Stem tip detoxification tissue culture seedling method for octaploid strawberry red

Abstract

The invention discloses a stem tip detoxification tissue culture seedling method for octaploid strawberry red. The method comprises the following steps: selecting an explant; stem tip treatment; performing primary culture; performing blank culture; carrying out subculture; performing rooting culture; transplanting and domesticating seedlings and planting. Through explant selection, application opportunity selection of a blank culture medium and subculture composite hormone proportion design, original seedlings of the virus-free seedlings of the Hongyan strawberries are successfully cultivated, the virus-free efficiency reaches 98% or above, and the transplanting survival rate reaches 99% or above. According to the method, the seedling culture time can be greatly saved, the blank that no stem tip tissue rapid propagation system exists in the Hongyan strawberries at present is filled up, higher detoxification efficiency and survival rate are achieved, and a foundation is laid for virus-free rapid culture of the Hongyan strawberries.

Description

Technical Field

[0001] The present invention relates to the technical field of strawberry cultivation, and particularly to a method for tissue culture and seedling raising of virus-free shoot tips of octoploid strawberry Hongyan. Background Art

[0002] As is well known, ordinary strawberry seedlings are mainly propagated vegetatively through strong stolons. However, years of vegetative propagation will cause viruses to accumulate in strawberries, and this kind of virus will be passed down from generation to generation, thus making the viral harm of strawberries increasingly severe. After strawberries are infected with viral diseases, the fruits will be significantly reduced in size, distorted in shape, and the quality will also decline accordingly. In addition, the leaves will show a wrinkled phenomenon, the growth rate of the plants will slow down, resulting in a substantial reduction in yield, bringing huge losses to strawberry growers.

[0003] According to incomplete statistics, there are as many as 62 kinds of strawberry viruses, and new viruses are constantly being reported. Among them, the strawberry viruses that are widely distributed and cause serious harm include strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV), strawberry vein band virus (SVBV), strawberry crinkle virus (SCV), and strawberry latent ringspot virus (SLRSV), etc.

[0004] Hongyan strawberries have a pivotal position in the strawberry cultivation market in China due to their fine pulp, proper sweet and sour taste, and strong aroma, and the planting area is increasing day by day. However, long-term vegetative propagation has led to virus accumulation, making commercial Hongyan strawberries carry viruses such as SMoV and SVBV, resulting in a slowdown in the growth rate of plants and a substantial reduction in yield, bringing huge losses to growers.

[0005] Strawberry plants themselves have no immunity to viruses. At present, there is no specific medicine or method to cure virus diseases. The only effective way to achieve virus-free cultivation of strawberries is to remove the viruses in the plants, obtain virus-free original mother plants, and carry out large-scale breeding of virus-free seedlings to meet the production needs. At present, the main methods for strawberry virus elimination rely on shoot tip meristem culture, anther tissue culture, and heat treatment, etc.

[0006] However, due to the sensitivity of the shoot tip meristem of Hongyan strawberries to peeling damage, and the lack of a culture medium optimized for octoploid strawberries in the existing technology, there are still problems of variety characteristic degradation in existing Hongyan strawberries. Summary of the Invention

[0007] The present invention provides a method for tissue culture and seedling raising of virus-free shoot tips of octoploid strawberry Hongyan.

[0008] To solve the above technical problems, the present invention provides a method for tissue culture and seedling raising of virus-free shoot tips of octoploid strawberry Hongyan, comprising the following steps:

[0009] (1) Selection of explants: Cut the apical bud on the healthy and pest-free stolons of Hongyan strawberry as the explant, and perform disinfection treatment;

[0010] (2) Shoot tip treatment: Cut the shoot tip growth point carrying leaf primordia from the disinfected explant above;

[0011] (3) Primary culture: Inoculate the shoot tip growth point cut in step (2) onto the primary culture medium for culture to induce the regeneration of cluster buds;

[0012] (4) Blank culture: Inoculate the cluster buds in step (3) into the blank culture medium, and let them grow in a hormone-free culture environment for a period of time to improve their growth and development ability;

[0013] (5) Subculture: Inoculate the healthy cluster buds in step (4) into the subculture medium, and proliferate tillering to obtain multiple clusters of cluster buds;

[0014] (6) Rooting culture: Divide the multiple clusters of cluster buds obtained from subculture into single cluster buds, and respectively cutt them into the rooting culture medium for root induction culture;

[0015] (7) Transplanting, acclimatizing seedlings and planting: Take out the seedlings with 3 - 5 true leaves and root length of 3 - 5 cm grown in the rooting culture medium, wash the root medium, plant them in the substrate seedbed for rooting outside the bottle, plant them, and cultivate the original seedling of virus-free strawberry seedlings.

