CA2322438A1 - Increasing levels of growth regulator and/or water stress during embryo development - Google Patents

Increasing levels of growth regulator and/or water stress during embryo development Download PDF

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Publication number
CA2322438A1
CA2322438A1 CA002322438A CA2322438A CA2322438A1 CA 2322438 A1 CA2322438 A1 CA 2322438A1 CA 002322438 A CA002322438 A CA 002322438A CA 2322438 A CA2322438 A CA 2322438A CA 2322438 A1 CA2322438 A1 CA 2322438A1
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embryos
growth regulator
treatment
water stressing
water
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CA002322438A
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CA2322438C (en
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Stephen M. Attree
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University of Saskatchewan
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University Of Saskatchewan Technologies Inc.
Stephen M. Attree
University Of Saskatchewan
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Priority to CA2322438A priority Critical patent/CA2322438C/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention comprises methods for producing mature desiccated and desiccation-tolerant somatic embryos, particularly conifer embryos. The methods include the application of a growth-promoting hormone such as abscisic acid (ABA) to the immature embryos during development. The embryos are also water stressed during development. The concentration of the ABA undergoes a net increase relative to its initial concentration to reach a peak prior to discontinuing ABA treatment when the embryos have attained a moisture content of between 32 and 55%. The water stressing may remain constant or may undergo a net increase in intensity over the period of time during which it is applied to the embryos. The method may optionally include the further water stressing of the mature embryos to further reduce moisture contents of the embryos preferably to the level at which the embryos are tolerant of freezer storage.

Claims (437)

What is claimed is:
1. A development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including:
(i) nourishment of the embryos by a suitable metabolizable carbon source, and (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that the concentration of the growth regulator is raised from a selected initial concentration to a selected peak concentration value before the completion of the growth regulator treatment.
2. The method of claim 1, wherein the embryos are conifer embryos.
3. The method of either of claims 1 or 2, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
4. The method of any of claims 1 to 3, wherein the water stressing and growth regulator treatments continue after the embryos attain desiccation tolerance.
5. The method of any of claims 1 to 3, wherein the water stressing or growth regulator treatments continues after the embryos attain desiccation tolerance.
6. The method of any of claims 1 to 5, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
7. The method of any cf claims 1 to 6, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
8. The method of claim 2, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
9. The method of any of claims 1 to 8, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
10. The method of any of claims 1 to 9, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
11. The method of any of claims 1 to 10, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
12. The method of any of claims 1, 10, or 11, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
13. The method of claim 12, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
14. The method of any of claims 1 to 13, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
15. The method of any of claims 1 to 14, wherein the initial concentration of the growth regulator at the commencement of the growth regulator treatment is selected to be less than about 40 µM,
16. The method of any of claims 1, 10, or 11, wherein the peak concentration of the growth regulator is selected to be about 30 µM to about 200 µM.
17. The method of claim 16, wherein the peak concentration of the growth regulator is selected to be about 30 µM
to 100 µM.
18. The method of claim 1, wherein the concentration of the growth regulator is raised progressively during at least part of the growth regulator treatment period.
19. The method of either of claims 1 or 18, wherein the concentration of the growth regulator is increased incrementally.
20. The method of claim 19, wherein the incremental increase of the growth regulator is at least about 5%
of the initial concentration of the growth regulator.
21. The method of either of claims 19 or 20, wherein the growth regulator is raised from its initial concentration to its peak concentration in a single increment.
22. The method of either of claims 19 or 20, wherein the growth regulator is raised from its initial concentration. to its peak concentration in a series of increments.
23. The method of claim 18, wherein the concentration of the growth regulator does not increase during the early part of the growth regulator treatment period.
24. The method of claim 18, wherein the concentration of the growth regulator does not increase during the middle part of the growth regulator treatment period.
25. The method of claim 18, wherein the concentration of the growth regulator does not increase during the late part of the growth regulator treatment period.
26. The method of claim 25, wherein there is no net increase or net decrease in the concentration of the growth regulator during the late part of the growth regulator treatment period.
27. The method of claim 25, wherein there is a net decrease of the growth regulator during the late part of the growth regulator treatment period.
28. The method of claim 27, wherein the growth regulator is reduced incrementally.
29. The method of claim 28, wherein the growth regulator is reduced from its peak concentration to its final concentration in a series of increments.
30. The method of claim 28, wherein the growth regulator is reduced from its peak concentration to its final concentration in a single increment.
31. The method of any cf claims 1, 10, or 11, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos' environment.
32. The method of any of claims 1, 10, 11, or 31, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
33. The method of any of claims 1, 10, 11, 31, or 32, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 40%.
34. The method any of claims 1, 10, 11, 31, 32, or 33, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 55%.
35. The method of any of claims 1, 10, 11, or 31, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
36. The method of any of claims 1, 10, 11, or 31, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
37. The method of any of claims 1 to 36, wherein she growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
38. The method of claim 37, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
39. The method of any of claims 1 to 38, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
40. The method of claim 39, wherein the growth regulator is abscisic acid.
41. The method of any of claims 1 to 90, wherein the water stressing creates a water potential of at least about -260 mmol/kg prior to the late cotyledonary stage of development.
42. The method of any of claims 1 to 41, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
43. The method of any of claims 1 to 42, wherein the water stressing is applied by means of a selected suitable environmental effect.
