METHOD FOR GROWING SOMATIC EMBRYOS OF CONIFERS INTO TREES
20200236884 ยท 2020-07-30
Assignee
Inventors
Cpc classification
A01H4/005
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a method for producing trees or wood from fully mature cotyledonary somatic embryos (SE) of conifer species within the genus Abies and to the use of plants thus obtained. The invention concerns a method for treating and growing somatic embryos under conditions that induce and stimulate root growth, that induce and stimulate shoot formation and increases the survival rate of the embryos, plantlets and emblings. The emblings can subsequently be grown into trees. Trees derived from somatic embryogenesis and grown according to the method of the invention are of a very homogeneous appearance and quality, which is preferred in the production of trees such as Christmas trees thereby reducing the wastage rate.
Claims
1-21. (canceled)
22. A method for development of plants from fully mature somatic embryos of the genus Abies comprising: a) subjecting fully mature somatic embryos to 2 C.-7 C. for 8-16 weeks; b) selecting the small plantlets obtained from step (a) that have developed a root and growing said small plantlets in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at LED light intensities of 70-300 mol/m.sup.2s for at least 3 weeks; and c) selecting the emblings obtained from step b) and growing these into plants.
23. The method according to claim 22 wherein said temperature in step a) is 3 C.-5 C.
24. The method according to claim 22 wherein said temperature in step a) is 4 C.
25. The method according to claim 22 wherein the fully mature embryos in step a) are subjected to said temperature for 9 to 11 weeks.
26. The method according to claim 22 wherein the fully mature embryos in step a) are subjected to said temperature for 12 weeks.
27. The method according to claim 22 wherein said LED light intensities applied in step b) are 150-250 mol/m.sup.2s.
28. The method according to claim 6 wherein said LED light intensities in step b) are 175-225 mol/m.sup.2s.
29. The method according to claim 22 wherein said plantlets in step b) are grown in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at said LED light intensities for 4 to 8 weeks.
30. The method according to claim 29 wherein said plantlets in step b) are grown in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at said LED light intensities for 6 weeks.
31. The method according to claim 22 comprising: a) subjecting fully mature somatic embryos to 2 C.-5 C. for 9 to 11 weeks; b) selecting the small plantlets from step (a) that have developed a root and growing said small plantlets in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at LED light intensities of 175-225 mol/m.sup.2s for 6 weeks; and c) selecting the emblings obtained from step b) and growing these into plants.
32. The method according to claim 22 comprising: a) subjecting fully mature somatic embryos to 3 C.-7 C. for 12 weeks; b) selecting the small plantlets from step (a) that have developed a root and growing said small plantlets in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at LED light intensities of 175-225 mol/m.sup.2s for 6 weeks; and c) selecting the emblings obtained from step b) and growing these into plants.
33. The method according to claim 22 comprising: a) subjecting fully mature somatic embryos to 4 C. for 12 weeks; b) selecting the small plantlets from step (a) that have developed a root and growing said small plantlets in a substrate that does not comprise a significant concentration of a plant accessible carbohydrate source; at LED light intensities of 200 mol/m.sup.2s for 6 weeks; and c) selecting the emblings obtained from step b) and growing these into plants.
34. The method according to claim 22 wherein said small plantlets in step (b) are grown in transparent boxes that allow aeration of said small plantlets.
35. The method according to claim 34 wherein said transparent box comprises a lid having a whole of 10-30% of the total area of the lid.
36. The method according to claim 22 wherein said somatic embryos of the genus Abies is selected from: Abies alba, Abies amabilis, Abies balsamea, Abies beshanzuensis, Abies bifolia, Abies borisii-regis, Abies bornmlleriana, Abies bracteata, Abies cephalonica, Abies chensiensis, Abies cilicica, Abies concolor, Abies delavayi, Abies densa, Abies duragensis, Abiesfabri, Abiesfargesii, Abies fanjingshanensis, Abies firma, Abies flinckii, Abies forrestii, Abies fraseri, Abies guatemalensis, Abies hickelii, Abies holophylla, Abies homolepis, Abies kawakamii, Abies koreana, Abies lasiocarpa, Abies lowiana, Abies magnifica, Abies mariesii, Abies nebrodensis, Abies nephrolepis, Abies nordmanniana, Abies numidica, Abies pardei, Abies pindrow, Abies pinsapo, Abies procera (Abies nobilis), Abies recurvata, Abies religiosa, Abies sachalinensis, Abies sibirica, Abies spectabilis, Abies squamata, Abies veitchii, Abies vejarii, Abies yuanbaoshanensis, Abies ziyuanensis or any hybrids obtained from inter-species hybridization between any of these species of the Abies genus.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1: Preparation of Somatic Embryos of Abies nordmanniana
[0176] Cones of Abies nordmanniana was collected from Ambrolauri, Georgian republic and cleaned and rinsed in water and disinfected with fungicide and ethanol. The cones were then stored at 5 C. for up to 60 days.
[0177] The seeds were then isolated from the cone by breaking the cone into pieces and collecting the seeds. The seeds were placed in a 35% hydrogen peroxide solution diluted 1:10 for 3-5 minutes while stirring. The seeds were then removed to a mild Presept solution (1 tablet, 0.5 g, comprising troclosene sodium in 1 liter of water and two drops of detergent (Tween)) for 24 hours while ensuring access to oxygen to the solution by stirring. The seeds were rinsed in 70% ethanol for 3 minutes. This will also separate vital seeds from dead seeds; the vital seeds will sink whereas the dead seeds flow. Only the vital seeds were collected for further processing. The vital seeds were removed to a Precept solution (1 tablet in 0.5 L sterile water and 3-4 drops of detergent (Tween)) and placed on a shaker for 10 minutes. The seeds were then rinsed 3 times in sterile water under sterile conditions. If seeds have been stored for a long time or if they appear unclean, the can be briefly immersed into 96% ethanol and flamed.
[0178] The cleaned seeds where cut open and the seed embryo removed. Each embryo was placed in a petri dish comprising growth medium 29.4 in order to initiate the somatic cell culture. The embryo and the initiating culture were kept and grown in dark at 20 C. for 8-12 weeks until sufficient culture was available. Then the cultures were grown at 20 C. in the dark for 10 to 16 more weeks, and re-placed on fresh medium every second week.
Medium 29.4: 1 Liter
[0179]
TABLE-US-00001 H2O 200 ml BLG - RAT 200 ml BAP 5.0 ml BLG amino 25 ml* Sucrose 10 g Water up to 975 ml Conductivity of solution
1.241-1.371 mS pH 5.7 Phytagel 1.8 g *Added after autoclaving
conductivity is measured in the solution of 975 ml, containing BLG-RAT, BAP and sucrose, after being set to pH 5.7
Recipes Used to Provide Medium 29.4
[0180]
TABLE-US-00002 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O topped to 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4, 7H2O 5.560 Na2EDTA, 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O topped to 2000 ml BAP 5 ml BAP 0.0225 g Cas no 1214-39-7 1.0N KOH 1.4 ml H2O topped to 100 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Sucrose 10 g H20 975 ml Phytagel 1.8 g
[0181] Samples of the somatic cell culture can optionally be prepared for cryopreservation or be used directly for embryo development and maturation.
