Stable efficient doubling and rapid propagation integrated method for sesbania cannabina

Abstract

The invention provides a sesbania cannabina doubling and rapid propagation integrated method, and relates to the technical field of sesbania cannabina breeding, and the method comprises the following steps: rubbing sesbania cannabina seeds to break seed coats, disinfecting the seeds, soaking the sesbania cannabina seeds for hydration, inducing genome doubling by colchicine, and then carrying out rooting culture; after cotyledons are unfolded, water culture seedling hardening is carried out; after compound leaves grow, genome ploidy identification is carried out, and the successfully doubled sesbania can be transplanted. The period of the whole method is 18 days, and the sesbania polyploidy induction rate can reach 50% or above. According to the method, sesbania genome doubling is achieved for the first time, a large number of polyploid plants meeting the breeding requirement can be directly obtained, the seedling raising time can be shortened, the plant survival rate can be increased, the production cost can be reduced, and integration of sesbania genome doubling and plant rapid propagation is achieved.

Description

Technical Field

[0001] The invention relates to a method for integrating stable and efficient doubling and rapid propagation of sesbania, and belongs to the technical field of sesbania breeding. Background Art

[0002] Sesbania cannabina belongs to the genus Sesbania of the subfamily Faboideae of the family Fabaceae. It is gradually recognized by the public because of its strong tolerance to environmental stress, its ability to grow normally on saline-alkali soil, its ability to symbiotically fix nitrogen, and its large plant biomass. It can be used as a green manure crop or forage crop. With the gradual expansion of the domestic market for green manure and forage on saline-alkali land, the country has gradually carried out the selection and breeding of excellent varieties (lines) of Sesbania, making Sesbania varieties (lines) diversified. At present, the selection and breeding of excellent varieties of Sesbania in China is mostly through hybridization or mutation under natural conditions, and varieties with multiple excellent trait combinations are screened out, but there are limitations such as seed abortion after hybridization of Sesbania with different ploidy and difficulty in fixing the traits of hybrid offspring.

[0003] Polyploidy is common in nature and is common in higher plants. According to statistics, about 38.71% of the 1,171 species of 528 genera in seed plants are polyploid, of which about 50% of angiosperms are polyploid. Polyploidy has one or more characteristics such as enhanced stress resistance, thickening of stems and leaves (increasing biomass of individual plants), and increased secondary metabolites (increasing the content of nutrients in individual plants), making polyploid breeding gradually become an important direction of plant breeding. For Sesbania sesbania, polyploidization may change its plant type, flowering period or nutrient content, making it more environmentally adaptable and valuable for feeding. In addition, the polyploidization technology combined with the haploid induction technology can achieve rapid homozygosity of the genome of Sesbania sesbania hybrid materials, efficiently fix the combination and traits of excellent alleles, and create new Sesbania sesbania varieties.

[0004] There is currently no research or report on the induction of polyploidy in sesbania. Summary of the invention

[0005] The purpose of the present invention is to provide a method for integrating stable and efficient doubling and rapid propagation of Sesbania sesbania, so as to realize the rapid production of Sesbania sesbania polyploids. The specific technical scheme is as follows:

[0006] A method for integrating stable and efficient doubling and rapid propagation of Sesbania sesbania, comprising the following steps:

[0007] S1 Sesbania seeds pretreatment: sterilize and disinfect the Sesbania seeds and soak and hydrate them to activate the Sesbania seeds to enter a cell division state;

[0008] S2 colchicine induced doubling: the sesbania seeds treated with S1 were placed in an induction solution, and incubated in a shaker at a frequency of 50 r / min at a temperature of 25±2°C and in a light-free environment for 6 to 24 hours. After the incubation, the sesbania seeds were taken out and cleaned with sterilized double distilled water, and the colchicine induction solution was removed to obtain induced doubling sesbania seeds; the induction solution was 0.05 to 0.30% w / v colchicine, and the solvent of the 0.05 to 0.30% w / v colchicine was 1% v / v dimethyl sulfoxide;

[0009] S3 rooting culture: the induced doubling Sesbania seeds obtained in S2 were inoculated into a rooting medium, placed at a temperature of 27±2°C, and cultured in the dark for 2-3 days. After the cotyledons unfolded, the temperature remained unchanged, the light was illuminated for 14 hours per day, and the rooting was cultured for 5-7 days. The rooting medium formula was: vitamin B6 0.4 mg / L, nicotinic acid 0.7 mg / L, 2-(N-morpholino)ethanesulfonic acid 0.7 mg / L, copper sulfate pentahydrate 2.9 mg / L, zeatin 4 mg / L, sucrose 20 g / L, plant gel 4 g / L, and the pH was adjusted to 5.8;

[0010] S4 hydroponic seedling hardening: transfer the sesbania plants that survived the S3 treatment and successfully took root to 1 / 2 Hoagland nutrient solution, place them at 27±2℃, light for 14h per day, light intensity of 2000-30001×, and culture them under alternating light and dark conditions for 5-7 days, until the first true leaf or compound leaf grows on the top of the plant and the fibrous roots elongate;

[0011] S5 genome ploidy identification.

