Cultivation method of polyploidy pennisetum alopecuroides

Abstract

The invention belongs to the technical field of polyploidy breeding, and provides a cultivation method of polyploidy pennisetum alopecuroides, the cultivation method comprises the operation of carrying out polyploidy induction culture on embryonic callus in a polyploidy induction culture solution to obtain callus after polyploidy induction; wherein the polyploidy induction culture solution contains colchicine with the mass concentration of 0.05%-0.2%. The method provided by the invention has a high polyploidy induction success rate, tissue browning is avoided in the tissue culture process, the obtained pennisetum tissue culture seedlings are normal in yield and high in quality, and the method is simple and convenient to operate, efficient and convenient to popularize and use.

Description

Technical Field

[0001] The present invention belongs to the technical field of polyploid breeding, and particularly relates to a method for cultivating polyploid Pennisetum alopecuroides. Background Art

[0002] Pennisetum alopecuroides is a perennial herb of the genus Pennisetum in the family Poaceae. It has the advantages of drought resistance, easy management, and low maintenance cost. It also has a beautiful shape and unique texture, and can form a natural and simple harmonious landscape. At present, Pennisetum plants are widely used as ornamental grasses in garden green spaces, and most of the ornamental Pennisetum varieties used are introduced from abroad. There are few reports on the breeding research of Pennisetum in China, and few indigenous varieties are cultivated with independent intellectual property rights. Therefore, it is particularly urgent to innovate the germplasm of native Pennisetum and carry out the breeding of new varieties.

[0003] The formation of polyploids is very important in the evolution of plants or crop breeding. However, the probability of naturally forming polyploid plants is small. At present, the main way to produce polyploids relies on artificial induction. The production of artificial induced polyploids is mainly affected by two major factors: physical and chemical. Among them, the method of using the chemical mutagen colchicine to induce polyploids is widely used, and its induction principle is clear, the induction efficiency is high, and the effect is stable. The morphological characteristics of artificially induced polyploid plants are significantly different from those of diploids. Generally, polyploid plants are shorter in plant height, thicker in stem, thicker in leaves, and stronger in resistance than diploid plants. In view of the above excellent characteristics of polyploid plants, using artificial induction to innovate the germplasm of Pennisetum can not only create polyploid Pennisetum germplasm resources, improve the ornamental value of Pennisetum, but also make it possible to solve the problem of interspecific distant hybridization incompatibility of Pennisetum through polyploid breeding. Therefore, it is of great significance to carry out the research on polyploid breeding of Pennisetum. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method for cultivating polyploid Pennisetum alopecuroides, and the cultivation method includes a polyploid induction step:

[0005] Carrying out polyploid induction culture on embryogenic callus in a polyploid induction culture solution to obtain callus after polyploid induction;

[0006] The polyploid induction culture solution is: a liquid medium containing colchicine with a mass concentration of 0.05% - 0.2%, preferably 0.1%.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] 1. The cultivation method of the present invention provides parameters for polyploid induction with strong pertinence and good effects, and has a high success rate of polyploid induction.

[0009] 2. The cultivation method of the present invention provides embryogenic callus induction media, polyploid induction culture solutions, differentiation media, etc. with reasonable component configurations, avoids tissue browning during tissue culture, and obtains normal yields and high-quality Pennisetum seedlings.

[0010] 3. Each step and parameter of the present invention act synergistically to jointly improve the induction efficiency of Pennisetum polyploids.

[0011] 4. The operation of the present invention is simple, efficient, and convenient for popularization and use.

[0012] 5. The present invention uses appropriate methods to carry out germplasm innovation and new variety cultivation of tetraploid Pennisetum, providing good technical support for the wide application of polyploid Pennisetum. At the same time, the newly obtained tetraploid Pennisetum germplasm is applied to provide better plant materials for landscape configuration of garden plants and park greening, and provides an opportunity to solve the problem of interspecific distant hybridization incompatibility of Pennisetum. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 FIG. is a diagram of the stomatal morphology of diploid and tetraploid plants of "Purple Light" Pennisetum in Test Example 1 (under the microscope); the left figure is the diploid, and the right figure is the tetraploid.

[0014] Figure 2 FIG. is the flow cytometry detection peak diagram of diploid and tetraploid plants of "Purple Light" Pennisetum in Test Example 1.

[0015] Figure 3 FIG. is a diagram of the root tip chromosome count of diploid and tetraploid plants of "Purple Light" Pennisetum in Test Example 1 (under the microscope); the left figure is the diploid, and the right figure is the tetraploid.

[0016] Figure 4 FIG. is a comparison diagram of the inflorescences of diploid and tetraploid plants of "Purple Light" Pennisetum in Test Example 1 (photo). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] To make the technical solutions, objectives, and advantages of the present invention clearer, the following further describes the present invention in detail through specific examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0018] The present invention provides a cultivation method for polyploid Pennisetum, and the method includes:

[0019] Polyploid induction step:

[0020] The embryogenic callus is subjected to polyploid induction culture in a polyploid induction culture medium to obtain the callus after polyploid induction.

