CN106069744B - A kind of method for seedless roxburgh rose tetraploid being induced to generate by mixing training method - Google Patents

Abstract

The invention discloses a method for inducing tetraploid production of seedless Rosa roxburghii through the mixed cultivation method of the present invention, comprising the following steps: Step 1, material preparation: the stem section of the sterile seedling is cut into 0.8-3cm long, and each section is at least Retain a leaf axil; step 2, pre-cultivation: pre-cultivate 0-3d in the medium MS+0.5mg/L 6-BA; step 3, colchicine light culture: the stem segments after the pre-cultivation are transferred into the stems with 0 ‑200mg/L colchicine in MS+0.5mg/L 6‑BA+ medium; step 4, culture without colchicine: step 3, after the above culture for 7 days, transfer to MS+0.5 without colchicine Continue to cultivate in mg/L 6-BA medium; Step 5, homogenization treatment of mutant buds: After step 4 is cultivated for 40 days, select the buds with obvious variation obtained from culture, and put them in the culture medium MS+0.15mg/L NAA+ In 0.5mg/L 6‑BA, the culture was repeated 5 times at an interval of 40 days. The induction method of the present invention obtains the new germplasm of the tetraploid Rosa roxburghii, which can increase the nutritional components of Rosa roxburghii fruit, such as Vc, VE and SOD, and increase the fruit diameter and edible part at the same time.

Description

A method for inducing tetraploid production of seedless Rosa roxburghii by mixed culture

technical field

The invention relates to the technical field of a method for producing polyploids of Rosa roxburghii by mixed cultivation, in particular to a method for inducing tetraploids of Rosa roxburghii by mixed cultivation.

Background technique

Seedless prickly pear (Rosa sterilis S.D.Shi) is a perennial climbing shrub belonging to the genus Rosa of the family Rosaceae. It is one of the endemic species in China, and it is a relative species of prickly pear (R.roxbunghii). Its wild resources are mainly distributed in the north of Anshun City, Guizhou Province and the south of Guiyang City. In recent years, a large number of species have been introduced and planted in many places. Seedless Rosa roxburghii has high medicinal health care, sightseeing, ecological protection and economic value. Compared with common prickly pears, the single fruit of seedless prickly pears is seedless or has 1-2 seeds, less tannin content, higher sugar content, thick flesh and sweet fruity taste, no obvious astringency, and fresh fruit sales are more advantageous . Its seedless or thin-seeded nature reduces processing costs. However, the nutrient components of the seedless Rosa roxburghii fruit such as Vc, VE and SOD content are less than Rosa roxburghii, and the fruit diameter is smaller than Rosa roxburghii, and the edible part is also less. This has greatly affected the production and utilization value of this characteristic plant to a certain extent. Therefore, utilizing modern biotechnology to select and breed the seedless Rosa roxburghii with large fruit and high nutritional content has become an urgent task for its industrial development and to improve the economic value of Rosa roxburghii. Among many plant breeding methods, the polyploid varieties bred by ploidy breeding can significantly increase fruit volume and nutrient content. Therefore, it is undoubtedly one of the effective methods to solve the above-mentioned problems to carry out polyploidy breeding on the seedless Rosa roxburghii and to breed the seedless Rosa roxburghii with higher ploidy. For the improvement of seedless Rosa roxburghii varieties, only Wei Jingfeng et al. have carried out studies on single plant selection, but there has been no report on ploidy breeding. In the invention, the young stems of the seedless roxburghii are used as materials, the "soaking method" and the "mixed cultivation method" are used to respectively induce the tetraploid of the seedless roxburghii, and identify the ploidy of the mutated materials.

Contents of the invention

In order to overcome the above-mentioned deficiencies in the prior art, the present invention provides a method for inducing tetraploid production of Rosa roxburghii through mixed cultivation, the purpose of which is to carry out polyploid breeding of Rosa roxburghii seedless, and to breed higher ploidy Seedless Rosa roxburghii, increase the nutritional components of the seedless Rosa roxburghii fruit such as Vc, VE and SOD, etc., increase the fruit diameter and edible part.

