Genetic transformation method of cold-resistant pennisetum alopecuroides

Abstract

The invention relates to the technical field of plant tissue culture and genetic engineering, in particular to a genetic transformation method of cold-resistant pennisetum alopecuroides. The cold-resistant pennisetum alopecuroides genetic transformation method provided by the invention has relatively high genetic transformation efficiency, the formula of the used culture medium is simple, and the method is easy to operate, simple and efficient; the method is suitable for existing cold-resistant pennisetum varieties or new germplasms, is not limited by genotypes, fills the blank of a cold-resistant pennisetum genetic transformation system, provides technical support for functional gene identification and molecular breeding, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture and genetic engineering, and in particular to a genetic transformation method of cold-resistant Pennisetum. Background Art

[0002] Pennisetum spp., belonging to the Panicoideae subfamily of the Poaceae family, is an annual or perennial C4 herb with strong drought and heat tolerance, resistance to barrenness, and few diseases and insect pests. Pennisetum glaucum ) and elephant grass ( Pennisetum purpureum Originating primarily in tropical and subtropical regions, it is one of the world's most productive high-quality forages (Hou Xincun et al., 2022). However, its poor cold tolerance limits its widespread cultivation in areas north of the Yangtze River in China.

[0003] It is worth noting that the cold-resistant Chinese Pennisetum, such as Pennisetum longipedum ( Pennisetum alopecuroides 'Changsui'), Pennisetum serrata ( Pennisetum alopecuroides 'Liren'), Pennisetum liqiuensis ( Pennisetum alopecuroides 'Liqiu')、Pennisetum no.3( Pennisetum alopecuroides 'No.3') have strong cold tolerance and can safely overwinter in high latitudes and high altitudes, but their yield is low and they are widely used as ornamental grasses in landscaping (Li Ping et al., 2024). Furthermore, by deciphering the high-quality chromosome-level genome of Pennisetum rufipogon, its origin and evolutionary patterns were clarified, confirming that Pennisetum rufipogon is a close relative of Pennisetum sutchuenensis and Elephant Grass, and is an important germplasm resource for genetically improving the cold tolerance of Pennisetum sutchuenensis or Elephant Grass (Teng et al., 2023). Elucidating the molecular mechanisms of cold tolerance in Pennisetum rufipogon is crucial for discovering superior cold-tolerant genetic resources, genetically improving the cold tolerance of Pennisetum sutchuenensis or Elephant Grass, and accelerating the development of new provenances of Pennisetum rufipogon with high cold tolerance. Although a genetic transformation system for Pennisetum amorphum has been established previously (Mu Tong et al., 2013), it is limited by genotype and cumbersome. Therefore, there is an urgent need to establish a genetic transformation method for cold-resistant Pennisetum that is not restricted by genotype and is simpler to operate, in order to provide technical support for the utilization of its excellent stress-resistant genes and molecular breeding. Summary of the Invention

[0004] The invention provides a genetic transformation method for cold-resistant Pennisetum.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for genetic transformation of cold-resistant Pennisetum, comprising the following steps: Using seeds or young spikes of cold-resistant Pennisetum as explants, inducing callus from the explants to obtain embryonic callus as a transformation recipient; The transformation recipient is infected with recombinant Agrobacterium containing the target gene vector, and then genetically transformed plants are obtained by co-cultivation, resistant callus screening culture, resistant bud differentiation culture, resistant cluster bud differentiation culture and resistant rooting culture; The co-cultivation uses a culture medium C, which contains a basic culture medium, a carbon source, a coagulant, 6-BA, 2,4-D, KT, and acetosyringone (AS); The resistance bud differentiation culture uses a culture medium F, which contains a basic culture medium, a carbon source, a coagulant, NAA, 6-BA, antibiotics, and adenosine monophosphate (AMP); The resistance cluster bud differentiation culture uses medium G, which contains a basic medium, a carbon source, a coagulant, NAA, 6-BA and antibiotics.