[0012] Furthermore, in S1, the specific steps of sterilization and disinfection treatment are: take the field sesbania seeds and place them in a seed rubbing machine, rub them for 30 to 40 seconds, soak the broken seeds in 75% v / v ethanol for 1 minute, rinse the seeds with sterile water 1 to 2 times, then shake and disinfect the seeds with 5 to 7% v / v sodium hypochlorite solution for 30 minutes, and rinse them with sterile water 3 to 5 times, each time for 5 minutes.

[0013] Furthermore, in S1, the soaking and hydration treatment time is 8 to 10 hours.

[0014] Furthermore, in S5, the genome ploidy identification step is: take the first true leaf or compound leaf obtained in S4 and immerse it in the commercial LB01 nuclear extraction reagent, quickly chop it with a blade on ice to release the cell nucleus into LB01, use the PI single staining method of flow cytometry to detect the genome size, and identify the rooting seedlings with doubled genome size in the cell nucleus.

[0015] The above-mentioned stable and efficient integrated doubling and rapid propagation method of Sesbania sesbania is applied in doubling and rapid propagation of Sesbania sesbania.

[0016] Beneficial effects of the present invention:

[0017] 1. Currently, there is no doubling technology in sesbania. The present invention uses colchicine in a suitable concentration range for short-term induction, which can achieve a polyploidization induction rate of 50% or more. Compared with the colchicine-induced doubling of embryos after removing the seed coat, the operation method of immersing the entire seed in the induction solution for induction doubling is more convenient, the material teratogenicity rate is lower, and the seedling rate is higher. The induction efficiency in different sesbania varieties (germplasms) is stable, and diploid sesbania can be induced into tetraploid, and tetraploid sesbania can be induced into octoploid.

[0018] 2. The rooting culture medium involved in the present invention can stabilize the seedling rate after colchicine doubling induction at 90% or above. When further hydroponically cultivated in a nutrient solution, the induced buds of Sesbania can grow normally and take root, bloom and produce seeds, thus realizing the integration of stable and efficient doubling and rapid propagation of Sesbania.

[0019] 3. After the genome of Sesbania sesbania is doubled by the method of the present invention, the Sesbania sesbania plants become compact, the flowering period is advanced, and the root nodules and stem nodules become larger, the leaves become larger and wider, the biomass increases, and the Sesbania sesbania has more production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The Sesbania tannica YS05094 plants are diploid, tetraploid from diploid doubled, and chimera from diploid incompletely doubled. The plant on the left is a diploid, the plant in the middle is a tetraploid from diploid doubled, and the plant on the right is a chimera from diploid incompletely doubled. They contain both diploid and tetraploid cell types.

[0021] Figure 2 This is a signal diagram of the PI single staining method for detecting genome size by flow cytometry to identify diploids, tetraploids doubled into diploids, chimeras with incomplete doubling of diploids, tetraploids, octoploids doubled into tetraploids, and chimeras with incomplete doubling of tetraploids.

[0022] Figure 3 This is the rooting condition of the diploid sesbania material YS05094 after cultivation in soil and rooting medium. DETAILED DESCRIPTION

[0023] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0024] Example 1 Rooting medium formulation screening test

[0025] Experimental materials: seeds of diploid sesbania YS05094, YS06002, YS06044 and YS06286, and seeds of tetraploid sesbania YS06004, YS05093 and YS06055. Germplasm resource information is shown in Table 1:

[0026] Table 1 Germplasm resource information

[0027] Germplasm name Germplasm Resource Bank of China Academy of Tropical Sciences Material ploidy YS05094 01k8010313000027 Diploid YS06002 01k8010313000006 Diploid YS06004 01k8010313000059 Tetraploid YS06044 01k8010313000001 Diploid YS06055 01k8010313000223 Tetraploid YS06286 01k8010313000020 Diploid Germplasm name Shandong Province Crop Germplasm Resources Center No. Material ploidy YS05093 Z6A00211 Tetraploid

[0028] Experimental steps:

