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US 5,312,801 AGranted
Somatic embryogenesis and plant regeneration of cacao
AssigneesDNA Plant Technology Corporation, Hershey Foods Corporation
InventorsMaro R. Sondahl, Zhenghua Chen, Thomas B. Sereduk, Claudia M. Bellato, Si-Jiu Liu, Alvina Bragin
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Abstract
The present invention provides a method for regeneration of somatic embryos from non-zygotic or zygotic tissue. The invention also provides a means for regeneration of cacao plantlets and plants from somatic embryos.
Claims32
A method for the production of a cacao plantlet capable of developing into a mature cacao plant from a non-zygotic somatic tissue of a mature cacao plant, which method comprises:
- (a) providing a somatic embryo derived from non-zygotic somatic tissue;
- (b) culturing the somatic embryo on a differentiation medium comprising effective amounts of at least growth regulator to obtain a differentiated embryo,
- wherein said at least one growth regulator is abscisic acid at a concentration of about 0.01 to 300 .mu.M, or a cytokinin at a concentration of about 0.1 to 300 .mu.M, or a gibberellin at a concentration of about 0.05 to 15 .mu.M, or a combination thereof;
- and
- (c) germinating the differentiated embryo on a germination medium comprising an auxin at a concentration of about 0.05 to 5 .mu.M, a gibberellin at a concentration of about 0.1 to 10 .mu.M, a cytokinin at a concentration of about 0.5 to 30 .mu.M, or a combination thereof to produce a cacao plantlet.
2 more elements
A method for the production of a cacao plantlet capable of developing into a mature cacao plant from a non-zygotic somatic tissue of mature cacao plant, which method comprises:
- (a) providing a somatic embryo derived from non-zygotic somatic tissue;
- (b) culturing the somatic embryo on a differentiation medium comprising a basal medium under dehydration conditions, which dehydration conditions are achieved by including in the medium a sugar or sugar alcohol osmoticum to provide an osmoticum equivalent in the range of about -5.8 bars to -11 bars, to obtain a differentiated embryo;
- and
- (c) germinating the differentiated embryo on a germination medium comprising an auxin at a concentration of about 0.05 to 5 .mu.M, a gibberellin at a concentration of about 0.1 to 10 .mu.M, a cytokinin at a concentration of about 0.5 to 30 .mu.M, or a combination thereof, to produce a cacao plantlet.
1 more element
A method for obtaining a somatic embryo from non-zygotic somatic tissue of a mature cacao plant which comprises:
- (a) culturing said cacao non-zygotic somatic tissue on a primary culture medium comprising effective amounts of at least one growth regulator to obtain an immature somatic embryo, wherein said at least one growth regulator in said primary culture medium is a cytokinin at a concentration of about 1 to 15 .mu.M and an auxin at a concentration of about 0.5 to 75 .mu.M;
- and
- (b) culturing the immature somatic embryo in a regeneration medium which comprises said primary culture medium, to complete the development of the somatic embryo.
A method for producing a cacao plantlet capable of becoming a mature cacao plant from a nonzygotic somatic tissue of a mature cacao plant, which method comprises:
- (a) culturing non-zygotic somatic tissue of a mature cacao plant on a primary culture medium comprising at least one auxin at a concentration of about 2.5 to 50 .mu.M and at least one cytokinin at a concentration of about 0.5 to 5 .mu.M to obtain an immature embryo;
- (b) culturing the immature embryo in an early differentiation medium comprising at least one cytokinin at a concentration of about 0.1 to 10 .mu.M, a gibberellin at a concentration of about 0.05 to 1 .mu.M, and abscisic acid at a concentration of about 0.05 to 2.5 .mu.M, to complete development of a somatic embryo;
- (c) culturing the somatic embryo on a late differentiation medium comprising
- (i) effective amounts of an osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars, or
- (ii) abscisic acid at a concentration of about 0.02 to 50 .mu.M, or
- (iii) a combination of (i) and (ii), or
- (iv) a combination of (iii) and a natural cytokinin at a concentration of about 0.1 to 10 .mu.M or a synthetic cytokinin at a concentration of about 0.1 to 20 .mu.M, and a gibberellin at a concentration of about 0.1 to 10 .mu.M, or
- (v) a combination of (i) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (vi) a combination of (ii) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (vii) a combination of (iii) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (viii) a combination of (iv) and an auxin at a concentration of about 0.5 to 3 .mu.M to produce a differentiated embryo;
- and
- (d) germinating the differentiated embryo on a germination medium comprising
- (i) at least one cytokinin at a concentration of about 0.5 to 15 .mu.M, and at least one auxin at a concentration of about 0.1 to 5 .mu.M, or
- (ii) at least one auxin at a concentration of about 0.1 to 5 .mu.M and a gibberellin at a concentration of about 0.1 to 5 .mu.M, or
- (iii) a combination of (i) and a gibberellin at a concentration of about 0.1 to 5 .mu.M, and abscisic acid at a concentration of about 0.01 to 1 .mu.M to produce a cacao plantlet capable of developing into a mature cacao plant.
