METHODS FOR IMPROVING PLANT EMBRYO DEVELOPMENT
20170175076 ยท 2017-06-22
Assignee
Inventors
Cpc classification
A01H4/005
HUMAN NECESSITIES
C12N5/0025
CHEMISTRY; METALLURGY
A01H4/006
HUMAN NECESSITIES
International classification
C12N5/00
CHEMISTRY; METALLURGY
Abstract
Provided is a method of improving plant embryo development and/or germination. The method comprises developing plant embryos in the presence of nitric oxide (NO). The method entails the step of incubating plant embryogenic suspensor mass (ESM) in, or on, a development medium containing a nitric oxide donor for a period of time to develop mature somatic embryos. The method disclosed herein improves the yield and/or the germination frequency of plant embryos as compared to plant embryos developed by conventional methods without a nitric oxide donor in the medium.
Claims
1. A method of improving plant embryo development and/or germination comprising incubating plant embryogenic suspensor mass (ESM) in, or on, a development medium comprising a nitric oxide donor for a period of time to develop mature somatic embryos, wherein the yield and/or the germination frequency of mature somatic embryos are improved as compared to embryos developed by conventional methods without a nitric oxide donor in the medium.
2. The method of claim 1, further comprising incubating the ESM in, or on, a maintenance medium for a period of time before incubating the ESM in, or on, the development medium comprising the nitric oxide donor.
3. The method of claim 2, wherein the maintenance medium comprises a nitric oxide donor.
4. The method of claim 2, wherein the ESM is incubated in, or on, the maintenance medium in dark.
5. The method of claim 1, wherein the ESM is incubated in, or on, the development medium in dark.
6. The method of claim 1, wherein the plant is a tree.
7. The method of claim 1, wherein the plant is a conifer.
8. The method of claim 1, wherein the nitric oxide donor is sodium nitroprusside (SNP).
9. The method of claim 1, wherein the nitric oxide donor is nitrosyl ethylenediaminetetraacetate ruthenium (II) ([Ru(NO)(Hedta0)]).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0008]
[0009]
[0010]
[0011]
DETAILED DESCRIPTION
[0012] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the disclosed technology.
[0013] As used herein, the term embryogenic suspensor mass (ESM) refers to early stage embryos in the process of multiplication by budding and cleavage.
[0014] As used herein, the term embryogenic tissue refers to an aggregate of tens to hundreds of embryogenic cells that form an embryogenic suspensor mass.
[0015] As used herein, the term somatic embryo refers to an embryo produced by culturing embryogenic tissue by standard methods under laboratory conditions in which the cells comprising the tissue are separated from one another and urged to develop into minute complete embryos.
[0016] As used herein, the phrase improving plant embryo development means increasing the yield of plant embryos, while the phrase improving plant embryo germination means increasing the germination frequency of plant embryos. As disclosed herein, developing embryos in or on a medium containing a nitric oxide donor unexpectedly improves plant embryo development and plant embryo germination versus embryos developed in or on a medium without a nitric oxide donor.
[0017] As used herein, the term germination frequency refers to the number, proportion, percentage or fraction of germinants in a particular population of somatic embryos.
[0018] The somatic embryogenesis process is a process to develop plant embryos in vitro. This process has significant uses in forestry and in making wood products. Methods for producing plant somatic embryos are known in the art and have been previously described (see, e.g., U.S. Pat. Nos. 4,957,866; 5,034,326; 5,036,007; 5,041,382; 5,236,841; 5,294,549; 5,482,857; 5,563,061; and 5,821,126). Generally, the somatic embryogenesis process includes the steps of (1) initiating formation of embryogenic tissue (initiation or induction), such as embryogenic suspensor mass (ESM), which is a white mucilaginous mass that includes early stage embryos having a long, thin-walled suspensor associated with a small head with dense cytoplasm and large nuclei; (2) multiplying and mass producing the embryogenic tissue (multiplication or maintenance); (3) developing and forming mature cotyledonary somatic embryos (development); and (4) post-development processes such as stratification, singulation, and conditioning over water (COW), and then germination or placement into manufactured seeds.