[0029] (1) Seeds of diploid sesbania YS05094, YS06002, YS06044 and YS06286 materials that are evolutionarily distant and genetically different, and seeds of tetraploid sesbania YS06004, YS05093 and YS06055 materials were selected. The seeds of each material were rubbed with a seed rubber for 30 to 40 seconds to break the seed coat, immersed in 75% v / v ethanol for 1 minute for disinfection, rinsed 1 to 3 times with sterile double distilled water, immersed in 5 to 7% v / v sodium hypochlorite solution with 2 drops of Tween-20 for shaking disinfection for 30 minutes, and rinsed 3 to 5 times with sterile double distilled water;

[0030] (2) Soak the disinfected seeds in sterile double-distilled water at 25±2°C for 8-10 hours to hydrate, break seed dormancy, activate embryonic cells to enter the cell cycle, and initiate cell division;

[0031] (3) immersing the hydrated seeds in a treatment solution with a concentration gradient of 0.05% to 0.30% w / v colchicine prepared with 1.0% v / v dimethyl sulfoxide as a solvent, and incubating at a temperature of 25±2°C in a light-free environment in a shaker at a frequency of 50 r / min for 6 to 24 hours to ensure that chromosome separation of the embryo is blocked by colchicine during the slow development process, and subsequently produce polyploidized sesbania;

[0032] (4) Rinse with sterile double-distilled water for 10 min, 5 to 6 times in total, to remove the colchicine induction solution;

[0033] (5) The cleaned seeds were inoculated into 5 different rooting culture media, placed at a temperature of 27±2°C, and cultured in the dark for 2 to 3 days. After the cotyledons unfolded, the temperature remained unchanged, the light was illuminated for 14 h per day, and cultured for 5 to 7 days for rapid rooting. The rooting test of the seeds of the above 7 types of Sesbania sesbania materials was carried out using 5 different rooting culture medium formulas (Table 2), with 3 biological replicates for each test condition.

[0034] Experimental results: According to statistics, the average rooting efficiency of the seven sesbania materials under formula 5 reached more than 90%, and the results are shown in Table 2. According to the comparison of the rooting efficiency of sesbania, formula 5 has the best rooting effect on sesbania. The formula of the rooting medium of formula 5 is: vitamin B6 0.4 mg / L, nicotinic acid 0.7 mg / L, 2-(N-morpholinyl)ethanesulfonic acid 0.7 mg / L, copper sulfate pentahydrate 2.9 mg / L, zeatin 4 mg / L, sucrose 20 g / L, plant gel 4 g / L, and the pH is adjusted to 5.8.

[0035] Table 2: Experimental design table for various rooting medium formulas

[0036] Medium conditions Recipe 1 Recipe 2 Recipe 3 Recipe 4 Recipe 5 MS basal medium 1X 1X 1X 1 / 2X - LS basal medium - - - - 1X Vitamin B6 - - - - 0.4mg / L niacin - - - - 0.7mg / L 2-(N-Morpholinyl)ethanesulfonic acid - - - - 0.7mg / L Copper sulfate pentahydrate - - - - 2.9mg / L 2，4D 3mg / L - 1mg / L - - Inositol - - 0.1g / L - - Kinetin 1mg / L 1.5mg / L - - - 6-Benzylaminoadenine - 1.5mg / L 0.5mg / L - - Thidiazuron - 1.5mg / L - - - Naphthaleneacetic acid - 0.25mg / L 1mg / L 0.05mg / L - Zeatin - - - - 4mg / L sucrose 30g / L 30g / L 30g / L 30g / L 20g / L Plant Gel 4g / L 4g / L 4g / L 4g / L 4g / L pH - - - 5.8 5.8 Rooting efficiency of Sesbania sesbania ～10％ ～12％ ～11％ ～10％ ≥90％

[0037] Example 2 Colchicine treatment and optimization of rapid propagation conditions

[0038] Using rooting medium formula 5 in Example 1, colchicine + dimethyl sulfoxide was used as a fixed induction solution, and the effects of multiple concentration conditions and incubation time conditions were compared. The specific experimental design is shown in Table 3, and numbers 1 to 30 correspond to 30 treatment condition combinations: YS05094 was used as a diploid treatment material to detect the efficiency of doubling into a tetraploid; YS06055 was used as a tetraploid treatment material to detect the efficiency of doubling into an octoploid.

[0039] After the sesbania sabdariffa took root on the culture medium, it was transferred to a commercial 1 / 2 Hoagland nutrient solution for hydroponic seedling hardening, and was placed at a temperature of 27±2°C, with 14 hours of light per day and a light intensity of 2000-30001× for 5-7 days under alternating light and dark conditions, until the first true leaf or compound leaf grew out of the top of the plant and the fibrous roots elongated; after the hydroponic sesbania sabdariffa developed the first true leaf or compound leaf, the first true leaf or compound leaf was taken and immersed in a commercial LB01 nuclear extraction reagent, and the cell nucleus was quickly chopped with a blade on ice to release the cell nucleus into LB01, and the PI single staining method for detecting the genome size by flow cytometry was used to identify the rooted seedlings with doubled genome size in the cell nucleus ( Figure 1-2 ).