[0007] The present invention found that adding acetosyringone to the culture medium C used for co-cultivation can significantly increase the Agrobacterium T-DNA fragment transferred into plant cells, thereby effectively improving the genetic transformation efficiency.

[0008] The present invention divides the differentiation of resistant embryogenic callus into two stages: resistance bud differentiation culture and resistance cluster bud differentiation culture, using different culture media for each. The present invention discovered that adding AMP to culture medium F used for resistance bud differentiation culture significantly improves the efficiency of resistance bud differentiation. During the differentiation stage, the combined action of NAA and 6-BA, two plant hormones, can further enhance the regeneration efficiency of resistance cluster buds.

[0009] Preferably, the explant is a seed.

[0010] Before callus induction, the explant is further disinfected. Preferably, the disinfection comprises: first disinfecting with 3-4% sodium hypochlorite and then disinfecting with 75% alcohol.

[0011] Preferably, the concentrations of 6-BA, 2,4-D, KT, and acetosyringone in medium C are as follows: 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, 0.5-1.5 mg / L KT, and 80-100 μM acetosyringone. Controlling the acetosyringone concentration within the range of 80-100 μM enhances the transfer of the Agrobacterium T-DNA fragment into plant cells.

[0012] Preferably, the concentrations of NAA, 6-BA, and adenosine monophosphate in medium F are as follows: 1.5-2.3 mg / L NAA, 1.5-3.2 mg / L 6-BA, and 1.5-3.5 mM adenosine monophosphate. Controlling the amount of adenosine monophosphate added to 1.5-3.5 mM has a better effect on increasing the differentiation rate of resistant buds.

[0013] Preferably, the concentrations of NAA and 6-BA in the culture medium G are as follows: 1.3-2.1 mg / L NAA, 1.6-3.4 mg / L 6-BA.

[0014] In the above-mentioned culture medium, the antibiotics are hygromycin (Hyg) and / or cefotaxime (Cef). The antibiotics in medium F include 5-8 mg / L hygromycin and 200-300 mg / L cefotaxime; the antibiotics in medium G include 3-5 mg / L hygromycin and 200-300 mg / L cefotaxime. Adjusting the Hyg concentration in medium F to 5-8 mg / L can significantly reduce the browning of resistant buds. To prevent browning of resistant clustered buds caused by long-term Hyg in the culture medium, the Hyg concentration in medium G is further adjusted to 3-5 mg / L. Gradually reducing the Hyg concentration in both mediums F and G can essentially eliminate browning.

[0015] In the present invention, the minimal culture medium is preferably MS medium. The carbon source can be selected from one or more sugars such as sucrose and glucose, preferably sucrose. The coagulant can be agar or plant gel, preferably agar.

[0016] Preferably, the culture medium C comprises the following components: 4.2-4.6 g / L MS, 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, 0.5-1.5 mg / L KT, 80-100 μM acetosyringone, 25-35 g g / L sucrose, and 6-10 g / L agar.

[0017] Further preferably, the culture medium C comprises the following components: 4.2-4.6 g / L MS, 0.08-0.12 mg / L 6-BA, 2.5-3.5 mg / L 2,4-D, 0.8-1.2 mg / L KT, 90-100 μM acetosyringone, 25-35 g g / L sucrose, and 6-10 g / L agar.

[0018] Preferably, the culture medium F comprises the following components: 4.2-4.6 g / L MS, 1.5-2.3 mg / L NAA, 1.5-3.2 mg / L 6-BA, 5-8 mg / L hygromycin, 200-300 mg / L cephalosporin, 1.5-3.5 mM adenosine monophosphate, 25-35 g / L sucrose, and 6-10 g / L agar.

[0019] Further preferably, the culture medium F comprises the following components: 4.2-4.6 g / L MS, 1.8-2.2 mg / L NAA, 2.5-3.2 mg / L 6-BA, 5-8 mg / L hygromycin, 200-250 mg / L cephalosporin, 2.5-3.5 mM adenosine monophosphate, 25-35 g / L sucrose, and 6-10 g / L agar.

