Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
1999-12-22
2002-05-21
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C800S298000, C800S300000, C435S469000, C435S418000, C435S430100, C435S320100
Reexamination Certificate
active
06392125
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for producing the stable transformants of coffee plants.
2. Description of Related Art
Coffee is a commercially important woody shrub planted in a large scale for harvesting its beans. Among more than 80 species, the most economically important are
Coffee arabica
(2n-44) and
C. canephora
(2n-22). In
C. arabica
, genetic diversity is limited by conventional breeding because of its self-pollination characteristic, and the plants are highly sensitive to pests and diseases.
C. canephora
, used for instant coffee powder products, is a cross-pollinated specie but has low production quality. Conventional breeding of coffee is difficult because of the long duration of cultivation to set seeds. Molecular breeding, therefore, is a desirable technique for the genetic improvement of coffee species, although production of transgenic coffee plants via gene transformation has generally been considered problematic.
Plant regeneration via in vitro tissue culture is a basic system for achieving genetic transformation, and there have been many reports involving somatic embryogenesis in coffee plants (Staritsky, 1970; Hatanaka et al. 1991; Menéndez-Yuffá and García, 1996). However, data for genetic transformation of coffee are limited. Barton et al. (1991) obtained transformants from electroporated protoplasts of
C. arabica
, but the cultured protoplasts did not develop into whole plants. Spiral et al. (1993) reported the transformation of coffee (
C. canephora
) by co-cultivation of Agrobacterium rhizogenes with microcut-somatic embryos. However, the efficiency of transformation was very low. Van Boxtel et al. (1995) reported only transient expression of GUS genes on the surfaces of coffee leaf tissues following biolistic delivery.
SUMMARY OF THE INVENTION
Agrobacterium tumefaciens-mediated transformation is considered to be best for plant transformation because of the availability of vectors. Despite such advantage, no report has been presented of successful coffee transformation using Agrobacterium tumefaciens strains, except for GUS positive transgenic callus induction at a low frequency reported from Ocampo and Manzanera (1991).
This invention provides the successful genetic transformation of
Coffea canephora
using Agrobacterium tumefaciens EHA101 harboring pIG121-Hm from embryogenic calli.
Embryogenic calli were induced from leaf explants of
Coffea canephora
on McCown's woody plant medium (WPM) supplemented with 5 &mgr;M N
6
-[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing &bgr;-glucuronidase (GUS)-, hygromycin phosphotransferase (HPT)- and neomycin phosphotransferase II (NPT II) genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (5, 50, 100 mg/l). The embryogenic calli surviving on a medium containing 100 mg/l hygromycin showed a strong GUS positive reaction with X-gluc solution. Somatic embryos were formed and germinated from these putative transgenic calli on WPM medium with 5 &mgr;M 2-iP. Regenerated small plantlets with shoots and roots were transferred to a medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction (PCR).
Moreover, the inventors have succeeded in production of a transgenic plant of
Coffea arabica
, wherein phosphinothricin acetyl transferase (BAR) gene was incorporated to render resistance against herbicide. Commercially,
Coffea arabica
is more valuable than
Coffea canephora
. Embryogenic calli derived from
Coffea arabica
were induced from leaf explants of coffee on Murashige and Skoog (MS) medium supplemented with 10 &mgr;M N
6
-[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pSMBuba, containing herbicide resistant BAR gene and hygromycin phosphotransferase (HPT) gene. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (25, 50 mg/l). The embryogenic calli maintained on MS medium with 50 mg/l hygromycin and 10 &mgr;M 2-iP. Prolonged culture of embryogenic callus induced somatic embryos. Germination of somatic embryos strongly enhanced by GA
3
treatment and developed into transgenic plantlets after 2 months of culture. Transgenic embryogenic callus, somatic embryos and small plantlets were tolerant to 2 mg/l Bialaphos. Whereas non-transformed ones were dead after 1 month. Prescence of HPT and BAR genes in those transgenic plantlets was confirmed by the genomic PCR and Northern assays.
This invention provides a method to incorporate an exogenous gene using Agrobacterium tumefaciens mediated method. Embryogenic calli were induced from leaf explants of coffee plants. The embryogenic calli thus obtained were infected by Agrobacterium tumefaciens, harboring a plasmid containing an exogenous gene to be incorporated and hygromycin phosphotransferase (HPT) gene. Putative transformed calli were selected using the hygromycin resistance as an indicator. And then somatic embryos were induced from the putative transformed calli. Transformed plantlets can be regenerated from the somatic embryos thus obtained.
Various species of coffee plants can be transformed using the method of this invention. The coffee plant species may preferably be cultivative coffee species such as
Coffea arabica, Coffea canephora, Coffea liberica
and
Coffea dewevrei.
Theoretically, any exogenous gene can be incorporated into coffee plants by the method of this invention. The exogenous genes to be incorporated may preferably be caffeine synthetase gene, herbicide resistance gene such as phosphinothricin acetyl transferase (BAR) gene, insect injury resistance gene such as Bacillus thuringiensis gene, and disease resistance gene such as chitinase gene and glucanase gene.
Other and further objects, features and advantages of the invention will appear more fully from the following descriptions. It is to be understood that, examples mentioned above and description of detailed embodiments are not to be intended to limit the range of this invention.
REFERENCES:
Prescott, A. et al., “Plant Transformation.” 1998, Molecular Biomethods Handbook, pp. 251-269.*
Sugiyama, M. et al., “Transformation of Coffee with Agrobacterium Rhizogenes.”, 1995, Colloq. Sci. Int. Café, vol. 2, pp. 853-859.*
Freire, A. V. et al., “Genetic Transformation of Coffee.”, 1994, HortScience, vol. 29 (5), p. 454.*
Leroy, T. et al., “Introduction of genes of interest in agronomy into the coffee canephora species (Pierre) by transformation with agrobacterium Sp.”, pp. 1-6.*
De Bondt, A. et al., “Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps.” 1994, Plant Cell Reports, vol. 13, pp. 587-593.*
Lulsdorf, M. M. et al., “Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains.” 1991, Plant Cell Reports, vol. 9, pp. 479-483.*
Hatanaka, T. et al., “Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium Tumefaciens-mediated transformation.” 1999, Plant Cell Reports, vol. 19, pp. 106-110.*
Spiral et al.,C.R. Acad. Sci. Paris, 316: 1-6 (1993).
Colloq. Sci. Int. Cafe 17 (1997) p. 439-446.
Antibiot Khimioter 35 [12] (1990) p. 24-26.
Simone S. Moesli Waldhauser et al, “Separation of the N-7-Methyltransferase, the key enzyme in caffeine biosynthesis”, Phytochemistry, vol. 45., No. 7, pp. 1407-1414, 1997.
Kusano Tomonobu
Sano Hiroshi
Fox David T.
Kubelik Anne
Nara Institute of Science and Technology
Oliff & Berridg,e PLC
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