[0182] If the samples were cryopreserved, approximately 2 gram of tissue from one specific genotype were placed in a sterile bottle and 20 g of medium 29.4 was added, and 1 ml Sorbitol. The cultures were placed on a shaker providing constant agitation for adequate aeration for 24 hours at 20 C. and 1 ml sorbitol was added. After 48 hours, samples were placed at 0 C. for at least 30 min. Then, 1 ml DMSO was added to each bottle. The solution were divided into 12 tubes and slowly frozen from 0.5 C. to 35 C. before being transferred to a cryo-tank at minus 180 C.
[0183] When applicable, the somatic cell cultures were thawed and the development of somatic embryos was initiated.
[0184] The samples were thawed in 40 C. water, disinfected in ethanol and poured onto filter paper in a petri dish with medium 29.4. After 1 hour the filter papers with cultures were moved to a new petri dish with medium 29.4, and again after 18 hours the filter papers with cultures were placed in a new petri dish comprising a growth medium, such as medium 29.4.
[0185] The cultures are grown at 20 C. and the viability of the cultures are checked every 2.sup.nd week. After 6 weeks, viable cultures are selected for embryo development and maturation. Every 2.sup.nd week cultures were moved onto fresh medium 29.4. After another 6-8 weeks the cultures were ready.
Example 2: Embryo Development and Maturation of Abies nordmanniana
[0186] Somatic cell cultures from two different genotypes of Abies nordmanniana obtained from example 1 were selected for embryo development and maturation.
[0187] For each sample of somatic cell culture, 4 g of culture were added to 100 g 29.4 medium and blended for 25 seconds. The samples were then left for 30 minutes and any surplus liquid was removed until 30 ml. One ml of the blend was pipetted onto filter paper in a petri dish comprising 15 ml 49.53 medium.
Medium 49.53: 1 Liter
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TABLE-US-00003 BLG - RAT 200 ml ABA 30 ml* BLG-Amino 25 ml* PEG-4000 50 g** Maltose 45 g** Water topped to 445 ml** Water up to 500 ml pH 5.7 Conductivity of solution
2.39-2.65 mS Phytagel 1.8 g *Added after autoclaving **PEG/the sugar, respectively, is mixed and autoclaved alone in the applied amount of water. The RAT-media can be obtained from common distributors such as Duchefa Biochemie.
conductivity is measured in the solution of 500 ml, containing BLG-RAT, after being set to pH 5.7
Recipes Used to Provide Medium 49.53
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TABLE-US-00004 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4, 7H2O 5.560 g Na2EDTA, 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O 2000 ml ABA 30 ml Abscisinsyre 0.2640 g Cas nr 14375-45-2 0.1N NaHCO3 140 ml H2O topped to 2000 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml PEG 4000 50 g H20 topped to 445 ml Maltose 45 g H20 topped to 500 ml Phytagel 1.8 g
[0190] The cultures were grown at 20 C. in the dark for 3 weeks. Filter papers holding suitable cultures were moved to medium 29.75 grown at 20 C. in the dark for 3 weeks.
Medium 29.75: 1 Liter
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TABLE-US-00005 BLG - RAT 200 ml ABA 40 ml* PCIB 25 ml BLG-amino 25 ml* Maltose 45 g** Water topped to 435 ml** Water op to 500 ml pH at 20-25 C. 5.7 Conductivity of Solution
2.39-2.65 mS Phytagel 1.8 g *Added after autoclaving **PEG/the sugar, respectively, is mixed and autoclaved alone in the applied amount of water.
conductivity is measured in the solution of 500 ml, containing BLG-RAT and PCIB, after being set to pH 5.7
Recipes Used to Provide Medium 29.75
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TABLE-US-00006 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O topped to 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O topped to 2000 ml ABA 40 ml Abscisinsyre 0.2640 g Cas nr 14375-45-2 0.1N NaHCO3 140 ml H2O topped to 2000 ml PCIB 25 ml MCPA 0.1070 g Cas no 882-09-7 0.1N KOH 25.00 ml H2O topped to 500 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Maltose 45 g H2O topped to 435 ml H20 topped to 500 ml Phytagel 1.8 g
[0193] Filter paper holding suitable cultures were then moved to medium 49.53 for 6 weeks for completing maturation of the embryos. Subsequently, the filter paper holding suitable embryos were removed to medium 8.95 for fattening and grown at 20 C. in the dark for two weeks in order to complete the maturation of the embryos.
Medium 8.95: 1 Liter
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TABLE-US-00007 KNV-8 RAT 200 ml BLG amino 25 ml* Sucrose 75.0 g Water topped to 975 ml pH 5.7 Conductivity of solution
2.20-2.44 mS Phytagel 1.80 g *Added after autoclaving
conductivity is measured in the solution of 975 ml, containing KNV-RAT and sucrose, after being set to pH 5.7
Recipes Used to Provide Medium 8.95:
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TABLE-US-00008 KNV-Rat 200 ml KNV-8 macro 2000 ml Ca(NO3)2 4H2O 28.340 g KNO3 20.220 g KH2PO4 10.888 g MgSO4 7H2O 19.720 g NH4NO3 14.400 g KCL 5.964 g H2O topped to 4.000 ml AXB- micro 20 ml UNDER STAM 10 ml AlCl3, 6H2O 0.024 g CoCl2, 6H2O 0.012 g KI 0.083 g NiCl2, 6H2O 0.024 g H2O topped to 100 ml H3BO3 0.464 g CuSO4 5H2O 0.025 g MnSO4 H2O 0.423 g Na2MoO4 2H2O 0.012 g ZnSO4 7H2O 0.144 g H2O topped to 100 ml FE EDTA 50 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-0 H2O topped to 1000 ml DCR-2 20 ml Thiamine-HCl 0.10 g Pyridoxine HCl 0.05 g Nicotinic Acid 0.05 g Glycine 0.20 g H2O topped to 100 ml Inositol 2.00 g H2O topped to 4000 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Sucrose 75 g H20 975 ml Phytagel 1.8 g
[0196] The thus obtained fully mature cotyledonary somatic embryos (as shown in
[0197] The fully mature somatic embryos selected for growing under root inducing conditions were characterized in being in general 4-5 mm long; having typically 2-5 or more small cotyledons; and a radicle (root primordia); and a diameter at the center of the stem of approximately 1 mm. Examples of fully mature embryos are shown in
Example 3: Rooting of Fully Mature Somatic Embryos of Abies nordmanniana
[0198] 300 fully mature cotyledonary embryos (corresponding to stage 7 as shown in
Medium 51.21: 1 Liter
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TABLE-US-00009 KNV Rat ml 200.0 Activated charcoal g 10.000 AgNO3 (250 mg/100 ml) ml 1.00 Sucrose g 20.0 Water up to ml 1000 Conductivity in solution
2.40-2.66 mS pH 5.7 Agar g 5.00
conductivity is measured in the solution of 1000 ml, containing KNV-RAT, AgNO3 and sucrose, after being set to pH 5.7
Recipes Used to Provide the 51.21 Medium:
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TABLE-US-00010 KNV-Rat 200 ml KNV-8 macro 2000 ml Ca(NO3)2 4H2O 28.340 g KNO3 20.220 g KH2PO4 10.888 g MgSO4 7H2O 19.720 g NH4NO3 14.400 g KCL 5.964 g H2O topped to 4.000 ml AXB- micro 20 ml UNDER STAM 10 ml AlCl3 6H2O 0.024 g CoCl2 6H2O 0.012 g KI 0.083 g NiCl2 6H2O 0.024 g H2O topped to 100 ml H3BO3 0.464 g CuSO4 5H2O 0.025 g MnSO4 H2O 0.423 g Na2MoO4 2H2O 0.012 g ZnSO4 7H2O 0.144 g H2O topped to 100 ml FE EDTA 50 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 20 ml Thiamine-HCl 0.10 g Pyridoxine HCl 0.05 g Nicotinic Acid 0.05 g Glycine 0.20 g H2O topped to 100 ml Inositol 2.00 g H2O topped to 4000 ml Activate 10.000 g charcoal AgNO3 1.000 ml (250 mg/100 ml) Sucrose 20.000 g H20 topped to 1000 ml Agar 5.000 g
[0201] After 8, 9, 10, 11, 12 and 16 weeks, the embryos were checked for root formation and embryos with a well developed root were selected for further growth under the shoot inducing conditions described in Example 4.