[0021] As a preferred embodiment, the above polyploid induction culture medium is a liquid medium containing colchicine with a mass concentration of 0.05% - 0.2%, preferably 0.1%.

[0022] As a further preferred embodiment, the above polyploid induction culture medium is based on MS medium and further contains: KT 0.5 - 1.5 mg / L, 6 - BA 0.05 - 0.15 mg / L, 2,4 - D 1.5 - 4.5 mg / L, PVP 3 - 10 mg / L, sucrose 15 - 45 g / L, and colchicine 0.05% - 0.2% (mass concentration). The pH value of this liquid medium is 5.8.

[0023] Specifically, the above polyploid induction culture medium is based on MS medium and further contains:

[0024] KT 0.5 - 1.5 mg / L, for example, it can be any one or the range between two of 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, and 1.5 mg / L, such as any one of 0.9 - 1.1 mg / L, 0.8 - 1.2 mg / L, 0.7 - 1.3 mg / L, 0.6 - 1.4 mg / L, and is preferably 1.0 mg / L;

[0025] 6 - BA 0.05 - 0.15 mg / L, for example, it can be any one or the range between two of 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.1 mg / L, 0.11 mg / L, 0.12 mg / L, 0.13 mg / L, 0.14 mg / L, 0.15 mg / L, such as any one of 0.09 - 0.11 mg / L, 0.08 - 0.12 mg / L, 0.07 - 0.13 mg / L, 0.06 - 0.14 mg / L, and is preferably 0.1 mg / L;

[0026] 2,4-D is 1.5 - 4.5 mg / L, for example, it can be any one or the range between two of 1.5 mg / L, 1.8 mg / L, 2.0 mg / L, 2.2 mg / L, 2.5 mg / L, 2.8 mg / L, 3.0 mg / L, 3.2 mg / L, 3.5 mg / L, 3.8 mg / L, 4.0 mg / L, 4.2 mg / L, 4.5 mg / L, for example, any one of 2.8 - 3.2 mg / L, 2.5 - 3.5 mg / L, 2.2 - 3.8 mg / L, 2.0 - 4.0 mg / L, 1.8 - 4.2 mg / L, and preferably it is 3.0 mg / L;

[0027] PVP is 3 - 10 mg / L, for example, it can be any one or the range between two of 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, for example, any one of 6 - 8 mg / L, 5 - 9 mg / L, 4 - 10 mg / L, 3 - 9 mg / L, and preferably it is 3.0 mg / L;

[0028] Sucrose is 15 - 45 g / L, for example, it can be any one or the range between two of 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, for example, any one of 28 - 32 g / L, 25 - 35 g / L, 22 - 38 g / L, 20 - 40 g / L, 18 - 42 g / L, and preferably it is 30 g / L;

[0029] Colchicine is 0.05% - 0.2% (mass concentration), for example, it can be any one or the range between two of 0.05%, 0.06%, 0.07%, 0.08%, 0.10%, 0.12%, 0.15%, 0.18%, 0.2%, for example, any one of 0.08% - 0.12%, 0.07% - 0.15%, 0.06% - 0.18%, and preferably it is 0.1%.

[0030] Preferably, the above polyploid induction culture medium is based on MS medium and also contains: KT 1.0 mg / L, 6 - BA 0.1 mg / L, 2,4 - D 3.0 mg / L, PVP 7.0 mg / L, sucrose 30 g / L, and colchicine 0.1%.

[0031] As a preferred embodiment, the above polyploid induction culture is carried out under dark conditions, the temperature is 23 - 27 °C (preferably 28 °C), the time is 24 - 72 hours (preferably 48 hours), and it is cultured with shaking at 100 - 150 rpm (preferably 120 rpm).

[0032] Among them, the embryogenic callus is obtained by the following method:

[0033] The explants of Pennisetum alopecuroides are cultured for inducing embryogenic callus in an embryogenic callus induction medium to obtain embryogenic callus.

[0034] As a preferred embodiment, the above explants are initial booting ears that are 1.0 - 1.5 cm long, longitudinally cut in half, and only the inner bracts are retained.

[0035] Preferably, the above explants have been disinfected with an ethanol solution and a mercuric chloride solution.

[0036] As a preferred embodiment, the above embryogenic callus induction medium is based on the MS basal medium and further contains:

[0037] KT 0.5 - 1.5 mg / L, 6 - BA 0.05 - 0.2 mg / L, 2,4 - D 1.5 - 4.5 mg / L, PVP 4 - 10 mg / L, plant gel or agar 5 - 10 g / L, sucrose 15 - 45 g / L. The pH value of the embryogenic callus induction medium is 5.8.

