A tissue culture rapid propagation method of salix mongolica

Abstract

The present application relates to the technical field of forestry asexual propagation, and particularly relates to a tissue culture and rapid propagation method of Salix psuedopectinata. The present application realizes the correction of the leaf color of Salix psuedopectinata tissue culture seedlings, solves the yellowing problem of the leaves in the process of Salix psuedopectinata tissue culture, and promotes the new shoots and rooting of Salix psuedopectinata, so that the Salix psuedopectinata tissue culture seedlings grow well. The method of the present application provides a new way for the preservation of the excellent germplasm resources of Salix psuedopectinata and the factory seedling.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of forestry asexual propagation, and particularly relates to a tissue culture and rapid propagation method of Salix psammophila. BACKGROUND

[0002] Salix psammophila (C.Wang et Ch.Y.Yang) is also named as north Salix and northwest Salix, and is a perennial deciduous shrub or small tree of Salicaceae Salix. The height is 2-5m, the branch is long and has hair, the leaf is alternate, strip or strip inversely lanceolate (up to 12cm), the flowering period is from March to April, and the fruiting period is from May to June. Salix psammophila has the characteristics of drought tolerance, cold tolerance, high temperature tolerance, sand burying tolerance and wind erosion resistance, is a tree species for afforestation in northwest China to prevent wind erosion, control desertification and restore vegetation, plays an important role in the local ecosystem, and is one of the preferred tree species of "Three North Shelter Forest".

[0003] Salix psammophila is mainly propagated by cutting and tissue culture. The cutting afforestation of Salix psammophila is the best afforestation method in the afforestation engineering practice, but the propagation quantity and speed are still limited by the number of cuttings, and the cutting without selection will also accelerate the degradation of seedling resources; the plant tissue culture technology is not affected by the season, is simple to operate, and can cultivate the tissue culture seedlings with excellent genetic traits in a short time.

[0004] At present, there are many research results on the tissue culture of Salix psammophila, but there are problems such as leaf yellowing and gradual death of Salix psammophila seedlings in the tissue culture in the experimental process.

[0005] Therefore, how to correct the leaf color of Salix psammophila tissue culture seedlings and quickly cultivate Salix psammophila tissue culture seedlings with healthy leaf color and excellent growth condition becomes a technical problem urgently to be solved in the technical field. SUMMARY

[0006] Firstly, the present application provides a Salix psammophila tissue culture method, comprising:

[0007] inducing and culturing the adventitious buds by inoculating the disinfected Salix psammophila stem segments into the culture medium; and inoculating the adventitious buds into the culture medium for rooting culture after the adventitious buds are formed.

[0008] The culture medium comprises WPM, 0.7-0.9mg / L EDDHA-Fe, 0.4-0.6mg / L IBA, 28-32g / L sucrose and 6-8g / L agar.

[0009] The present application aims at the problem of leaf yellowing of sand willow tissue culture seedlings in the growth process. It is found that there is no obvious improvement effect by trying to replace MS, 1 / 2MS, SH, WPM, DKW and other culture media. Further attempts to adjust the components in the culture medium show that when 0.7-0.9 mg / L of EDDHA-Fe is additionally added to the WPM basic medium, the sand willow tissue culture seedlings can have dark green leaves, and the new bud growth and rooting effect of sand willow stem segment explants are excellent, the growth condition is excellent, and it is beneficial to the healthy growth of plants. When NRM and MNRM culture media containing EDDHA-Fe are used, although the sand willow tissue culture seedlings have dark green leaves, the rooting rate and root length of the sand willow tissue culture seedlings are significantly reduced.

[0010] Preferably, the sand willow stem segment is a tender sand willow stem segment (explant).

[0011] The above explant is convenient to take, and effectively saves operation time in the experiment.

[0012] Preferably, the pH of the culture medium is 5.8-6.0.

[0013] Preferably, the conditions of the adventitious bud induction culture and / or rooting culture include:

[0014] The temperature is 25±1℃, the light is 14-18h / d, and the light intensity is 2200-2600LX.

[0015] Preferably, the step of disinfection includes:

[0016] First, the sand willow stem segment is washed with 75% alcohol, then washed with sterile water, then disinfected with a mixture of 2-4% sodium hypochlorite and Tween 20, and finally washed with sterile water.