44. The method of any of claims 1 to 42, wherein the water stressing is applied by means of at least one selected suitable water stressing agent.
45. The method of any of claims 1 to 42, wherein the water stressing is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
46. The method of either of claims 44 or 45, wherein the water stressing is applied at least in part by means of at least one gel.
47. The method of either of claims 44 or 45, wherein the water stressing is applied at least in part by means of at least one osmoticum.
48. The method of claim 47, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
49. The method of claim 47, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
50. The method of claim 49, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
51. The method of either of claims 48 and 50, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo for nutrition.
52. The method cf claim 49, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (A).
53. The method of either of claims 49 or 52, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
54. The method of any of claims 49, 52, or 53, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
55. The method of either of claims 43 or 45, wherein the environmental water stressing is applied at least in part by relative humidity of less than 100%.
56. The method of claim 55, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
57. The method of any of claims 1 to 56, wherein the embryos are cultured in a bioreactor.
58. The method of any of claims 1 to 57, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
59. The method of claim 58, wherein the adsorbent means comprises activated charcoal.
60. The method of claim 59, wherein the activated charcoal is pre-saturated with the growth regulator.
61. The method of claim 58, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
62. The method of claim 1, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
63. The method of claim 62, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
64. The method of any of claims 1 to 61, wherein the embryos are in contact with medium.
65. The method of claim 64, wherein the embryos are removed from medium after they have attained a moisture content of about 55%.
66. The method of any of claims 1 to 61, wherein the embryos are supported by nutrient-permeable support means in contact with a medium.
67. The method of claim 66, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 55%.
68. The method of any of claims 1 to 67, comprising the further step of rehydrating the mature embryos in preparation for germination.
69. The method of claim 68, comprising the further step of imbibing the embryos in 0-15°C.
70. The method of claim 68, wherein the rehydrating step occurs following a rest period following termination of water stressing the embryos.
71. The method of any of claims 1 to 70, further comprising germinating the mature embryos to form somatic seedlings.
72. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 1 to 70.
73. A somatic seedling produced in accordance with the method of claim 70.
74. A development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metaboiizable carbon source, and (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that:
(a) the concentration of the growth regulator is raised from a selected initial concentration to a selected peak concentration value before the completion of the growth regulator treatment period, and (b) the intensity of the water stressing is raised from a selected initial intensity to a selected higher intensity before the completion of the water stressing period.
75. The method of claim 74, wherein the embryos are conifer embryos.
76. The method of either of claims 74 or 75, wherein the water stressing and growth regulator treatments continue after the embryos attain desiccation tolerance.
77. The method of either of claims 74 or 75, wherein the water stressing or growth regulator treatments continues after the embryos attain desiccation tolerance.
78. The method of any of claims 74 to 77, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
79. The method of any of claims 74 to 78, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
80. The method of any of claims 74 to 79, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
81. The method of claim 75, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
82. The method of any of claims 74 to 81, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
83. The method of any of claims 74 to 82, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
84. The method of any of claims 74 to 83, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
85. The method of any of claims 74, 83, or 84, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
86. The method of claim 85, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
87. The method of any of claims 79 to 86, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
88. The method of any of claims 74 to 87, wherein the initial concentration of the growth regulator at the commencement of the growth regulator treatment is selected to be less than about 40 µM.
89. The method of any of claims 74, 83, or 84, wherein the peak concentration of .the growth regulator is selected to be about 30 µM to about 200 µM.
90. The method of claim 89, wherein the peak concentration of the growth regulator is selected to be about 30 µM
to 100 µM.
91. The method of claim 74, wherein the concentration of the growth regulator is raised progressively during at least part of the growth regulator treatment period.
92. The method of either of claims 74 or 91, wherein the concentration of the growth regulator is increased incrementally.
93. The method of claim 92, wherein the incremental increase of the growth regulator is at least about 5%
of the initial concentration of the growth regulator.
94. The method of either of claims 92 or 93, wherein the growth regulator is raised from its initial concentration to its peak concentration in a single increment.
95. The method of either of claims 92 or 93, wherein the growth regulator is raised from its initial concentration to its peak concentration in a series of increments.
96. The method of claim 91, wherein the concentration of the growth regulator does not increase during the early part of the growth regulator treatment period.
97. The method of claim 91, wherein the concentration of the growth regulator does not increase during the middle part of the growth regulator treatment period.
98. The method or claim 91, wherein the concentration of the growth regulator does not increase during the late part of the growth regulator treatment period.
99. The method of claim 98, wherein there is no net increase or net decrease in the concentration of the growth regulator during the late part of the growth regulator treatment period.
100. The method of claim 98, wherein there is a net decrease of the growth regulator during the late part of the growth regulator period.
101. The method of claim 100, wherein the growth regulator is reduced incrementally.
102. The method of claim 101, wherein the growth regulator is reduced from its peak concentration to its final concentration in a series of increments.
103. The method of claim 101, wherein the growth regulator is reduced from its peak concentration to its final concentration in a single increment.
