc agt gct ggt cgt aac
 110
                Met Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn
                  1               5                  10
cgt cga cgt cgt ccg cgt cgt ggt tcc cgc tcc gcc ccc tcc tcc gcg
 158
Arg Arg Arg Arg Pro Arg Arg Gly Ser Arg Ser Ala Pro Ser Ser Ala
         15                  20                  25
gat gct aac ttt aga gtc ttg tcg cag cag ctt tcg cga ctt aat aag
 206
Asp Ala Asn Phe Arg Val Leu Ser Gln Gln Leu Ser Arg Leu Asn Lys
    30                  35                  40
acg tta gca gct ggt cgt cca act att aac cac cca acc ttt gta ggg
 254
Thr Leu Ala Ala Gly Arg Pro Thr Ile Asn His Pro Thr Phe Val Gly
 45                  50                  55                  60
agt gaa cgc tgt aga cct ggg tac acg ttc aca tct att acc cta aag
 302
Ser Glu Arg Cys Arg Pro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys
                 65                  70                  75
cca cca aaa ata gac cgt ggg tct tat tac ggt aaa agg ttg tta cta
 350
Pro Pro Lys Ile Asp Arg Gly Ser Tyr Tyr Gly Lya Arg Leu Leu Leu
             80                  85                  90
cct gat tca gtc acg gaa tat gat aag aag ctt gtt tcg cgc att caa
 398
Pro Asp Ser Val Thr Glu Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln
         95                 100                 105
att cga gtt aat cct ttg ccg aaa ttt gat tct acc gtg tgg gtg aca
 446
Ile Arg Val Asn Pro Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr
    110                 115                 120
gtc cgt aaa gtt cct gcc tcc tcg gac tta tcc gtt gcc gcc atc tct
 494
Val Arg Lys Val Pro Ala Ser Ser Asp Leu Ser Val Ala Ala Ile Ser
125                 130                 135                 140
gct atg ttc gcg gac gga gcc tca ccg gta ctg gtt tat cag tat gcc
 542
Ala Met Phe Ala Asp Gly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala
                145                 150                 155
gca tct gga gtc caa gcc aac aac aaa ctg ttg tat gat ctt tcg gcg
 590
Ala Ser Gly Val Gln Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala
                160                 165                 170
atg cgc gct gat ata ggt gac atg aga aag tac gcc gtc ctc gtg tat
 638
Met Arg Ala Asp Ile Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr
        175                 180                 185
tca aaa gac gat gcg ctc gag acg gac gag cta gta ctt cat gtt gac
 686
Ser Lys Asp Asp Ala Leu Glu Thr Asp Glu Leu Val Leu His Val Asp
    190                 195                 200
atc gag cac caa cgc att ccc aca tct gga gtg ctc cca gtc
 728
Ile Glu His Gln Arg Ile Pro Thr Ser Gly Val Leu Pro Val
205                 210                 215
tgattccgtg ttcccagaat cctccctccg atctctgtgg cgggagctga gttggcagtt
 788
ctgctataaa ctgtctgaag tcactaaacg ttttttacgg tgaacgggtt gtccatccag
 848
cttacggcta aaatggtcag tcgtggagaa atccacgcca gcagatttac aaatctctga
 908
ggcgcctttg aaaccatctc ctaggtttct tcggaaggac ttcggtccgt gtacctctag
 968
cacaacgt
 976.

