Tissue culture breeding method for amaranthus caudatus

Abstract

The invention discloses a tissue culture breeding method for amaranthus caudatus, which is characterized in that a stem with a bud point is used as an explant, an effective bud breeding system and a plant regeneration system are successfully established, 1.0 mg / LBA and 0.1 mg / LNAA are added to an MS culture medium to serve as a proliferation culture medium to culture for 30 days, the maximum bud breeding coefficient can reach 7.2, 0.1-1.0 mg / L IBA or NAA or IAA is added to the MS culture medium to serve as a root induction culture medium to culture for 30 days, and the maximum bud breeding coefficient can reach 7.2. The rooting rate can reach 100%, and the survival rate can reach 100% when the seedlings are transplanted into a matrix containing vermiculite and sand.

Description

Technical field:

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

[0002] Amaranthus splendidus is native to Brazil (Alternanthera bettzickiana (Regel) Nichols.), a perennial herb of the Amaranthaceae family. It likes high temperatures and grows best under conditions between 22-32°C. Amaranthus splendidus, also known as five-color grass, is mostly short, neatly shaped, luxuriantly foliaged, resistant to pruning, with strong branching, rich colors, and strong contrast. It can be made into various patterns, designs, texts, and other flat or three-dimensional images in different colors. It is most suitable for decorative landscapes and flower beds in green scenic spots. It is a good material for arranging felt flower beds. Amaranthus splendidus potted plants are suitable for balconies, windowsills, and flower troughs. Amaranthus splendidus can also be eaten directly, fried or shabu-shabu, and has medicinal effects. The whole plant has the effects of clearing away heat and detoxification, cooling blood and stopping bleeding, eliminating accumulation and removing blood stasis, clearing the liver and improving eyesight, and can treat symptoms such as conjunctivitis, bloody stools, and dysentery. Amaranthus splendidus is also a salt-tolerant plant that can survive on islands and reefs. There is a lack of specific research reports on the biological propagation technology of Amaranthus splendidus, especially its tissue culture technology. Summary of the invention:

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

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

[0005] A tissue culture breeding method for Amaranthus scabra, comprising the following steps: using Amaranthus scabra stem segments as explants, sterilizing and inoculating them into an induction culture medium for induction culture to obtain axillary buds, elongating and culturing the axillary buds and placing them in a proliferation culture medium for proliferation culture to obtain proliferation buds, transferring the proliferation buds into a root induction culture medium for rooting culture to obtain tissue culture seedlings, and transplanting them into a substrate after hardening the seedlings.

[0006] Preferably, the Amaranthus splendidus stem segment has buds and is about 1 cm in length.

[0007] Preferably, the disinfection step is: soak the stem segments in detergent for 30 minutes, rinse with running water, and absorb the surface moisture, put them in 75% alcohol for disinfection on an ultra-clean workbench for 30 seconds, rinse with sterile water 3 times, soak and disinfect with 0.1wt% mercuric chloride for 5 minutes, rinse with sterile water 5 times, and absorb the surface moisture on sterilized filter paper.

[0008] Preferably, the induction medium is: MS medium containing 3wt% sucrose, 0.7wt% agar + 0.5mg / L BA + 0.1mg / L NAA, pH 5.8; the induction culture conditions are: the culture bottle has a diameter of 8cm and a height of 10cm, and the culture room is lit at 26±2℃ with two cool white fluorescent lamps, providing 80μmol m -2 s -1 Light density.

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

[0010] Preferably, the axillary buds are cut into small segments of about 1 cm with a pair of leaves after elongation culture, and inoculated into a proliferation culture medium for proliferation culture.

[0011] Preferably, the proliferation medium is: MS medium containing 3wt% sucrose, 0.7wt% agar + 0.5-2.0mg / L BA, pH 5.8; the proliferation culture conditions are: the culture bottle has a diameter of 8cm and a height of 10cm, and the culture room is lit at 26±2℃ with two cool white fluorescent lamps, providing 80μmol m -2 s -1 Light density.

[0012] Further preferably, BA and other plant growth regulators are used in combination in the proliferation medium, and the plant growth regulator combination is: 1.0 mg / L BA + 0.1-1.0 mg / L NAA; or, 1.0 mg / L BA + 0.1-1.0 mg / L IBA; or, 1.0 mg / L BA + 0.1-1.0 mg / L IAA.

[0013] Preferably, the proliferation culture time is 30 days.

[0014] Preferably, the root induction medium is: MS medium containing 3wt% sucrose, 0.7wt% agar + 0.1-1.0mg / L NAA or 0.1-1.0mg / L IBA or 0.1-1.0mg / L IAA, pH 5.8; the rooting 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.