[0016] In a preferred embodiment of the present invention, in step (1), the disinfectant used for the disinfection and sterilization treatment is ethanol with a volume concentration of 75% and sodium hypochlorite with a concentration of 2 - 5%.

[0017] In a preferred embodiment of the present invention, in step (2), the length of the shoot tip growth point is 0.2 - 0.3 mm.

[0018] In a preferred embodiment of the present invention, in step (3), the primary culture medium is MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.6 mg / L 6 - BA + 0.1 mg / L NAA.

[0019] In a preferred embodiment of the present invention, in step (4), the blank culture medium is MS medium + 3 g / L phytagel + 30 g / L sucrose.

[0020] In a preferred embodiment of the present invention, in step (5), the subculture medium is MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.5 - 0.6 mg / L 6-BA + 0.1 mg / L IAA.

[0021] In a preferred embodiment of the present invention, in step (6), the rooting medium is MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.1 mg / L IBA.

[0022] The beneficial effects of the present invention are as follows: The method for tissue culture and virus-free seedling propagation of the octoploid strawberry Hongyan of the present invention successfully cultivates the original virus-free seedlings of Hongyan strawberry through the selection of explants, the selection of the application timing of the blank medium, and the design of the compound hormone ratio of the subculture medium, and the virus-free efficiency is up to more than 98%, and the transplanting survival rate is up to more than 99%. The present invention can greatly save the seedling raising time, fill the blank of the current lack of a rapid propagation system for the shoot tips of 'Hongyan' strawberries, achieve higher virus-free efficiency and survival rate, and lay a foundation for the virus-free rapid cultivation of Hongyan strawberries. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is an example diagram of the method for tissue culture and virus-free seedling propagation of the octoploid strawberry Hongyan of the present invention during cultivation in the subculture medium;

[0024] Figure 2 It is an example diagram of the initial stage of cultivation of the method for tissue culture and virus-free seedling propagation of the octoploid strawberry Hongyan of the present invention in the rooting medium;

[0025] Figure 3 It is an example diagram of the later stage of cultivation of the method for tissue culture and virus-free seedling propagation of the octoploid strawberry Hongyan of the present invention in the rooting medium. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0027] Example 1

[0028] A method for tissue culture and virus-free seedling propagation of the octoploid strawberry Hongyan includes the following steps:

[0029] April to May is the end of the fruiting period of Hongyan strawberries, a season when a large number of stolons are sprouting. When the stolons of Hongyan strawberries grow to about 20 cm, select healthy and pest-free Hongyan strawberry stolons, and cut 3 cm apical buds as explants for tissue culture.

[0030] The cut apical buds were disinfected with 75% ethanol by volume for 30 s, washed 2 - 3 times with sterile water, then transferred to a 2% sodium hypochlorite solution by volume for disinfection for 10 min, washed 4 - 5 times with sterile water, blotted dry on sterile filter paper, and the large leaves on the explants were removed.

[0031] Under an electron microscope, the young leaves of the shoot tip explants were removed with a precision scalpel and stainless - steel pointed forceps, and a 0.2 - 0.3 mm shoot tip meristem carrying leaf primordia was excised with a scalpel.

[0032] After the excised shoot tip meristems were inoculated onto the primary medium and cultured for 30 days, multiple shoots were induced to regenerate; they were dark - treated for 7 days in a constant - temperature light - incubator at 22 °C, and then cultured in a constant - temperature light - incubator at 22 °C with 16 h of light, 8 h of darkness, and a light intensity of 3000 lx. The primary medium was MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.6 mg / L 6 - BA (6 - benzylaminopurine) + 0.1 mg / L NAA (1 - naphthylacetic acid).