104. The method of any of claims 74, 83, or 84, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos' environment.
105. The method of any of claims 74, 83, 84, or 104, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
106. The method of any of claims 74, 83, 84, 109, or 105, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 40%.
107. The method of any of claims 74, 83, 84, 104, 105, or 106, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 55%.
108. The method of any of claims 74, 83, 84, or 104, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
109. The method of any of claims 74, 83, 89, or 104, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
110. The method of any of claims 74 to 109, wherein the growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
111. The method of claim 110, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
112. The method of claim 74, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
113. The method of claim 112, wherein the growth regulator is abscisic acid.
114. The method of any of claims 74 to 113, wherein the initial intensity of the water stressing creates a water potential of at least about -120 mmol/kg.
115. The method of any of claims 74 to 114, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
116. The method of any of claims 74 to 115, wherein the water stressing creates a water potential of at least about -800 mmol/kg prior to the late cotyledonary stage of development.
117. The method of any of claims 74 to 116, wherein the water stressing is applied by means of a selected suitable environmental effect.
118. The method of any of claims 74 to 116, wherein the water stressing is applied by means of at least one selected suitable water stressing agent.
119. The method of any of claims 74 to 116, wherein the water stressing is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
120. The method of either of claims 118 or 119, wherein the water stressing is applied at least in part by means of at least one gel.
121. The method of either of claims 118 or 119, wherein the water stressing is applied at least in part by means of at least one osmoticum.
122. The method of claim 121, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
123. The method of claim 121, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
124. The method of claim 123, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
125. The method of either of claims 122 or 124, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo for nutrition.
126. The method of claim 123, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (.ANG.).
127. The method of either of claims 123 or 126, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
128. The method of any of claims 123, 126 or 127, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
129. The method of either of claims 117 or 119, wherein the environmental water stressing is applied at least in part by relative humidity of less than 100%.
130. The method of claim 129, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
131. The method of claim 74, wherein there is a net increase in the intensity of water stressing of the embryos over the duration of the water stressing treatment period.
132. The method of claim 131, wherein the net increase in the intensity of water stressing of the embryos is effected by changing the value of the water potential from a selected initial value to a selected final value.
133. The method of any of claims 74 to 132, wherein the embryos are cultured in a bioreactor.
134. The method of any of claims 74 to 133, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
135. The method of claim 134, wherein the adsorbent means comprises activated charcoal.
136. The method of claim 135, wherein the activated charcoal is pre-saturated with the growth regulator.
137. The method of claim 134, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
138. The method of claim 74, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
139. The method of claim 138, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
140. The method of any of claims 74 to 137, wherein the embryos are in contact with medium.
141. The method of claim 140, wherein the embryos are removed from medium after they have attained a moisture content of about 55%.
142. The method of any of claims 74 to 137, wherein the embryos are supported by nutrient-permeable support means in contact with a medium.
143. The method of claim 142, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 55%.
144. The method of any of claims 74 to 143, comprising the further step of rehydrating the mature embryos in preparation for germination.
145. The method of claim 144, comprising the further step of imbibing the embryos in 0-15°C.
146. The method of claim 144, wherein the rehydrating step occurs following a rest period following completion of water stressing the embryos.
147. The method of any of claims 74 to 146, further comprising germinating the mature embryos to form somatic seedlings.
148. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 74 to 146.
149. A somatic seedling produced in accordance with the method of claim 147.
150. A method of producing viable mature cotyledonary desiccated somatic embryos, comprising:
(a) a development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metabolizable carbon source, (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that the concentration of the growth regulator is raised from a selected initial concentration to a selected peak concentration value before the completion of the growth regulator treatment period; and (b) further water stressing the mature somatic embryos to reach a severely desiccated state characterised by a moisture content of less than about 30% to 36% following the completion of the growth regulator treatment in (a).
151. The method of claim 150, wherein the embryos are conifer embryos.
152. The method of either of claims 150 or 151, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
153. The method of any of claims 150 to 152, wherein the growth regulator treatment continues after the embryos attain desiccation tolerance.
154. The method of any of claims 150 to 153, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
155. The method of any of claims 150 to 154, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
156. The method of claim 151, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
157. The method of any of claims 150 to 156, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
158. The method of any of claims 150 to 157, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
159. The method of any of claims 150 to 157, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
160. The method of any of claims 150, 158, or 159, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
161. The method of claim 160, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
162. The method of any of claims 150 to 161, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
163. The method of any of claims 150 to 162, wherein the initial concentration of the growth regulator at the commencement of the growth regulator treatment is selected to be less than about 40 µM.
164. The method of any of claims 150, 158, or 159, wherein the peak concentration of the growth regulator is selected to be about 30 µM to about 200 µM.
165. The method of claim 164, wherein the peak concentration of the growth regulator is selected to be about 30 µM to 100 µM.
166. The method of claim 150, wherein the concentration of the growth regulator is raised progressively during at least part of the growth regulator treatment period.
167. The method of either of claims 150 or 166, wherein the concentration of the growth regulator is increased incrementally.
168. The method of claim 167, wherein the incremental increase of the growth regulator is at least about 5%
of the initial concentration of the growth regulator.
169. The method of either of claims 167 or 168, wherein the growth regulator is raised from its initial concentration to its peak concentration in a single increment.
170. The method of either of claims 167 or 168, wherein the growth regulator is raised from its initial concentration to its peak concentration in a series of increments.
171. The method of claim 166, wherein the concentration of the growth regulator does not increase during the early part of the growth regulator treatment period.
172. The method of claim 166, wherein the concentration of the growth regulator does not increase during the middle part of the growth regulator treatment period.
