Tissue culture breeding method of vitex trifolia

Abstract

The invention discloses a vitex umbellata tissue culture breeding method, which is characterized in that stem segments of vitex umbellata are used as explants, axillary buds are successfully induced, effective breeding is carried out, rooting is further induced, and an effective bud breeding system and a plant regeneration system are established. 0.8 mg / LBA is added to an MS culture medium to serve as a proliferation culture medium, and the bud proliferation coefficient reaches 6.3 after 45 days of culture; iBA or NAA is added to a 1 / 2 MS culture medium to serve as a root induction culture medium, and the rooting rate can reach 100% after 40 days of culture; when the seedlings are transferred into a plastic basin filled with vermiculite and peat soil (1: 1, v / v) to be cultured for one month, the survival rate reaches up to 98.4%. According to the invention, a set of tissue culture and rapid propagation system of the vitex umbellata is established, and a foundation is laid for large-scale planting of the vitex umbellata.

Description

Technical field:

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a tissue culture and breeding method for Vitex foetida. Background technology:

[0002] Premna serratifolia Linn. (synonyms: P.corymbosa (Burm.f.) Merr., P.integrifolia L.and P.obtusifolia R.Br.), (synonym Premna serratifolia, Premnacorymbosa (Burm.F.) Rottl.et Willd) is a plant of the Lamiaceae family and the genus Tofu Shiitake. It is named because of the strong odor that can be smelled after rubbing its leaves. Premna serratifolia Linn. is distributed in coastal areas of India, Sri Lanka, Malaysia, and even the South Pacific islands, as well as Taiwan, Guangxi, and the Xisha Islands in China. It mostly grows in forests by the sea, plains, or mountains. Premna serratifolia is a common plant among coral reefs and rocks. Because of its fast growth and drought, wind, and barrenness resistance, it is an excellent tree species for windbreaks, hedges, and potted plants. Its drupe is spherical, and turns from yellow-green to dark brown when ripe, like a bunch of green and purple grapes, connected and stretched. The bud of the ... Summary of the invention:

[0003] In view of the shortcomings of the prior art, the present invention provides a tissue culture and breeding method for Vitex foetida, which lays a foundation for the rapid propagation and large-scale planting of Vitex foetida.

[0004] The present invention is achieved through the following technical solutions:

[0005] A tissue culture and breeding method of Vitex foetida, comprising the following steps: using young stem segments of Vitex foetida with axillary buds as explants, slicing and inoculating into an induction medium for induction culture after disinfection, continuing to perform subculture after budding to obtain clustered buds, placing the clustered buds in a proliferation medium for proliferation culture; transferring the clustered buds to a root induction medium for rooting culture to obtain tissue culture seedlings, and transplanting them to a substrate after hardening.

[0006] Preferably, the stem segment is derived from a plant that is at least one year old, and the axillary buds in the stem segment are 2-3 cm long.

[0007] Preferably, the specific steps of disinfection are: after the stem segments are washed with tap water, the surface is cleaned with cotton wool and 75% ethanol for 15 seconds, then transferred to a 0.1wt% HgCl2 solution for surface disinfection for 15 minutes, and finally washed 4 times with sterile distilled water.

[0008] Preferably, the induction medium is: MS medium containing 3wt% sucrose, 0.7wt% agar + 1.0mg / L BA + 0.1mg / L NAA, pH 5.8; the induction culture conditions are: the temperature is 26±2°C, and the culture room is illuminated by two cool white fluorescent lamps to provide 80μmol m -2 s -1 Light density.

[0009] Preferably, the induction culture time is 30 days.

[0010] Preferably, the proliferation medium is: MS medium containing 3wt% sucrose, 0.7wt% agar + 0.5-1.5mg / L BA, pH 5.8; the proliferation culture conditions are: the temperature is 26±2°C, the culture room is illuminated by two cool white fluorescent lamps, providing 80μmol m -2 s -1 Light density.

[0011] Preferably, the proliferation culture time is 45 days.

[0012] Preferably, the root induction medium is: 1 / 2MS medium containing 3wt% sucrose and 0.7wt% agar + 0.5mg / LIBA and / or 0.5mg / LNAA, pH 5.8; the rooting culture conditions are: the temperature is 26±2°C, the culture chamber is illuminated by two cool white fluorescent lamps, providing 80μmol m -2 s -1 Light density.

[0013] Preferably, the rooting culture time is 20-40 days.

[0014] Preferably, the hardening process is to transfer the seedlings to a greenhouse and place them under natural light for 7 days.

[0015] Preferably, the matrix consists of vermiculite and peat soil (1:1, v / v).