[0020] Preferably, the culture medium G comprises the following components: 4.2-4.6 g / L MS, 1.3-2.1 mg / L NAA, 1.6-3.4 mg / L 6-BA, 3-5 mg / L hygromycin, 200-300 mg / L cephalosporin, 25-35 g / L sucrose, and 6-10 g / L agar.

[0021] Further preferably, the culture medium G comprises the following components: 4.2-4.6 g / L MS, 1.7-2.1 mg / L NAA, 2.8-3.2 mg / L 6-BA, 3-5 mg / L hygromycin, 200-250 mg / L cephalosporin, 25-35 g / L sucrose, and 6-10 g / L agar.

[0022] In the above method, the resistant callus screening culture comprises sequentially using medium D and medium E for resistant callus screening; wherein the medium D and medium E both contain a basic medium, a carbon source, a coagulant, and 6-BA, 2,4-D, KT, antibiotics, and ascorbic acid; In the culture medium D, the concentrations of 6-BA, 2,4-D, KT, antibiotics, and ascorbic acid are: 0.08-0.21 mg / L 6-BA, 2.2-3.6 mg / L 2,4-D, 0.6-1.8 mg / L KT, 15-20 mg / L hygromycin, 200-300 mg / L cephalosporin, and 160-220 mg / L ascorbic acid; In the culture medium E, the concentrations of 6-BA, 2,4-D, KT, antibiotics, and ascorbic acid are: 0.06-0.18 mg / L 6-BA, 1.2-2.4 mg / L 2,4-D, 0.6-1.8 mg / L KT, 8-10 mg / L hygromycin, 200-300 mg / L cephalosporin, and 150-210 mg / L ascorbic acid.

[0023] Adding ascorbic acid as an antioxidant to medium D and E significantly inhibited the browning of embryogenic callus induced by the antibiotic Hyg, achieving the best effect compared to other antioxidants. Adjusting the concentration of the antibiotic Hyg in medium E to 8-10 mg / L induced more resistant embryogenic callus.

[0024] Preferably, the culture medium D comprises the following components: 4.2-4.6 g / L MS, 0.08-0.21 mg / L 6-BA, 2.2-3.6 mg / L 2,4-D, 0.6-1.8 mg / L KT, 15-20 mg / L hygromycin, 200-300 mg / L cephalosporin, 160-220 mg / L ascorbic acid, 25-35 g / L sucrose, and 5-10 g / L agar.

[0025] Further preferably, the culture medium D comprises the following components: 4.2-4.6 g / L MS, 0.08-0.12 mg / L 6-BA, 2.8-3.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 15-20 mg / L hygromycin, 200-250 mg / L cephalosporin, 180-220 mg / L ascorbic acid, 25-35 g / L sucrose, and 5-10 g / L agar.

[0026] The culture medium E contains the following components: 4.2-4.6 g / L MS, 0.06-0.18 mg / L 6-BA, 1.2-2.4 mg / L 2,4-D, 0.6-1.8 mg / L KT, 8-10 mg / L hygromycin, 200-300 mg / L cephalosporin, 150-210 mg / L ascorbic acid, 25-35 g / L sucrose, and 6-10 g / L agar.

[0027] Further preferably, the culture medium E comprises the following components: 4.2-4.6 g / L MS, 0.08-0.12 mg / L 6-BA, 1.8-2.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 8-10 mg / L hygromycin, 200-250 mg / L cephalosporin, 180-210 mg / L ascorbic acid, 25-35 g / L sucrose, and 6-10 g / L agar.

[0028] In the above method, the resistance rooting culture uses culture medium H, which contains a basic culture medium, a carbon source, a coagulant, NAA, IBA and antibiotics; wherein the concentrations of NAA, IBA and antibiotics are 0.3-0.6 mg / L NAA, 0.4-0.7 mg / L IBA, and 200-300 mg / L cephalosporin.