Results:
[0202] Only a small share of the embryos of both genotypes that were grown at 2 C. or 9 C. developed a root after 8-16 weeks.
[0203] Very few of the embryos of both genotypes that were grown at 10 C. developed a root after 8-16 weeks.
[0204] The majority of the embryos of both genotypes that were grown at 4 C. or at 3-7 C. had developed a root after 12 weeks, a smaller number already after 8 weeks and a smaller number only after 16 weeks.
[0205] Examples of suitable small plantlets that were chosen for further growth under shoot inducing conditions are shown in
Example 4: Shoot Development in Rooted Somatic Embryos of Abies nordmanniana
[0206] From each of the two Abies nordmanniana genotypes, 30-40 small plantlets that had developed a root when grown under each of the root inducing conditions described in Example 3 were selected for growing under shoot inducing conditions. Most of the selected small plantlets had been grown at 3-7 C. for 12-14 weeks as described in Example 3 since this was the temperature by which most of the somatic embryos successfully developed a root.
[0207] As mentioned in Example 3, small plantlets included in this experiment were characterized in having a root, the cotyledons were 1-5 mm long, and the hypocotyl was about 5-15 mm long.
[0208] The small plantlets were moved into plastic boxes (Eco2box, oval, Duchefa Bichemie) of 125 mm length65 mm width80 mm height having a lid. The lid was partially open for air-movement. The humidity RH % was kept above 90.
[0209] Each box contained 100 ml of the sugar-free medium 47.07.
Medium 47.07: 1 Liter
[0210]
TABLE-US-00011 Duchefa WPM M0219 0.590 g DCR 2 0.250 ml AgNO3 (250 mg/100 ml) 1.000 ml Inositol 0.100 g Water topped to 1000.0 ml Conductivity of solution
751-830 S pH 5.7 Agar 7.00 g Phytagel 0.70 g
conductivity is measured in the solution of 1000 ml, containing WPM, DCR 2, AgNO3 and inositol, after being set to pH 5.7
Recipes Used to Provide Medium 47.07:
[0211]
TABLE-US-00012 Duchefa WPM 0.590 g M0219 DCR-2 250 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml AgNO3 1 ml (250 mg/100 ml) Inositol 0.1 g Phytagel 0.7 g Gelrite, Duchefa Biochemie, CAS 71010-52-1 Italiensk Agar 7 g Plantager S1000, B&V Srl, Italy, CAS 9002-18-0 H2O topped to 1000 ml (pH = 5.7)
[0212] Each box comprised approximately 20-30 small plantlets of the same genotype, that had been grown under the same root inducing conditions.
[0213] The small plantlets were grown for 4 to 8 weeks at 15 in 24 h periods of white LED light of 70 to 400 mol/m.sup.2s.
[0214] The light intensities were measured on top of each box as close to the cotyledons as possible, and thereafter accounting for the light reducing effect of the lit. The light intensities ranged from approximately 100 to 400 mol/m.sup.2s depending on the distance from the nearest light source.
Results:
[0215] The majority of the small plantlets of both genotypes had developed into emblings having new cotyledons, which also had turned darker green showing a normal surface of a fully developed conifer needle; after 6 weeks. The total height of these emblings above the root were generally 8-22 mm and the root was generally of a length in the range of 1-30 mm. Examples of such emblings are shown in
[0216] A smaller amount of the plants of both genotypes had developed same characteristics after only 4-5 weeks, and a smaller amount of the plants of both genotypes had developed same after 7-8 weeks.
[0217] The majority of the plants of both genotypes that had developed new cotyledons, which also had turned darker green showing a normal surface of a fully developed conifer needle after 4 to 8 weeks were plantlets that had been subjected to growth at 3-7 C. in the previous root inducing step
Example 5: Development of Small Plants of Abies nordmanniana
[0218] All well developed emblings with new cotyledons obtained from Example 4 were transferred to small forest C7 Jiffy pots of 025 mm plug for continued growth and were grown for 6-8 weeks at 25 C. in 24 h periods of white LED light of 100 to 400 mol/m.sup.2s. The humidity RH % was slowly decreased to 70.
[0219] Almost all plants survived and were subsequently selected for continued nursery production.
[0220] Examples of emblings growing into small plants at this stage are shown in
Example 6: Nursery Production of Small Plants of Abies nordmanniana
[0221] Plants obtained from Example 5 were potted into larger pots and grown in a greenhouse for 1-2 years until a suitable size for growing outside (for instance in a field for Christmas tree production) was reached.
[0222] Examples of commercial emblings of Abies nordmanniana obtained from conditions described in Example 5 and the previous Examples are shown in
Example 7: Preparation of Somatic Embryos of Abies bornmlleriana
[0223] Cones of Abies bornmlleriana was collected from the Danish seed orchard FP.267 Kongsre, north of Holbeek. Seed trees were selected in a Danish Christmas tree stand originating from the area near Bolu Kkez in Turkey. The cones were cleaned and rinsed in water and disinfected with fungicide and ethanol. The cones were then stored at 5C for up to 60 days.