[0015] Preferably, the rooting culture time is 15-30 days.

[0016] Preferably, the seedling hardening process is: take the Amaranthus splendidus tissue culture seedlings with good growth, about 5 cm high, open the culture bottle cap, and harden the seedlings for 3-7 days to allow the seedlings to initially adapt to the external environment.

[0017] Preferably, the matrix consists of vermiculite and sand (volume ratio 1:1).

[0018] Beneficial effects:

[0019] The present invention successfully establishes a tissue culture breeding method for Amaranthus splendidus. After culturing in an MS medium supplemented with 1.0 mg / BA+0.1 mg / LNAA as a proliferation medium for 30 days, the bud propagation coefficient can reach up to 7.2. After culturing in an MS medium supplemented with 0.1-1.0 mg / L BA or NAA or IAA as a root induction medium for 30 days, the rooting rate can reach 100%. When the seedlings are transplanted into a matrix composed of vermiculite and sand, the survival rate is also as high as 100%. Description of the drawings:

[0020] Figure 1 The invention discloses a tissue culture propagation and rooting process of Amaranthus splendidus, wherein: a: buds are cultured on MS medium supplemented with 1.0 mg / L BA+0.1 mg / L NAA; b: buds are cultured on MS medium supplemented with 0.1 mg / L NAA for 30 days to take root; c: tissue culture seedlings are transplanted into a matrix composed of vermiculite and sand (volume ratio 1:1) and cultured for 30 days. Specific implementation method:

[0021] 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.

[0022] Abbreviations used in the following examples are: BA, 6-benzyladenine; IAA, indoleacetic acid; 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.

[0023] Example 1

[0024] 1. Explant acquisition and sterilization

[0025] In order to reduce the contamination rate during the tissue culture of Amaranthus splendidus, materials were collected in the afternoon on a sunny day. Cut the stem segments of Amaranthus splendidus, remove the leaves, and cut the stem segments with buds of about 1 cm as explants. Take the stem segments of Amaranthus splendidus, soak them in detergent for 30 minutes, rinse them with running water, and dry the surface moisture. Put the explants in 75% alcohol on the clean bench for 30 seconds, rinse them with sterile water 3 times, soak them in 0.1% mercuric chloride for 5 minutes, rinse them with sterile water 5 times, and dry the surface moisture on sterilized filter paper.

[0026] 2. Axillary Bud Induction

[0027] The explants after disinfection are inoculated on the culture medium, and the axillary buds on the stem segments of Amaranthus splendidus are allowed to germinate.

[0028] Culture medium: MS medium containing 3% sucrose by mass, 0.7% agar by mass + 0.5mg / LBA + 0.1mg / LNAA, 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℃ and 104k Pa for 18 minutes. The preparation method of other culture media is similar. Culture conditions: The culture bottle is 8cm in diameter and 10cm in height. The culture room at 26±2℃ is illuminated by two cool white fluorescent lamps, providing 80μmol m in a 12-hour light cycle. -2 s -1 Light density. Cultivation time: 30 days.

[0029] 3. Effects of different plant growth regulators (PGRs) on bud propagation

[0030] The axillary buds after elongation culture were cut into small segments of about 1 cm with a pair of leaves, and inoculated on MS medium supplemented with different types and concentrations of plant growth regulators for culture. Four buds were inoculated in each bottle, and the bud reproduction coefficient was calculated after 30 days of culture.

[0031] Culture medium: MS medium containing 3% sucrose and 0.7% agar + different types and concentrations of PGRs (KIN, BA, TDZ; 0.5-2.0 mg / L), pH 5.8. Culture conditions: The culture bottle was 8 cm in diameter and 10 cm in height, and the culture room was 26±2℃ with two cool white fluorescent lamps providing 80 μmol m in a 12-hour light cycle. -2 s -1 The results are shown in the table below.

[0032] Table 1 Effects of different types and concentrations of plant growth regulators on the bud propagation of Amaranthus splendidus

[0033]

[0034] As shown in Table 1, when different types and concentrations of plant growth regulators were added to the MS medium, the addition of BA could significantly increase the bud propagation coefficient. When the concentration was 1.0 mg / L, the bud propagation coefficient reached 5.8 after 30 days of culture.

[0035] On this basis, other plant growth regulators were added to the above culture medium and used in combination with BA to conduct bud breeding experiments. The combination was: 1.0mg / L BA + 0.1-1.0mg / L NAA; or, 1.0mg / L BA + 0.1-1.0mg / L IBA; or, 1.0mg / L BA + 0.1-1.0mg / L IAA. The other components of the culture medium and the culture conditions refer to the above experiments. The results are shown in the table below.