[0029] Preferably, the antibiotic used in culture medium H is cephalosporin. The present invention has found that removing the Hyg selection pressure in culture medium H can significantly improve the rooting efficiency of resistant seedlings and induce rooting of resistant seedlings more quickly.

[0030] Preferably, the culture medium H comprises the following components: 2.0-2.4 g / L MS, 0.3-0.6 mg / L NAA, 0.4-0.7 mg / L IBA, 200-300 mg / L cephalosporin, 20-30 g / L sucrose, and 6-10 g / L agar.

[0031] Further preferably, the culture medium H comprises the following components: 2.0-2.4 g / L MS, 0.4-0.6 mg / L NAA, 0.4-0.6 mg / L IBA, 200-250 mg / L cephalosporin, 20-30 g / L sucrose, and 6-10 g / L agar.

[0032] In the above method, the callus induction uses culture medium A, which contains a basic culture medium, a carbon source, a coagulant, and 6-BA, 2,4-D, and KT; wherein the concentrations of 6-BA, 2,4-D, and KT are as follows: 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, and 0.5-1.5 mg / L KT.

[0033] Preferably, the culture medium A comprises the following components: 4.2-4.6 g / L MS, 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, 0.5-1.5 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

[0034] Further preferably, the culture medium A comprises the following components: 4.2-4.6 g / L MS, 0.08-0.12 mg / L 6-BA, 2.8-3.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

[0035] In the above method, the embryonic callus is subcultured 1-2 times and then used as a transformation recipient or directly used as a transformation recipient; wherein the subculture uses medium B, which contains a basic medium, a carbon source, a coagulant, and 6-BA, 2,4-D, and KT; the concentrations of 6-BA, 2,4-D, and KT are as follows: 0.06-0.18 mg / L 6-BA, 1.2-2.2 mg / L 2,4-D, and 0.8-1.5 mg / L KT.

[0036] Preferably, the culture medium B comprises the following components: 4.2-4.6 g / L MS, 0.06-0.18 mg / L 6-BA, 1.2-2.2 mg / L 2,4-D, 0.8-1.5 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

[0037] Further preferably, the culture medium B comprises the following components: 4.2-4.6 g / L MS, 0.08-0.12 mg / L 6-BA, 1.8-2.2 mg / L 2,4-D, 0.8-1.2 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

[0038] In the above method, the callus induction is carried out by culturing in the dark at 23-26° C. for 40-50 days.

[0039] The co-cultivation is carried out at 23-26° C. in the dark for 4-6 days.

[0040] The resistant callus induction screening culture is sequentially cultured in culture medium D at 23-26° C. in the dark for 13-15 days and cultured in culture medium E at 23-26° C. in the dark for 20-30 days.

[0041] The resistant bud differentiation is cultured at 23-26° C. in the light for 20-30 days.

[0042] The resistant clustered buds are differentiated by culturing at 23-26° C. for 20-30 days under light conditions.

[0043] The resistance rooting culture is carried out at 23-26° C. under light conditions for 14-20 days.

[0044] In the above method, after the resistance rooting is cultured, the following step may be further included: hardening the obtained resistance rooting seedlings in a sterile environment for 2-3 days, and then transplanting them into 1 / 2 Hogland nutrient solution for culture for 3-4 weeks.

[0045] In the present invention, the cold-resistant Pennisetum is preferably Pennisetum longissima, Pennisetum beauty, Pennisetum beauty autumn or Pennisetum No. 3.

[0046] In some embodiments of the present invention, the genetic transformation method comprises the following steps: (1) Explant preparation: Select mature and plump seeds as explants and sterilize them with sodium hypochlorite solution and alcohol to obtain sterile explants. (2) Callus induction: The sterile explants described in step (1) are placed in culture medium A for callus induction and cultured in the dark at 25°C for 40-50 days to obtain dense granular yellow embryonic callus as the transformation recipient; wherein, the embryonic callus with good growth condition is placed in subculture medium B for 1-2 subcultures or directly used as the transformation recipient; (3) Agrobacterium infection and co-cultivation: Use recombinant Agrobacterium culture containing the target gene expression vector (OD 600 The transformed receptor described in step (2) is preferably inoculated with Agrobacterium infection solution for 15-25 minutes, and then transferred to medium C for co-cultivation, and cultured in the dark at 25°C for 4-6 days; (4) Resistant callus screening: The co-cultured callus tissue in step (3) was transferred to medium D for resistant callus induction screening and cultured in the dark at 25°C for 13-15 days. The resistant callus tissue was then transferred to medium E for further resistant callus screening and cultured in the dark at 25°C for 20-30 days. White or light yellow dense granular resistant callus appeared.

