Method for improving chromosome doubling efficiency of rice haplobiont

Abstract

The invention discloses a method for improving chromosome doubling efficiency of a rice haplobiont, and belongs to the technical field of plant breeding. The method comprises the following steps: (1) taking and pretreating young ears; (2) stripping young ears, disinfecting and carrying out induction culture; (3) carrying out callus subculture; (4) differentiation culture; (5) rooting culture; (6) acclimatization and transplantation; and (7) regenerating the rice stubble. According to the method, callus induction is initiated for the first time, double paths of tiller bud soaking cooperate, and the total doubling rate is increased to 80% or above; the rice stubble regeneration technology is used for doubling the plants which are not successfully doubled, and material waste is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a method for improving chromosome doubling efficiency of haploid rice plants. Background Art

[0002] Haploid doubling technology is a core breakthrough in crop breeding. It artificially doubles the chromosome set of a haploid plant (n chromosomes) into a homozygous diploid (2n), achieving complete genotyping in a single step. This technology completely solves the time-consuming problem of traditional hybrid breeding, which requires 6-8 generations of selfing to obtain homozygous lines, and shortens the breeding cycle by over 70%. This technology not only accelerates the fixation of superior genes and the creation of stable lines, but can also be combined with modern technologies such as gene editing and molecular marker-assisted selection to play an irreplaceable role in the development of disease-resistant germplasm, the utilization of hybrid vigor, and the cultivation of customized new varieties.

[0003] Traditional colchicine immersion methods (such as tillering bud treatment) suffer from low doubling rates (typically <30%), a short treatment window, and susceptibility to phytotoxicity. Single callus induction methods, while more efficient, are prone to somatic cell variation and have difficulty inducing callus differentiation in some species. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method for improving chromosome doubling efficiency in haploid rice plants, comprising the following steps: (1) Collection and pretreatment of young spikelets Haploid rice from rice hybrid plant anther culture regeneration seedlings was selected, and the panicles were extracted at the 3rd to 4th stage of rice panicle differentiation when the panicle length was 1.1 to 2 cm, and pretreated at 3 to 5°C for 4 to 6 days; (2) Peeling off young ears, disinfection and induction culture Remove the outer layer and leaf sheath of the main stem, retaining two layers of young leaf coverings, wrap with sterile gauze, soak in 70-75% alcohol for 0.5-1.5 minutes, then sterilize with 0.5% sodium hypochlorite for 15-25 minutes through shaking, rinse several times with sterile water, remove the outer young leaves, cut the young spikes into 3-5 mm segments, place on induction medium YSA, YSB, or YSC, and culture in the dark at 25-27°C. The YSA medium is NB + 2,4-D 1.8-2.2 mg / L + 6-BA 0.4-0.6 mg / L + NAA 0.4-0.6 mg / L + sucrose 28-32 g / L + phytogenin 2.3-2.7 g / L, pH = 5.8; The YSB medium is MS+2,4-D 1.8-2.2 mg / L+sucrose 38-42 g / L+phytagel 2.3-2.7 g / L, pH=5.8; The YSC culture medium is NB+2,4-D 1.8-2.2 mg / L+sucrose 43-47 g / L+phytagel 2.3-2.7 g / L, pH=5.8; (3) Callus subculture After 20-22 days of dark culture, callus tissue was induced at the spikelet site of the young panicle. The callus tissue was peeled off and transferred to callus subculture medium YSJ1. The light intensity was 1000 lux for 13-15 hours per day and the culture temperature was 25-27°C. After one week, the callus tissue became light yellow in color and dense in structure, ready for differentiation culture. The callus subculture medium YSJ1 is NB + glutamic acid 0.4-0.6 g / L + proline 2.6-3 g / L + hydrolyzed casein 0.2-0.4 g / L + 2,4-D 1.8-2.2 mg / L + sucrose 28-32 g / L + plant gel 2.8-3.2 g / L, pH=5.8; (4) Differentiation culture After subculture, the callus tissue of the young spikelets was transferred to MS differentiation medium and cultured at 13-15 h / d with a light intensity of 3000 lux at 25-27°C. Green spots appeared after 7-9 days of culture and differentiation was continued for 19-21 days. The MS differentiation medium is MS + glutamic acid 480-520 mg / L + proline 480-520 mg / L + hydrolyzed casein 280-320 mg / L + 6-BA 2.8-3.2 mg / L + NAA 0.4-0.6 mg / L + sucrose 28-32 g / L + phytogenin 6.8-7.2 g / L, pH 5.8; (5) Rooting culture Transfer seedlings 4 to 6 cm in length to rooting medium for growth; The rooting medium is 1 / 2MS + paclobutrazol 1.8-2.2 mg / L + colchicine 2.8-3.2 mg / L + sucrose 28-32 g / L + agar 6-8 g / L, pH 5.8.