173. The method of claim 166, wherein the concentration of the growth regulator does not increase during the late part of the growth regulator treatment period.
174. The method of claim 173, wherein there is no net increase or net decrease in the concentration of the growth regulator during the late part of the growth regulator treatment period.
175. The method of claim 173, wherein there is a net decrease of the growth regulator during the late part of the growth regulator period.
176. The method of claim 175, wherein the growth regulator is reduced incrementally.
177. The method of claim 176, wherein the growth regulator is reduced from its peak concentration to its peak concentration in a series of increments.
178. The method of claim 176, wherein the growth regulator is reduced from its peak concentration to its final concentration in a single increment.
179. The method of any of claims 150, 158, or 159, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos environment.
180. The method of any of claims 150, 158, 159, or 179, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
181. The method of any of claims 150, 158, 159, 179, or 180, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 40%.
182. The method of any of claims 150, 158, 159, 179, 180, or 181, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 55%.
183. The method of any of claims 150, 158, 159, or 179, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
184. The method of any of claims 150, 158, 159, or 179, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
185. The method of any of claims 150 to 189, wherein the growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
186. The method of claim 185, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
187. The method of claim 150, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
188. The method of claim 187, wherein the growth regulator is abscisic acid.
189. The method of any of claims 150 to 188, wherein the water stressing creates a water potential of at least about -260 mmol/kg prior to the late cotyledonary stage of development.
190. The method of any of claims 150 to 189, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
191. The method of claim 150, wherein the water stressing of the embryos to reach a severely desiccated state is selected to reduce the amount of free unbound water to a level sufficient to induce freezing tolerance in the embryos.
192. The method of any of claims 150 to 191, wherein the rate of water loss in water stressing the embryos to reach a severely desiccated state is greater than the rate of water loss in water stressing the embryos to a mature cotyledonary stage.
193. The method of any of claims 150 to 190, wherein the water stressing to reach a mature cotyledonary stage is applied by means of a selected suitable environmental effect.
194. The method of any of claims 150 to 192, wherein the water stressing to reach a severely desiccated state is applied by means of a selected suitable environmental effect.
195. The method of any of claims 150 to 190, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of at least one selected suitable water stressing agent.
196. The method of any of claims 150 to 192, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of at least one selected suitable water stressing agent.
197. The method of any of claims 150 to 190, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
198. The method of any of claims 150 to 192, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
199. The method of any of claims 195 to 198, wherein the water stressing is applied at least in part by means of at least one gel.
200. The method of any of claims 195 to 198, wherein the water stressing is applied at least in part by means of at least one osmoticum.
201. The method of claim 200, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
202. The method of claim 200, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
203. The method of claim 202, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
204. The method of either of claims 201 or 203, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo for nutrition.
205. The method of claim 202, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (A).
206. The method of either of claims 202 or 205, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
207. The method of any of claims 202, 205, or 206, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
208. The method of any of claims 193, 194, 197, or 198, wherein the environmental water stressing is applied at least in part by relative humidity of less than 1000.
209. The method of claim 208, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
210. The method of any of claims 150 to 209, wherein the embryos are cultured in a bioreactor.
211. The method of any of claims 150 to 210, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
212. The method of claim 211, wherein the adsorbent means comprises activated charcoal.
213. The method of claim 212, wherein the activated charcoal is pre-saturated with the growth regulator.
214. The method of claim 211, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
215. The method of claim 150, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
216. The method of claim 215, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
217. The method of claim 150, wherein the somatic embryos are frozen following the water stressing treatment in (b).
218. The method of claim 150, wherein the embryos are in contact with medium during the development treatment in (a).
219. The method of claim 150, wherein the embryos are in contact with medium during the water stressing treatment in (b).
220. The method of claim 150, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the development treatment in (a).
221. The method of claim 150, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the water stressing treatment in (b).
222. The method of claim 218, wherein the embryos are removed from medium after they have attained a moisture content cf about 550.
223. The method of claim 220, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 550.
224. The method of any of claims 150 to 223, comprising the further step of rehydrating the mature embryos in preparation for germination.
225. The method of claim 224, wherein the rehydrating step occurs following a rest period following completion of water stressing the embryos.
226. The method of any of claims 150 to 225, further comprising germinating the mature embryos to form somatic seedlings.
227. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 150 to 225.
228. A somatic seedling produced in accordance with the method of claim 226.
229. A method of producing viable mature cotyledonary desiccated somatic embryos, comprising:
(a) a development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metabolizable carbon source, (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that:
(i) the concentration of the growth regulator is raised from a selected initial concentration to a selected peak concentration value before the completion of the growth regulator treatment period, and (ii) the intensity of the water stressing is raised from a selected initial intensity to a selected higher intensity before the completion of the water stressing period;
(b) further water stressing the mature somatic embryos to reach a severely desiccated state characterised by a moisture content of less than about 36o following the completion of the growth regulator treatment in (a).
230. The method of claim 229, wherein the embryos are conifer embryos.
231. The method of either of claims 229 or 230, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
232. The method of any of claims 229 to 231, wherein the growth regulator treatment continues after the embryos attain desiccation tolerance.