[0038] Specifically, the above polyploid induction culture solution is based on the MS basal medium and further contains:

[0039] KT 0.5 - 1.5 mg / L, for example, it can be any one or the range between two of 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, and 1.5 mg / L, such as any one of 0.9 - 1.1 mg / L, 0.8 - 1.2 mg / L, 0.7 - 1.3 mg / L, 0.6 - 1.4 mg / L, preferably 1.0 mg / L;

[0040] 6 - BA 0.05 - 0.2 mg / L, for example, it can be any one or the range between two of 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.1 mg / L, 0.11 mg / L, 0.12 mg / L, 0.13 mg / L, 0.14 mg / L, 0.15 mg / L, 0.16 mg / , 0.17 mg / , 0.18 mg / , 0.19 mg / , 0.2 mg / , such as any one of 0.09 - 0.11 mg / L, 0.08 - 0.12 mg / L, 0.07 - 0.13 mg / L, 0.06 - 0.14 mg / L, 0.08 - 0.16 mg / L, 0.09 - 0.18 mg / L, preferably 0.1 mg / L;

[0041] 2,4-D is 1.5 - 4.5 mg / L, for example, it can be any one within the range of 1.5 mg / L, 1.8 mg / L, 2.0 mg / L, 2.2 mg / L, 2.5 mg / L, 2.8 mg / L, 3.0 mg / L, 3.2 mg / L, 3.5 mg / L, 3.8 mg / L, 4.0 mg / L, 4.2 mg / L, 4.5 mg / L or the range between any two of them, such as any one within the range of 2.8 - 3.2 mg / L, 2.5 - 3.5 mg / L, 2.2 - 3.8 mg / L, 2.0 - 4.0 mg / L, 1.8 - 4.2 mg / L, and preferably it is 3.0 mg / L;

[0042] PVP is 4 - 10 mg / L, for example, it can be any one within the range of 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L or the range between any two of them, such as any one within the range of 6 - 8 mg / L, 5 - 9 mg / L, 4 - 10 mg / L, and preferably it is 7.0 mg / L;

[0043] Phytagel or agar is 5 - 10 g / L, for example, it can be any one within the range of 5 g / L, 5.5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or the range between any two of them, such as any one within the range of 5.5 - 7 g / L, 5.5 - 8 g / L, 5.5 - 9 g / L, 5 - 8 g / L, and preferably it is 6 g / L;

[0044] Sucrose is 15 - 45 g / L, for example, it can be any one within the range of 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45 or the range between any two of them, such as any one within the range of 28 - 32 g / L, 25 - 35 g / L, 22 - 38 g / L, 20 - 40 g / L, 18 - 42 g / L, and preferably it is 30 g / L.

[0045] Preferably, the above embryogenic callus induction medium is based on MS basal medium and also contains:

[0046] KT is 1.0 mg / L, 6 - BA is 0.1 mg / L, 2,4 - D is 3.0 mg / L, PVP is 7.0 mg / L, phytagel is 6 g / L, and sucrose is 30 g / L.

[0047] The above PVP (Polyvinyl pyrrolidone) is a phenolic substance adsorbent, which adsorbs phenolic substances through complexation to prevent the formation of quinone substances and can more effectively inhibit the browning of the culture; especially, callus is prone to browning after subculture, and adding PVP can prevent browning. The molecular weight of the above PVP is 111.14, and conventional products on the market can be used.

[0048] As a preferred embodiment, the above-mentioned embryogenic callus induction culture is carried out under dark conditions, at a temperature of 21-25°C (preferably 23°C), for 50-90 days.

[0049] During the above-mentioned embryogenic callus induction culture process, after the embryogenic callus appears, it is subcultured once with the above-mentioned embryogenic callus induction medium for 15-25 days (preferably 20 days), and then used for polyploid induction culture after 2-3 subcultures.

[0050] After the above-mentioned polyploid induction step, it further includes:

[0051] Adventitious bud differentiation step:

[0052] The callus after the above-mentioned polyploid induction is transferred to a differentiation medium for adventitious bud differentiation culture to obtain adventitious buds.

[0053] As a preferred embodiment, the above-mentioned differentiation medium is based on MS medium and further contains: NAA 0.5-1.5 mg / L, 6-BA 1.5-4.5 mg / L, phytagel or agar 5-10 g / L, sucrose 15-45 g / L. The pH value of this differentiation medium is 5.8.

[0054] Preferably, the above-mentioned differentiation medium is based on MS medium and further contains: NAA 1.0 mg / L, 6-BA 3.0 mg / L, phytagel 7 g / L, sucrose 30 g / L.

[0055] As a preferred embodiment, the above-mentioned adventitious bud differentiation culture is carried out under light-dark alternating conditions, with a light time of 16 h / d, a dark time of 8 h / d, and a temperature of 24±1°C.

[0056] When the above-mentioned adventitious buds grow to 1.5-2.5 cm, root culture is carried out.

[0057] Rooting step:

[0058] The above-mentioned adventitious buds are inoculated into a rooting medium for rooting culture to obtain regenerated plants.

[0059] As a preferred embodiment, the above-mentioned rooting medium is based on 1 / 2MS basal medium and further contains:

[0060] NAA 0.1-1.0 mg / L, IBA 0.1-1.0 mg / L, phytagel or agar 5-10 g / L, sucrose 15-45 g / L. The pH value of this rooting medium is 5.8.

[0061] Preferably, the above-mentioned rooting medium is based on 1 / 2MS basal medium and further contains:

[0062] NAA 0.5 mg / L, IBA 0.5 mg / L, phytagel 6.8 g / L, sucrose 30 g / L.