[0047] (5) Resistance bud differentiation: Transfer the dense granular white or light yellow resistant calli to medium F for resistance bud differentiation. Culture at 25°C for 20-30 days until green resistance buds appear. (6) Differentiation of resistant cluster buds: The callus with green resistant buds in step (5) was transferred to culture medium G to continue differentiation of resistant cluster buds. Cultured at 25°C for 20-30 days under light, the resistant seedlings grew to about 3-4 cm in diameter.

[0048] (7) Resistance rooting: The resistant seedlings of about 3-4 cm in step (6) were transferred to culture medium H for rooting, and cultured at 25°C for 14-20 days to produce resistant roots.

[0049] In some embodiments of the present invention, the vector is pCAMBIA1300; and the Agrobacterium is Agrobacterium tumefaciens GV3101 or EHA105.

[0050] In a second aspect, the present invention provides a culture medium set for genetic transformation of cold-resistant Pennisetum, comprising a culture medium A for callus induction, a culture medium C for co-cultivation after infection with recombinant Agrobacterium, a culture medium D and a culture medium E for resistant callus screening culture, a culture medium F for resistant bud differentiation culture, a culture medium G for resistant cluster bud differentiation culture, and a culture medium H for resistant rooting culture; Wherein, the culture medium A comprises the following components: 4.2-4.6 g / L MS, 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, 0.5-1.5 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar; The medium C contains the following components: 4.2-4.6 g / L MS, 0.05-0.15 mg / L 6-BA, 2.0-4.0 mg / L 2,4-D, 0.5-1.5 mg / L KT, 80-100 μM acetosyringone, 25-35 g g / L sucrose, and 6-10 g / L agar; The culture medium D comprises the following components: 4.2-4.6 g / L MS, 0.08-0.21 mg / L 6-BA, 2.2-3.6 mg / L 2,4-D, 0.6-1.8 mg / L KT, 15-20 mg / L hygromycin, 200-300 mg / L cephalosporin, 160-220 mg / L ascorbic acid, 25-35 g / L sucrose, and 5-10 g / L agar; The culture medium E comprises the following components: 4.2-4.6 g / L MS, 0.06-0.18 mg / L 6-BA, 1.2-2.4 mg / L 2,4-D, 0.6-1.8 mg / L KT, 8-10 mg / L hygromycin, 200-300 mg / L cephalosporin, 150-210 mg / L ascorbic acid, 25-35 g / L sucrose, and 6-10 g / L agar; The medium F comprises the following components: 4.2-4.6 g / L MS, 1.5-2.3 mg / L NAA, 1.5-3.2 mg / L 6-BA, 5-8 mg / L hygromycin, 200-300 mg / L cephalosporin, 1.5-3.5 mM adenosine monophosphate, 25-35 g / L sucrose, and 6-10 g / L agar; The medium G comprises the following components: 4.2-4.6 g / L MS, 1.3-2.1 mg / L NAA, 1.6-3.4 mg / L 6-BA, 3-5 mg / L hygromycin, 200-300 mg / L cephalosporin, 25-35 g / L sucrose, and 6-10 g / L agar; The culture medium H comprises the following components: 2.0-2.4 g / L MS, 0.3-0.6 mg / L NAA, 0.4-0.7 mg / L IBA, 200-300 mg / L cephalosporin, 20-30 g / L sucrose, and 6-10 g / L agar.

