CN114041463B

CN114041463B - Agents and method for reduplicating and reducing damage and improving mutagenesis rate of gynura bicolor chromosome

Info

Publication number: CN114041463B
Application number: CN202111317063.6A
Authority: CN
Inventors: 张永福; 曹魏; 陈娇; 徐仕琴; 王凯; 谢莹莹
Original assignee: Kunming University
Current assignee: Kunming University
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2023-04-07
Anticipated expiration: 2041-11-09
Also published as: CN114041463A

Abstract

The invention relates to a medicament and a method for reduplicating chromosome of holboellia latifolia to reduce damage and improve mutagenesis rate, wherein salicylic acid and sodium nitroprusside are adopted as medicaments to treat the root of the holboellia latifolia, the upper part of the root of the holboellia latifolia is treated by a low-concentration chromosome reduplicating agent for a long time, the medicament is sprayed on the whole holboellia latifolia plant, the contact area of the medicament and the plant is large, the absorption effect of the young and tender parts of leaves and stems is good, the injury of the chromosome reduplicating agent to the holboellia latifolia is greatly reduced due to the fact that the root is irrigated with the salicylic acid and the sodium nitroprusside, and the mutagenesis efficiency is greatly improved.

Description

Agents and method for reducing damage and improving mutagenesis rate by doubling of akebia japonica chromosomes

Technical Field

The invention relates to the technical field of plant breeding, in particular to a medicament and a method for reduplicating and reducing damage and improving mutagenesis rate of a gynura bicolor chromosome.

Background

The Holboellia latifolia wall is a new third-generation fruit tree which is popular in recent years, and is a evergreen woody vine, and 3 small leaves are arranged on palm-shaped compound leaves. The August melon and fruit is nutritious, the pulp is milk white, the flavor is unique, the August melon and fruit is fragrant, sweet, soft and glutinous, and the August melon and fruit can be used for brewing wine and making fruit pulp and beverage besides fresh food. However, the holboellia latifolia is still at the beginning of development and application at present, and the whole industry is slow in development due to the lack of artificially bred excellent varieties, so that breeding of new high-quality and high-efficiency holboellia latifolia varieties is imperative.

The major defects of the current planted holboellia latifolia are that the seeds are too many, the pulp is less, the edible rate is low, and the main method for solving the problem is to obtain tetraploid through chromosome doubling treatment, then to use the tetraploid to be hybridized with diploid, and finally to obtain seedless triploid. However, the traditional chromosome doubling agent treatment method has low mutagenesis efficiency, high aberration rate and high lethality rate, and is difficult to obtain satisfactory mutagenesis effect. Therefore, the effects of reducing plant damage and improving mutagenesis efficiency are of great significance to accelerating breeding of the holboellia latifolia.

Disclosure of Invention

The invention provides a medicament and a method for reduplicating chromosome of holboellia latifolia to reduce damage and improve mutagenesis rate, which greatly reduce damage of the chromosome reduplicating agent to holboellia latifolia, and greatly improve mutagenesis efficiency.

The technical purpose of the invention is realized by the following technical scheme:

an agent for reduplicating chromosome of holboellia latifolia to reduce injury and increase mutagenesis rate is one or a mixture of salicylic acid and sodium nitroprusside.

Further preferably, the concentration of the salicylic acid is 50-200 mu mol/l, and the concentration of the sodium nitroprusside is 50-200 mu mol/l.

Further preferably, the agent is salicylic acid at a concentration of 100. Mu. Mol/l.

Further preferably, the medicament is sodium nitroprusside with the concentration of 100 mu mol/l.

Further preferably, the medicament is a mixture of salicylic acid with the concentration of 100 mu mol/l and sodium nitroprusside with the concentration of 100 mu mol/l.

The invention also provides a method for reduplicating the chromosome of the holboellia latifolia to reduce the damage and improve the mutagenesis rate, which comprises the following steps:

s1, after transplanting seedlings of the holboellia latifolia for two weeks, after sufficiently delaying the seedlings, infiltrating a nutrition pot in which the holboellia latifolia is planted with the medicament, enabling the culture medium in the nutrition pot to sufficiently absorb the medicament, and then treating (irrigating) once a week;

s2, spraying the chromosome doubling agent once every day at 18 pm from the day of agent treatment, wherein the spraying amount is that the leaves and stems of each august melon seedling are completely wet.