[0017] Preferably, the sand willow stem segment is washed with 75% alcohol for 30s-50s, then washed with sterile water for 4-5 times, then disinfected with a mixture of 2-4% sodium hypochlorite and Tween 20 for 2-3 times, each time for 15-25min, and finally washed with sterile water for 6-8 times, and the water is absorbed with sterile filter paper.

[0018] Preferably, the step of disinfection further includes: rinsing with running water before washing with 75% alcohol.

[0019] Preferably, the running water is rinsed for more than 3 hours.

[0020] Preferably, the sand willow tissue culture method further includes: when the root length is 3-4cm, the culture medium is removed, and the explant is transplanted into a substrate with a volume ratio of peat to perlite of (1.5-2.5):1.

[0021] Preferably, the induction rate of the adventitious bud induction culture reaches more than 95%.

[0022] Preferably, the rooting rate of the rooting culture reaches 100%.

[0023] Preferably, the survival rate after transplanting reaches 100%.

[0024] Further, the present application provides the use of the Salix organization culture method in any of the above embodiments in Salix cultivation or establishment of an organization culture regeneration system.

[0025] Compared with the prior art, the present application has the beneficial effects that:

[0026] The present application realizes the correction of the leaf color of Salix tissue culture seedlings by using the WPM basic culture medium containing EDDHA-Fe in the whole process of Salix tissue culture, solves the problem of leaf yellowing in the process of Salix tissue culture, and can promote the new buds and rooting of Salix, and the growth condition is very good, so as to ensure the healthy growth and breeding of Salix tissue culture seedlings. The method of the present application is convenient in material taking, simple in operation, short in seedling growth period, and not affected by seasons, and provides a new way for Salix excellent germplasm resource cultivation, gene transformation, genetic resource preservation and factory seedling raising. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Fig. 1 is a diagram of the growth of the new buds of Salix in Example 1 of the present application.

[0028] Figure 2 Fig. 2 is a diagram of the adventitious buds of Salix in Example 1 of the present application.

[0029] Figure 3 Fig. 3 is a diagram of the rooting of Salix in Example 1 of the present application.

[0030] Figure 4 Fig. 4 is a diagram of the transplanting of the regenerated seedlings of Salix in Example 1 of the present application in the first week.

[0031] Figure 5 Fig. 5 is a diagram of the transplanting of the regenerated seedlings of Salix in Example 1 of the present application in the fifth week.

[0032] Figure 6 Fig. 6 is a photo of the Salix tissue culture seedlings of Comparative Example 1.

[0033] Figure 7 Fig. 7 is a photo of the Salix tissue culture seedlings of Example 2 with different amounts of EDDHA-Fe added; A in the figure indicates that 0.2 mg / L of EDDHA-Fe is added to the WPM culture medium, B indicates that 0.4 mg / L of EDDHA-Fe is added to the WPM culture medium, C indicates that 0.6 mg / L of EDDHA-Fe is added to the WPM culture medium, and D indicates that 0.8 mg / L of EDDHA-Fe is added to the WPM culture medium.

[0034] Figure 8The photo of the tissue culture seedling of the Salix mongolica for the comparative example 2.

[0035] Figure 9 The photo of the tissue culture seedling of the Salix mongolica for the comparative example 3. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0037] In the following examples, the formula of the WPM culture medium is as follows:

[0038]

[0039]

[0040] Example 1

[0041] The present embodiment provides a tissue culture method of Salix mongolica, and the specific steps are as follows:

[0042] (1) Disinfection of explants

[0043] The young stem segments of Salix mongolica are cut into short branches, washed with flowing water for 3 hours, taken out, placed in a clean bench, washed with 75% alcohol for 40 seconds, washed with sterile water for 4-5 times, 2% sodium hypochlorite is added with 2 drops of Tween 20 and shaken for 2 times, each time for 20 minutes, washed with sterile water for 6-8 times, and the water is absorbed with sterile filter paper;

[0044] (2) Induction culture of primary adventitious buds

[0045] The disinfected stem segments are inoculated into the primary culture medium with the formula of WPM+0.8mg / L EDDHA-Fe+0.5mg / L IBA+30g / L sucrose+7g / L agar, the pH is adjusted to 5.9, the temperature is 25±1℃, the illumination is 16h / d, the illumination intensity is 2500LX, the rooting starts at the 5th day, the adventitious buds are formed at the 7th day, and the induction rate reaches 95%;