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention uses the stem segments of the cymbidium stinkingii as explants, successfully induces axillary buds and performs effective reproduction, and further induces rooting, establishes an effective bud propagation system and plant regeneration system for the first time, and lays a foundation for large-scale planting. Using MS culture medium supplemented with 0.8 mg / L BA as a proliferation culture medium, the bud propagation coefficient reaches 6.3 after 45 days of cultivation; using 1 / 2MS culture medium supplemented with 0.5 mg / L IBA or NAA as a root induction culture medium, the rooting rate can reach 100% after 40 days of cultivation; the seedlings are transferred to a plastic pot filled with vermiculite and peat soil (1:1, v / v) and cultured for one month, and the survival rate is as high as 98.4%. Description of the drawings:

[0018] Figure 1 The invention discloses different processes of tissue culture and propagation of the bud of Vitex foetida; wherein, a: the stem segment is induced and cultured on an MS medium supplemented with 1.0 mg / L BA and 0.1 mg / L NAA for 30 days to grow two axillary buds; b: the axillary buds are proliferated and cultured on an MS medium supplemented with 0.8 mg / L BA for one month to grow clusters of buds; c: the buds are cultured on a rooting medium 0.5 mg / L IBA for one month to form roots; and d: the tissue culture seedlings are transplanted to a substrate for one month. Specific implementation method:

[0019] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0020] Abbreviations used in the following examples are: BA, 6-benzyladenine; IBA, indole-3-butyric acid; KIN, kinetin; MS medium, Murashige and Skoog (1962) medium; NAA, α-naphthylacetic acid; PGR, plant growth regulator; SPC, proliferation coefficient; TDZ, thiazolidine.

[0021] Example 1

[0022] 1. Explant acquisition and sterilization

[0023] The plant material (Vitex foetida) was originally sourced from the Nansha Islands in Hainan. The plantlets were stem cuttings grown in plastic pots at the Wenchang Propagation Center in Hainan for more than a year and were brought to the South China Botanical Garden in Guangzhou, Guangdong in 2017. From plants at least one year old, young stem segments with 2-3 cm long axillary buds were cut, washed with tap water, the surface of the explants was cleaned with cotton wool and 75% ethanol for 15 seconds, transferred to a 0.1% HgCl2 solution and surface sterilized for 15 minutes, and then washed 4 times with sterile distilled water.

[0024] 2. Axillary Bud Induction

[0025] Surface sterilized stems were sliced ​​and inoculated onto culture medium to induce axillary buds ( Figure 1 a).

[0026] Culture medium: MS medium containing 3% sucrose by mass, 0.7% agar by mass + 1.0 mg / L BA + 0.1 mg / L NAA, pH 5.8. The preparation method is to add sucrose and agar to MS medium. The plant growth regulators used are directly added to the medium that has been sterilized at 121°C and 104 kPa for 18 minutes. The preparation method of other culture media is similar. Culture conditions: The culture bottle is 8 cm in diameter and 10 cm in height. The culture room at 26±2°C is illuminated by two cool white fluorescent lamps, providing 80 μmol m -2 s -1 The light density was 30 days.

[0027] After the induced shoots were subcultured for 6 times on the same culture medium, clustered axillary buds were formed, and the clustered buds that proliferated in large quantities were used for subsequent experiments.

[0028] 3. Effects of different plant growth regulators (PGRs) on shoot proliferation

[0029] The clusters of buds were cut into small pieces of 2-3 cm in height, each with three buds, and inoculated into medium containing different types and concentrations of PGRs for axillary bud proliferation (Table 1). Each culture bottle contained three clusters of buds. Each treatment contained 10 culture bottles. After 45 days of culture, all clusters of buds were divided into smaller buds, each with three buds, and then the bud proliferation line SPC (the number of axillary buds after 45 days of proliferation / the number of clusters of axillary buds before proliferation) was calculated.

[0030] Culture medium: MS medium containing 3% sucrose and 0.7% agar by mass + different types and concentrations of PGRs (BA, 0.5-1.5 mg / L; KIN, 0.5-1.5 mg / L; KIN 0.5-1.5 mg / L + 0.1 mg / L NAA; BA 0.5-1.5 mg / L + 0.1 mg / L NAA), pH 5.8, the plant growth regulators used were directly added to the culture medium that had been sterilized at 121°C and 104 kPa for 18 minutes. Culture conditions: The culture bottle was 8 cm in diameter and 10 cm in height, and the 26±2°C culture room was illuminated by two cool white fluorescent lamps, providing 80 μmol m -2 s -1 The results are shown in the table below.

[0031] Table 1 Effects of plant growth regulators in MS medium on the bud proliferation coefficient (SPC) of the bud base of Vitex foetida

[0032]

[0033] Results: On MS medium without PGR, almost no new axillary buds multiplied within 45 days (Table 1). When MS medium was supplemented with BA alone, low concentration (0.5 mg / L) of BA did not induce callus at the base of the shoot, while when 0.8 mg / L BA was used, SPC increased to 6.3, but some callus was induced at the base of the shoot. When the BA concentration was increased to 1.5 mg / L, SPC decreased, and more callus was induced at the base of the shoot. When 0.5 mg / L BA was combined with 0.1 mg / L NAA, some axillary buds were formed, and a small amount of callus appeared at the base of the shoot. However, when 0.8 mg / L BA and 0.1 mg / L NAA were combined, SPC reached 4.7 within 45 days. When 1.5 mg / L BA was used, callus increased and SPC decreased (Table 1). Therefore, the use of NAA and higher concentrations of BA is not recommended for shoot proliferation. In the culture medium supplemented with different concentrations of KIN, even when used in combination with 0.1 mg / L NAA, the SPC was relatively lower than that of BA, and the induced callus was also smaller than that of BA.