233. The method of any of claims 229 to 232, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
234. The method of any of claims 229 to 233, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
235. The method of claim 230, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
236. The method of any of claims 229 to 235, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
237. The method of any of claims 229 to 236, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
238. The method of any of claims 229 to 237, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
239. The method of any of claims 229, 237, or 238, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
240. The method of claim 239, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
241. The method of any of claims 229 to 240, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
242. The method of any of claims 229 to 241, wherein the initial concentration of the growth regulator at the commencement of the growth regulator treatment is selected to be less than about 40 µM.
243. The method of any of claims 229, 237, or 238, wherein the peak concentration of the growth regulator is selected to be about 30 µM to about 200 µM.
249. The method of claim 243, wherein the peak concentration of the growth regulator is selected to be about 30 µM to 100 µM.
295. The method of claim 229, wherein the concentration of the growth regulator is raised progressively during at least part of the growth regulator treatment period.
246. The method of either of claims 229 or 295, wherein the concentration of the growth regulator is increased incrementally.
247. The method of claim 246, wherein the incremental increase of the growth regulator is at least about 5%
of the initial concentration of the growth regulator.
248. The method of claim 246, wherein the growth regulator is raised from its initial concentration to its peak concentration in a single increment.
249. The method of claim 246, wherein the growth regulator is raised from its initial concentration to its peak concentration in a series of increments.
250. The method of claim 245, wherein the concentration of the growth regulator does not increase during the early part of the growth regulator treatment period.
251. The method of claim 245, wherein the concentration of the growth regulator does not increase during the middle part of the growth regulator treatment period.
252. The method of claim 245, wherein the concentration of the growth regulator does not increase during the late part of the growth regulator treatment period.
253. The method of claim 252, wherein there is no net increase or net decrease in the concentration of the growth regulator during the late part of the growth regulator treatment period.
254. The method of claim 252, wherein there is a net decrease of the growth regulator during the late part of the growth regulator treatment period.
255. The method of claim 254, wherein the growth regulator is reduced incrementally.
256. The method of claim 255, wherein the growth regulator is reduced from its peak concentration to its final concentration in a series of increments.
257. The method of claim 255, wherein the growth regulator is reduced from its peak concentration to its final concentration in a single increment.
258. The method of any of claims 229, 237, or 238, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos' environment.
259. The method of any of claims 229, 237, 238, or 258, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
260. The method of any of claims 229, 237, 238, 258, or 259, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 400.
261. The method of any of claims 229, 237, 238, 258, 259, or 260, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 550.
262. The method of any of claims 229, 237, 238, or 258, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
263. The method of any of claims 229, 237, 238, or 258, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
264. The method of any of claims 229 to 263, wherein the growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
265. The method of claim 264, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
266. The method of claim 229, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
267. The method of claim 266, wherein the growth regulator is abscisic acid.
268. The method of any of claims 229 to 267, wherein the initial intensity of water stressing creates a water potential of about -120 mmol/kg.
269. The method of any cf claims 229 to 268, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
270. The method of any of claims 229 to 269, wherein the water stressing creates a water potential of at least about -800 mmol/kg prior to the late cotyledonary stage of development.
271. The method of claim 229, wherein the water stressing of the embryos to reach a severely desiccated state is selected to reduce the amount of free unbound water to a level sufficient to induce freezing tolerance in the embryos.
272. The method of any of claims 229 to 271, wherein the rate of water loss in water stressing the embryos to reach a severely desiccated state is greater than the rate of water loss in water stressing the embryos to a mature cotyledonary stage.
273. The method of any of claims 229 to 270, wherein the water stressing to reach a mature cotyledonary stage is applied by means of a selected suitable environmental effect.
274. The method of any of claims 229 to 272, wherein the water stressing to reach a severely desiccated state is applied by means of a selected suitable environmental effect.
275. The method of any of claims 229 to 270, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of at least one selected suitable water stressing agent.
276. The method of any of claim 229 to 272, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of at least one selected suitable water stressing agent.
277. The method of any of claims 229 to 270, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
278. The method of any of claims 229 to 272, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of a combination.
of a selected suitable water stressing agent and a selected suitable environmental effect.
279. The method of any of claims 275 to 278, wherein the water stressing is applied at least in part by means of at least one gel.
280. The method of any of claims 275 to 278, wherein the water stressing is applied at least in part by means of at least one osmoticum.
281. The method of claim 280, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
282. The method of claim 280, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
283. The method of claim 282, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
284. The method of either of claims 281 or 283, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo nor nutrition.
285. The method of claim 282, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (.ANG.).
286. The method of either of claims 282 or 285, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
287. The method of any of claims 282, 285, or 286, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
288. The method of any of claims 273, 274, 277, and 278, wherein the environmental water stressing is applied at least in part by relative humidity of less than 100%.
289. The method of claim 288, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
290. The method of claim 229, wherein there is a net increase in the intensity of water stressing of the embryos over the duration of the water stressing treatment period.
291. The method of claim 290, wherein the net increase in the intensity of water stressing of the embryos is effected by changing the value of the water potential from a selected initial value to a selected final value.