[0063] As a preferred embodiment, the above rooting culture is carried out under alternating light and dark conditions, with a light duration of 16 h / d, a dark duration of 8 h / d, and a temperature of 24 ± 1 °C.

[0064] Steps for acclimatizing and transplanting seedlings:

[0065] Acclimatize and transplant the above regenerated plants to obtain tissue-cultured seedlings.

[0066] The composition of the above MS basal medium (without agar and sucrose) is as follows:

[0067] Macronutrients, including:

[0068] Potassium nitrate (KNO3): 1900 mg / L, ammonium nitrate (NH4NO3): 1650 mg / L,

[0069] Magnesium sulfate (MgSO4·7H2O): 370 mg / L, potassium dihydrogen phosphate (KH2PO4): 170 mg / L,

[0070] Calcium chloride (CaCl2·2H2O): 440 mg / L;

[0071] Micronutrients, including:

[0072] Manganese sulfate (MnSO4·H2O): 16.9 mg / L, zinc sulfate (ZnSO4·7H2O): 8.6 mg / L,

[0073] Boric acid (H3BO3): 6.2 mg / L, potassium iodide (KI): 0.83 mg / L,

[0074] Sodium molybdate (Na2MoO4·2H2O): 0.25 mg / L, copper sulfate (CuSO4·5H2O): 0.025 mg / L,

[0075] Cobalt chloride (CoCl2·6H2O): 0.025 mg / L;

[0076] Iron salts, including:

[0077] Disodium ethylenediaminetetraacetate dihydrate (Na2-EDTA): 37.3 mg / L,

[0078] Ferrous sulfate (FeSO4·4H2O): 27.8 mg / L;

[0079] Organics, including:

[0080] Glycine: 2.0 mg / L, Pyridoxine Hydrochloride: 0.5 mg / L, Thiamine Hydrochloride: 0.1 mg / L,

[0081] Nicotinic Acid: 0.5 mg / L, Creatine: 100 mg / L;

[0082] The above 1 / 2 MS basal medium (without agar and sugar) is based on the above MS basal medium, and the contents of macroelements, microelements, iron salts, and organic substances are all reduced to 1 / 2, while other components remain unchanged.

[0083] For all kinds of reagents, materials, etc. used in the following examples, if not otherwise specified, they are all products that can be obtained from commercial channels; for all kinds of testing and detection methods used in the following examples, if not otherwise specified, they are all conventional testing and detection methods in this field and can be obtained from textbooks, reference books or academic journals.

[0084] Example 1

[0085] This example is used to illustrate a cultivation method of polyploid Pennisetum americanum, including the following steps.

[0086] S1: Preparation of explants

[0087] Cut the nodes of "Ziguang" Pennisetum americanum with initial booting, and use a scalpel to cut the nodes into initial young spikes about 2 - 5 cm long, leaving only the inner bracts.

[0088] Then place the initial young spikes with only the inner bracts in a laminar flow hood, disinfect them with 75% ethanol solution for 30 s, rinse them with sterile water 3 times, soak them in 0.1% mercuric chloride solution for 5 - 8 min, and then rinse them with sterile water 3 - 5 times and place them on sterile paper to obtain disinfected young spikes.

[0089] Then cut the disinfected young spikes into small segments about 1 cm long, and longitudinally cut each small segment into two halves to obtain explants.

[0090] The improved variety number of this "Ziguang" Pennisetum americanum is: Guo R-SV-PA-005-2007, provided by the Institute of Prataculture and Ornamental Horticulture, Beijing Academy of Agriculture and Forestry Sciences. Anyone can freely obtain "Ziguang" Pennisetum americanum for the purpose of implementing the present invention.

[0091] S2: Preparation of embryogenic callus

[0092] After blotting the above explants dry, inoculate them into the embryogenic callus induction medium, and carry out embryogenic callus induction culture at a temperature of 23°C under dark conditions.

[0093] When the above-mentioned embryogenic callus induction culture is carried out for 2-3 weeks, embryogenic callus begins to appear; subculture once every 20 days, and after 2-3 subculture times (using the same embryogenic callus induction medium), a sufficient amount of embryogenic callus is obtained.

[0094] The above-mentioned embryogenic callus induction medium is based on the MS basic medium and also contains:

[0095] KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 7.0 mg / L, Phytagel 6 g / L, sucrose 30 g / L. The pH value of this embryogenic callus induction medium is 5.8.

[0096] The above-mentioned PVP has a molecular weight of 111.14; it is purchased from Sigma Company, agency item number: 81440-250G, polyvinylpyrrolidone, CAS number 9003-39-8.

[0097] The above-mentioned Phytagel is Sigma Phytagel TM , item number is P8169-500g, numbers are 0000270530, 0000280602.

[0098] S3: Polyploid induction

[0099] Using the above-mentioned embryogenic callus as the receptor, polyploid induction is carried out by the colchicine soaking method: inoculate the above-mentioned embryogenic callus into a liquid medium (i.e., polyploid induction culture solution) containing 0.1% colchicine by mass concentration for soaking, and carry out polyploid induction culture to obtain the callus after polyploid induction.