[0005] Preferably, the YSA culture medium in step (2) is NB + 2,4-D 2 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 2.5 g / L, pH = 5.8.

[0006] More preferably, the YSB culture medium in step (2) is MS+2,4-D 2 mg / L+sucrose 40 g / L+phytagel 2.5 g / L, pH=5.8.

[0007] More preferably, the YSC culture medium in step (2) is NB+2,4-D 2 mg / L+sucrose 45 g / L+phytagel 2.5 g / L, pH=5.8.

[0008] More preferably, the callus subculture medium YSJ1 in step (3) is NB + 0.5 g / L glutamic acid + 2.8 g / L proline + 0.3 g / L hydrolyzed casein + 2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L plant gel, pH = 5.8.

[0009] More preferably, the MS differentiation medium in step (4) is MS + glutamic acid 500 mg / L + proline 500 mg / L + hydrolyzed casein 300 mg / L + 6-BA 3 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 7 g / L, pH 5.8.

[0010] More preferably, the rooting medium in step (5) is 1 / 2MS + paclobutrazol 2 mg / L + colchicine 3 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0011] More preferably, the step (5) further includes the step (6) of hardening and transplanting the seedlings.

[0012] More preferably, the step (6) further comprises the following step (7): cutting the undoubled regenerated seedlings 4 to 6 cm above the ground; digging out the rice stumps and drying them in a cool place for 1 day, and hydroponically cultivating them for 18 to 12 days; peeling out the regenerated tiller buds, immersing them in a solution containing 0.07 to 0.08 g / L colchicine at 27 to 29° C. for 46 to 50 hours; and rinsing with clean water and transplanting them to the field.

[0013] The present invention also provides application of the above method in improving chromosome doubling efficiency of rice haploid plants.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention pioneered the dual-path synergy of callus induction and tiller bud soaking, increasing the total doubling rate to over 80%; (2) Rice pile regeneration technology reuses unsuccessful plants to reduce material waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the haploid panicle material in Example 1.

[0016] Figure 2This figure shows the callus induction process of young spikelet callus in Example 1. A shows a young spikelet segment placed on induction medium YSA; B shows callus induced after 21 days of incubation; and C shows callus induced from the young spikelet one week after subculture. Scale bar length is 1 cm.

[0017] Figure 3 These are the rice regenerated seedlings on the differentiation medium in Example 1.

[0018] Figure 4 This is the regenerated tiller bud treated with colchicine in Example 1.

[0019] Figure 5 The morphology of the haploid and doubled rice plants in Example 2 is shown. A and D are haploid rice plants and ears; B and E are incompletely doubled rice plants and ears; C and F are fully doubled rice plants and ears. The scale is 10 cm long. DETAILED DESCRIPTION

[0020] Example 1 1. Collection and pretreatment of young spikelets Haploid rice from anther culture of regenerated seedlings of rice hybrid plants cultivated by Hubble Company was used. There were 4 groups of rice, numbered WHN402, WHN403, WHN404, and WHN411. At the 3rd and 4th stages of rice panicle differentiation, the panicles were extracted when the panicle length was 1.1-2 cm. Figure 1 ), placed in a 4℃ refrigerator for 5 days; 2. Peeling off young ears, disinfection and induction culture Peel off the outer layer of the main stem and the leaf sheath, retaining two layers of young leaves, and wrap them with sterile gauze. Soak in 70% alcohol for 1 minute, then sterilize with 0.5% sodium hypochlorite for 20 minutes, rinse with sterile water 5-6 times, peel off the outer young leaves in the culture dish, cut the young spike into 3-5mm small segments and place them on the induction medium YSA ( Figure 2 A), YSB, and YSC medium and cultured in the dark at 26°C.

[0021] 3. Callus subculture After about 21 days of dark culture, callus tissue was induced at the spikelet position of the young spike ( Figure 2 B) The callus tissue was peeled off and transferred to callus subculture medium YSJ1, with a light intensity of 1000 lux for 14 hours per day and a culture temperature of 26°C. After one week, the callus tissue became light yellow and dense in structure, ready for differentiation culture ( Figure 2 C). Data on panicle induction rates are shown in Table 1. Callus differentiation efficiency showed little difference among YSA, YSB, and YSC media, with average induction rates ranging from 48.12% to 56.64%.

