Melon (Cucumis melo)

Summary

The patents granted to Biosem (assignments changed to Groupe Limagrain Holding) in the United States and in Europe claim

  • the production of transgenic diploid melon (C. melo) plants having a DNA molecule introduced by A. tumefaciens;
  • a method to transform cotyledons of C. melo by contacting with A. tumefaciens;
  • the insertion of a sequence conferring resistance to cucumber mosaic virus; and
  • media for inducing shoot formation from transformed cotyledons and for regeneration of transformed plantlets.

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
US 5422259

  • Earliest priority – 11 August 1989
  • Filed – 5 March 1993
  • Granted – 6 June 1995
  • Expected expiry – 6 June 2012
Title – Transgenic plants belonging to the species Cucumis melo

Claim 1
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.
Claim 2
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.

Process for the production of transgenic diploid melon plantlets by transforming cotyledons of C. melohaving a gene of interest introduced via A. tumefaciens. The process includes media components and protocols for inducing formation of transformed shoot buds and formation of plantlets.

Biosem (now owned by Groupe Limagrain Holding)

US 5789656

  • Earliest priority – 11 August 1989
  • Filed – 2 March 1995
  • Granted – 4 August 1998
  • Expected expiry – 6 June 2012
Title – Transgenic plants belonging to the species Cucumis melo

Claim 1
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.
Claim 2
Transgenic plant tissue having a diploid phenotype belonging to the species Cucumis melocomprising at least one DNA sequence that confers resistance to cucumber mosaic virus.
Claim 9
Transgenic plants having a diploid phenotype and belonging to the species Cucumis melocomprising at least one DNA sequence that confers resistance to Cucumber mosaic virus.
Claim 15
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.

Granted US 5789656 is a divisional of now granted US 5422259.

Process for the production of transgenic diploid melon plantlets by transforming cotyledons of C. melohaving a gene of interest introduced via A. tumefaciens. Transgenic diploid melon having the gene of interest, i.e. gene for cucumber mosaic virus, are also claimed.

US 6198022

  • Earliest priority – 11 August 1989
  • Filed – 3 August 1998
  • Granted – 6 March 2001
  • Expected expiry – 6 June 2012
Title – Transgenic plants belonging to the species Cucumis melo

Claim 1
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).

Claim 5
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.

Granted US 6198022 is a divisional of now granted US 5789656 (see above), which is a divisional of now granted US 5422259 (see above).

The process described above applied to the production of cucumber mosaic virus-resistant plants by expression of the viral capsid protein (this is a divisional application of the patent listed immediately above).

EP 412912 B1

  • Earliest priority – 11 August 1989
  • Filed – 9 August 1990
  • Granted – 16 March 1994
  • Expected expiry – 9 August 2010
Title – Transgenic plants of the species Cucumis melo

Claim 1Process 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
Claim 15Cell 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.
Claim 18
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.

Designated contracting States at the time of grant are: Belgium (patent lapsed as reported by INPADOC), Germany (patent lapsed as reported by INPADOC), Spain, France (patent lapsed as reported by INPADOC), United Kingdom (patent lapsed as reported by INPADOC), Greece, Italy, Luxembourg (patent lapsed as reported by INPADOC), Netherlands

The process for the production of transgenic diploid melon plantlets is very similar to the process claimed in the related United States patents. Media components for shoot buds induction and plantlet development are also part of the claims.

Remarks
  1. Related patent in Japan (JP 3174048 B2) was granted on 11 June 2001. A divisional application of now granted JP 3174048 was filed (JP H11/320981), which was rejected on 13 November 2002.
  2. Other related patent documents include Israel (IL 95334) and Portugal (PT 94967).

Note: Patent information on this page was last updated on 21 March 2006.