[0100] The time of the above-mentioned polyploid induction culture is 48 h, and the culture conditions are: dark, constant temperature shaker at 28 °C, 120 rpm.

[0101] The above-mentioned polyploid induction culture solution is based on the MS medium and also contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 7.0 mg / L, sucrose 30 g / L, colchicine 0.1%. The pH value of this polyploid induction medium is 5.8.

[0102] S4: Adventitious bud differentiation culture

[0103] Transfer the above-mentioned callus after polyploid induction to the differentiation medium for adventitious bud differentiation culture until adventitious buds about 2 cm long are obtained.

[0104] The above differentiation medium group is based on MS medium and also contains: NAA 1.0 mg / L, 6-BA 3.0 mg / L, phytagel 7 g / L, and sucrose 30 g / L. The pH value of this differentiation medium is 5.8.

[0105] A total of 4 culture dishes were inoculated for the above adventitious bud differentiation culture, and the culture conditions were: alternating light and dark culture, with a light duration of 16 h / d, a dark duration of 8 h / d, no limit on light intensity, and an environmental temperature of 24 ± 1°C.

[0106] S5: Rooting culture

[0107] The adventitious buds obtained from the culture in step S4 were inoculated into the rooting medium for rooting culture, and regenerated plants were obtained after 21 d. This rooting medium was placed in a culture bottle with a height of 6 - 10 cm.

[0108] The above rooting medium uses 1 / 2MS medium as the basic medium and also contains: NAA 0.5 mg / L, IBA 0.5 mg / L, phytagel 6.8 g / L, and sucrose 30 g / L. The pH value of this rooting medium is 5.8.

[0109] The culture conditions for the above rooting culture were: alternating light and dark culture, with a light duration of 16 h / d, a dark duration of 8 h / d, no limit on light intensity, and an environmental temperature of 24 ± 1°C.

[0110] S6: Hardening off and transplanting

[0111] The above regenerated plants were first hardened off (i.e., opening the culture bottle mouth to break the sterile environment) for 3 d, and then the complete regenerated plants were taken out and transplanted into a plug tray for culture to obtain tissue culture seedlings.

[0112] In this example, 35 tissue culture seedlings of Pennisetum alopecuroides 'Ziguang' were obtained after transplantation; as shown in Table 3, 27 plants were taken and subjected to ploidy identification according to the method of Test Example 1, among which 15 plants were tetraploid plants, and the induction rate was 55.56%.

[0113] Detection Example 1

[0114] This detection example is used to describe the detection results of ploidy identification of the tissue culture seedlings obtained in Example 1.

[0115] 1. Stomatal morphology identification

[0116] Samples were taken between 9 and 10 am on a sunny day. Select vigorous polyploid plants and control plants (i.e., plants obtained by directly differentiating adventitious buds, rooting, acclimatizing, and transplanting embryogenic callus without polyploid induction in step S3), and then cut the middle part of the leaves with scissors, about 2 cm in length. To obtain the traces of stomata and the epidermal cell layer, apply a layer of clear nail polish on the back of the leaf. After 20 minutes, wait for the nail polish to dry, gently peel off the nail polish layer with forceps, place it on a glass slide, observe the size of the stomata and the number of stomata in the field of view with a 20X optical microscope, and take pictures for recording.

[0117] Test results:

[0118] Through the observation of stomatal morphology ( Figure 1 , where the left figure is diploid and the right figure is tetraploid), it can be seen that there are extremely significant differences in the stomatal density between the tetraploid and diploid plants of "Purple Light" Pennisetum, and there are significant differences in the stomatal size between the tetraploid and diploid plants of "Purple Light" Pennisetum. The results show that the tetraploid plants of "Purple Light" Pennisetum show the characteristics of smaller stomatal density and larger stomatal size in stomatal morphology (as shown in Table 1).

[0119] Table 1: Analysis table of stomatal morphology of diploid and tetraploid plants of "Purple Light" Pennisetum

[0120] Plant type Stomatal length (μm) Stomatal width (μm) Stomatal density (number per unit field of view) "Violet light" 2x <![CDATA[33.15±3.44 * > 20.72±3.13 41.67±1.53 "Violet light" 4x 48.19±6.13 23.33±3.11 <![CDATA[17.67±1.15 ** >

[0121] 2. Flow cytometry

[0122] Take 50 mg of young leaves of the plants to be tested, put them into a petri dish, add 500 μL of cell extraction solution, and chop the leaves with a blade. Then add another 500 μL of cell extraction solution and let it stand for 5 minutes. Then filter the extraction solution through a 400-mesh filter membrane into a 1.5-mL centrifuge tube, place it on ice for storage, let it stand for a moment, add 200 μL of staining solution, and stain it in the dark at 4°C for 20 minutes. Then use a flow cytometer (MoFlo XDF) for detection.

[0123] The cell extraction solution Galbraith’s Buffer used in the operation process is: 45 mmol / L MgCl2, 30 mmol / L sodium citrate, 20 mmol / L 3-(N-morpholino)propanesulfonic acid (pH 7.0), 0.1% Triton X-100, 50 μg / L RNase A, stored at 4°C. The staining solution used is: 50 μg / L propidium iodide solution, stored at 4°C.