[0051] Preferably, the culture medium set further comprises a culture medium B for subculture of embryonic callus, wherein the culture medium B comprises the following components: 4.2-4.6 g / L MS, 0.06-0.18 mg / L 6-BA, 1.2-2.2 mg / L 2,4-D, 0.8-1.5 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

[0052] Based on the culture medium with different hormone and antibiotic ratios, the present invention further adds ascorbic acid to the resistant callus screening culture medium and adds adenosine monophosphate to the resistant bud differentiation culture medium, breaking the genotype restriction barrier of the genetic transformation of cold-resistant Pennisetum (long-spiked Pennisetum, beautiful Pennisetum, beautiful autumn Pennisetum, No. 3 Pennisetum) varieties or new germplasm.

[0053] In a third aspect, the present invention provides the above-mentioned genetic transformation method of cold-resistant Pennisetum or the application of the culture medium set for genetic transformation of cold-resistant Pennisetum in gene function identification, gene editing, genetic breeding, variety improvement or transgenic plant construction of cold-resistant Pennisetum.

[0054] Preferably, the cold-resistant Pennisetum is Pennisetum longissima, Pennisetum beauty, Pennisetum beauty autumn or Pennisetum No. 3.

[0055] The beneficial effects of the present invention include at least: the genetic transformation method for cold-resistant Pennisetum provided by the present invention has a high genetic transformation efficiency, a simple culture medium formula, and is easy to operate, simple and efficient; the method is applicable to existing cold-resistant Pennisetum varieties or new germplasm, is not restricted by genotype, fills the gap in the genetic transformation system for cold-resistant Pennisetum, provides technical support for its functional gene identification and molecular breeding, and has broad application prospects. Cold-resistant Pennisetum has strong cold-resistant characteristics. Using its genetic transformation system, it is possible to study the molecular mechanism of adaptation to low temperatures, explore and identify key cold-resistant genes related to it, and apply its cold-resistant genes to the cold-resistant genetic improvement of American Pennisetum, elephant grass, or crops through genetic engineering methods to improve their ability to withstand low temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0057] Figure 1 Identification of positive clones transformed with Agrobacterium in Example 2 of the present invention. A is a schematic diagram of the plant expression vector pCAMBIA1300; B is PCR detection of positive clones transformed with Agrobacterium, where M is DNA marker DL1000, and 1-4 are hygromycin-labeled amplification products.

[0058] Figure 2 This is the screening of callus induction of mature embryos of cold-resistant Pennisetum in Example 3 of the present invention. Among them, A is the callus induction of resistant embryos of Pennisetum longissima; B is the callus induction of resistant embryos of Pennisetum beauty; C is the callus induction of resistant embryos of Pennisetum liqiu; and D is the callus induction of resistant embryos of Pennisetum no. 3.

[0059] Figure 3 This is the differentiation of cold-resistant Pennisetum resistant clustered buds in Example 4 of the present invention. Among them, A is the differentiation of resistant clustered buds of Pennisetum longispinum; B is the differentiation of resistant clustered buds of Pennisetum liren; C is the differentiation of resistant clustered buds of Pennisetum liqiu; and D is the differentiation of resistant clustered buds of Pennisetum no. 3.

[0060] Figure 4 The cold-resistant Pennisetum resistant seedlings in Example 5 of the present invention are rooted. Among them, A is the rooting resistance of Pennisetum longissima; B is the rooting resistance of Pennisetum beauty; C is the rooting resistance of Pennisetum liqiu; and D is the rooting resistance of Pennisetum No. 3.

[0061] Figure 5 The hydroponic transplantation of cold-resistant Pennisetum resistant seedlings in Example 5 of the present invention is shown in Figure 5. A is the hydroponic transplantation of long-spiked Pennisetum resistant seedlings; B is the hydroponic transplantation of beauty Pennisetum resistant seedlings; C is the hydroponic transplantation of beautiful autumn Pennisetum resistant seedlings; and D is the hydroponic transplantation of No. 3 Pennisetum resistant seedlings.