Further preferably, the holboellia latifolia is treated with the agent for a total of four weeks.

Further preferably, the chromosome doubling agent is prepared from 0.01% pendimethalin, 0.01% trifluralin, 0.01% pretilachlor and 0.01% colchicine mixed liquor (the first three are in volume ratio, and the last one is in mass-volume ratio).

Compared with the prior art, the invention has the following beneficial effects:

firstly, the invention adopts the low-concentration long-time chromosome doubling agent for treatment, the medicament is sprayed on the whole august plant, the contact area of the medicament and the plant is large, the absorption effect through the young and tender parts of the leaves and the stems is good, the damage of the chromosome doubling agent to the august is greatly reduced because the roots are irrigated with the salicylic acid and the sodium nitroprusside, and the mutagenesis efficiency is greatly improved.

And secondly, under the stress of the chromosome doubling agent, the salicylic acid and sodium nitroprusside treatment of the holboellia latifolia seedlings can effectively improve the content of osmotic adjusting substances of the holboellia latifolia seedlings and slow down cell dehydration, so that the stress resistance of the holboellia latifolia seedlings is improved.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described in detail with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.

Since 2019, mutation breeding of the holboellia latifolia is carried out in a greenhouse of an agricultural practice garden of Kunming academy of academic, mutation treatment of the holboellia latifolia is carried out according to the method provided by the invention, and the following operations are carried out:

example 1

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate comprise the following steps:

1) Seedling raising of holboellia latifolia

Adopts the modes of layering and sowing to carry out seedling culture. The layering method comprises the steps of pulling new branches of the holboellia latifolia which are extracted from the base part to the ground, then digging soil to cover the branches, adopting interval layering, covering each branch by soil at intervals of about 20cm, not covering the branch at intervals of about 20cm, covering the branch by soil at intervals of about 20cm, then covering the branch by soil at intervals of about 20cm, and repeating the steps until the whole branch is obtained. The sowing method comprises the steps of picking fruits after the fruits are ripe for 8-9 months, taking out seeds and pulp, cleaning the pulp on the seeds, immediately sowing the seeds in a greenhouse, and sowing 200-300 seeds per square meter. Generally, the seedlings begin to sprout after being sown for 1 month, heat preservation is carried out in winter, and the absolute low temperature of the overwintering seedlings is not lower than 4 ℃.

2) Transplanting and soilless culture of holboellia latifolia

Transplanting the seedlings into nutrition pots with the diameter and the height of 25cm when the seedlings grow to about 10cm, fully and uniformly mixing peat, vermiculite, perlite and other culture media in the nutrition pots, and transplanting 3 seedlings into each nutrition pot. After the seedlings are transplanted, hoagland's nutrient solution (pH value is 5.6) is poured once a week.

3) Chemical mutagen resistance improving agent and treatment method

After 2 weeks of transplanting, after sufficient seedling delaying, selecting strong-growing Bayue melon seedlings and putting the Royue melon seedlings together with nutrition bowls into watertight long-strip pots (the length, the width and the height of each long-strip pot are 120cm, 35m and 30cm respectively), putting 4 nutrition bowls into each long-strip pot, then adding 8L of salicylic acid with the concentration of 100 mu mol/L into each corresponding long-strip pot respectively, enabling the culture medium in the nutrition bowls to fully absorb the treatment solution, and finally leaving about 5cm of the treatment solution at the bottoms of the long-strip pots (T9 is managed normally without adding any treatment solution). The treatment solution was replaced every week, that is, the excess treatment solution in the long tub was poured out and 8L of a new treatment solution was added. Repeat 4 times.

4) Chemical mutagen formula of holboellia latifolia and processing method

The first sampling was performed on the day of treatment 3), and the samples were healthy, mature, disease and pest free leaves. From the day of treatment 3), the chromosome doubling agent was sprayed once every day at 18 pm in an amount such that the leaves and stems of each august seedling were completely wet, for 4 weeks (28 days). The formulation of the chromosome doubling agent is 0.01% pendimethalin, 0.01% trifluralin, 0.01% pretilachlor and 0.01% colchicine mixed solution (the former three are in volume ratio, the last one is in mass-volume ratio).

Example 2

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of the embodiment 2 which is the same as that of the embodiment 1 is not repeated, and the difference lies in that: the agent in 3) is salicylic acid at a concentration of 100. Mu. Mol/l.