[0124] The test results are as Figure 2 shown. Compared with the diploid plants of "Purple Light" Pennisetum, the DNA fluorescence intensity absorption peak of the tetraploid plants appears at twice that of the diploid plants, showing an obvious relative ratio, and thus the diploid and tetraploid plants can be identified.

[0125] 3. Root tip chromosome counting

[0126] Take about 1 cm of young root tips between 7 and 9 am, put them into 2 mL of Carnoy's fixative (absolute ethanol: glacial acetic acid = 3:1), and refrigerate them in a 4°C refrigerator. After about 24 hours, pick out the root tips with white dots at the front end and put them into distilled water for 20 minutes. Then take them out, place them on a glass slide, cut off the white dots at the front end, and put them into about 50 μL of lysate (cellulase: pectinase = 1:1). Place the root tips in a 37°C oven (or metal bath) for 2 hours of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, add 1 mL of distilled water to promote the absorption and rupture of the cell membrane by water. After 20 minutes, suck out the root tips into distilled water, then suck them out and place them on a glass slide. Blot the water with absorbent paper to avoid drying of the root tips. Add a drop of fixative and tap the root tips until they are invisible to the naked eye. Then add another drop of fixative and let it stand for a while. Then add 30 μL of gentian violet staining solution, cover with a cover glass, and observe the chromosome number under a microscope.

[0127] The detection results are as Figure 3 (The left figure is diploid, and the right figure is tetraploid) shown. Using the root tip chromosome counting method to identify the plants obtained after colchicine treatment, it was found that the chromosome number of the diploid plants of "Ziguang" Pennisetum alopecuroides was 18, and the chromosome number of the tetraploid plants after doubling was 36.

[0128] 4. Analysis of phenotypic characteristics of regenerated plants

[0129] In step S6 of Example 1, after transplanting the regenerated plants into plug trays and culturing them for 2 months, the diploid plants and the tetraploid plants obtained by doubling of "Ziguang" Pennisetum alopecuroides were compared, and significant differences were shown in multiple traits between the two (Table 2). Through phenotypic observation, as Figure 4 (The left figure is diploid, the middle figure is tetraploid, and the right figure is the comparison of the inflorescences of diploid and tetraploid) shown, compared with the diploid plants, the tetraploid plants showed the characteristics of reduced plant height, shorter inflorescence, fewer inflorescence numbers, shorter and thicker internodes, and thicker and wider leaves.

[0130] Table 2: Phenotypic analysis of diploid and tetraploid plants (annual) of "Ziguang" Pennisetum alopecuroides

[0131] Index / Plant type "Violet light" 2X "Violet light" 4X Plant height (cm) 98±6.29 <![CDATA[67.3±7.99 ** > Canopy width (cm) 88.85±8.88 <![CDATA[75.7±7.68 * > Inflorescence length (cm) 16.38±2.00 <![CDATA[13.13±1.25 ** > Inflorescence width (cm) <![CDATA[4.84±0.36 ** > 5.85±0.54 Number of inflorescences (number per plant) 38.7±6.75 <![CDATA[15.1±6.42 ** > Leaf length (cm) 51.14±9.89 <![CDATA[39.27±8.47 ** > Leaf width (mm) <![CDATA[7.4±0.86 * > 8.75±1.50 Number of tillers (number per plant) 33.33±3.51 <![CDATA[19±6.24 * >

[0132] Experimental example

[0133] This experimental example is a parallel experiment (including Examples 1 to 4), used to describe the effects of different colchicine concentrations and different times on polyploid induction in step S3 of Examples 1 to 4.

[0134] According to the operation methods of S1 and S2 in Example 1, embryogenic callus was obtained.

[0135] Using this callus as the receptor, induction was carried out by the impregnation method (refer to S3 of Example 1): the embryogenic callus was respectively immersed in liquid culture media containing 0.05%, 0.1%, and 0.2% colchicine for 24 h, 48 h, and 72 h, for a total of 9 treatments.

[0136] Then, according to the operation method of S4 in Example 1, the induced callus was transferred to an adventitious bud differentiation medium for adventitious bud differentiation.

[0137] Then, according to the operation method of S5 in Example 1, the adventitious buds were inoculated into a rooting medium to obtain regenerated plants.

[0138] Then, according to the operation method of S6 in Example 1, the regenerated plants were acclimatized and transplanted to obtain tissue culture seedlings.

[0139] Taking 258 tissue culture seedlings of the above Pennisetum alopecuroides, their ploidy was identified by flow cytometry in Detection Example 1, and the induction rate of the regenerated plants was statistically analyzed. It was shown that among the 9 induction conditions for Pennisetum alopecuroides polyploids, the 5th induction condition (i.e., the culture condition of Example 1) was the best, that is, when the colchicine concentration was 0.1% and the treatment time was 48 h, the induction rate was the highest, and the induction rate could reach 55.56% under this condition. The sub-optimal induction condition was the 7th (i.e., the culture condition of Example 2), that is, the colchicine concentration was 0.2% and the treatment time was 24 h, and the induction rate under this condition was 40%. The research results showed that among the 258 plants subjected to polyploid identification, a total of 87 tetraploid plants with successful doubling were obtained, and the overall induction rate was 33.72%. Finally, through identification, a new germplasm of tetraploid Pennisetum alopecuroides was obtained in this study.