292. The method of any of claims 229 to 291, wherein the embryos are cultured in a bioreactor.
293. The method of any of claims 229 to 292, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
294. The method of claim 293, wherein the adsorbent means comprises activated charcoal.
295. The method of claim 294, wherein. the activated charcoal is pre-saturated with the growth regulator.
296. The method of claim 293, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
297. The method of claim 229, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
298. The method of claim 297, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
299. The method of claim 229, wherein the somatic embryos are frozen following the water stressing treatment in (b).
300. The method of claim 229, wherein the embryos are in contact with medium during the development treatment in (a).
301. The method of claim 229, wherein the embryos are in contact with medium during the water stressing treatment in (b).
302. The method of claim 229, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the development treatment in (a).
303. The method of claim 229, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the water stressing treatment in (b).
304. The method of claim 300, wherein the embryos are removed from medium after they have attained a moisture content of about 55%.
305. The method of claim 302, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 55%.
306. The method of any of claims 229 to 305, comprising the further step of rehydrating the mature embryos in preparation for germination.
307. The method of claim 306, wherein the rehydrating step occurs following a rest period following completion of water stressing the embryos.
308. The method of any of claims 229 to 307, further comprising germinating the mature embryos to form somatic seedlings.
309. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 229 to 307.
310. A somatic seedling produced in accordance with the method of claim 308.
311. A development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metabolizable carbon source, and (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that:
(a) the concentration of the growth regulator remains substantially constant throughout the growth regulator treatment, and (b) the intensity of the water stressing is raised from a selected initial intensity to a selected higher intensity before the completion of the water stressing period.
312. The method of claim 311, wherein the embryos are conifer embryos.
313. The method or either of claims 311 or 312, wherein the water stressing and growth regulator treatments continue after the embryos attain desiccation tolerance.
314. The method of either of claims 311 or 312, wherein the water stressing or growth regulator treatments continues after the embryos attain desiccation tolerance.
315. The method of any of claims 311 to 314, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
316. The method of any of claims 311 to 315, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
317. The method of any of claims 311 to 316, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
318. The method of claim 312, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
319. The method of any of claims 311 to 318, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
320. The method of any of claims 311 to 319, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
321. The method of any of claims 311 to 320, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
322. The method of any of claims 311, 320, or 321, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
323. The method of claim 322, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
324. The method of any of claims 311 to 323, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
325. The method of any of claims 311, 320, or 321, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos' environment.
326. The method of any of claims 311, 320, 321, or 325, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
327. The method of any of claims 311, 320, 321, 325, or 326, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 40%.
328. The method of any of claims 311, 320, 321, 325, 326, or 327, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 55%.
329. The method of any of claims 311, 320, 321, or 325, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
330. The method of any of claims 311, 320, 321, or 325, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
331. The method of any of claims 311 to 330, wherein the growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
332. The method of claim 331, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
333. The method of claim 311, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
334. The method of claim 333, wherein the growth regulator is abscisic acid.
335. The method of any of claims 311 to 334, wherein the initial intensity of the water stressing creates a water potential of at least about -120 mmol/kg.
336. The method of any of claims 311 to 335, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
337. The method of any of claims 311 to 336, wherein the water stressing creates a water potential of at least about -800 mmol/kg prior to the late cotyledonary stage of development.
338. The method of any of claims 311 to 337, wherein the water stressing is applied by means of a selected suitable environmental effect.
339. The method of any of claims 311 to 337, wherein the water stressing is applied by means of at least one selected suitable water stressing agent.
340. The method of any of claims 311 to 337, wherein the water stressing is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
341. The method of either of claims 339 or 340, wherein the water stressing is applied at least in part by means of at least one gel.
342. The method of either of claims 339 or 340, wherein the water stressing is applied at least in part by means of at least one osmoticum.
393. The method of claim 342, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
344. The method of claim 342, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
345. The method of claim 344, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
346. The method of either of claims 343 or 345, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo for nutrition.
347. The method of claim 344, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (.ANG.).
348. The method of either of claims 344 or 347, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
349. The method of any of claims 399, 347, or 348, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
350. The method of either of claims 338 or 340, wherein the environmental water stressing is applied at least in part by relative humidity of less than 100%.
351. The method of claim 350, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
352. The method of claim 311, wherein there is a net increase in the intensity of water stressing of the embryos over the duration of the water stressing treatment period.
353. The method of claim 352, wherein the net increase in the intensity of water stressing of the embryos is effected by changing the value of the water potential from a selected initial value to a selected final value.
354. The method of any of claims 311 to 353, wherein the embryos are cultured in a bioreactor.
355. The method of any of claims 311 to 354, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
356. The method of claim 35, wherein the adsorbent means comprises activated charcoal.
357. The method of claim 356, wherein the activated charcoal is pre-saturated with the growth regulator.
358. The method of claim 355, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
359. The method of claim, 311, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
360. The method of claim 359, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
361. The method of any of claims 311 to 358, wherein the embryos are in contact with medium.
362. The method of claim 361, wherein the embryos are removed from medium after they have attained a moisture content of about 55%.
363. The method of any of claims 311 to 358, wherein the embryos are supported by nutrient-permeable support means in contact with a medium.
364. The method of claim 363, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 55%.
365. The method of any of claims 311 to 364, comprising the further step of rehydrating the mature embryos in preparation for germination.
366. The method of claim 365, comprising the further step of imbibing the embryos in 0-15°C.
367. The method of claim 365, wherein, the rehydrating step occurs following a rest period following completion of water stressing the embryos.
368. The method of any of claims 311 to 367, further comprising germinating the mature embryos to form somatic seedlings.
369. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 311 to 367.
370. A somatic seedling produced in accordance with the method of claim 368.
371. A method of producing viable mature cotyledonary desiccated somatic embryos, comprising:
(a) a development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metabolizable carbon source, (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that:
(i) the concentration of the growth regulator remains substantially constant throughout the growth regulator treatment, and (ii) the intensity of the water stressing is raised from a selected initial intensity to a selected higher intensity before the completion of the water stressing period;
(b) further water stressing the mature somatic embryos to reach a severely desiccated state characterised by a moisture content of less than about 36% following the completion of the growth regulator treatment in (a).
372. The method of claim 371, wherein the embryos are conifer embryos.
373. The method of either of claims 371 or 372, wherein the duration of the development time period selected to develop the embryos to a cotyledonary stage is selected to be sufficiently long to permit the embryos to store substantial reserves of nourishment and to enhance desiccation tolerance.
374. The method of any of claims 371 to 373, wherein the growth regulator treatment continues after the embryos attain desiccation tolerance.
375. The method of any of claims 371 to 374, wherein the temperature is selected to be in the range of about 0°C to about 35°C.
376. The method of any of claims 371 to 375, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the globular stage.
377. The method of claim 372, wherein at least one of the growth regulator and the water stressing is applied to the embryos before the club-shaped stage.
378. The method of any of claims 371 to 377, wherein the commencement of the growth regulator treatment and the commencement of the water stressing treatment are concurrent.
379. The method of any of claims 371 to 378, wherein the effective termination of the influence of the growth regulator on embryo development determines the completion of the growth regulator treatment.
380. The method of any of claims 371 to 379, wherein the completion of the growth regulator treatment comprises the removal of the embryos from the influence of the growth regulator.
381. The method of any of claims 371, 379, or 380, wherein the completion of the growth regulator treatment and the completion of the water stressing treatment are not concurrent.
382. The method of claim 381, wherein the water stressing treatment continues after the completion of the growth regulator treatment.
383. The method of any of claims 371 to 382, wherein the concentration of the growth regulator is selected to be about 0.1 µM to about 300 µM.
384. The method of any of claims 371, 379, or 380, wherein the means of completing the growth regulator treatment is selected from the class comprising the discontinuation of the growth regulator treatment and the removal of the growth regulator from the embryos' environment.
385. The method of any of claims 371, 379, 380, or 384, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 30%.
386. The method of any of claims 371, 379, 380, 384, or 385, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 40%.
387. The method of any of claims 371, 379, 380, 384, 385, or 386, wherein the growth regulator treatment is completed before the moisture content of the embryos reaches about 55%.
388. The method of any of claims 371, 379, 380, or 384, wherein the growth regulator treatment is completed before the embryos reach the late cotyledonary stage.
389. The method of any of claims 371, 379, 380, or 384, wherein the growth regulator treatment is completed when the embryos reach the late cotyledonary stage.
390. The method of any of claims 371 to 389, wherein the growth regulator is supplemented with at least one selected suitable growth promoter selected from the group comprising auxin, cytokinin, gibberellin, and functional equivalents thereof.
391. The method of claim 390, wherein the growth promoters are selected from the group comprising auxin, cytokinin, and functional equivalents thereof.
392. The method of claim 371, wherein the growth regulator is selected from the group comprising abscisic acid, precursors, derivatives, and analogs thereof.
393. The method of claim 392, wherein the growth regulator is abscisic acid.
394. The method of any of claims 371 to 393, wherein the initial intensity of water stressing creates a water potential of about -120 mmol/kg.
395. The method of any of claims 371 to 394, wherein the water stressing creates a water potential of at least about -400 mmol/kg prior to the late cotyledonary stage of development.
396. The method of any of claims 371 to 395, wherein the water stressing creates a water potential of at least about -800 mmol/kg prior to the late cotyledonary stage of development.
397. The method of claim 371, wherein the water stressing of the embryos to reach a severely desiccated state is selected to reduce the amount of free unbound water to a level sufficient to induce freezing tolerance in the embryos.
398. The method of any of claims 371 to 397, wherein the rate of water loss in water stressing the embryos to reach a severely desiccated state is greater than the rate of water loss in water stressing the embryos to a mature cotyledonary stage.
399. The method of any of claims 371 to 396, wherein the water stressing to reach a mature cotyledonary stage is applied by means of a selected suitable environmental effect.
400. The method of any of claims 371 to 398, wherein the water stressing to reach a severely desiccated state is applied by means of a selected suitable environmental effect.
401. The method of any of claims 371 to 396, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of at least one selected suitable water stressing agent.
402. The method of any of claim 371 to 398, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of at least one selected suitable water stressing agent.
403. The method of any of claims 371 to 396, wherein the water stressing of the embryos to a mature cotyledonary stage is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
404. The method of any of claims 371 to 398, wherein the water stressing of the embryos to reach a severely desiccated state is applied by means of a combination of a selected suitable water stressing agent and a selected suitable environmental effect.
405. The method of any of claims 401 to 404, wherein the water stressing is applied at least in part by means of at least one gel.
406. The method of any of claims 401 to 404, wherein the water stressing is applied at least in part by means of at least one osmoticum.
407. The method of claim 406, wherein the water stressing is applied in part by an embryo cell-permeating osmoticum.
408. The method of claim 406, wherein at least one osmoticum is selected from the group comprising non-permeating osmotica.