The technical solution adopted in the present invention is: a method for inducing the tetraploid production of seedless Rosa roxburghii by the mixed cultivation method, comprising the steps of:

Step 1, material preparation: Remove the leaves and tops of the stems of sterile seedlings, cut them into 0.8-3cm long, and keep at least one leaf axil for each section,

Step 2, pre-cultivation: pre-cultivate in medium MS+0.5mg/L 6-BA for 0-3d;

Step 3, colchicine light culture: the pre-cultured stem segments are transferred to MS+0.5mg/L 6-BA+ medium supplemented with 0-200mg/L colchicine respectively, and the stem segments are slightly submerged in the medium. Make the axillary bud fully contact with the culture medium;

Step 4, culture without adding colchicine: after step 3 for 7 days, transfer to MS+0.5mg/L6-BA medium without adding colchicine to continue the culture;

Step 5: Homogenization treatment of mutated buds: After step 4 is cultivated for 40 days, the sprouts with obvious variation obtained from the culture are selected and cultured in medium MS+0.15mg/L NAA+0.5mg/L 6-BA, repeated at intervals of 40d Cultivate 5 times.

Further, the stem segments pre-cultured in Step 3 were transferred to MS+0.5 mg/L 6-BA+ medium supplemented with 100 mg/L colchicine.

Further, when the pre-cultivation time in step 2 is 0, that is, when no pre-cultivation is carried out, the stem segment obtained in step 1 is directly inoculated in MS+0.5mg/L of 100mg/L colchicine in said step 3. 6-BA medium.

Further, carry out ploidy identification on the mutant seedlings after homogenization treatment: when the buds of homogenization treatment grow to 2-3cm, transfer the seedless Rosa roxburghii sprouts with obvious variation to 1/2MS+0.1mg/ Rooting in the culture medium of L 6-BA+0.2mg/L IBA+0.3g/L activated carbon. When the root length was 1 cm, the root tip material was intercepted, and the chromosomes were counted by the conventional film-making method to determine its ploidy.

Further, in step 1, the leaves and tops of the stems of the sterile seedlings are removed, cut into 1 cm lengths, and at least one leaf axil is reserved for each section.

Compared with the prior art, the beneficial effect of the present invention is: after the individual seedlings treated with colchicine are transferred to the culture medium without additional colchicine for a period of time, compared with the control, most of them show leaf Dark green and obviously thickened, the stems become thicker, etc., showing the advantages of polyploid materials in terms of growth compared with diploid materials. The induction method of the present invention doubles the number of chromosomes of Rosa roxburghii and induces tetraploid Rosa roxburghii fruit, which can increase the nutritional components of Rosa roxburghii fruit such as Vc, VE and SOD, and increase the fruit diameter and edible part.

Description of drawings

Fig. 1 is a kind of flow chart of the induction process of the induction method that the tetraploid of seedless Rosa roxburghii is produced by the mixed cultivation method;

Fig. 2 is a flow chart of the induction process of an induction method for producing tetraploids of Rosa roxburghii by soaking.

Detailed ways

In order to deepen the understanding of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments, which are only used to explain the present invention and do not limit the protection scope of the present invention.

Example The seedless Rosa roxburghii sterile seedling established by the key laboratory of biodiversity conservation of a certain university was selected for polyploid induction test and ploidy identification

1 Materials and methods

1.1 Test material

As the test material, the aseptic seedlings of Rosa roxburghii established by the Key Laboratory of Biodiversity Conservation of Southwest Forestry University were selected.