[0224] The seeds were then isolated from the cone by breaking the cone into pieces and collecting the seeds. The seeds were placed in a 35% hydrogen peroxide solution diluted 1:10 for 3-5 minutes while stirring. The seeds were then removed to a mild Presept solution (1 tablet comprising troclosene sodium in 1 liter of water and two drops of detergent (Tween)) for 24 hours while ensuring access to oxygen to the solution by stirring. The seeds were then wrapped in gaze briefly for drying and subsequently rinsed in 70% ethanol for 3 minutes. This will also separate vital seeds from dead seeds; the vital seeds will sink whereas the dead seeds flow. Only the vital seeds were collected for further processing. The vital seeds were removed to a Precept solution (1 tablet in 0.5 L sterile water and 3-4 drops of detergent (Tween)) and placed on a shaker for 10 minutes. The seeds were then rinsed 3 times in sterile water under sterile conditions. If seeds have been stored for a long time or if they appear unclean, the can be briefly immersed into 96% ethanol and flamed.
[0225] The cleaned seeds where cut open and the seed embryo removed. Each embryo was placed in a petri dish comprising growth medium 29.4 in order to initiate the somatic cell culture. The cultures were grown at 20 C. in the dark for 10 to 16 weeks.
Medium 29.4: 1 Liter
[0226]
TABLE-US-00013 H2O 200 ml BLG - RAT 200 ml BAP 5.0 ml BLG amino 25 ml * Sucrose 10 g Water up to 975 ml Conductivity of solution
1.241-1.371 mS pH 5.7 Phytagel 1.8 g * Added after autoclaving
: conductivity is measured in the solution of 975 ml, containing BLG-RAT, BAP and sucrose, after being set to pH 5.7.
Recipes Used to Provide Medium 29.4:
[0227]
TABLE-US-00014 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O topped to 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4, 7H2O 5.560 Na2EDTA, 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O topped to 2000 ml BAP 5 ml BAP 0.0225 g Cas no 1214-39-7 1.0N KOH 1.4 ml H2O topped to 100 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Sucrose 10 g H20 975 ml Phytagel 1.8 g
[0228] Samples of the somatic cell culture can optionally be prepared for cryopreservation or be used directly for embryo development and maturation.
[0229] If the samples were cryopreserved, approximately 2 gram of tissue from one specific genotype were placed in a sterile bottle and 20 g of medium 29.4 was added, and 1 ml Sorbitol. The cultures were placed on a shaker providing constant agitation for adequate aeration for 24 hours at 20 C. and 1 ml sorbitol was added. After 48 hours samples were placed at 0 C. for at least 30 min. Then, 1 ml DMSO was added to each bottle. The solution were divided into 12 tubes and slowly frozen from 0.5 C. to 35 C. before being transferred to a cryo-tank at minus 180 C.
[0230] When applicable, the somatic cell cultures were thawed and the development of somatic embryos was initiated.
[0231] The samples were thawed in 40 C. water, disinfected in ethanol and poured onto filter paper in a petri dish. After 1 hour the cultures were removed to a clean filter paper, and again after 18 hours the cultures were placed in a new petri dish comprising a growth medium, such as medium 29.4.
[0232] The cultures are grown at 20 C. and the viability of the cultures are checked every 2.sup.nd week. After 6 weeks, viable cultures are selected for embryo development and maturation. Every 2nd week cultures were moved onto fresh medium 29.4. After another 6-8 weeks the cultures were ready.
Example 8: Embryo Development and Maturation of Abies bornmlleriana
[0233] Somatic cell cultures from two different genotypes of Abies bornmlleriana obtained from example 7 were selected for embryo development and maturation.
[0234] For each sample of somatic cell culture, 4 g of culture were added to 100 g 29.4 medium and blended for 25 seconds. The samples were then left for 30 minutes and any surplus liquid was removed until 30 ml. One ml of the blend was pipetted onto filter paper in a petri dish comprising 15 ml 49.53 medium.
Medium 49.53: 1 Liter
[0235]
TABLE-US-00015 BLG - RAT 200 ml ABA 30 ml* BLG-Amino 25 ml* PEG-4000 50 g** Maltose 45 g** Water topped to 445 ml** Water up to 500 ml pH 5.7 Conductivity of solution
2.39-2.65 mS Phytagel 1.8 g *Added after autoclaving **PEG/the sugar, respectively, is mixed and autoclaved alone in the applied amount of water. The RAT-media can be obtained from common distributors such as Duchefa Biochemie.
: conductivity is measured in the solution of 500 ml, containing BLG-RAT, after being set to pH 5.7
Recipes Used to Provide Medium 49.53
[0236]
TABLE-US-00016 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4, 7H2O 5.560 g Na2EDTA, 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O 2000 ml ABA 30 ml Abscisinsyre 0.2640 g Cas nr 14375-45-2 0.1N NaHCO3 140 ml H2O topped to 2000 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml PEG 4000 50 g H20 topped to 445 ml Maltose 45 g H20 topped to 500 ml Phytagel 1.8 g
[0237] The cultures were grown at 20 C. in the dark for 3 weeks. Filter papers holding suitable cultures were moved to medium 29.75 grown at 20 C. in the dark for 3 weeks.
Medium 29.75: 1 Liter
[0238]
TABLE-US-00017 BLG - RAT 200 ml ABA 40 ml* PCIB 25 ml BLG - amino 25 ml* Maltose 45 g** Water topped to 435 ml** Water op to 500 Ml pH at 20-25 C. 5.7 Conductivity of Solution
2.39-2.65 mS Phytagel 1.8 g *Added after autoclaving **PEG/the sugar, respectively, is mixed and autoclaved alone in the applied amount of water.
: conductivity is measured in the solution of 500 ml, containing BLG-RAT and PCIB, after being set to pH 5.7
Recipes Used to Provide Medium 29.75
[0239]
TABLE-US-00018 BLG-Rat 200 ml BLG 29 makro 500 ml KNO3 1.000 g KCL 7.450 g MgSO4 7H2O 3.200 g KH2PO4 1.700 g CaCl2 2H2O 4.400 g H2O topped to 1000 ml BLG 29 mikro/MS 7 5 ml UNDER STAM 10 ml CuSO4 5H2O 0.025 g CoCl2 6H2O 0.025 g H2O topped to 100 ml H3BO3 0.62 g MnSO4 H2O 1.69 g ZnSO4 7H2O 0.86 g KI 0.083 g Na2MoO4 2H2O 0.025 g H2O topped to 100 ml FE EDTA 25.0 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 10.0 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml Inositol 1.000 g H2O topped to 2000 ml ABA 40 ml Abscisinsyre 0.2640 g Cas nr 14375-45-2 0.1N NaHCO3 140 ml H2O topped to 2000 ml PCIB 25 ml MCPA 0.1070 g Cas no 882-09-7 0.1N KOH 25.00 ml H2O topped to 500 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Maltose 45 g H2O topped to 435 ml H20 topped to 500 ml Phytagel 1.8 g
[0240] Filter paper holding suitable cultures were then moved to medium 49.53 for 6 weeks for completing maturation of the embryos. Subsequently, the filter paper holding suitable embryos were removed to medium 8.95 for fattening and grown at 20 C. in the dark for two weeks in order to complete the maturation of the embryos.