[0033] The above - mentioned multiple shoots were inoculated into a hormone - free blank medium and allowed to grow in a hormone - free culture environment for 30 d. Conditions of the constant - temperature light - incubator: 22 °C, 16 h of light, 8 h of darkness, and a light intensity of 3000 lx. The blank medium can prevent or inhibit the growth of callus, avoid stress responses of multiple shoots due to long - term hormone stress, and make the multiple shoots grow stronger. Specifically, the blank medium was MS medium + 3 g / L phytagel + 30 g / L sucrose.

[0034] The strong multiple shoots in the blank medium were respectively inoculated onto sub - culture media containing different concentrations of the hormone 6 - BA and cultured for 40 d; specifically, the sub - culture medium was MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.1 mg / L IAA, and the concentrations of 6 - BA are shown in Table 1 below.

[0035] Table 1

[0036] IAA (mg / L) 0.1 0.1 0.1 0.1 0.1 6-BA (mg / L) 0.3 0.4 0.5 0.6 0.7 Inoculation number (pcs) 6 6 6 6 6 Number of cluster buds (pcs) 3 4 18 12 1 Callus (pcs) 4 2 0 0 0 Number of roots (pcs) 0 0 0 2 5

[0037] It can be seen from the data in Table 1 that 0.5 mg / L 6 - BA + 0.1 mg / L IAA is the optimal ratio, which can maximize the proliferation of multiple shoots and avoid callusing.

[0038] Multiple clusters of multiple shoots in the sub - culture medium containing 0.5 mg / L 6 - BA + 0.1 mg / L IAA were divided into single - plant multiple shoots and respectively cuttaged into a rooting medium for root induction culture; the rooting medium was MS medium + 3 g / L phytagel + 30 g / L sucrose + 0.1 mg / L IBA.

[0039] Take out the seedlings that have grown 3 true leaves and have roots 3-4 cm long in the rooting medium, wash the root medium, and plant them in the substrate seedbed for ex vitro rooting. During the transplanting process, remove the unhealthy seedlings and leave the healthy ones to continue growing for 60 days. Finally, plant the vigorously growing plug seedlings in the A-frame substrate to cultivate the original virus-free strawberry seedlings.

[0040] Comparative Example 1

[0041] The difference from Example 1 is that after primary culture, blank culture is not carried out, and subculture is directly carried out.

[0042] Comparative Example 2

[0043] The difference from Example 1 is that when the primary culture reaches half, it is transferred to the blank medium, and the subsequent culture conditions and time are the same.

[0044] The situations of the Hongyan strawberry seedlings obtained by culturing in Example 1, Comparative Example 1 and Comparative Example 2 are shown in Table 2 below.

[0045] Table 2

[0046] Example 1 Comparative Example 1 Comparative Example 2 Callus None Large amount None Quality of cluster buds Robust Poor Growth stagnation Rooting rate 99％ 55％ 0％

[0047] It can be seen from the comparison data in Table 2 that when the cluster buds are aging, timely use of the blank medium can effectively inhibit the callus and improve the seedling quality and rooting rate.

[0048] Comparative Example 3

[0049] The difference from Example 1 is that the length of the shoot apical meristem with leaf primordia cut by the scalpel is 0.1 mm.

[0050] Comparative Example 4

[0051] The difference from Example 1 is that the length of the shoot apical meristem with leaf primordia cut by the scalpel is 0.5 mm.

[0052] Comparative Example 5

[0053] The difference from Example 1 is that the length of the shoot apical meristem with leaf primordia cut by the scalpel is 1.0 mm.

[0054] The situations of the Hongyan strawberry seedlings obtained by culturing in Example 1, Comparative Examples 3-5 are shown in Table 3 below.

[0055] Table 3

[0056] Example 1 Comparative Example 3 Comparative Example 4 Comparative Example 5 Shoot tip size 0.2 - 0.3 mm 0.1 mm 0.5 mm 1.0 mm Survival rate 97％ 25％ 90％ 95％ Virus elimination rate Above 98% 100％ 80％ 60％ Number of cluster buds 18 pcs 1 - 2 pcs 10 pcs 8 pcs

[0057] It can be seen from the data comparison in Table 3 that 0.2-0.3 mm is the optimal shoot apical size, which can not only ensure the survival rate but also achieve the maximum virus elimination effect.