[0140] Table 3: Statistical table of polyploid induction results of "Ziguang" Pennisetum alopecuroides

[0141] Serial number Example Treatment time / h Colchicine concentration / % Number of plants detected Number of tetraploid plants Induction rate / % 1 24 0.05 27 9 33.33 2 48 0.05 25 5 20.00 3 72 0.05 25 6 24.00 4 4 24 0.1 41 16 39.02 5 1 48 0.1 27 15 55.56 6 3 72 0.1 28 11 39.29 7 2 24 0.2 35 14 40.00 8 48 0.2 25 9 36.00 9 72 0.2 25 2 8.00 Total 258 87 33.72

[0142] Example 2

[0143] The cultivation method of this example was the same as that of Example 1 in all other aspects except for the following operations and parameters:

[0144] In step S3:

[0145] The time for polyploid induction culture was 24 h, and the polyploid induction culture solution contained colchicine with a mass concentration of 0.2%.

[0146] 808 tissue culture seedlings of Pennisetum alopecuroides "Ziguang" were obtained after transplantation in this example.

[0147] As shown in Table 3, 35 plants were randomly selected for ploidy identification, and 14 of them were tetraploids, with an induction rate of 40%.

[0148] Example 3

[0149] The cultivation method of this example is the same as that of Example 1 in all aspects except for the following operations and parameters:

[0150] In step S3:

[0151] The time for polyploid induction culture is 72 h, and the polyploid induction culture medium contains colchicine with a mass concentration of 0.1%.

[0152] After transplantation in this example, 34 tissue culture seedlings of Pennisetum alopecuroides "Ziguang" were obtained. As shown in Table 3, 28 plants were randomly selected for ploidy identification, among which 11 were tetraploids, and the induction rate was 39.29%.

[0153] Example 4

[0154] The cultivation method of this example is the same as that of Example 1 in all aspects except for the following operations and parameters:

[0155] In step S3:

[0156] The time for polyploid induction culture is 24 h, and the polyploid induction culture medium contains colchicine with a mass concentration of 0.1%.

[0157] After transplantation in this example, 453 tissue culture seedlings of Pennisetum alopecuroides "Ziguang" were obtained.

[0158] As shown in Table 3, 41 plants were randomly selected for ploidy identification, among which 16 were tetraploids, and the induction rate was 39.02%.

[0159] Example 5

[0160] The cultivation method of this example is the same as that of Example 1 in all aspects except for the following operations and parameters:

[0161] In step S2:

[0162] The embryogenic callus induction medium is based on the MS basal medium and also contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 4.0mg / L , phytagel 6 g / L, sucrose 30 g / L. The pH value of this embryogenic callus induction medium is 5.8.

[0163] In step S3:

[0164] The polyploid induction culture medium is based on MS as the basal medium and also contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 4.0mg / L , sucrose 30 g / L, colchicine 0.1%. The pH value of this polyploid induction medium is 5.8.

[0165] In this example, after transplantation, tissue culture seedlings of Pennisetum alopecuroides "Ziguang" were obtained. 40 plants were randomly selected for ploidy identification. Among them, 20 plants were tetraploids, and the induction rate was 50%.

[0166] Example 6

[0167] The cultivation method of this example is the same as that of Example 1 in all aspects except for the following operations and parameters:

[0168] In step S2:

[0169] The embryogenic callus induction medium is based on the MS basal medium and also contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 10.0mg / L , phytagel 6 g / L, and sucrose 30 g / L. The pH value of this embryogenic callus induction medium is 5.8.

[0170] In step S3:

[0171] The polyploid induction culture solution is based on MS as the basal medium and also contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, PVP 10.0mg / L , sucrose 30 g / L, and colchicine 0.1%. The pH value of this polyploid induction medium is 5.8.

[0172] In this example, after transplantation, tissue culture seedlings of Pennisetum alopecuroides "Ziguang" were obtained. 40 plants were randomly selected for ploidy identification. Among them, 19 plants were tetraploids, and the induction rate was 47.5%.

[0173] Comparative Examples 1 - 4

[0174] The cultivation methods of Comparative Examples 1 - 4 are the same as that of Example 1 in all aspects except for the following operations and parameters:

[0175] As shown in Table 4, different polyploid culture times and different colchicine mass concentrations were set in step S3. After obtaining the tissue culture seedlings, ploidy identification was carried out.

[0176] Table 4: Statistical table of polyploid induction results of Pennisetum alopecuroides "Ziguang" in Comparative Examples 1 - 4

[0177] Control example Treatment time / h Colchicine concentration / % Number of plants detected Number of tetraploid plants Induction rate / % 1 19 0.07 25 5 20 2 19 0.26 25 6 24 3 76 0.07 25 4 16 4 76 0.26 25 5 8

[0178] As can be seen from Table 4, shortening the polyploid induction treatment time or increasing the colchicine concentration will significantly reduce the induction rate.