Medium 8.95: 1 Liter
[0241]
TABLE-US-00019 KNV-8 RAT 200 ml BLG amino 25 ml* Sucrose 75.0 g Water topped to 975 ml pH 5.7 Conductivity of solution
2.20-2.44 mS Phytagel 1.80 g *Added after autoclaving
: conductivity is measured in the solution of 975 ml, containing KNV-RAT and sucrose, afer being set to pH 5.7
Recipes Used to Provide Medium 8.95:
[0242]
TABLE-US-00020 KNV-Rat 200 ml KNV-8 macro 2000 ml Ca(NO3)2 4H2O 28.340 g KNO3 20.220 g KH2PO4 10.888 g MgSO4 7H2O 19.720 g NH4NO3 14.400 g KCL 5.964 g H2O topped to 4.000 ml AXB- micro 20 ml UNDER STAM 10 ml AlCl3, 6H2O 0.024 g CoCl2, 6H2O 0.012 g KI 0.083 g NiCl2, 6H2O 0.024 g H2O topped to 100 ml H3BO3 0.464 g CuSO4 5H2O 0.025 g MnSO4 H2O 0.423 g Na2MoO4 2H2O 0.012 g ZnSO4 7H2O 0.144 g H2O topped to 100 ml FE EDTA 50 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 20 ml Thiamine-HCl 0.10 g Pyridoxine HCl 0.05 g Nicotinic Acid 0.05 g Glycine 0.20 g H2O topped to 100 ml Inositol 2.00 g H2O topped to 4000 ml BLG amino 25 ml L-glutamin 29.000 g L-asparagin 2.000 g Barnsteadwater 800 ml H20 topped to 1000 ml Sucrose 75 g H20 975 ml Phytagel 1.8 g
[0243] The thus obtained fully mature cotyledonary somatic embryos (as shown in
[0244] The fully mature somatic embryos selected for growing under root inducing conditions were characterized in being in general 4-5 mm long; having typically 2-5 or more small cotyledons; and a radicle (root primordia); and a diameter at the center of the stem of approximately 1 mm
Example 9: Rooting of Fully Mature Somatic Embryos of Abies bornmlleriana
[0245] Approximately 300 fully mature cotyledonary embryos (corresponding to stage 7 as shown in
[0246] After 8, 9, 10, 11, 12 and 16 weeks, the embryos are checked for root formation and embryos with a well developed root are selected for further growth under the shoot inducing conditions described in Example 10.
Medium 51.21: 1 Liter
[0247]
TABLE-US-00021 KNV Rat ml 200.0 Activated charcoal g 10.000 AgNO3 (250 mg/100 ml) ml 1.00 Sucrose g 20.0 Water up to ml 1000 Conductivity in solution
2.40-2.66 mS pH 5.7 Agar g 5.00
: conductivity is measured in the solution of 1000 ml, containing KNV-RAT, AgNO3 and sucrose, after being set to pH 5.7
Recipes Used to Provide the 51.21 Medium:
[0248]
TABLE-US-00022 KNV-Rat 200 ml KNV-8 macro 2000 ml Ca(NO3)2 4H2O 28.340 g KNO3 20.220 g KH2PO4 10.888 g MgSO4 7H2O 19.720 g NH4NO3 14.400 g KCL 5.964 g H2O topped to 4.000 ml AXB- micro 20 ml UNDER STAM 10 ml AlCl3 6H2O 0.024 g CoCl2 6H2O 0.012 g KI 0.083 g NiCl2 6H2O 0.024 g H2O topped to 100 ml H3BO3 0.464 g CuSO4 5H2O 0.025 g MnSO4 H2O 0.423 g Na2MoO4 2H2O 0.012 g ZnSO4 7H2O 0.144 g H2O topped to 100 ml FE EDTA 50 ml FeSO4 7H2O 5.560 g Na2EDTA 2H2O 7.450 g Cas no 6381-92-6 H2O topped to 1000 ml DCR-2 20 ml Thiamine-HCl 0.10 g Pyridoxine HCl 0.05 g Nicotinic Acid 0.05 g Glycine 0.20 g H2O topped to 100 ml Inositol 2.00 g H2O topped to 4000 ml Activate 10.000 g charcoal AgNO3 1.000 ml (250 mg/100 ml) Sucrose 20.000 g H20 topped to 1000 ml Agar 5.000 g
Results:
[0249] Only a small share of the embryos of both genotypes that are grown at 2 C. or 9 C. will have developed a root after 8-16 weeks.
[0250] Very few of the embryos of both genotypes that are grown at 10 C. will have developed a root after 8-16 weeks.
[0251] The majority of the embryos of both genotypes that are grown at 4 C. or at 3-7 C. will have developed a root after 12 weeks, a smaller number already after 8 weeks and a smaller number only after 16 weeks.
[0252] These small plantlets thus obtained that are suitable for further growth under the shoot inducing conditions are characterized in that the cotyledonary embryo has developed a radicle (root), the cotyledons are 1-5 mm long and green, and the hypocotyl is about 10-20 mm long.
Example 10: Shoot Development of Small Plantlets of Abies bornmlleriana
[0253] From each of the two Abies bornmlleriana genotypes, 30-40 small plantlets that will have developed a root when grown under each of the root inducing conditions described in Example 3 are selected for growing under shoot inducing conditions. Most of the selected plantlets will be plants that have grown at a cold period of 3-7 C. as described in Example 9 since this will be the temperature by which most of the somatic embryos successfully develops a root.
[0254] As mentioned in Example 3, the small plantlets included in this experiment are characterized in having a root, the cotyledons are 1-5 mm long and green, and the hypocotyl is about 5-15 mm long.
[0255] The small plantlets are moved into plastic boxes (Eco2box, oval, Duchefa Bichemie) of 125 mm length65 mm width80 mm height having a lid. The lid was partially open for air-movement. The humidity RH % was kept above 90.
[0256] Each box contained 100 ml of the sugar-free medium 47.07.