Example 3

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of example 3 which is the same as that of example 1 is not repeated, and the difference lies in that: the agent in 3) is salicylic acid at a concentration of 200. Mu. Mol/l.

Example 4

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of example 4 which is the same as that of example 1 is not repeated, and the difference lies in that: the agent in 3) is sodium nitroprusside with a concentration of 50. Mu. Mol/l.

Example 5

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of example 5 which is the same as that of example 1 is not repeated, and the difference lies in that: the agent in 3) is sodium nitroprusside with the concentration of 100 mu mol/l.

Example 6

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of example 6 which is the same as that of example 1 is not repeated, and the difference lies in that: the agent in 3) is sodium nitroprusside with the concentration of 200 mu mol/l.

Example 7

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, and the technical scheme of embodiment 7 which is the same as that of embodiment 1 is not repeated, and the differences are as follows: the agent in 3) is a mixture of salicylic acid at a concentration of 100. Mu. Mol/l and sodium nitroprusside at a concentration of 100. Mu. Mol/l.

Example 8

A medicament and a method for reduplicating chromosome of holboellia latifolia to reduce injury and improve mutagenesis rate are provided, the technical scheme of the embodiment 8 which is the same as that of the embodiment 1 is not repeated, and the difference lies in that: the medicament in 3) is replaced by distilled water.

Settings Contrast (CK)

The same technical solution of the Control (CK) as that of the embodiment 1 is not repeated, but the differences are as follows: without the treatment of 3), the chromosome doubling agent sprayed in 4) was replaced by distilled water, and the spraying method and time were the same as in example 1.

Statistics of the damage of the Bayue melon seedlings after the treatment of examples 1-8 and Control (CK)

The damage degree grading standard of the pumpkin seedlings sprayed with the chromosome doubling agent under different treatments is as follows: the 0-grade plant grows normally without any injury sign; the 1 st class young leaves have injured spots, and the adult leaves have no obvious change; 2, the injured spots of the young leaves are enlarged and drop a little, and the spots also appear on the adult leaves; a large amount of 3-grade young leaves wither and fall off, and the injured spots of the adult leaves are enlarged and have a small amount of withering and fall off; most adult leaves of grade 4 die and fall off.

The damage index = [ Σ (number of damaged plants × number of stages of diseases)/(total number of plants × highest damage stage) ] × 100%.

TABLE 1 Effect of salicylic acid and sodium nitroprusside treatment on the injury index of August melon seedlings under chromosome doubling agent stress (%)

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Note that different letters on the same column indicate significant differences at the P <0.05 level; data are the average of 4 replicates. The following table is the same.

As can be seen from Table 1, with the increase of the stress time of the chromosome doubling agent, the damage degree of the chromosome doubling agent to the August melon seedlings can be effectively reduced by the treatment of the salicylic acid and the sodium nitroprusside. Injury index was 0 for all treatments on day 0 of treatment; on day 7 of treatment, the injury index for distilled water treatment reached 21.12%, significantly higher than other treatments, while the injury index for 100 μmol/l sodium nitroprusside +100 μmol/l sodium nitroprusside treatment was only 5.81%, significantly lower than other treatments (with the exception of CK); the same is true at day 14, day 21 and day 28 of treatment; by day 28 of treatment, the injury index for distilled water treatment was already as high as 93.34% significantly higher than other treatments, while the injury index for 100 μmol/l sodium nitroprusside +100 μmol/l sodium nitroprusside treatment was only 48.47% significantly lower than other treatments (except CK).

It can be seen that, although 50. Mu. Mol/l, 100. Mu. Mol/l and 200. Mu. Mol/l salicylic acid and sodium nitroprusside can effectively improve the capability of the pachyrhizus to resist the stress of the chromosome doubling agent, the effect is best when 100. Mu. Mol/l salicylic acid and 100. Mu. Mol/l sodium nitroprusside are used.

Examples 1-8 and Control (CK) treatment of the physiological and biochemical indices of the August melon seedlings

Soluble sugar, proline and protein are important osmoregulation substances in plants, and under the stress of adverse circumstances, the accumulation of the substances in a large amount can slow down the degree of injury caused by cell dehydration, thereby improving the stress resistance of crops.