[0040] Table 3: Experimental design table for multiple induction doubling conditions

[0041]

[0042] Experimental results: With 1% v / v dimethyl sulfoxide as solvent, the concentration of colchicine inducer 0.1% w / v has the highest polyploidy rate, and the incubation time is not more than 12h, and the effect is the best. The genome size is identified through the ploidy identification of the present invention. The specific results are shown in Table 4:

[0043] Table 4: Induction results of various induction doubling conditions

[0044]

[0045] Example 3 Comparison of survival of sesbania plants grown on rooting medium and in soil

[0046] After the diploid sesbania material YS05094 and the tetraploid sesbania material YS06055 were doubled treated with colchicine, the treatment conditions were the 30 conditions in Table 3 of Example 2, each condition had 30 plant samples, and the survival of the sesbania plants rooted in the rooting medium (Formula 5) provided in Example 1 and soil cultured was compared; the total sample size of each material soil cultured was 900 plant samples for rooting survival test.

[0047] Experimental results: After the double treatment of colchicine, the average survival rate of diploid sesbania material YS05094 in soil culture was 1.22%, and the average survival rate in rooting medium culture was 85.33% ( Figure 3 ); the average survival rate of tetraploid sesbania material YS06055 in soil culture was 2.22%, and the average survival rate in rooting medium culture was 92.22%. In summary, the survival rate of sesbania in soil culture was much lower than that of sesbania in rooting medium culture provided by the present invention.

[0048] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for integrating stable and efficient doubling and rapid propagation of Sesbania sesbania, characterized in that: The following steps are involved: S1 Sesbania seeds pretreatment: sterilize and disinfect the Sesbania seeds and soak and hydrate them to activate the Sesbania seeds to enter a cell division state; S2 colchicine induced doubling: the sesbania seeds treated with S1 were placed in an induction solution, and incubated in a shaker at a frequency of 50 r / min at a temperature of 25±2°C and in a light-free environment for 6 to 24 hours. After the incubation, the sesbania seeds were taken out and cleaned with sterilized double distilled water, and the colchicine induction solution was removed to obtain induced doubling sesbania seeds; the induction solution was 0.05 to 0.30% w / v colchicine, and the solvent of the 0.05 to 0.30% w / v colchicine was 1% v / v dimethyl sulfoxide; S3 rooting culture: the induced doubling Sesbania seeds obtained in S2 were inoculated into a rooting medium, placed at a temperature of 27±2°C, and cultured in the dark for 2-3 days. After the cotyledons unfolded, the temperature remained unchanged, the light was illuminated for 14 hours per day, and the rooting was cultured for 5-7 days. The rooting medium formula was: vitamin B6 0.4 mg / L, nicotinic acid 0.7 mg / L, 2-(N-morpholino)ethanesulfonic acid 0.7 mg / L, copper sulfate pentahydrate 2.9 mg / L, zeatin 4 mg / L, sucrose 20 g / L, plant gel 4 g / L, and the pH was adjusted to 5.8; S4 hydroponic seedling hardening: transfer the sesbania plants that survived the S3 treatment and successfully took root to 1 / 2 Hoagland nutrient solution, place them at 27±2℃, light for 14h per day, light intensity of 2000-30001×, and culture them under alternating light and dark conditions for 5-7 days, until the first true leaf or compound leaf grows on the top of the plant and the fibrous roots elongate; S5 genome ploidy identification.

2. The method according to claim 1, characterized in that: In S1, the specific steps of sterilization and disinfection are: take the field sesbania seeds and place them in a seed rubbing machine, rub them for 30 to 40 seconds, soak the broken seeds in 75% v / v ethanol for 1 minute, rinse the seeds with sterile water 1 to 2 times, then shake and disinfect the seeds with 5 to 7% v / v sodium hypochlorite solution for 30 minutes, and rinse them with sterile water 3 to 5 times, each time for 5 minutes.

3. The method according to claim 1, characterized in that: In S1, the soaking and hydration treatment time is 8 to 10 hours.

4. The method according to claim 1, characterized in that: In S5, the genome ploidy identification step is: take the first true leaf or compound leaf obtained in S4 and immerse it in the commercial LB01 nuclear extraction reagent, quickly chop it with a blade on ice to release the cell nucleus into LB01, use the PI single staining method of flow cytometry to detect the genome size, and identify the rooting seedlings with doubled genome size in the cell nucleus.

5. Application of the method according to any one of claims 1 to 4 in doubling and rapid propagation of Sesbania sesbania.

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