Medium 47.07: 1 Liter
[0257]
TABLE-US-00023 Duchefa WPM M0219 0.590 g DCR 2 0.250 ml AgNO3 (250 mg/100 ml) 1.000 ml Inositol 0.100 g Water topped to 1000.0 ml Conductivity of solution
751-830 S pH 5.7 Agar 7.00 g Phytagel 0.70 g
: conductivity is measured in the solution of 1000 ml, containing WPM, DCR 2, AgNO3 and inositol, after being set to pH 5.7
Recipes Used to Provide Medium 47.07:
[0258]
TABLE-US-00024 Duchefa WPM 0.590 g M0219 DCR-2 250 ml Thiamine-HCl 0.100 g Pyridoxine HCl 0.050 g Nicotinic Acid 0.050 g Glycine 0.200 g H2O topped to 100 ml AgNO3 1 ml (250 mg/100 ml) Inositol 0.1 g Phytagel 0.7 g Gelrite, Duchefa Biochemie, CAS 71010-52-1 Italiensk Agar 7 g Plantager S1000, B&V Srl, Italy, CAS 9002-18-0 H2O topped to 1000 ml (pH = 5.7)
[0259] Each box comprises approximately 24-30 small plantlets of the same genotype that will have been grown under the same root inducing conditions.
[0260] The small plantlets are grown for 4 to 8 weeks at 15 in 24 h periods of white LED light of 70 to 400 mol/m.sup.2s.
[0261] The light intensities are measured inside each box as close to the cotyledons as possible. The light intensities ranges from approximately 100 to 400 mol/m.sup.2s depending on the distance from the nearest light source.
Results:
[0262] The majority of the small plantlets of both genotypes will have developed new cotyledons, which also have turned darker green showing a normal surface of a fully developed conifer needle after 6 weeks. The total height of these emblings above the root are generally 8-22 mm and the root is generally of a length in the range of 1-30 mm.
[0263] A smaller amount of the emblings of both genotypes will have developed same characteristics after only 4-5 weeks, and a smaller amount of the plants of both genotypes will have developed same after 7-8 weeks.
[0264] The majority of the emblings of both genotypes that will have developed new cotyledons, which also have turned darker green showing a normal surface of a fully developed conifer needle after 4 to 8 weeks will be plantlets that have been grown at 3-7 C. in the previous root inducing step
Example 11: Development of Small Plants of Abies bornmlleriana
[0265] All well developed emblings with new cotyledons obtained from Example 10 will be transferred to small forest C7 Jiffy pots of 25 mm plug for continued growth and will be grown for 6-8 weeks at 25 C. in 24 h periods of white LED light of 100 to 400 mol/ms. The humidity RH % will slowly be decreased to 70.
[0266] Almost all plants will have survived and will subsequently be selected for continued nursery production.
Example 12: Comparison of Developmental Stage of Somatic Embryos or Small Plantlets Grown without any Root Inducing Conditions
[0267] A number of mature somatic embryos of two different genotypes of Abies nordmanniana obtained from Example 2 and two different genotypes of Abies bornmlleriana obtained from Example 8 is grown at the shoot inducing conditions described in Example 4 and in Example 10, respectively, i.e. without previous growth under the root inducing conditions described in Example 3 and in Example 11, respectively.
[0268] For all 4 genotypes, the survival rate of the plantlets will be significantly lower compared to genotypes of the same kind, that are to be grown under both the root inducing conditions described in Example 3 and in Example 9, respectively, and under the shoot inducing conditions described in Example 4 and in Example 10, respectively.
Example 13: Comparison of Developmental Stage of Small Plantlets Grown without any Shoot Inducing Conditions
[0269] A number of small rooted plantlets of two different genotypes of Abies nordmanniana and of Abies bornmlleriana, respectively, obtained from Example 3 and from Example 9, respectively, will be grown into small plantlets under the conditions described in Example 5 and in Example 11, respectively, i.e. without previous growth under the shoot inducing conditions described in Example 4 and in Example 10, respectively.
[0270] For all 4 genotypes, the survival rate of the plantlets will be significantly lower compared to genotypes of the same kind, that are to be grown under both the root inducing conditions described in Example 3 and in Example 9, respectively, and under the shoot inducing conditions described in Example 4 and in Example 10, respectively.
Example 14: Features Influencing the Survival Rate
[0271] The impact of growing small plantlets in air-tight boxes vs. growing these in boxes wherein part of the lid had been removed was tested in order to find out whether this had any influence on the development and survival rate of the small plantlets. Further, different media were tested.
[0272] The test was performed on 240 rooted embryos/small plantlets of Abies nordmanniana of two different genotypes that had been provided, developed and subsequently grown as described in Examples 1 to 3, respectively, where the conditions described in Example 3 were selected to growth of 3-7 C. for 12 weeks.
[0273] The small plantlets had thus all developed a root, cotyledons of 1-5 mm long and green, and a hypocotyl of about 5-15 mm. At this developmental stage, the small plantlets were separated into two groups of 120 plantsone group for growing in air-tight boxes and the other group for growing in boxes where continuous access to air was secured by removing approximately 20% of the lid.
[0274] Each group of 120 rooted embryos were divided into four groups of 30 plants each and grown in a box containing one of the following four media:
[0275] 47.037: Double strength of media 47.07 as described in Examples 4 and 10, respectively
[0276] 47.06: Double strength of media 47.07 as described in Examples 4 and 10, respectively with the addition of 3 types of auxins and Cu
[0277] 47.07: This media is described in Examples 4 and 10, respectively
[0278] 47.08: Media 47.07 as described in Examples 4 and 10, respectively with the addition of 3 types of auxins and Cu
[0279] The plants were then grown in the same chamber for 6 weeks at 15 in 24 h periods of white LED light of 100 to 400 mol/ms.
[0280] After the 6 weeks, the survival rate was determined and on the survived plantlets, the percentage of the plantlets having developed a green top, a bud and having a white root was determined. In some of the survived plantlets the root had turned black. A black root indicates that the plantlets will have less chance of surviving when transferred into a plug or soil.
Results:
[0281] As evident from the below table, the a significantly higher percentage of the small plantlets that were grown in boxes where part of the lid was open to air survived compared to the small plantlets that were grown in air-tight boxes with the same media. The access to air did, however, not appear to influence the percentages of the survived plantlets having a green top, a bud or a white root.
TABLE-US-00025 Media % living % green top % with bud % white root 47.037 51 95 72 16 47.06 18 78 22 12 47.07 56 100 73 8 47.08 26 49 13 0 47.037 + air 77 97 68 12 47.06 + air 33 55 24 3 47.07 + air 77 93 75 6 47.08 + air 35 54 12 0
[0282] It can thus be concluded, that it is beneficial for the survival rates that plants grown under the shoot inducing growth conditions according to the method of the present invention are grown under circumstances where access to air is secured.
[0283] It is also evident from the results that the media wherein the rooted embryos are grown greatly influences both the percentage of the small plantlets that survive, and also the percentage of plantlets having a green top, a bud and a white root.
[0284] Thus, finding the optimum media for a specific species may require some routine optimization tests.