Claim 6

An isolated nucleotide sequence coding for the capsid protein of the cucumber mosaic virus, comprising the coding region of the following sequence (SEQ ID:3):
agagagtgtg tgtgctgtgt tttctctttt gtgtcgtaga attgagtcga gtc atg
 56
                                                                                     Met
                                                             1
gac aaa tct gaa tca acc agt gct ggt cgt aac cgt cga cgt cgt ccg
 104
Asp Lys Ser Glu Ser Thr Ser Ala Gly Arg Asn Arg Arg Arg Arg Pro
              5                  10                  15
cgt cgt ggt tcc cgc tcc gcc ccc tcc tcc gcg gat gct aac ttt aga
 152
Arg Arg Gly Ser Arg Ser Ala Pro Ser Ser Ala Asp Ala Asn Phe Arg
         20                  25                  30
gtc ttg tcg cag cag ctt tcg cga ctt aat aag acg tta gca gct ggt
 200
Val Leu Ser Gln Gln Leu Ser Arg Leu Asn Lys Thr Leu Ala Ala Gly
     35                  40                  45
cgt cca act att aac cac cca acc ttt gta ggg agt gaa cgc tgt aga
 248
Arg Pro Thr Ile Asn His Pro Thr Phe Val Gly Ser Glu Arg Cys Arg
 50                  55                  60                  65
cct ggg tac acg ttc aca tct att acc cta aag cca cca aaa ata gac
 296
Pro Gly Tyr Thr Phe Thr Ser Ile Thr Leu Lys Pro Pro Lys Ile Asp
                 70                  75                  80
cgt ggg tct tat tac ggt aaa agg ttg tta cta cct gat tca gtc acg
 344
Arg Gly Ser Tyr Tyr Gly Lys Arg Leu Leu Leu Pro Asp Ser Val Thr
             85                  90                  95
gaa tat gat aag aag ctt gtt tcg cgc att caa att cga gtt aat cct
 392
Glu Tyr Asp Lys Lys Leu Val Ser Arg Ile Gln Ile Arg Val Asn Pro
        100                 105                 110
ttg ccg aaa ttt gat tct acc gtg tgg gtg aca gtc cgt aaa gtt cct
 440
Leu Pro Lys Phe Asp Ser Thr Val Trp Val Thr Val Arg Lys Val Pro
    115                 120                 125
gcc tcc tcg gac tta tcc gtt gcc gcc atc tct gct atg ttc gcg gac
 488
Ala Ser Ser Asp Leu Ser Val Ala Ala Ile Ser Ala Met Phe Ala Asp
130                 135                 140                 145
gga gcc tca ccg gta ctg gtt tat cag tat gcc gca tct gga gtc caa
 536
Gly Ala Ser Pro Val Leu Val Tyr Gln Tyr Ala Ala Ser Gly Val Gln
                 150                 155                 160
gcc aac aac aaa ctg ttg tat gat ctt tcg gcg atg cgc gct gat ata
 584
Ala Asn Asn Lys Leu Leu Tyr Asp Leu Ser Ala Met Arg Ala Asp Ile
            165                 170                 175
ggt gac atg aga aag tac gcc gtc ctc gtg tat tca aaa gac gat gcg
 632
Gly Asp Met Arg Lys Tyr Ala Val Leu Val Tyr Ser Lys Asp Asp Ala
        180                 185                 190
cta gag acg gac gag cta gta ctt cat gtt gac atc gag cac caa cgc
 680
Leu Glu Thr Asp Glu Leu Val Leu His Val Asp Ile Glu His Gln Arg
    195                 200                 205
att ccc acg tct gga gtg ctc cca gtc tgattcgtgt tcccagaatc
 727
Ile Pro Thr Ser Gly Val Leu Pro Val
210                 215
ctccctccga tctctgtggc gggagctgag ttggcagttc tgctataaac tgtctgaagt
 787
cactaaacgt ttttacggtg aacgggttgt ccatccagct tacggctaaa atggtcagtc
 847
gtggagaaat ccacgccagt agatttacaa atctctgagg cgcctttgaa accatctcct
 907
aggtttcttc ggaaggactt cggtccgtgt acctctagca caacgtgcta gtttcagggt
 967
acgggtgccc ccccactttc gtgggggcct ccaaaaggag
 1007.

Process for production of transgenic, phenotypically normal plantlets from genetically transformed explants, said plantlets belonging to the species Cucumis melo and containing at least one gene, which has been introduced through Agrobacterium tumefaciens, characterized by the following steps: A) induction of genetically transformed shoot buds from cotyledons of Cucumis melo which have germinated for 0 to 4 days and, after this period, have been brought into contact with A. tumefaciens, the induction being carried out on an induction medium for genetically transformed shoot buds which comprises all of the minerals, salts and vitamins normally required for the induction of shoot buds from genetically non-transformed explants and containing, amongst the mineral salts, approximately 440 to approximately 2,200 mg/L of calcium chloride calculated as CaCl₂R₂H₂O, and approximately 0.8 to approximately 1.2% of bacto-agar or agar-agar, said induction medium being supplemented with approximately 0.3 to about 1.13 mg/L of 6-benzyl aminopurine (BAP); and approximately 0 to approximately 1.3 mg/L indole-3-acetic acid (IAA);
B) culturing the resulting genetically transformed shoot buds in two successive stages, the first of these culture stages taking place on a plant cell culture medium containing a cytokinin and, more particularly, 6-benzyl aminopurine (BAP), and the second stage, which is carried out when the shoot buds have reached a length of at least 3 mm, taking place on a plant cell culture medium containing, as macroelements:

• KH₂ PO₄ approximately 50 to approximately 100 mg/L
• MgSO₄ approximately 75 to approximately 300 mg/L
• CaCl₂R₂H₂O approximately 500 to approximately 2500 mg/L
• KNO₃ approximately 750 to approximately 1200 mg/L
• NH₄NO₃ approximately 150 to approximately 200 mg/L

Cell culture medium suitable for the development of shoot buds into plantlets in the course of the regeneration of a plant, characterized in that it contains, as macro-elements:

• KH₂ PO₄ approximately 50 to approximately 100 mg/L
• MgSO₄R2H₂O approximately 75 to approximately 300 mg/L
• CaCl₂R₂H₂O approximately 1000 to approximately 2500 mg/L
• KNO₃ approximately 750 to approximately 1200 mg/L
• NH₄NO₃ approximately 150 to approximately 200 mg/L.

Shoot bud induction medium composed of a plant cell culture medium, which comprises all the minerals, salts and vitamins normally required for inducing shoot buds from non-genetically transformed explants and containing, amongst its mineral salts, calcium chloride, and bacto-agar or agar-agar, characterized in that the CaCl₂ content of this medium is 1000 to 2200 mg/L calculated as CaCl₂ R₂H₂O, and the bacto-agar or agar-agar content is 0.8 to 1.2%, said medium being supplemented with 0.3 to about 2.0 mg/L of BAP and 0 to 1.3 mg/L of IAA.