[0022] 4. Differentiation Culture The callus tissue of the young spikelets after subculture was transferred to MS differentiation medium, and cultured at 26℃ with a light intensity of 3000 lux for 14 hours per day. After 8 days of culture, green spots appeared. After 20 days of differentiation, the grown seedlings ( Figure 3 ) were transferred to rooting medium. The efficiency of callus differentiation into shoots ranged from 48.12% to 56.64% (Table 2).

[0023] 5. Rooting culture and seedling hardening and transplanting Transfer seedlings about 5 cm in length into rooting medium to strengthen the seedlings. Open the lid in time after two weeks, add appropriate amount of water to harden the seedlings, wash off the agar on the roots and transplant them.

[0024] 6. Phenotypic Observation Observe the phenotype of regenerated seedlings in the field and identify haploids by plant height, glume size and fruiting condition ( Figure 4 The haploid doubling rates were calculated (Table 3). The haploid doubling rates ranged from 16.4% to 78%.

[0025] Table 1 Young spike induction rate

[0026] Table 2 Callus differentiation efficiency

[0027] Table 3 Statistics of doubling rates of haploid panicle callus regeneration method

[0028] The culture medium formula used in the above experiments is as follows: 1. Induction medium formula 1.1 YSA culture medium NB + 2,4-D 2 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 2.5 g / L, pH=5.8.

[0029] 1.2 YSB culture medium MS + 2,4-D 2 mg / L + sucrose 40 g / L + plant gel 2.5 g / L, pH=5.8.

[0030] 1.3 YSC culture medium NB + 2,4-D 2 mg / L + sucrose 45 g / L + plant gel 2.5 g / L, pH=5.8.

[0031] 2. YSJ1 subculture medium NB + glutamic acid 0.5 g / L + proline 2.8 g / L + hydrolyzed casein 0.3 g / L + 2,4-D 2 mg / L + sucrose 30 g / L + plant gel 3 g / L, pH=5.8.

[0032] 3. Differentiation medium MS + glutamic acid 500 mg / L + proline 500 mg / L + hydrolyzed casein 300 mg / L + 3 mg / L 6-BA + 0.5 mg / L NAA + sucrose 30g / L + 7 g / L plant gel, pH 5.8.

[0033] 4. Rooting medium 1 / 2MS + 2 mg / L paclobutrazol + 3 mg / L colchicine + 30 g / L sucrose + 7 g / L agar, pH 5.8.

[0034] Table 4 Abbreviations

[0035] Example 2 (1) Cut the regenerated seedlings that were not doubled in Example 1 5 cm above the ground (using WHN411 as an example); (2) Dig out the rice piles and dry them in a cool place for one day, then hydroponically cultivate them for 10 days; (3) The regenerated tiller buds were peeled out and immersed in colchicine solutions containing 0g / L, 0.05g / L, 0.075g / L, 0.1g / L and 0.2g / L (containing 2vol% DMSO for penetration) at 28℃ for 48 hours ( Figure 4 ); (4) After washing with clean water, the plants were transplanted to the field and the doubling rate was calculated based on the phenotype (Table 5). The results showed that under the treatment concentration range of 0.05g / L~0.2g / L, the chromosome doubling rate of haploid plants reached the highest level of 42.35% when the regenerated tillers were treated with 0.075g / L colchicine solution for 48 hours. Some of the haploid plants had partial doubling, and a small number of the whole plants had doubled ( Figure 5 Furthermore, under this optimized treatment system, the overall survival rate of haploid seedlings reached over 70%, with no significant physiological damage or developmental abnormalities observed. Therefore, this treatment concentration ensured doubling efficiency while effectively avoiding plant damage caused by phytotoxicity, demonstrating that the treatment scheme employed exhibited excellent doubling stability.