13 more elements
A method for producing a mature cacao plant from a non-zygotic somatic tissue of a mature cacao plant, which method comprises:
- (a) culturing non-zygotic somatic tissue of a mature cacao plant on a primary culture medium comprising at least one synthetic auxin at a concentration of about 2.5 to 50 .mu.M and at least one cytokinin at a concentration of about 0.5 to 5 .mu.M to obtain an immature embryo;
- (b) culturing the immature embryo in an early differentiation medium comprising at least one cytokinin at a concentration of about 0.1 to 10 is M, a fibberellin at a concentration of about 0.05 to 1 .mu.M, and abscisic acid at a concentration of about 0.05 to 2.5 .mu.M, to complete development of a somatic embryo;
- (c) culturing the somatic embryo on a late differentiation medium comprising
- (i) effective amounts of an osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars, or
- (ii) abscisic acid at a concentration of about 0.02 to 10 .mu.M, or
- (iii) a combination of (i) and (ii), or
- (iv) a combination of (iii) and a natural cytokinin at a concentration of about 0.1 to 10 .mu.M or a synthetic cytokinin at a concentration of about 0.1 to 20 .mu.M, and a gibberellin at a concentration of about 0.1 to 10 .mu.M, or
- (v) a combination of (i) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (vi) a combination of (ii) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (vii) a combination of (iii) and an auxin at a concentration of about 0.5 to 3 .mu.M, or
- (viii) a combination of (iv) and an auxin at a concentration of about 0.5 to 3 .mu.M to produce a differentiated embryo;
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- (d) grafting the differentiated embryo and obtained in step (c) on to cacao seedling root stock to produce a mature cacao plant.
10 more elements
A method for producing a cacao plantlet capable of developing into a mature cacao plant from a non-zygotic somatic tissue of a mature cacao plant, which method comprises:
- (a) culturing non-zygotic somatic tissue of a mature cacao plant on a primary culture medium comprising at least one auxin at a concentration of about 1.5-45 .mu.M to obtain an immature somatic embryo;
- (b) culturing the immature somatic embryo in a regeneration medium comprising at least one cytokinin at a concentration of about 0.1 to 10 .mu.M to induce completion of development of a primary somatic embryo;
- (c) culturing the primary somatic embryo in said regeneration medium to induce the development of a secondary somatic embryo;
- (d) culturing the secondary somatic embryo of step (c) on an early differentiation medium comprising effective amounts of an osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars, at least one cytokinin at a concentration of about 15 to 100 .mu.M and abscisic acid at a concentration of about 50 to 300 .mu.M to induce differentiation on the secondary somatic embryo;
- (e) culturing the somatic embryo obtained in step (d) on a second differentiation medium comprising effective amounts of an osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars and abscisic acid at a concentration of about 0.02 to 10 .mu.M to obtain a differentiated embryo;
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- (f) germinating the differentiated embryo on a germination medium comprising at least one auxin at a concentration of about 0.1 to 5 .mu.M and a gibberellin at a concentration of about 0.1 to 6 .mu.M to produce a cacao plantlet capable of developing into a mature cacao plant.