[0179] Comparative Example 5

[0180] The operation of step S1 in this comparative example is the same as that of Example 1:

[0181] In step S2: The embryogenic callus induction medium is based on the MS basal medium and further contains: KT 1.0 mg / L, 6-BA 0.1 mg / L, 2,4-D 3.0 mg / L, phytagel 6 g / L, and sucrose 30 g / L. The pH value of the embryogenic callus induction medium is 5.8. The embryogenic callus induction medium Without adding PVP .

[0182] According to the culture conditions of Example 1, after 60 days of callus induction culture, most of the calli became brown and it was difficult to carry out the next adventitious bud differentiation.

[0183] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for cultivating polyploid Pennisetum americanum, characterized in that: The cultivation method includes the operation of performing polyploid induction culture on embryogenic callus in a polyploid induction culture solution to obtain callus after polyploid induction; Among them, the polyploid induction culture solution contains colchicine with a mass concentration of 0.05% - 0.2%.

2. The method for cultivating polyploid Pennisetum americanum according to claim 1, characterized in that: The polyploid induction culture solution contains colchicine with a mass concentration of 0.1%.

3. The method for cultivating polyploid Pennisetum americanum according to claim 1 or 2, characterized in that: The polyploid induction culture solution uses MS as the basic medium and also contains: KT 0.5 - 1.5 mg / L, 6 - BA 0.05 - 0.15 mg / L, 2,4 - D 1.5 - 4.5 mg / L, PVP 4 - 10 mg / L, sucrose 15 - 45 g / L, colchicine 0.05% - 0.2%; Preferably, the polyploid induction culture solution uses MS as the basic medium and also contains: KT 1.0 mg / L, 6 - BA 0.1 mg / L, 2,4 - D 3.0 mg / L, PVP 7.0 mg / L, sucrose 30 g / L, colchicine 0.1%.

4. The method for cultivating polyploid Pennisetum americanum according to claim 3, characterized in that: The time of the polyploid induction culture is 24 - 72 hours, preferably 48 hours; Preferably, the conditions for the polyploid induction culture include: under dark conditions, the temperature is 23 - 27 °C, preferably 28 °C, and it is cultured with shaking at 100 - 150 rpm, preferably 120 rpm.

5. The method for cultivating polyploid Pennisetum americanum according to claim 3, characterized in that: The embryogenic callus is obtained in the following manner: The explant of Pennisetum americanum is subjected to embryogenic callus induction culture in an embryogenic callus induction medium to obtain the embryogenic callus; Preferably, the explant is the initial ear in the booting stage with a length of 1.0 - 1.5 cm, longitudinally cut in half, and only the inner bracts are left.

6. The method for cultivating polyploid Pennisetum americanum according to claim 5, characterized in that: The embryogenic callus induction medium is based on the MS basic medium and also contains: KT 0.5 - 1.5 mg / L, 6 - BA 0.05 - 0.2 mg / L, 2,4 - D 1.5 - 4.5 mg / L, PVP 4 - 10 mg / L, phytagel or agar 5 - 10 g / L, sucrose 15 - 45 g / L; Preferably, the embryogenic callus induction medium is based on the MS basic medium and also contains: KT 1.0 mg / L, 6 - BA 0.1 mg / L, 2,4 - D 3.0 mg / L, PVP 7.0 mg / L, phytagel 6 g / L, sucrose 30 g / L.

7. The method for cultivating polyploid Pennisetum americanum according to claim 6, characterized in that: The time of the embryogenic callus induction culture is 50 - 90 days; Preferably, the conditions for inducing embryogenic callus culture include: under dark conditions, the temperature is 21-25 °C, preferably 23 °C.

8. The cultivation method of the polyploid Pennisetum according to claim 7, characterized in that: After the polyploid induction step, it further includes: Adventitious bud differentiation step: Transfer the callus after polyploid induction to a differentiation medium for adventitious bud differentiation culture to obtain adventitious buds; Preferably, in the adventitious bud differentiation step, the differentiation medium is based on MS medium and further contains: NAA 0.5-1.5 mg / L, 6-BA 1.5-4.5 mg / L, phytagel or agar 5-10 g / L, sucrose 15-45 g / L.

9. The cultivation method of the polyploid Pennisetum according to claim 8, characterized in that: After the polyploid induction step, it further includes: Rooting step: Inoculate the adventitious buds into a rooting medium for rooting culture to obtain regenerated plants; In the rooting step, the rooting medium is based on 1 / 2MS basal medium and further contains: NAA 0.1-1.0 mg / L, IBA 0.1-1.0 mg / L, phytagel or agar 5-10 g / L, sucrose 15-45 g / L; Preferably, the rooting medium is based on 1 / 2MS basal medium and further contains: NAA 0.5 mg / L, IBA 0.5 mg / L, phytagel 6.8 g / L, sucrose 30 g / L.

10. The cultivation method of the polyploid Pennisetum setaceum according to claim 9, characterized in that: The variety of the Pennisetum is "Ziguang".

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