TABLE 2 influence of salicylic acid and sodium nitroprusside treatment on the soluble sugar content of Bacopa monnieri (mg/g) under the stress of chromosome doubling agents

As can be seen from table 2, on day 0 of the treatment, the soluble sugar content of each treatment did not differ greatly, did not reach the level of significance of the difference, and then the soluble sugar content of each treatment increased greatly (except CK). The soluble sugar content after treatment with 50, 100, 200. Mu. Mol/l salicylic acid and sodium nitroprusside was significantly higher than that of distilled water and CK on days 7, 14 and 21 of the treatment; by day 28 of treatment, the soluble sugar content was higher for the 100. Mu. Mol/l salicylic acid + 100. Mu. Mol/l sodium nitroprusside treatment than for each of the other treatments, while the distilled water treatment was significantly lower than for each of the other treatments (CK excluded).

TABLE 3 influence of salicylic acid and sodium nitroprusside treatment on the content of Gynurenine under the stress of chromosome doubling agents (. Mu.g/g)

As can be seen from table 3, on day 0 of the treatment, the proline content of each treatment did not differ much, but the proline content of each treatment increased greatly with the passage of treatment time (except for CK). On the 14 th, 21 st and 28 th days of treatment, the proline content after the 100. Mu. Mol/l salicylic acid + 100. Mu. Mol/l sodium nitroprusside composite treatment is the highest and is significantly higher than that of the distilled water treatment and CK.

TABLE 4 influence of salicylic acid and sodium nitroprusside treatment on the protein content of Baccharis augustata (mg/g) under chromosome doubling stress

As can be seen from table 4, on day 0 of the treatment, the protein contents of the treatments were not greatly different and were not significantly different, but the protein contents of the treatments were greatly increased with the passage of the treatment time (except for CK). On the 14 th, 21 st and 28 th days of treatment, the protein content after the compound treatment of 100 mu mol/l salicylic acid and 100 mu mol/l sodium nitroprusside is the highest and is obviously higher than that of the distilled water treatment and CK. Therefore, under the stress of the chromosome doubling agent, the salicylic acid and the sodium nitroprusside can effectively improve the content of osmoregulation substances of the holboellia latifolia seedlings and slow down cell dehydration, so that the stress resistance of the holboellia latifolia seedlings is improved, wherein the compound treatment effect of 100 mu mol/l salicylic acid and 100 mu mol/l sodium nitroprusside is optimal.

Malondialdehyde is a product of lipid peroxidation of cell membranes and its content directly reflects the degree of oxidative damage to cells. Under the stress of adversity, the higher malondialdehyde is accumulated in plants, which proves that the damage to cell membranes is more serious, and the stress resistance is worse.

TABLE 5 influence of salicylic acid and sodium nitroprusside treatment on the malondialdehyde content of Bactrocera octovalvis (μmol/g) under the stress of chromosome doubling agents

As can be seen from table 5, on the 0 th day of the treatment, the malonaldehyde content of each treatment did not differ greatly, and did not reach the significance level of difference, and then the malonaldehyde content of each treatment started to increase (except CK); on the 7 th, 14 th, 21 st and 28 th days of treatment, the malondialdehyde content of the 100. Mu. Mol/l salicylic acid + 100. Mu. Mol/l sodium nitroprusside composite treatment was significantly lower than that of the other treatments, while the malondialdehyde content of the distilled water treatment was higher than that of the other treatments.

Superoxide dismutase (SOD) and Peroxidase (POD) are two important antioxidant enzymes in plants, and their actions are to scavenge oxygen free radicals and hydrogen peroxide to protect cells from oxidative damage.

TABLE 6 influence of salicylic acid and sodium nitroprusside treatment on the SOD Activity of Baccharis augustata (U/g) under chromosome doubling agent stress

As can be seen from table 6, on day 0 of the treatment, the SOD activities of the treatments were not greatly different, and all reached a level of significance of the difference, but then increased to different extents (except for CK); SOD activity was lowest (CK excluded) in distilled water treatment at day 7, 14, 21 and 28 of treatment; on the 21 st day and the 28 th day of the treatment, the SOD activity of the 100. Mu. Mol/l salicylic acid + 100. Mu. Mol/l sodium nitroprusside compound treatment was the highest, and particularly, the difference significance level was reached at the 28 th day of the treatment compared with the other treatments.