[0034] 4. Rooting of buds

[0035] The clumps of shoots (5 cm high) obtained from any of the above tests were separated into single shoots, combined and inoculated on root induction medium. Each culture bottle contained 3 shoots and there were 10 culture bottles for each treatment. After 20 and 40 days of cultivation under light, the rooting rate was calculated.

[0036] Culture medium: 1 / 2MS medium containing 3% sucrose and 0.7% agar, pH 5.8. Plant growth regulators were added directly to the culture medium sterilized at 121°C and 104 kPa for 18 minutes. Culture conditions: The culture bottle was 8 cm in diameter and 10 cm in height. The culture room was 26±2°C and provided with 80 μmol m in a 12-hour light cycle by two cool white fluorescent lamps. -2 s -1 The results are shown in the table below.

[0037] Table 2 Effects of 1 / 2MS medium and medium auxin on rooting of single buds of Vitex foetida

[0038]

[0039] Results: On the 1 / 2MS medium without PGR, no roots appeared within 20 days, but after 40 days, 39.3% of the shoots induced roots, but the roots were relatively short. On the 1 / 2MS medium containing 0.1 mg / L IBA, only 18.3% of the shoots induced roots after 20 days. On the 1 / 2MS medium containing 0.1 mg / L NAA, only 17.1% of the shoots induced roots after 20 days, and on the 1 / 2MS medium containing 0.5 mg / L IBA, only 49.4% of the shoots induced roots after 20 days, and 100% after 40 days, and the roots were thin and long. On the 1 / 2MS medium containing 0.5 mg / L NAA, only 58.6% of the shoots induced roots within 20 days, but after 40 days of culture, 100% of the shoots induced roots, and the roots were thick and long ( Figure 1 c) When 0.5 mg / L NAA and 0.5 mg / L IBA were mixed in 1 / 2 MS medium, 100% of the shoots induced roots after 20 or 40 days of culture.

[0040] 5. Transplantation of plants

[0041] The culture bottles cultured in the rooting induction medium supplemented with 0.5 mg / L NAA and / or 0.5 mg / L IBA for 40 days were transferred to the greenhouse and placed under natural light for 7 days. The agar on the roots was gently washed off under tap water, and then the seedlings were transferred to plastic pots (15 cm high, 15 cm in diameter; 1 plantlet / bottle) filled with vermiculite and peat soil (1:1, v / v). Water was sprayed on the leaves and soil at 8 am every day, and 1 / 2MS medium was sprayed once a week. After 30 days, the survival percentage of the transplanted seedlings was determined: (number of live plants after transplantation / number of live plants before transplantation) × 100%.

[0042] Results: After acclimation in pots for 30 days, 98.4% of the young plants survived ( Figure 1 d).

Claims

1. A tissue culture and breeding method for Vitex foetida, characterized in that: The method comprises the following steps: using young stem segments of Vitex foetida with axillary buds as explants, sterilizing and slicing them and inoculating them into an induction medium for induction culture, continuing to perform subculture after budding to obtain clustered buds, placing the clustered buds in a proliferation medium for proliferation culture; transferring the clustered buds into a root induction medium for rooting culture to obtain tissue culture seedlings, and transplanting them into a substrate after hardening.

2. The method according to claim 1, characterized in that The induction medium is: MS medium containing 3wt% sucrose and 0.7wt% agar + 1.0mg / L BA + 0.1mg / L NAA, pH 5.8; the proliferation medium is: MS medium containing 3wt% sucrose and 0.7wt% agar + 0.5-1.5mg / L BA, pH 5.8; the root induction medium is: 1 / 2MS medium containing 3wt% sucrose and 0.7wt% agar + 0.5mg / L IBA and / or 0.5mg / L NAA, pH 5.8; the conditions of the induction culture, proliferation culture or rooting culture are: the temperature is 26±2°C, 80μmol m -2 s -1 Light density.

3. The method according to claim 1 or 2, characterized in that: The stem segment is derived from a plant of at least one year old, and the axillary buds in the stem segment are 2-3 cm long.

4. The method according to claim 1 or 2, characterized in that: The specific steps of the disinfection are: the stem segments are first washed with tap water and then cleaned with cotton wool and 75% ethanol for 15 seconds, transferred to a 0.1wt% HgCl2 solution for surface disinfection for 15 minutes, and finally washed with sterile distilled water for 4 times.

5. The method according to claim 1 or 2, characterized in that: The induction culture time is 30 days.

6. The method according to claim 1 or 2, characterized in that: The proliferation culture time is 45 days.

7. The method according to claim 1 or 2, characterized in that: The rooting culture time is 20-40 days.

8. The method according to claim 1 or 2, characterized in that: The seedling hardening process is to transfer the tissue culture seedlings to a greenhouse and place them under natural light for 7 days; the matrix is ​​composed of vermiculite and peat soil in a volume ratio of 1:1.

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