Example 15: Impact of Temperature at the Shoot Inducing Step
[0285] 8 boxes wherein the lid was partially open comprising approximately 30 small plantlets of Abies nordmanniana of different genotypes was obtained from a process comprising the steps as described in Examples 1, 2 and 3, wherein the cold treatment period in the root inducing step was 3-7 C. for 12 weeks were grown at different temperatures in order to find the more beneficial temperature for inducing top shoot formation while at the same time stimulating further root development.
[0286] The boxes were placed at one of the following 8 temperatures for 4 weeks in 24 h light periods from LED at light intensities from 100-300 mol/m.sup.2s and subsequently, the percentages of the small plantlets having developed into emblings having shoots and further rooting was evaluated.
[0287] The results are shown in the below table and in
TABLE-US-00026 No. having % having No. having % having No. of developed developed developed developed Temperature Box no. plants top shoot top shoot white roots white roots 10 1 30 3 10.0 19 63.3 12.1 2 38 6 15.8 22 57.9 14.3 3 30 4 13.3 17 56.7 16.4 4 31 7 22.6 5 16.1 18.6 5 33 9 27.3 0 0.0 20.7 6 17 12 70.6 0 0.0 22.9 7 29 7 24.1 1 3.4 25 8 24 5 20.8 0 0.0
[0288] It is evident from these results that at temperatures ranging from 10 C. to approximately 16 C., the percentage of plantlets that develops a top shoot is lower than at temperatures ranging from approximately 16 C. to 25 C.
[0289] It is further evident that at temperatures ranging from 10 C. to approximately 16 C., the percentage of plantlets that develops new white roots is higher than at temperatures ranging from approximately 16 C. to 25 C.
[0290] Thus, if both the rooting and the development of a top shoot is to be prioritized, then the most suitable temperature is approximately 14-17 C.
Example 16: Production of Abies nordmanniana Christmas Trees
[0291] A total of 327 trees from 9 clones of small Abies nordmanniana emblings obtained from the method described in Example 6 (i.e. as a result of being developed in accordance with examples 1, 2, 3, 4, 5, wherein the root inducing step comprised growing the somatic embryos at 3-7 C. for 8 to 12 weeks and at the shoot inducing step at light intensities of 150-350 mol/m.sup.2s) were planted in soil in forest in Denmark in areas that were already used for the production of Christmas trees. The appearance and size of the trees were registered regularly. After 8 years of growth in the field, it was concluded that the majority of the trees had survived and the appearance of the trees was compared between the genotypes thereby identifying the genotypes that are most suitable for the production of Christmas trees. It was moreover concluded that trees of the same genotypes were nearly identical of shape and size.
Example 17: Rooting of Fully Mature Somatic Embryos of Abies nordmanniana and Abies bornmlleriana
[0292] In total 3459 fully mature cotyledonary embryos (corresponding to stage 7 as shown in
[0293] Number of embryos employed in experiment by species and genotype:
TABLE-US-00027 Temperature C. Total Total Clone 2 4 5 8 10 15 20 clone species Abies nordmanniana 0.221 40 60 100 40 40 40 40 360 0.690 40 80 120 40 40 40 40 400 0.710 40 60 101 40 40 40 40 361 0.715 40 59 100 40 40 40 40 359 0.721 40 60 100 40 40 40 40 360 1840 Abies bornmlleriana 0.854 40 140 200 40 40 40 40 540 0.856 40 79 119 40 40 40 40 398 938 Total 360 658 1002 360 360 360 359 3459
[0294] After 8, 9, 10 and 11 weeks, the embryos (that had now developed into small plantlets) were checked for root formation and embryos with a well-developed root (longer than 5 mm, such as those shown in
[0295] Subject to the shoot inducing conditions described in Example 19, the obtained Good emblings (such as those shown in
Results:
[0296] As shown in
[0297] Initial rooting has a strong impact (a significant correlation of 0.96) on the final number of good emblings after finalizing step b) (data not shown) and further growing in step c) for additional 8 weeks into well developed emblings of acceptable quality (having at least one rosette of mostly green needles),
[0298] Significant effect of genotype on rooting was seen. The tested genotypes were in general variable (significant interaction clone by temperature) and showed deviating rooting patterns as function of rooting temperaturesome more sensitive to temperature than others, although mostly all clones showed the average pattern of declining rooting by increased temperature,
[0299] The two tested species Abies nordmanniana and Abies bornmlleriana showed the same general response to temperature in rooting embryos as well as good emblings after finalizing step b) and growing the small emblings in step c) for additional 8 weeks into well developed emblings of acceptable quality, as shown in
[0300] The percent of rooted embryos increased in general from week 8 to 11, whereas nearly no new rooting was seen in week 12 and thereafter. The lowest temperatures 2 C., 4 C. and 5 C. had also the steadiest increase in rooted individuals and reached the highest level of accumulated rooting after 11 weeks as shown in
[0301] The number of weeks needed to achieve rooting depended also on genotypeat least 9 weeks was needed for some of the tested clones to reach maximum, whereas others benefited from 11 weeks,
[0302] Examples of suitable small plantlets that were chosen for further growth under shoot inducing conditions are shown in
Example 18. Rooting of Fully Mature Somatic Embryos of Abies nordmanniana and Abies bornmllerianaEffect of Initial Embryo Quality
[0303] The 3459 fully mature cotyledonary embryos used in Example 17 were prior to any treatment individually evaluated using an initial embryo score assigning a quality score from 4 to 9. Embryos that had obtained scores 1 to 3 were not included in the 3459 embryos selected for growth.
Initial Embryo Quality Score at Time of Initiating Rooting:
[0304] Score Description
TABLE-US-00028 Embryos deformed 1 Very deformed embryos, embryos accreted, cotyledon's grow from center of hypocotyl, cotyledons deformed 2 Just long hypocotyl, either straight, bent or shrimp-like 3 Looks like and embryo, but to many cotyledons, swollen hypocotyl, small and bent Slightly swollen hypocotyl, minimum 2 cotyledons 4 hypocotyl less than or equal 1.99 mm 5 hypocotyle 2.0-3.99 mm 6 hypocotyle longer or equal 4.0 mm Slim hypocotyle minimum, 2 cotyledons 7 hypocotyle less than or equal 3.99 mm 8 hypocotyle 4.0-5.99 mm 9 hypocotyle longer or equal 6.0 mm
[0305] After 8, 9, 10 and 11 weeks of growth at the conditions described in Example 17, the embryos were checked for root formation and embryos with a well-developed root (longer than 5 mm) were selected (when the individual embryo first meet the criteria and week of transfer was noted) for further growth under the shoot inducing conditions described in Example 19.