TABLE 7 influence of salicylic acid and sodium nitroprusside treatment on POD Activity of Bacopa monnieri [ U/(mg-min) ]under chromosome doubling stress

As can be seen from table 7, on day 0 of the treatment, the POD activities of the treatments were not greatly different, and none of them reached the level of significance of the difference, but then the treatments were increased to different extents (except for CK); POD activity was lowest (except CK) for distilled water treatment on days 7, 14 and 28 of treatment; the POD activity of the compound treatment of 100 mu mol/l salicylic acid and 100 mu mol/l sodium nitroprusside is the highest.

Examples 1-8 and Control (CK) treatment survival and mutation rates of Bayue melon seedlings two months after cessation of treatment

The number of seedlings of each treated august was 48, and 28 days after the chromosome doubling agent was sprayed, the chromosome doubling agent was stopped for each treatment, the watering of salicylic acid and sodium nitroprusside was also stopped in the individual pots, and only 500mL of Hoagland's nutrient solution was poured into each nutrition pot every 2 days to slow the seedlings and restore the growth.

TABLE 8 influence of salicylic acid and sodium nitroprusside treatment under the stress of chromosome doubling agents on the survival and mutation rates of Bacopa monnieri

Treatment of	Total plant number (plant)	Number of surviving plants	Survival rate (%)	Number of variant strains	Rate of variation (%)
						Example 1	48	20	41.67	12	25.00
Example 2	48	24	50.00	13	27.08
						Example 3	48	20	41.67	8	16.67
Example 4	48	24	50.00	11	22.92
						Example 5	48	24	50.00	14	29.17
Example 6	48	20	41.67	12	25.00
						Example 7	48	32	66.67	20	41.67
Example 8	48	8	16.67	6	12.50
						Control (CK)	48	48	100	0	0

As can be seen from Table 8, after two months of seedling rejuvenation and growth recovery, the number of survived strains and the survival rate of each treatment are greatly different, wherein the number of survived strains and the survival rate are the highest after the treatment of 100 mu mol/l salicylic acid and 100 mu mol/l sodium nitroprusside in a composite way, the number of survived strains is 32, and the survival rate is 66.67%; the lowest number of surviving strains and survival rate were obtained by distilled water treatment, the number of surviving strains was only 8, and the survival rate was only 16.67%. And preliminarily judging the number of the variant plants treated according to the morphological characteristics of the holboellia latifolia plants, and calculating the variant rate. The number and the mutation rate of the mutant strains subjected to the composite treatment of 100 mu mol/l salicylic acid and 100 mu mol/l sodium nitroprusside are respectively 20 strains and 41.67 percent; the number of variants and the mutation rate in distilled water treatment were the minimum, 6 and 12.50% respectively.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims

1. The medicament for reduplicating the chromosomes of the holboellia latifolia to reduce the damage and improve the mutagenesis rate is characterized by being the mixture of salicylic acid and sodium nitroprusside, wherein the concentration of the salicylic acid is 50-200 mu mol/l, and the concentration of the sodium nitroprusside is 50-200 mu mol/l.

2. The agent for reduplicating chromosomes of Citrullus augustum as claimed in claim 1, wherein the agent is effective for reducing injury and increasing mutagenesis rate: the medicament is a mixture of salicylic acid with the concentration of 100 mu mol/l and sodium nitroprusside with the concentration of 100 mu mol/l.

3. The use of the agent for reduplicating the chromosomes of the holboea augustata according to any one of claims 1-2, wherein the method comprises the following steps:

s1, after transplanting seedlings of the holboellia latifolia for two weeks, after sufficiently delaying the seedlings, infiltrating a nutrition pot in which the holboellia latifolia is planted with the medicament, enabling the culture medium in the nutrition pot to sufficiently absorb the medicament, irrigating once every week later, and treating the holboellia latifolia with the medicament for four weeks;

s2, from the day of medicament treatment, spraying a chromosome doubling agent once every day at 18 pm, wherein the spraying amount is that the leaves and stems of each august melon seedling are completely wet, and the formula of the chromosome doubling agent is a mixed solution of 0.01% of pendimethalin, 0.01% of trifluralin, 0.01% of pretilachlor and 0.01% of colchicine, wherein the pendimethalin, the trifluralin and the pretilachlor are in a volume ratio, and the colchicine is in a mass-volume ratio.