1.2 Test method

1.2.1 Soaking method, see Figure 2

Remove the leaves and tops of the stems of sterile seedlings, cut them into about 1 cm long, keep at least one leaf axil for each segment, and transfer them to the medium MS+0.5mg/L 6-BA for pre-cultivation. The pre-cultivation time is 1, 2, and 3d. After the pre-cultivation, light culture was carried out. During light culture, the pre-cultured stem segments were soaked in sterile water containing different concentrations of colchicine (200, 300, 400, 500mg/L), and placed on a shaker at 120r/min, 25-30 ℃, protected from light and shaking, and the treatment time was 12, 24, 48 and 72 hours respectively. The stem segments of the control group were placed in sterile water for 12, 24, 48 and 72 hours, and the method was the same as above. After the treatment, put the stem section on the ultra-clean workbench, absorb the residual liquid on the surface with sterile filter paper, and then transfer it to the solution of MS+0.15mg/LNAA+0.5mg/L 6-BA (without adding colchicine). Continue to grow in culture medium. There were 52 groups of treatments in total, each group was inoculated with 20 bottles, and each bottle was inoculated with 5 stem segments, repeated 3 times.

1.2.2 Mixed cultivation method

Remove the leaves and tops of the stems of sterile seedlings, cut them into about 1 cm long, keep at least one leaf axil for each segment, and pre-culture them in medium MS+0.5mg/L 6-BA for 0, 1, 2, 3 days. The pre-cultivated stem segments were transferred to MS+0.5 mg/L 6-BA+ medium supplemented with 0, 50, 100, 150, and 200 mg/L colchicine respectively, and the stem segments were slightly submerged in the medium to make the axillary buds and medium in full contact. After 7 days, they were transferred to MS+0.5 mg/L 6-BA medium without colchicine and continued to culture. One group was set up as the control group, which was inoculated in the axillary bud induction medium. There were 13 treatments in total, each treatment group was inoculated with 20 bottles, each bottle was inoculated with 5 stem segments, and repeated 3 times. 1.2.3 Homogenization treatment of mutant buds

After 40 days, select the sprouts that became obvious in the above 1.2.1 and 1.2.2, and culture them repeatedly 5 times in medium MS+0.15mg/L NAA+0.5mg/L 6-BA at intervals of 40 days.

1.2.4 Ploidy identification

When the homogeneously treated buds grow to 2-3cm, compared with the control group, the seedless Rosa roxburghii sprouts with obvious variation are transferred to 1/2MS+0.1mg/L 6-BA+0.2mg/L IBA+0.3 g/L activated carbon medium. And using the method proposed by Lin Yuan et al., the number of chromosomes in the root tip of the mutant seedless Rosa roxburghii and diploid material was counted to determine the ploidy.

2. Results and Analysis

2.1 Dipping method

The pre-cultivation time, colchicine concentration and treatment time had obvious effects on the generation of polyploid in Rosa roxburghii (Table 2).

Table 1 The effect of colchicine concentration and treatment time on stem segment induced polyploidy

It can be seen from Table 1 that under different treatments, the mutation rate of Rosa roxburghii was significantly different. In general, under the same pretreatment time, with the increase of treatment concentration, the mutagenesis rate has an upward trend in the same treatment days; while at the same pretreatment time and the same concentration but treatment No. 26, that is: after 1 day of pre-culture , then soaked in 400mg/L colchicine solution for 24h, its average mutagenesis rate was the highest (30.0%), followed by pre-cultivation for 3d, and treated with 300mg/L colchicine for 12h, its mutation rate was 26.7% ( 21 processing). In all controls, no variable material appeared. The results of multiple comparisons showed that there was no significant difference between No. 21 treatment and No. 26 treatment. Therefore, both No. 21 and No. 26 treatments are suitable for stem soaking method to induce polyploid adventitious buds of seedless Rosa roxburghii. However, considering the amount of reagents, etc., the No. 21 treatment should be the best, that is, after pre-cultivation for 3 days, treat with 300mg/L colchicine for 12 hours, which is the result of polyploidy induced by the seedless Rosa roxburghii soaking method. The best induction regimen.

2.2 Mixed cultivation method, as shown in Figure 1, the mutagenic effects of the mixed cultivation method are listed in Table 2 after the stem segments are treated differently.