[0046] (3) Rooting culture

[0047] The adventitious buds obtained in step (2) are inoculated into a medium of WPM+0.8 mg / L EDDHA-Fe+0.5 mg / L IBA+30 g / L sucrose+7 g / L agar, pH is adjusted to 5.9, temperature is 25±1℃, illumination is 16 h / d, illumination intensity is 2500 LX, rooting is started at the 5th day, and whole and robust tissue culture seedlings can be seen after 30 days, and the rooting rate is 100%;

[0048] (4) Seedling regeneration

[0049] When the radicles of the adventitious buds in step (3) grow to 3-4 cm, the bottle cap is opened, the medium is washed, and then the adventitious buds are transplanted into a substrate of peat:perlite=2:1 in volume ratio for culture, and the survival rate is 100%.

[0050] The growth photos of the primary new shoots of Salix mongolica in this example are shown in Figure 1 , the photos of the adventitious buds of Salix mongolica are shown in Figure 2 , the photos of the rooting of Salix mongolica are shown in Figure 3 , the photos of the transplanted regenerated seedlings of Salix mongolica in the first week are shown in Figure 4 , and the photos of the transplanted regenerated seedlings of Salix mongolica in the fifth week are shown in Figure 5 .

[0051] Example 2

[0052] In this example, the culture method of Example 1 is referred to, and only the added amount of EDDHA-Fe added to the WPM medium is changed to 0.2 mg / L, 0.4 mg / L, 0.6 mg / L and 0.8 mg / L respectively, and the influence of different concentrations of EDDHA-Fe on the growth of Salix mongolica tissue culture seedlings is analyzed.

[0053] The results are shown in Table 1 and Figure 7 . The length statistics of the new shoots show that in the medium with 0.8 mg / L EDDHA-Fe, the new leaves are small and many, the leaf color is dark green Figure 7 (D-1), and the length of the new shoots is the longest, reaching 53.00 mm; followed by 0.4 mg / L EDDHA-Fe and 0.6 mg / L EDDHA-Fe, the lengths of the new shoots are 48.27 mm and 44.43 mm respectively, the leaves of the explants are fewer but larger, and the leaves are green Figure 7 (B-1, 7C-1); when 0.2 mg / L EDDHA-Fe is added, the lateral buds of the explants grow slowly, the seedling height is only 40.07 mm, the leaves of the explants are large and many, and the leaves are green Figure 7 (A-1).

[0054] Statistical analysis of rooting numbers revealed that the rooting rate of *Salix psammophila* shoots decreased with increasing EDDHA-Fe concentration. Under the culture condition of 0.2 mg / L EDDHA-Fe, the number of roots in the explants reached as high as 9.40, while under the culture condition of 0.8 mg / L EDDHA-Fe, the number of roots in the explants was the lowest, at only 5.2.

[0055] Root length analysis revealed a negative correlation between the length of adventitious roots and the number of roots on young branches of *Salix psammophila*. The length of adventitious roots increased with increasing EDDHA-Fe concentration. There were no significant differences in adventitious root length among the four treatment groups. The treatment group with the longest roots was treatment 4 (addition of 0.8 mg / L EDDHA-Fe), with roots reaching 45.65 mm. These adventitious roots were slender and had few root hairs. Figure 7 D-2); Under 0.2 mg / L EDDHA-Fe culture conditions, the explant roots were relatively short, only 39.84 mm, with robust adventitious roots and numerous root hairs. Figure 7 A-2).

[0056] The above analysis shows that the optimal concentration of EDDHA-Fe for the growth of young branches of Salix psammophila is 0.8 mg / L, and the young branches of Salix psammophila exhibit the best growth under this treatment condition.

[0057] Table 1 Comparison of explant growth at different concentrations of EDDHA-Fe

[0058]

[0059] Note: The data in the table are mean ± standard error (M ± SD); the same letter indicates that the difference between treatments is not significant, and different letters indicate that the difference between treatments is significant.

[0060] Comparative Example 1

[0061] This comparative example provides a method for tissue culture of Salix psammophila, the only difference from Example 1 being that EDDHA-Fe is not added to any of the culture media.