[0036] Table 2 Effects of BA, NAA, IBA and IAA on the bud propagation of Amaranthus splendidus

[0037]

[0038]

[0039] It can be seen from Table 2 that when the plant growth regulator combination is 1.0 mg / LBA + 0.1 mg / LNAA, the bud propagation coefficient can reach up to 7.2.

[0040] 4. Rooting Induction

[0041] The rootless buds with a plant height of about 3 cm and 2-4 fully expanded leaves were inoculated into the culture medium for cultivation. Six buds were inserted into each culture bottle, and the rooting of the plants was observed after 15 and 30 days of cultivation.

[0042] Culture medium: MS medium containing 3% sucrose, 0.7% agar, 0.1-1.0 mg / L NAA or 0.1-1.0 mg / L IBA or 0.1-1.0 mg / L IAA, pH 5.8; Culture conditions: The culture bottle was 8 cm in diameter and 10 cm in height, and the culture room was lit at 26±2℃ with two cool white fluorescent lamps providing 80 μmol m -2 s -1 The results are shown in the table below.

[0043] Table 3 Rooting on different rooting media

[0044]

[0045] As shown in Table 3, roots will form after 15 days of cultivation, and the rooting rate can reach 100% after 30 days of cultivation.

[0046] 5. Hardening and transplanting

[0047] Take the well-growing Amaranthus tissue culture seedlings, about 5cm tall. Open the cap of the culture bottle and harden the seedlings for 3-7 days to allow the seedlings to initially adapt to the external environment. Take them out and wash off the solid culture medium attached to the roots with tap water, then transplant them into a black seedling bag containing vermiculite and sand (volume ratio 1:1), transplanting one plant per bag. The survival rate of the transplanted seedlings after 30 days of transplantation was 100%, and the leaves of the tissue culture seedlings were emerald green, the stems were thick, and they were growing well ( Figure 1 c).

Claims

1. A method for tissue culture and breeding of Amaranthus splendidus, characterized in that: The following steps are involved: The stem segments of Amaranthus splendidus were used as explants, which were sterilized and inoculated into induction medium for induction culture to obtain axillary buds. The axillary buds were elongated and then placed in proliferation medium for proliferation culture to obtain proliferation buds. The proliferation buds were transferred to root induction medium for rooting culture to obtain tissue culture seedlings, which were then transplanted to 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 + 0.5mg / L BA + 0.1mg / L NAA, pH 5.8; the proliferation medium is: MS medium containing 3wt% sucrose and 0.7wt% agar + 0.5-2.0mg / L BA, pH 5.8; the root induction medium is: MS medium containing 3wt% sucrose and 0.7wt% agar + 0.1-1.0mg / L NAA or 0.1-1.0mg / L IBA or 0.1-1.0mg / L IAA, pH 5.8; the conditions of the induction culture, proliferation culture or rooting culture are: 26±2°C, providing 80μmolm -2 s -1 Light density.

3. The method according to claim 2, characterized in that In the proliferation culture medium, BA and other plant growth regulators are used in combination, and the combination is: 1.0 mg / L BA + 0.1-1.0 mg / L NAA; or, 1.0 mg / L BA + 0.1-1.0 mg / L BA; or, 1.0 mg / L BA + 0.1-1.0 mg / L IAAA.

4. The method according to claim 1, 2 or 3, characterized in that: The Amaranthus splendidus stem segment has buds and is 1 cm in length.

5. The method according to claim 1, 2 or 3, characterized in that: The disinfection steps are as follows: soak the stem segments in detergent for 30 minutes, rinse with running water and dry the surface moisture, put them in 75% alcohol for disinfection for 30 seconds on a clean bench, rinse with sterile water for 3 times, soak and disinfect with 0.1wt% mercuric chloride for 5 minutes, rinse with sterile water for 5 times, and dry the surface moisture on sterilized filter paper.

6. The method according to claim 1, 2 or 3, characterized in that: After elongation culture, the axillary buds were cut into 1 cm segments with a pair of leaves and inoculated into proliferation medium for proliferation culture.

7. The method according to claim 1, 2 or 3, characterized in that: The seedling hardening process is as follows: opening the cap of the culture bottle, taking out 5 cm high Amaranthus splendidus tissue culture seedlings with good growth, and hardening the seedlings for 3-7 days to make the tissue culture seedlings initially adapt to the external environment.

8. The method according to claim 1, 2 or 3, characterized in that: The matrix is ​​a mixture of vermiculite and sand in a volume ratio of 1:1.

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