[0062] Figure 6 This figure illustrates the screening and molecular identification of transgenic cold-tolerant Pennisetum plants from Example 6 of the present invention. The electropherograms show hygromycin-labeled PCR amplification of selected transgenic lines. M: DNA marker DL1000; WT1-WT4 represent wild-type Pennisetum serrata (Longsui), Liren, Liqiu, and No. 3 plants, respectively; 1-3: transgenic Pennisetum serrata (Longsui), 4-6: transgenic Pennisetum serrata (Liren), 7-9: transgenic Pennisetum serrata (Liqiu), and 10-12: transgenic Pennisetum serrata (No. 3). DETAILED DESCRIPTION

[0063] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0064] Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples can be obtained from commercial channels, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0065] The MS culture medium used in the present invention is MS culture medium powder.

[0066] Example 1 Disinfection of cold-resistant Pennisetum explants and callus induction 1. Explant disinfection Select mature and plump cold-resistant Pennisetum (Long-spiked Pennisetum, Beauty Pennisetum, Beautiful Autumn Pennisetum, No. 3 Pennisetum) seeds as explants. First, peel off the seed shells with tweezers and soak them in pure water for 16 hours. Then, disinfect them in a sterile environment with 4% sodium hypochlorite for 4 minutes and 75% alcohol for 30 seconds. Rinse with sterile water 3-5 times to obtain sterilized cold-resistant Pennisetum seeds, which are sterile explants.

[0067] 2. Callus induction Place the sterile explants from step 1 on medium A for callus induction and culture in the dark at 25°C for 40-50 days to obtain dense, granular, yellow embryogenic calli as the transformation recipient; The embryonic callus with good growth status was subcultured 1-2 times on medium B under light conditions of 25°C (16 hours light / 8 hours dark) or directly used for Agrobacterium transformation.

[0068] The formula of the above-mentioned culture medium A is as follows: 4.4 g / L MS, 0.1 mg / L 6-BA, 3 mg / L 2,4-D, 1 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0069] The formula of the above-mentioned medium B is as follows: 4.4 g / L MS, 0.1 mg / L 6-BA, 2 mg / L 2,4-D, 1 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0070] Example 2 Preparation of Agrobacterium infection solution 1. Agrobacterium transformation and identification The plant expression vector pCAMBIA1300 was transformed into Agrobacterium tumefaciens GV3101 competent cells using the freeze-thaw method. The transformed bacterial solution was evenly spread on YEP solid medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif). After inverted culture at 28°C in the dark for 2-3 days, single colonies grew. A single colony was picked with an inoculation needle and inoculated into 100 mL YEP liquid medium containing 50 mg / L Kan and 50 mg / L Rif. The culture was shaken at 28°C and 200 rpm until the OD 600 The plasmid was extracted and PCR amplified using hyg-tagged primers P1 (hygF: 5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and P2 (hygR: 5'-TTCCGGAAGTGCTTGACATTGGGGA-3'), and a 289 bp target fragment was detected ( Figure 1 ). Confirm that the expression vector has been successfully introduced into Agrobacterium GV3101 and use it directly or store it at -20℃.

[0071] 2. Preparation of Agrobacterium infection solution The recombinant Agrobacterium carrying the plant expression vector was streaked and cultured. A single colony was picked from the solid YEP medium plate and added to the liquid YEP medium containing 50 mg / L Kan and 50 mg / L Rif. The culture was shaken at 200 rpm at 28°C until the OD 600 The value reached 0.8-1.0; centrifuged at 10000 rpm for 8 min at 28°C, discarded the supernatant, and collected the bacteria; then diluted with suspension (0.44 g / L MS + 0.1 g / L MES + 3 mg / L 2,4-D + 25 g / L sucrose + 100 μM AS) to obtain OD 600 Agrobacterium infection solution with a value of 0.3-0.4.