[0019] Nitric oxide (NO) is an endogenous signaling molecule involved in many physiological processes in plants and acts as a mediator in the developmental and physiological processes including embryogenesis. Simontacchi et al., Nitric oxide as a key component in hormone-regulated processes, Plant Cell Rep. 32: 853-866 (2013). Plants can synthesize NO from nitrite by nitrate reductase (NR) or by oxidation of arginine and polyamines by enzymes. de Osti et al. reported that sodium nitroprusside (SNP) is an effect NO donor for plant cell culture and that growing plant cultures in liquid media containing SNP promotes cellular growth. de Osti et al., Nitrosyl ethylenediaminetetraacetate ruthenium (II) complex promotes cellular growth and could be used as nitric oxide donor in plants, Plant Science 178: 448-453 (2010). Others reported that SNP promotes multiplication and regeneration of certain plants. Han et al., Sodium nitroprusside promotes multiplication and regeneration of Malus hupehensis in vitro plantlets, Plant Cell Tiss. Organ. Cult. 96: 29-34 (2009). However, it has not been reported that nitric oxide acts as a starting signal in regulation of growth and morphogenesis in somatic embryogenesis of plants, particularly for conifer species.
[0020] As described herein, it has been unexpectedly discovered that providing nitric oxide to a medium by adding a nitric oxide donor, such as sodium nitroprusside or nitrosyl ethylenediaminetetraacetate ruthenium (II) ([Ru(NO)(Hedta0)]), to a standard maintenance medium and/or development medium during embryo development stage improves the yield and/or germination frequency of plant embryos as compared to the embryos developed in or on standard media without a nitric oxide donor.
[0021] The present technology provides a method of improving plant embryo development and/or germination. In several embodiments, methods in accordance with the present technology include incubating the ESM in, or on, a development medium containing a nitric oxide donor for a period of time to develop mature somatic embryos, wherein the yield and/or the germination frequency of mature somatic embryos are improved as compared to embryos developed by conventional methods without a nitric oxide donor in the medium. Optionally, the methods include incubating the ESM in, or on, a maintenance medium for a period of time before incubating in, or on the development medium containing a nitric oxide donor. The maintenance medium may or may not contain a nitric oxide donor.
[0022] The plant embryos are maintained and developed in dark for certain period of time according to standard protocols. In certain embodiments, the ESM is incubated in, or on a maintenance medium for about 3 days, about 5 days, about 1 week, or about 10 days. In certain embodiments, the ESM is incubated in, or on a development medium for about 8 weeks, about, 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks.
[0023] The nitric oxide in a medium can be provided by including a nitric oxide donor in the medium. It is within the purview of one skilled in the art to select a suitable nitric oxide donor and a suitable concentration of the nitric oxide donor. For example, nitric oxide donors that can be used include sodium nitroprusside (SNP) and nitrosyl ethylenediaminetetraacetate ruthenium (II) ([Ru(NO)(Hedta0)]). The plant ESM is maintained and/or developed in a medium containing a nitric oxide donor at a concentration ranging from about 5 M to about 250 M.
[0024] In certain embodiments, when a nitric oxide donor is included in a development medium, the concentration of the nitric oxide donor is from about 10 M to about 250 M, from about 30 M to about 200 M, from about 50 M to about 150 M, from about 75 M to about 100 M, about 10 M, about 50 M, about 75 M, about 100 M, about 125 M, about 150 M, or about 200 M. However, it has been unexpectedly discovered that a higher concentration of nitric oxide does not necessarily result in a greater yield or a higher germination frequency.
[0025] The optimal concentration of a nitric oxide donor in the development medium may be different for different species of plants. For example, loblolly pine ESM is incubated in, or on a development medium containing a nitric oxide donor at a concentration from about 30 M to about 250 M, from about 50 M to about 100 M, about 50 M, about 100 M, or about 200 M. Douglas-fir ESM is incubated in, or on a development medium containing a nitric oxide donor at a concentration from about 5 M to about 50 M, from about 10 M to about 30 M, about 5 M, about 10 M, or about 30 M
[0026] In certain embodiments, the maintenance medium also contains a nitric oxide donor. The yield and/or germination frequency of the plant somatic embryos may be further increased when the maintenance medium also contains nitric oxide, provided by a nitric oxide donor. In some embodiments, the maintenance medium contains a nitric oxide donor in a concentration lower than the concentration of the nitric oxide donor in the development medium. For example, the concentration of a nitric oxide donor in a maintenance medium is about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, or about 10 M.
[0027] Surprisingly, not only the yield but also the germination frequency of plant embryos is increased when embryos are developed in the presence of nitric oxide. Nitric oxide is not needed during germination stage to achieve the improvement described in this disclosure.
[0028] In some embodiments, the nitric oxide donor in the maintenance medium is the same as the nitric oxide donor in the development medium. In other embodiments, the nitric oxide donor in the maintenance medium is different from the nitric oxide donor in the development medium.