[0062] Photos of Salix psammophila tissue culture seedlings Figure 6 As shown, on day 30, the leaves of the *Salix psammophila* tissue culture seedlings began to turn yellow and the terminal buds turned black. After another 60 days of subculture, the entire plant gradually withered and died.

[0063] Comparative Example 2

[0064] This comparative example provides a method for tissue culture of Salix psammophila, the only difference from Example 1 being that the WPM medium is replaced with 1 / 2 MS medium.

[0065] Photos of Salix psammophila tissue culture seedlings Figure 8As shown in the figure, the experimental results show that: compared with Example 1, after 30 days of culture, the statistical results of the newly grown shoots show that the newly grown shoots reach 57.50±2.47mm when WPM is used as the basic medium, which is significantly higher than the length of the newly grown shoots in 1 / 2MS medium, which is 48.79±3.40mm. The statistics of rooting rate and root length show that the rooting rate in WPM medium is 100.00±0.00, which is consistent with the rooting rate in 1 / 2MS medium, which is 100.00±0.00, and there is no significant difference. The root length in WPM medium is 49.17±2.54mm, which is significantly higher than the root length in 1 / 2MS medium, which is 36.48±3.93mm.

[0066] The overall growth condition analysis shows that WPM as the basic medium is significantly better than 1 / 2MS medium.

[0067] Comparative Example 3

[0068] This comparative example provides a Salix mongolica tissue culture method, and the only difference between the specific steps and Example 1 is that EDDHA-Fe is not added in all culture media, and the WPM medium is replaced by NRM medium.

[0069] The Salix mongolica tissue culture seedling photos are as shown in the figure Figure 9 As shown in the figure, the experimental results show that: compared with Example 1, after 30 days of culture, the statistical results of the newly grown shoots show that the newly grown shoots reach 57.50±2.47mm when WPM is used as the basic medium, which is significantly higher than the length of the newly grown shoots in 1 / 2MS medium, which is 48.79±3.40mm. The statistics of rooting rate and root length show that the rooting rate in WPM medium is 100.00±0.00, which is consistent with the rooting rate in 1 / 2MS medium, which is 100.00±0.00, and there is no significant difference. The root length in WPM medium is 49.17±2.54mm, which is significantly higher than the root length in 1 / 2MS medium, which is 36.48±3.93mm.

[0070] The overall growth condition analysis shows that WPM as the basic medium is significantly better than 1 / 2MS medium.

[0071] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for tissue culture of Salix psammophila, characterized in that, include: Disinfected Salix psammophila stem segments were inoculated into a culture medium for adventitious bud induction culture; After adventitious buds are formed, they are inoculated into a culture medium for rooting culture. The culture medium consists of the following components: WPM, 0.7-0.9 mg / L EDDHA-Fe, 0.4-0.6 mg / L IBA, 28-32 g / L sucrose, and 6-8 g / L agar.

2. The method for tissue culture of Salix psammophila according to claim 1, characterized in that, The pH of the culture medium is 5.8 to 6.

0.

3. The method for tissue culture of Salix psammophila according to claim 1, characterized in that, The conditions for adventitious bud induction culture and / or rooting culture include: The temperature is 25±1℃, the light intensity is 14~18h / d, and the light intensity is 2200~2600LX.

4. The method for tissue culture of Salix psammophila according to claim 1, characterized in that, The disinfection steps include: First, clean the sand willow stem segments with 75% alcohol, then clean them with sterile water, then disinfect them with a mixture of 2-4% sodium hypochlorite and Tween 20, and finally clean them with sterile water.

5. The method for tissue culture of Salix psammophila according to claim 4, characterized in that, First, wash the sand willow stem segments with 75% alcohol for 30-50 seconds, then wash with sterile water 4-5 times. Next, disinfect with a mixture of 2-4% sodium hypochlorite and Tween 20 by shaking 2-3 times, each time for 15-25 minutes. Finally, wash with sterile water 6-8 times and blot dry with sterile filter paper.

6. The method for tissue culture of Salix psammophila according to claim 1, characterized in that, Also includes: Once the roots have grown to 3-4 cm in length, remove the culture medium and transplant them into a substrate with a peat moss to perlite volume ratio of (1.5-2.5):

1.

7. The application of the Salix psammophila tissue culture method according to any one of claims 1 to 6 in the cultivation of Salix psammophila or the establishment of a tissue culture regeneration system.

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