[0036] Table 5 Doubling rates of haploid rice treated with different concentrations of colchicine

[0037] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for improving chromosome doubling efficiency in haploid rice plants, characterized in that: The following steps are involved: (1) Collection and pretreatment of young spikelets Haploid rice from rice hybrid plant anther culture regeneration seedlings was selected, and the panicles were extracted at the 3rd to 4th stage of rice panicle differentiation when the panicle length was 1.1 to 2 cm, and pretreated at 3 to 5°C for 4 to 6 days; (2) Peeling off young ears, disinfection and induction culture Remove the outer layer and leaf sheath of the main stem, retaining two layers of young leaf coverings, wrap with sterile gauze, soak in 70-75% alcohol for 0.5-1.5 minutes, then sterilize with 0.5% sodium hypochlorite for 15-25 minutes through shaking, rinse several times with sterile water, remove the outer young leaves, cut the young spikes into 3-5 mm segments, place on induction medium YSA, YSB, or YSC, and culture in the dark at 25-27°C. The YSA medium is NB + 2,4-D 1.8-2.2 mg / L + 6-BA 0.4-0.6 mg / L + NAA 0.4-0.6 mg / L + sucrose 28-32 g / L + phytogenin 2.3-2.7 g / L, pH = 5.8; The YSB medium is MS+2,4-D 1.8-2.2 mg / L+sucrose 38-42 g / L+phytagel 2.3-2.7 g / L, pH=5.8; The YSC culture medium is NB+2,4-D 1.8-2.2 mg / L+sucrose 43-47 g / L+phytagel 2.3-2.7 g / L, pH=5.8; (3) Callus subculture After 20-22 days of dark culture, callus tissue was induced at the spikelet site of the young panicle. The callus tissue was peeled off and transferred to callus subculture medium YSJ1. The light intensity was 1000 lux for 13-15 hours per day and the culture temperature was 25-27°C. After one week, the callus tissue became light yellow in color and dense in structure, ready for differentiation culture. The callus subculture medium YSJ1 is NB + glutamic acid 0.4-0.6 g / L + proline 2.6-3 g / L + hydrolyzed casein 0.2-0.4 g / L + 2,4-D 1.8-2.2 mg / L + sucrose 28-32 g / L + plant gel 2.8-3.2 g / L, pH=5.8; (4) Differentiation culture After subculture, the callus tissue of the young spikelets was transferred to MS differentiation medium and cultured at 13-15 h / d with a light intensity of 3000 lux at 25-27°C. Green spots appeared after 7-9 days of culture and differentiation was continued for 19-21 days. The MS differentiation medium is MS + glutamic acid 480-520 mg / L + proline 480-520 mg / L + hydrolyzed casein 280-320 mg / L + 6-BA 2.8-3.2 mg / L + NAA 0.4-0.6 mg / L + sucrose 28-32 g / L + phytogenin 6.8-7.2 g / L, pH 5.8; (5) Rooting culture Transfer seedlings 4 to 6 cm in length to rooting medium for growth; The rooting medium is 1 / 2MS+paclobutrazol 1.8-2.2 mg / L+colchicine 2.8-3.2 mg / L+sucrose 28-32 g / L+agar 6-8 g / L, pH 5.

8.

2. The method according to claim 1, characterized in that The YSA culture medium in step (2) is NB + 2,4-D 2 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 2.5 g / L, pH = 5.

8.

3. The method according to claim 2, characterized in that The YSB culture medium in step (2) is MS+2,4-D 2 mg / L+sucrose 40 g / L+phytagel 2.5 g / L, pH=5.

8.

4. The method according to claim 3, characterized in that The YSC culture medium in step (2) is NB+2,4-D 2 mg / L+sucrose 45 g / L+phytagel 2.5 g / L, pH=5.

8.

5. The method according to claim 4, characterized in that In step (3), the callus subculture medium YSJ1 is NB + 0.5 g / L glutamic acid + 2.8 g / L proline + 0.3 g / L hydrolyzed casein + 2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L plant gel, pH = 5.

8.

6. The method according to claim 5, characterized in that The MS differentiation medium in step (4) is MS + glutamic acid 500 mg / L + proline 500 mg / L + hydrolyzed casein 300 mg / L + 6-BA 3 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 7 g / L, pH 5.

8.

7. The method according to claim 6, characterized in that The rooting medium in step (5) is 1 / 2MS + paclobutrazol 2 mg / L + colchicine 3 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.

8.

8. The method according to claim 7, characterized in that After step (5), the method further includes step (6) of hardening and transplanting the seedlings.

9. The method according to claim 8, characterized in that After step (6), the method further comprises step (7): cutting the undoubled regenerated seedlings at 4 to 6 cm from the ground; digging out the rice stumps and drying them in a cool place for 1 day, and hydroponically cultivating them for 18 to 12 days; peeling out the regenerated tiller buds, immersing them in a solution containing 0.07 to 0.08 g / L colchicine at 27 to 29° C. for 46 to 50 hours; and rinsing them with clean water and then transplanting them to the field.

10. Use of the method according to any one of claims 1 to 9 for improving chromosome doubling efficiency in haploid rice plants.

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