[0029] The methods disclosed herein can be applied to develop different plant somatic embryos, for example, somatic embryos of trees, such as conifers. In some embodiments, the conifer is loblolly pine or Douglas-fir. In certain embodiments, the maintenance medium or the development medium used in this technology is a liquid medium, a semi-solid medium, or a solid medium.
[0030] After the development period, the somatic embryos can optionally be transferred to a maturation medium, and then subjected to post-development steps such as singulation, stratification, germination, placement into manufactured seeds, and transferring to soil for further growth and development.
[0031] As shown in the working examples, the presence of nitric oxide in the development medium and/or the maintenance medium increased the yield of plant embryos by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 80% as compared to the yield of embryos developed by conventional methods without nitric oxide. Additionally or alternatively, the presence of nitric oxide in the development medium and/or the maintenance medium increased the germination frequency of plant embryos by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 80% as compared to the germination frequency of embryos developed by conventional methods without nitric oxide.
[0032] The following examples are provided for the purpose of illustration and are not intended to limit the disclosure in any way.
EXAMPLES
Example 1
[0033] Prior patents, for example, U.S. Pat. Nos. 5,036,007; 5,563,061; and 8,999,713, disclose culturing conifers. The contents of these patents are incorporated herein by reference in their entireties. This example shows the compositions of the media used in the examples that follow.
TABLE-US-00001 TABLE 1 Douglas-fir Basic Culture Media (BM) Constituent mg/L Basal Salts KNO.sub.3 varies CaCl.sub.26H.sub.2O 200.0 Ca(NO.sub.3).sub.22H.sub.2O varies KH.sub.2PO.sub.4 340.0 MgSO.sub.47H.sub.2O 400.0 MnSO.sub.4H.sub.2O 20.8 ZnSO.sub.47H.sub.2O 8.0 CuSO.sub.45H.sub.2O 0.024 FeSO.sub.47H.sub.2O 27.85 Na.sub.2EDTA 37.25 H.sub.3BO.sub.3 5.0 NaMoO.sub.42H.sub.2O 0.20 CoCl.sub.26H.sub.2O 0.025 KI 1.00 Organic Additives Myo-Inositol varies ThiamineHCl 1.00 Nicotinic acid 0.50 PyridoxineHCl 0.50 Glycine 2.00 L-Glutamine varies Casamino acids 500.0 Sugar varies pH 5.7
TABLE-US-00002 TABLE 2 Formulations of Douglas-fir Media BM-2 BM-3 BM-4 BM-5 Mainte- Singu- Devel- Germi- nance lation opment nation KNO.sub.3 1250 1050 2500 1170 Ca(NO.sub.3).sub.22H.sub.2O 200 220 Myo-Inositol 5000 100 100 100 L-Glutamine 1000 1000 750 Amino 290 acid mixture.sup.(2) Maltose 30,000 20,000 25000 Sucrose 20000 PEG 8000 190,000 2.4-D 1.1 N6-Benzyladenine 0.22 Kinetin 0.22 Abscisic acid 10/5/5 10 Gibberellin 4/7 7.5 Activated charcoal 1000 2500 Agar 5000 .sup.8000.sup.(3) Gelrite 3000 .sup.(1)All units are in mg/L (or ppm). .sup.(2)L-Proline - 100, L-Asparagine - 100, L-Arginine - 50, L-Alanine - 20, L-Serine - 20. .sup.(3)Tissue culture agar.
The pH of all media are adjusted to 5.7.
Stratification medium BM-5 without Agar.
GELRITE: gellan gum (an agar substitute);
2,4-D: 2,4-dichlorophenoxyacetic acid;
Kinetin: 6-furfurylaminopurine.