4 more elements
A method of obtaining a somatic embryo from non-zygotic somatic tissue of a mature cacao plant which comprises:
- (a) culturing said cacao non-zygotic somatic tissue on a primary culture medium, which primary culture medium is solidified with a gelling agent and comprises a cytokinin at a concentration of about 0.1 to 30 .mu.M to obtain friable embryogenic tissue;
- (b) suspending the friable embryogenic tissue of step (a) in a liquid medium comprising a cytokinin at a concentration of about 0.5 to 15 .mu.M to obtain an immature embryo;
- (c) culturing the suspended immature embryo of step (b) in a regeneration medium comprising effective amounts of a osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars and an auxin at a concentration at about 0.1 to 10 .mu.M to complete the development of a somatic embryo.
A method of obtaining a somatic embryo from non-zygotic somatic tissue of a mature cacao plant which comprises:
- (a) culturing said cacao non-zygotic somatic tissue on a primary culture medium, which primary culture medium is solidified with a gelling agent and comprises a cytokinin at a concentration of about 0.5 to 30 .mu.M or an auxin at a concentration of about 0.2 to 20 .mu.M to obtain friable embryogenic tissue;
- (b) suspending the friable embryogenic tissue of step (a) in a liquid regeneration medium comprising a cytokinin at a concentration of about 0.5 to 30 .mu.M or an auxin at a concentration of about 0.2 to 20 .mu.M to complete the development of a somatic embryo.
A method for the production of a cacao plantlet from a non-zygotic somatic tissue of a parent cacao plant, said plantlet being capable of developing into a mature cacao plant, which method comprises:
- (a) providing a somatic embryo derived from non-zygotic somatic tissue;
- (b) culturing the somatic embryo on a first differentiation medium comprising
- (i) a cytokinin at a concentration of about 0.1 to 10 .mu.M,
- (ii) a gibberellin at a concentration of about 0.05 to 1 .mu.M,
- (iii) abscisic acid at a concentration of about 0.05 to 2.5 .mu.M, and
- (iv) IAA at a concentration of about 0.1 to 1 .mu.M to obtain a first stage differentiated embryo;
- (c) culturing the first stage differentiated embryo in a second differentiation medium comprising abscisic acid at a concentration of about 5-50 .mu.M, to obtain a second stage differentiated embryo;
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- (d) germinating the second stage differentiated embryo on a germination medium comprising an auxin at a concentration of about 0.1 to 5 .mu.M, a fibberellin at a concentration of about 0.05 to 6 .mu.M, a cytokinin at a concentration of about 0.5 to 30 .mu.M, or a combination thereof, thereby producing a cacao plantlet.
6 more elements
A method for the production of cacao plantlet from a non-zygotic somatic tissue of a mature cacao plant, said plantlet being capable of developing into a mature cacao plant, which method comprises:
- (a) providing a somatic embryo derived from non-zygotic somatic tissue;
- (c) culturing the somatic embryo on series of three differentiation media to obtain a differentiated embryo, wherein:
- (i) the first differentiation medium comprises 6-BA at a concentration of about 2.5 .mu.M, zeatin at a concentration of about 2.5 .mu.M, NAA at a concentration of about 1.5 .mu.M, gibberellic acid at a concentration of about 1.5 .mu.M and abscisic acid at a concentration of about 0.02 .mu.M;
- (ii) the second differentiation medium comprises 6-BA at a concentration of about 5.0 .mu.M, zeatin at a concentration of about 2.0 .mu.M, NAA at a concentration of about 1.5 .mu.M, gibberellic acid at a concentration of about 1.5 .mu.M and abscisic acid at a concentration of about 0.02 .mu.M;
- and
- (iii) the third differentiation medium comprises 6-BA at a concentration of about 5.0 .mu.M, zeatin at a concentration of about 2.0 .mu.M, NAA at a concentration of about 1.5 .mu.M, gibberellic acid at a concentration of about 1.5 .mu.M, abscisic acid at a concentration of about 0.02 .mu.M, and charcoal at a concentration of about 5 grams/liter,
- each of said three differentiation media further comprising effective amounts of an osmoticum having an osmotic potential in the range of about -5.8 bars to -11 bars;
- and
- (d) germinating the differentiated embryo on a germination medium comprising an auxin at a concentration of about 1 to 1.5 .mu.M, a gibberellin at a concentration of about 1.5 .mu.M, a cytokinin at a concentration of about 2 to 5 .mu.M, or a combination thereof,
- thereby producing a cacao plantlet.
7 more elements
Dependencies are inferred from the claim text.