Table 2 The effect of colchicine concentration and treatment time on stem segment induced polyploid

It can be seen from Table 2 that the mutagenesis rate of the method of inducing stem segments by the mixed culture method is generally low, and the analysis of variance shows that there are significant differences among different treatments. The mutagenesis rate of the control group without any treatment was 0.0%, and the mutagenesis rate decreased significantly with the increase of the treatment concentration. When the pre-cultivation is optional for 1 day, the mutagenesis rates of the materials inoculated in the medium supplemented with 100mg/L colchicine were higher, respectively 5.8% and 5.6% (treatment 1 and 2). And there was no significant difference between the two treatments. This shows that in the treatment of adding 100mg/L colchicine, the presence or absence of pretreatment has little effect on the mutagenic effect. Considering the simplicity of the experiment, it is considered that directly inserting the stem segments into the medium supplemented with 100 mg/L colchicine without pretreatment is an ideal method for inducing polyploidy of Rosa roxburghii by mixed culture.

2.3 Ploidy identification

After 5 homogenization treatments, the number of chromosomes in the root tip of the seedless Rosa roxburghii rooted tissue culture seedlings was observed. The results showed that the number of diploid chromosomes in Rosa roxburghii was 2n=2x=14. Among the mutant plants, some materials have 28 chromosomes at the root tip, which can be judged as tetraploid; while some materials have both 14 and 28 chromosome cells, which can be judged as mosaic. No triploid cells were found in the experiment.

The invention relates to a method for inducing tetraploids of Rosa roxburghii by mixed cultivation method, which involves polyploid breeding of Rosa roxburghii seedless and breeding tetraploid Rosa roxburghii seedless, which can increase the roxa seedless roxburghii Fruit nutrients such as Vc, VE and SOD, etc., while increasing the fruit diameter and edible part.

The embodiments of the present invention disclose preferred embodiments, but are not limited thereto. Those skilled in the art can easily comprehend the spirit of the present invention based on the above-mentioned embodiments, and make different extensions and changes. But as long as it does not deviate from the spirit of the present invention, it is within the protection scope of the present invention.

Claims (2)

1. a method for inducing seedless Rosa roxburghii tetraploid to produce by mixed culture method, is characterized in that, comprises the following steps Steps: Step 1, material preparation: remove the leaves and tops of the stem section of the sterile seedling, cut it into 0.8-3cm long, and keep at least one leaf axil for each section; Step 2, pre-cultivation: pre-cultivate in medium MS+0.5mg/L 6-BA for 2-3 days; Step 3, colchicine light culture: the stem section after the pre-cultivation is transferred to respectively supplemented with 100mg/L colchicine In MS+0.5mg/L 6-BA medium, the stem segment is submerged in the medium, so that the axillary buds are fully in contact with the medium; Step 4, culturing without adding colchicine: after step 3 for 7 days, transfer to MS+0.5 mg/L 6-BA medium without adding colchicine to continue culturing; Step 5: Homogenization treatment of mutated buds: After step 4 is cultivated for 40 days, select the buds with obvious variations obtained from the culture, and repeat in medium MS+ 0.15mg/L NAA+ 0.5mg/L 6-BA at intervals of 40d Cultivate 5 times; Carry out ploidy identification of the mutant seedlings after homogenization treatment: when the homogenization treatment buds grow to 2-3cm, transfer the seedless Rosa roxburghii sprouts with obvious variation to 1/2MS +0 .1mg/L 6 -BA+0 .2mg/L IBA+0 .3g/L activated carbon medium to take root, when the root length is 1cm, cut off the root tip material, use the routine method to count the chromosomes and determine its ploidy. 2. a kind of method that induces seedless Rosa roxburghii tetraploid to produce by mixed cultivation method according to claim 1 is characterized in that, in described step 1, blade and top are removed from the stem section of aseptic seedling, cut into 1cm Long, with at least one leaf axil per segment.