TABLE-US-00003 TABLE 3 Formulations of Loblolly Pine Media BM 2 BM 4 BM 5 BM 6 Mainte- BM 3 Devel- Strati- Germi- nance Rinse opment fication nation Salts (mg/L) NH.sub.4NO.sub.3 150 150 150 150 206.25 KNO.sub.3 909.9 909.9 909.9 909.9 1170 Ca(NO.sub.3).sub.24H.sub.2O 236.15 236.15 236.15 236.15 MgSO.sub.47H.sub.2O 246.5 246.5 246.5 246.5 185 Mg(NO.sub.3).sub.26H.sub.2O 256.5 256.5 256.5 256.5 MgCl.sub.26H.sub.2O 50 50 50 50 KH.sub.2PO.sub.4 136 136 136 136 85 CaCl.sub.22H.sub.2O 50 50 50 50 220 KI 4.15 4.15 4.15 4.15 0.415 H.sub.3BO.sub.3 15.5 15.5 15.5 15.5 3.1 MnSO.sub.4H.sub.2O 10.5 10.5 10.5 10.5 8.45 ZnSO.sub.47H.sub.2O 14.4 14.4 14.4 14.4 4.3 Na.sub.2MoO.sub.42H.sub.2O 0.125 0.125 0.125 0.125 0.125 CuSO.sub.45H.sub.2O 0.125 0.125 0.125 0.125 0.0125 CoCl.sub.26H.sub.2O 0.125 0.125 0.125 0.125 0.0125 FeSO.sub.47H.sub.2O 27.87 27.87 27.87 13.93 13.93 Na.sub.2EDTA 37.26 37.26 37.26 18.63 18.63 Vitamins/Amino Acids (mg/L) Nicotinic Acid 0.5 0.5 0.5 0.5 0.5 Pyridoxine HCl 0.5 0.5 0.5 0.5 0.5 Thiamine HCl 1 1 1 1 1 Glycine 2 2 2 2 2 L-proline 100 100 100 L-asparagine 100 100 100 L-arginine 50 50 50 L-alanine 20 20 20 L-serine 20 20 20 PEG 8000 mw 10000 Sugar/Agar mg/L Myo-Inositol 200 100 100 100 100 Casein 500 500 500 500 hydrolysate L-glutamine 1000 1000 1000 1000 Sucrose 25000 20000 Maltose 30000 25000 25000 Glucose 10000 GELRITE 2500 2500 TC Agar 8000 Activated carbon 1000 1000 2500 Hormones mg/L ABA 0 25 2,4-D 1.1 BAP 0.63 0.1 Kinetin 0.61 0.1 epi-brassinolide 0.96 GELRITE: gellan gum (an agar substitute); ABA: abscisic acid 2,4-D: 2,4-dichlorophenoxyacetic acid; BAP: 6-benzylaminopurine; Kinetin: 6-furfurylaminopurine.
Example 2
[0034] This example shows the effects of increased concentrations of nitric oxide on somatic embryo development and germination of loblolly pine.
Materials and Method
[0035] Loblolly pine embryogenic suspensor mass (ESM) from four different genotypes were maintained in a standard liquid maintenance medium with 5 M SNP or without SNP (control) at room temperature in dark for one week. One mL of 1:1 mix of settled cells and rinse medium of each genotype was plated with Decotex 22 membranes to standard semi-solid development media containing various concentrations of SNP, 50 M, 200 M and 500 M. A standard development medium containing no SNP was used as a control. The plating procedure was performed as described in U.S. Pat. No. 8,925,245, the content of which is incorporated by reference in its entirety. Each plating consisted of 6 plates of each genotype. Development was carried out for 12 weeks at room temperature in dark. The plates were visually assessed at 9 weeks.
[0036] Symmetrical embryos without obvious defects that had four or more cotyledons without any fused cots or cots sprouting from the center were chosen. Embryos selected had all three parts, cotyledons, hypocotyl, and radicle regions although the sizes of embryos varied. A slight curve to the hypocotyl region was acceptable. Embryos with split radicle regions were avoided. Embryos chosen were opaque, and acceptable colors were shades of white, yellow or green. No translucent embryos or vitrified green embryos were chosen. The good quality embryos are classified in two categories: Category 1 embryos have developed root (about 1 mm), hypocotyl, epicotyl (about 5 mm) and at least 5 leaves; and Category 2 embryos have developed root, hypocotyl and epicotyl.
[0037] Post-development, the loblolly pine somatic embryos developed with various concentrations of SNP were subjected to stratification on BM-5 medium for 4 weeks at 2-4 C. in dark, followed by conditioning over water (COW) for two weeks. Subsequently, the somatic embryos were hand transferred for germination using the method as described in U.S. Pat. No. 9,090,872, the content of which is incorporated by reference herein in its entirety.
Results
[0038] Visual Inspection Results
[0039] Volume of SCV (settled cell volume) was recorded for each flask and is shown in Table 4 below. Although the treatment with SNP in the maintenance media slightly improved by visual inspection in three out of four genotypes compared to control, there was no clear difference in settled cell volume.