409. The method of claim 408, wherein a second osmoticum is present selected from the group comprising permeating osmotica.
410. The method of either of claims 407 or 409, wherein the water stressing is applied at least in part by a concentration of the metabolizable carbon source in excess of that utilized by the embryo for nutrition.
411. The method of claim 408, wherein the osmoticum has a molecular size of at least about 30 Angstrom units (.ANG.).
412. The method of either of claims 408 or 411, wherein the water stressing agent is selected from the group comprising polyalkylene glycols.
413. The method of any of claims 408, 411, or 412, wherein the water stressing agent comprises at least one polyalkylene glycol having a minimum molecular weight of about 1,000.
414. The method of any of claims 399, 400, 403, and 404, wherein the environmental water stressing is applied at least in part by relative humidity of less than 100%.
415. The method of claim 414, wherein the water stressing is applied at least in part by a controlled decrease in relative humidity.
416. The method of claim 371, wherein there is a net increase in the intensity of water stressing of the embryos over the duration of the water stressing treatment period.
417. The method of claim 416, wherein the net increase in the intensity of water stressing of the embryos is effected by changing the value of the water potential from a selected initial value to a selected final value.
418. The method of any of claims 371 to 417, wherein the embryos are cultured in a bioreactor.
419. The method of any of claims 371 to 418, wherein the embryos are in contact with medium with which is associated toxin removal means, said means being selected from the group comprising adsorbent means and filtration means.
420. The method of claim 419, wherein the adsorbent means comprises activated charcoal.
421. The method of claim 420, wherein the activated charcoal is pre-saturated with the growth regulator.
422. The method of claim 419, wherein the filtration means is selected from the group comprising molecular sieves and dialysis.
423. The method of claim 371, further comprising, as a step preceding the development treatment, culturing the immature embryos in a medium containing at least one of:
(i) auxin, or a functional equivalent thereof, in the amount of 0 µM to about 9 µM;
(ii) cytokinin, or a functional equivalent thereof, in the amount of 0 µM to about 4.5 µM.
424. The method of claim 423, further comprising water stressing the immature embryos to create a water potential of at least about -40 mmol/kg.
425. The method of claim 371, wherein the somatic embryos are frozen following the water stressing treatment in (b).
426. The method of claim 371, wherein the embryos are in contact with medium during the development treatment in (a).
427. The method of claim 371, wherein the embryos are in contact with medium during the water stressing treatment in (b).
428. The method of claim 371, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the development treatment in (a).
429. The method of claim 371, wherein the embryos are supported by nutrient-permeable support means in contact with a medium during the water stressing treatment in (b).
430. The method of claim 426, wherein the embryos are removed from medium after they have attained a moisture content of about 55%.
431. The method of claim 428, wherein the embryos are removed from contact with the support means after the embryos have attained a moisture content of about 55%.
432. The method of any of claims 371 to 431, comprising the further step of rehydrating the mature embryos in preparation for germination.
433. The method of claim 432, wherein the rehydrating step occurs following a rest period following completion of water stressing the embryos.
434. The method of any of claims 371 to 433, further comprising germinating the mature embryos to form somatic seedlings.
435. A viable mature cotyledonary desiccation-tolerant embryo produced in accordance with the method of any of claims 371 to 433.
436. A somatic seedling produced in accordance with the method of claim 434.
437. A development treatment over a selected development time period of somatic embryos for producing viable mature cotyledonary desiccation-tolerant somatic embryos, said development treatment including (i) nourishment of the embryos by a suitable metabolizable carbon source, and (ii) for a selected growth regulator treatment time period, treatment of the embryos by at least one growth regulator influencing embryo development selected from the class comprising stress hormones, and (iii) for a selected water stressing treatment time period, water stressing the embryos;
wherein the duration of the development period, the type and intensity of the water stressing, the duration of the water stressing treatment time period, the duration of the growth regulator treatment time period, and the type and concentration of the metabolizable carbon source and growth regulator are selected to reduce the moisture content of the embryos to a level of less than about 55% and to render the embryos desiccation-tolerant, and wherein the water stressing is non-plasmolysing;
characterized in that (a) the concentration of the growth regulator is raised from a selected initial concentration to a selected peak concentration value before the completion of the growth regulator treatment, and (b) the intensity of the water stressing over the water stressing treatment period is selected to be at least at its initial level.
CA2322438A 1998-06-05 1999-06-03 Increasing levels of growth regulator and/or water stress during embryo development Expired - Fee Related CA2322438C (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9078427B1 (en) 2014-08-29 2015-07-14 Pioneer Hi Bred International Inc Method of storing plant embryos
US10278345B2 (en) 2014-08-29 2019-05-07 Pioneer Hi-Bred International, Inc. Methods and devices for creating doubled haploid embryos using oil matrices

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9078427B1 (en) 2014-08-29 2015-07-14 Pioneer Hi Bred International Inc Method of storing plant embryos
US10278345B2 (en) 2014-08-29 2019-05-07 Pioneer Hi-Bred International, Inc. Methods and devices for creating doubled haploid embryos using oil matrices
US10477859B2 (en) 2014-08-29 2019-11-19 Pioneer Hi-Bred International, Inc. Plant embryo storage and manipulation

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