[0058] The method for tissue culture of virus-free shoot tips of octoploid strawberry Hongyan of the present invention has the following beneficial effects:

[0059] (1) Through virus-free tissue culture of the stolon shoot tips of Hongyan strawberry, the purification and update of the 'Hongyan' strawberry variety are promoted, and the efficient propagation of high-quality virus-free seedlings is achieved. In addition, this technology can significantly improve the yield and quality of 'Hongyan' strawberry.

[0060] (2) Virus-free seedlings of 'Hongyan' strawberry are quickly obtained through tissue culture, filling the blank of the current lack of a shoot tip tissue rapid propagation system for 'Hongyan' strawberry.

[0061] (3) The culture medium of the present invention is rationally prepared, not only with low cost, but also can extend the time of the explants of 'Hongyan' strawberry in the tissue culture bottle.

[0062] (4) The optimal proliferation conditions are determined through systematic experiments on hormone ratios.

[0063] (5) The virus-free efficiency of the present invention is over 98%, which can greatly save the seedling raising time, and the transplanting survival rate is over 99%.

[0064] The tissue culture method of the present invention establishes a complete technical chain from explant selection to planting, achieving higher virus-free efficiency and survival rate, and laying a foundation for the rapid virus-free culture of Hongyan strawberry.

[0065] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A method for virus-free tissue culture of stem tip of octoploid strawberry red, characterized in that: The steps include: (1) Selection of explants: The terminal buds were cut from healthy and pest-free red strawberry runners as explants and disinfected; (2) Shoot tip treatment: Cut the stem tip growth point carrying the leaf primordium from the above-mentioned disinfected explants; (3) Primary culture: inoculating the stem tip growth point cut in step (2) onto the primary culture medium for culture to induce regeneration of clustered buds; (4) Blank culture: inoculating the clustered buds in step (3) into a blank culture medium and allowing them to grow in a hormone-free culture environment for a period of time to improve their growth and development ability; (5) Subculture: inoculating the robust clustered buds in step (4) into a subculture medium to proliferate and tiller to obtain multiple clustered buds; (6) Rooting culture: The multiple clusters of buds obtained by subculture are separated into individual clusters of buds, and each of them is inserted into a rooting medium for rooting induction culture; (7) Transplanting and planting: Take out the seedlings with 3-5 true leaves and root length of 3-5 cm in the rooting medium, wash the root medium, plant them in the matrix seedling bed for out-of-bottle rooting, and plant them to cultivate the original seedlings of virus-free strawberry seedlings.

2. The method for cultivating octoploid strawberry red beauty's stem tip virus-free tissue seedlings according to claim 1, characterized in that: In step (1), the disinfectant used for the disinfection and sterilization treatment is 75% ethanol and 2-5% sodium hypochlorite in a volume concentration.

3. The method for stem tip virus-free tissue culture seedling of octoploid strawberry red according to claim 1, characterized in that, In step (2), the length of the stem tip growth point is 0.2-0.3 mm.

4. The method for cultivating octoploid strawberry red beauty's stem tip virus-free tissue seedlings according to claim 1, characterized in that: In step (3), the primary culture medium is MS culture medium + 3g / L plant gel + 30g / L sucrose + 0.6mg / L 6-BA + 0.1mg / L NAA.

5. The method for stem tip virus-free tissue culture seedling of octoploid strawberry red beauty according to claim 1, characterized in that, In step (4), the blank culture medium is MS culture medium + 3 g / L plant gel + 30 g / L sucrose.

6. The method for stem tip virus-free tissue culture seedling of octoploid strawberry red according to claim 1, characterized in that, In step (5), the subculture medium is MS medium + 3g / L phytagel + 30g / L sucrose + 0.5-0.6mg / L 6-BA + 0.1mg / L IAA.

7. The method for stem tip virus-free tissue culture seedling of octoploid strawberry red according to claim 1, characterized in that, In step (6), the rooting medium is MS medium + 3g / L plant gel + 30g / L sucrose + 0.1mg / L IBA.

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