TABLE-US-00004 TABLE 4 SCV growth after one week in maintenance media Mainte- Initial Final nance Volume Volume SCV Genotype Medium (mL) (mL) Appearance Genotype A Control (no SNP) 50 120 Looks good 5 M SNP 50 120 Looks a little better than control Genotype B Control (no SNP) 50 59 Stressed 5 M SNP 50 85 Less stressed Genotype C Control (no SNP) 50 60 Similar to treatment 5 M SNP 50 61 Similar to control Genotype D Control (no SNP) 50 70 Calloused 5 M SNP 50 57 Less calloused than control
[0040] Eight treatments with various concentrations of SNP in maintenance media and/or development media were used in this experiment, detailed in Table 5 below.
TABLE-US-00005 TABLE 5 SNP Concentrations in Different Treatments SNP Concentration SNP Concentration in Maintenance in Development Treatment Medium (M) Medium (M) 1 0 0 2 0 50 3 0 200 4 0 500 5 5 0 6 5 50 7 5 200 8 5 500
[0041] At the end of development, it was observed that treatments with SNP in development media had reduced extraneous ESM than the controls. The effect of reduced extraneous ESM increased with higher concentration of SNP in the development medium.
[0042] Yield
[0043] As shown in
[0044] These visual observations suggest that SNP in maintenance media reduces ESM and improves embryo quality, and that SNP in development media improves embryo quality up to a concentration of 200 M but not at higher concentration, such as at 500 M.
[0045] Germination
[0046] Good quality germinants having all three parts, root, hypocotyl and epicotyl, visible were selected and plotted.
[0047] All genotypes with embryos developed in the presence of SNP demonstrated improved germination percentage compared to the control with embryos developed in the absence of SNP: Genotype A improved by 18%, Genotype B improved by 39%, Genotype C improved by 16%, and Genotype D improved by 5%.
[0048] Adding 5 M SNP to maintenance media resulted in a mild but statistically significant increase in germination percentage. Adding 50 M to 200 M SNP to development media resulted in a stronger and statistically significant increase in germination percentage. However, adding 500 M SNP to development media did not result in any significant difference in germination relative to control. The root length of the germinants did not show improvements at the various SNP concentrations tested.
Example 3
[0049] This example shows the effects of increased concentrations of nitric oxide on somatic embryo development and germination of Douglas-fir. Unless specified below, the procedure is similar to Example 2.
Materials and Method
[0050] Douglas-fir embryogenic suspensor mass (ESM) from five different genotypes were maintained in a standard liquid maintenance medium, BM-2 medium, at room temperature in dark for one week. After singulation, all cultures were grown in a singulation medium, BM-3 medium, through ABA I, II and III stages. 3 mL of settled cells of each genotype was plated with Decotex 22 membranes to standard semi-solid development media (BM-4) containing various concentrations of SNP, 10 M, 50 M, 100 M and 200 M. A standard development medium (BM-4) containing no SNP was used as a control. The plating procedure was performed as described in U.S. Pat. No. 8,925,245, the content of which is incorporated by reference in its entirety. Each plating consisted of 6 plates of each genotype. Development was carried out for 10 weeks at room temperature in dark. The plates were visually assessed at 9 weeks.
[0051] After completion of 10 weeks incubation on development medium, cultures were transferred to a standard germination medium, BM-5 medium, for stratification treatment in dark at 2-4 C. for 4 weeks. Embryos were then sorted and kept for germination on the standard germination medium for 8 weeks at room temperature with light. Good quality germinants having all three parts, root, hypocotyl and epicotyl, visible were selected and plotted.
Results
[0052] Effects of different concentrations of SNP in the development medium on Douglas-fir embryo development are shown in Table 6 below and in
TABLE-US-00006 TABLE 6 Effects of SNP on Douglas-fir embryo development (embryos per ml) SNP SNP SNP SNP SNP Genotype (0 M) (10 M) (50 M) (100 M) (200 M) A 20 30 25 10 22 B 101 91 51 26 32 C 40 42 15 18 14 D 30 60 47 28 11 E 20 23 20 9 12 Average 42.2 49.2 31.6 18.2 18.2
[0053] The germination percentage of all genotypes is shown in Table 7 and
TABLE-US-00007 TABLE 7 Effects of SNP on Douglas-fir embryo germination percentage SNP SNP SNP SNP SNP Genotype (0 M) (10 M) (50 M) (100 M) (200 M) A 37.3 55.4 51.7 54.2 54 B 60.9 66.3 57.4 48.4 51.5 C 42 46.7 27 21.4 25.8 D 22.5 27.5 19.2 23.8 13.1 E 32.5 26.1 42 37 30 Average 39.04 44.4 39.46 36.96 34.88