[0306] Good emblings (such as those shown in
Results:
[0307] Percent rooting embryos and percent good emblings increased significantly from embryos having an initial embryo quality score of 4 until 9, respectively a range of rooting from 24 percent to 82 percent and for good emblings 3 percent to 24 percent,
[0308] Clones showed some minor deviating patterns in rooting as function of initial embryo score (significant interaction clone by initial embryo score)although mostly all showed the average pattern of increasing rooting due to increased initial embryo quality,
[0309] Percent rooting embryospooled into groups based on initial embryo scores (scores 4 to 9, respectively) performed very similar across temperatures despite significant interaction (scale effect) between initial rooting score and rooting temperature,
Example 19: Shoot Development in Rooted Somatic Embryos of Abies nordmanniana and Abies bornmlleriana Under Light Treatment
[0310] From the five Abies nordmanniana and two Abies bornmlleriana genotypes, 1455 small plantlets (of a developmental stage similar to those shown in
TABLE-US-00029 Score Number of plants per clone and Initial embryo score Clone 4 5 6 7 8 9 Total 0.221 1 10 24 67 39 17 158 0.690 8 31 57 117 83 37 333 0.710 4 15 35 83 65 19 221 0.715 1 4 17 33 44 7 106 0.721 9 19 37 41 48 34 188 0.854 4 5 29 58 77 32 205 0.856 17 25 45 77 63 17 244 Total 44 109 244 476 419 163 1455
[0311] As mentioned in Example 17, small plantlets selected for being included in this experiment were characterized in having a root, the cotyledons were 1-5 mm long, and the hypocotyl was about 5-15 mm long.
[0312] The small plantlets were moved into plastic boxes (Eco2box, oval, Duchefa Bichemie) of 125 mm length65 mm width80 mm height having a lid. The lid was partially open for air-movement. The humidity RH % was kept above 90.
[0313] Each box contained 100 ml of the sugar-free medium 47.07 (same recipe as shown in Example 3).
[0314] As mentioned in Example 17, small plantlets included in this experiment were characterized in having a root. Randomization of this step of the experiment was approximated by transferring rooted embryos from a given clone and petri dish (treatment) into separate boxes. This procedure was repeated during weeks 8 to 11.
[0315] At each week, 8-11, boxes were randomized to seven light conditions: of white LED light of 50, 100, 150, 200, 250, 300 and 400 mol/m.sup.2s and were kept at that light condition for 8 weeks at 15 in 24 h light periods.
[0316] The light intensities were measured on top of each box as close to the cotyledons as possible, and thereafter accounting for the light reducing effect of the lit. The light intensities ranged from 50 to 400 mol/m.sup.2s depending on the distance from the nearest light source.
Results:
[0317] The majority of the small plantlets of both species had developed into emblings having new cotyledons, which also had turned darker green showing a normal surface of a fully developed conifer needle; after 8 weeks. Examples of such emblings are shown in
[0318] The number of cotyledons developed after 8 weeks varied significantly between light intensities, from 4.0 to 5.4,
[0319] The length of the longest cotyledon on each of the small plantlets were measured after 8 weeks and ranged across light intensities from 7.8 mm to 8.6 mm. The length of the longest cotyledon seems to have a maximum around a light intensity of 100 to 200 mol/m.sup.2s,
Example 20: Development of Emblings of Abies nordmanniana and Abies bornmlleriana Grown in Plugs for 8 Weeks
[0320] 1455 emblings (of a developmental stage as shown in
[0321] Good emblings were recorded after finalizing step b) and further growing the emblings in step c) for additional 8 weeks into well developed emblings of acceptable quality, (i.e. having at least one rosette of mostly green needles.
Results:
[0322] Although grown under even conditions for eight weeks in standardized plugs, there was a strong and significant effect on mortality due to the previous light treatment as described in Example 19. The mortality was most severe for the plants that had been grown at the lowest light intensity, 50 mol/m.sup.2s, which was significantly different from all other treatments and mortality was at its lowest at a light intensity of 200 mol/m.sup.2s,
[0323] Also for the percentage of good emblings, there was a strong and significant effect of the previous light treatment. Increasing light intensity from 50 mol/m.sup.2s to 200 mol/m.sup.2s increased the percentage of good emblings from 13 percent to 30 percent,
[0324] Combining the observed figures for the desired good emblings and realized mortality there is an optimum for the number of good emblings obtained when growing the small plantlets in step b) at 100 to 200 mol/m.sup.2s, with an optimum closest to 200 mol/m.sup.2s.
[0325] The percent of successful good emblings obtained from the total of embryos started increased with lowering the temperature and showed a maximum in the range of 2 C. to 4 C. in step (a) (the root inducing conditions) and subsequently growing the emblings a LED light intensity of at least 200 mol/m.sup.2s in step (b) (the shoot inducing conditions), as seen in the response surface shown in
[0326] The percent of successful good emblings obtained from the total of embryos started was moreover strongly influenced by the temperature applied in the (a)the rooting temperature and by the initial embryo quality. Overall, the maximum number of successful good emblings obtained after growth at first the root-inducing conditions and subsequently at the shoot inducing conditions was achieved by growing the fully mature cotyledonary embryos at a temperature of 4 C. and by using embryos having the best initial embryo quality (score 9) as seen in the response surface shown in
LITERATURE
[0327] Farjon and Rushfort, 1989. A classification of Abies miller (Pinaceae). Notes of the Royal Botanic Garden Edinburgh 46(1):59-79. [0328] Hggman et al., 1999, Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction, Journal of Experimental Botany, Vol. 50, pp. 1769-1778 [0329] Liu 1971. A monograph of the genus Abies. Taipei, Taiwan: Department of Forestry, College of Agriculture, National Taiwan University. [0330] Malabadi and Nataraja, 2007, Plant Regeneration via Somatic Embryogenesis Using Secondary Needles of Mature Trees of Pinus roxburghii Sarg, International Journal of Botany 3, pp. 40-47 [0331] Nawrot-Chorabik, 2012, Somatic Embryogenesis in Forest Plants, Chapter 20 in Embryogenesis, book edited by Ken-ichi Sato, available online on https://www.intechopen.com/books/embryogenesis/somatic-embryogenesis-in-woody-plants [0332] Nawrot-Chorabik, 2016, Plantlet regeneration through somatic embryogenesis in Nordmann's fir (Abies nordmanniana), J. For. Res. 27, pp. 1219-1228 [0333] Nrgaard J V, 1997, somatic embryo maturation and plant regeneration in Abies nordmanniana Lk, Plant Science vol 124, pp. 211-22 [0334] Pullman et al., 2016, Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation, New Forests 47, pp. 453-480 [0335] US 2009/0280566 [0336] U.S. Pat. No. 5,187,092 [0337] U.S. Pat. No. 5,731,204 [0338] U.S. Pat. No. 6,897,065 [0339] von Arnold and Clapham, 2008, Spruce Embryogenesis, Plant Embryogenesis, Volume 427 of the series Methods In Molecular Biology pp 31-47 [0340] von Arnold et al., Norway spruce as a model for studying regulation of somatic embryo development in conifers, Vegetative Propagation of Forest Trees pp. 351-372 in Somatic EmbryogenesisFundamental Aspects and Applications. Springer International Publishing 2016