Regeneration of somatic embryos from plant tissues

• US 6,071,746 A
• Filed: 02/02/1998
• Issued: 06/06/2000
• Est. Priority Date: 02/02/1998
• Status: Expired due to Term

• Current Assignee: Agriculture And Agri-Food Canada
• Original Assignee: Agriculture And Agri-Food Canada
• Inventors: Douglass, L. Katheryn, Seabrook, Jane
• Primary Examiner(s): Fox, David T.
• Assistant Examiner(s): Grunberg, Anne Marie
• Application Number:
  US09/017,648
• Time in Patent Office: 855 Days
• Field of Search: 435/420, 435/429, 435/430, 435/430.1, 435/431, 800/265, 800/268, 800/317.2
• US Class Current: 435/429
• CPC Class Codes: A01H 4/002 Culture media for tissue culture
• CPC Class Codes: A01H 4/005 Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

Abstract

This invention is directed to a method for producing somatic embryos on plant tissue. Furthermore, somatic embryos may be produced using explants obtained from a broad range of plant species, and using either juvenile or mature tissues. The method involves obtaining a stock tissue culture plantlet by exposing the plant tissue to a medium comprising salts, vitamins and an energy source; preparing an explant from the stock tissue culture plantlet; transferring the explant to a proliferation medium comprising salts, vitamins, an energy source and at least one growth regulator for a period of time sufficient to produce a callused explant; and transferring the callused explant to a medium comprising salts, vitamins, an energy source and at least two growth regulators for a period of time sufficient to produce somatic embryos. Following this method somatic embryos are produce in significantly less time that observed using other somatic embryogenesis protocols. Plants produced from the somatic embryos are easily prepared. Since somatic embryos can be obtained from mature tissues, specific plants of agronomic importance comprising desirable traits, may now be clonally propagated. Furthermore, somatic embryos produced following the method of this invention may be used for the preparation of synthetic seed.

Claims

1. A method for producing somatic embryos on potato plant tissue obtained from a non-juvenile plant comprising:

i) obtaining a stock tissue culture plantlet by exposing potato plant tissue obtained from a non-juvenile plant to a growth regulator-free medium comprising salts, vitamins and an energy source;

ii) preparing an explant from the stock tissue culture plantlet;

iii) transferring the explant to a proliferation medium comprising salts, vitamins, an energy source and at least one growth regulator selected from the group consisting of a cytokinin or a cytokinin-like compound; and

Indole acetic acid combined with said cytokinin or cytokinin-like compound, for about 3 days to less than 4 weeks, sufficient to produce a callused explant; and

iv) transferring the callused explant to a medium comprising salts, vitamins, an energy source and at least two growth regulators comprising at least one auxin and at least one cytokinin or cytokinin-like compound for a period of time sufficient to produce somatic embryos.

2. The method of claim 1 wherein, the stock tissue culture plantlet is maintained in the medium of step i) from about 2 to about 20 weeks.

3. The method of claim 2 wherein, the period of time in step iv) is from about 2 to about 6 weeks.

4. The method of claim 1 wherein the cytokinin or cytokinin-like compound of the proliferation medium of step iii) is selected from the group consisting of Thidiazuron and BAP.

5. The method of claim 1, wherein the growth regulators of the medium of step iv) are selected from the group consisting of Zeatin and Indole acetic acid;

and a combination of Zeatin, Indole acetic acid and Gibberellic acid.

6. The method of claim 1 wherein the explant is selected from the group consisting of stem internode, root, leaf, and microtuber tissue.

7. The method of claim 1 wherein the somatic embryo obtained from step iv) is grown into a plant.

8. The method of claim 1, wherein the stock tissue culture plantlets are maintained on a Medium #1 comprising:

space="preserve" listing-type="tabular">______________________________________ Murashige and Skoog salts, full strength sucrose 30 g/l myo-inositol 100 g/l vitamins Nicotinic acid 0.5 mg/l Pantothenic acid 2.5 mg/l Pyridoxine.HCl 1.0 mg/l Thiamine.HCl 0.5 mg/l pH 5.8 agar 8 g/l;

______________________________________

the proliferation medium, medium #2, comprises;

space="preserve" listing-type="tabular">______________________________________ Murashige and Skoog salts, full strength sucrose 30 g/l myo-inositol 100 g/l vitamins Nicotinic acid 0.5 mg/l Pantothenic acid 2.5 mg/l Pyridoxine 1.0 mg/l Thiamine.HCl 0.5 mg/l pH 5.8 agar 6.5 g/l;

______________________________________

and further comprises the constituents selected from the group consisting of;

space="preserve" listing-type="tabular">______________________________________ Medium 2a;

Thidiazuron 0.15 μ

M Indole acetic acid 19 μ

M Medium 2b Thidiazuron 0.15 μ

M [mg/l];

Medium 2c BAP 0.15 μ

M IAA 19 μ

M; and

Medium 2d BAP 0.15 μ

M; and

______________________________________

the medium of step iv), medium #3, comprises;

space="preserve" listing-type="tabular">______________________________________ Murashige and Skoog salts, full strength sucrose 30 g/l myo-inositol 100 mg/l vitamins nicotinic acid 1.0 mg/l thiamine.HCl 0.5 mg/l pantothenic acid 0.5 mg/l pyridoxine 1.0 mg/l Zeatin 12.0 μ

M Indole acetic acid 0.05 μ

M Gibberellic acid 0.55 μ

M pH 5.8 agar. 8 g/l ______________________________________

9. A method of maintaining a potato plant comprising a desired set of characteristics comprising:

i) screening a library of potato plants for a desired set of characteristics;

ii) selecting a potato plant having the desired set of characteristics;

iii) preparing a somatic embryo, from tissue obtained from the selected plant of step ii), using the method of claim 1; and

iv) either preserving the somatic embryo, or growing the somatic embryo into a potato plant.