[0072] Example 3 Screening of cold-resistant embryogenic calli of Pennisetum To screen for cold-tolerant Pennisetum through embryogenic callus induction, we first screened the antibiotic concentrations and found that 200 mg / L Cef completely inhibited the growth of Agrobacterium GV3101. Addition of 20 mg / L Hyg caused the embryonic tissue to completely brown and lose viability. Therefore, 200 mg / L Cef and 20 mg / L Hyg were selected as the antibiotic selection pressures for cold-tolerant Pennisetum genetic transformation.

[0073] The specific resistant callus screening process is as follows: (1) Place the transformed receptor on OD 600The cells were inoculated with Agrobacterium infection solution with a pH value of 0.3-0.4, cultured at 28°C with shaking at 80 rpm for 20 min, and then the residual bacterial solution was dried with sterile filter paper. The cells were then rinsed three times with 200 mg / L Cef, placed on sterile filter paper and allowed to stand for 10 min, and then transferred to medium C for co-cultivation and cultured in the dark at 25°C for 5 days. Adding 100 μM AS to medium C can effectively increase the amount of Agrobacterium T-DNA fragments transferred to plant cells, thereby improving the efficiency of genetic transformation. (2) The transformed recipients after co-cultivation were transferred to medium D used for resistant callus screening culture for resistant callus induction screening. The best effect was achieved by adding 200 mg / L ascorbic acid as an antioxidant to medium D, which could significantly inhibit the browning of embryonic callus caused by 20 mg / L Hyg. Under dark culture at 25°C, pinpoint-shaped white or light yellow resistant embryonic callus appeared after about 14 days. (3) The white or light yellow embryonic callus with pinpoints was cut off and placed in medium E for resistant callus screening and further resistant callus induction screening was performed. In order to induce more resistant embryonic callus, the Hyg concentration in medium E was adjusted to 10 mg / L, and 200 mg / L ascorbic acid was added. Under dark culture at 25°C, dense granular white or light yellow resistant embryonic callus appeared after about 20-30 days. Figure 2 ). Medium E was changed every 2 weeks.

[0074] The formula of the above-mentioned medium C is as follows: 4.4 g / L MS, 0.1 mg / L 6-BA, 3 mg / L 2,4-D, 1 mg / L KT, 100 μM acetosyringone (AS), 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0075] The formula of the above-mentioned medium D is as follows: 4.4 g / L MS, 0.1 mg / L 6-BA, 3 mg / L 2,4-D, 1 mg / L KT, 20 mg / L hygromycin (Hyg), 200 mg / L cephalosporin (Cef), 200 mg / L ascorbic acid (Vc), 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0076] The formula of the above-mentioned culture medium E is as follows: 4.4 g / L MS, 0.1 mg / L 6-BA, 2 mg / L 2,4-D, 1 mg / L KT, 10 mg / L Hyg, 200 mg / L Cef, 200 mg / L Vc, 30 g / L sucrose, 7 g / L agar, and pH 5.8.

[0077] Example 4 Differentiation of cold-resistant Pennisetum resistant buds and resistant clustered buds The dense granular white or light yellow resistant embryonic callus with a size of 1-2 cm and good growth condition in Example 3 was transferred to culture medium F for resistance bud differentiation culture, and then transferred to culture medium G for resistance cluster bud differentiation culture. The specific implementation process is divided into two steps: (1) The dense granular white or light yellow resistant embryonic callus was transferred to culture medium F for resistance bud differentiation culture. In order to obtain more resistance differentiation buds, adding AMP to culture medium F can significantly promote the efficiency of resistance bud differentiation. It was further found that the addition of 3 mM AMP had the best effect, which greatly increased the resistance bud differentiation rate compared with the control, and the Hyg concentration was adjusted to 8 mg / L to reduce the browning of the resistance buds. Under 25°C light (16 hours light / 8 hours dark) culture, green resistance buds appeared in about 20-30 days. Culture medium F was changed every 2 weeks. (2) The callus with green resistance buds was placed in culture medium G for resistance cluster bud differentiation. To further prevent the browning of resistant buds caused by long-term Hyg in the culture medium, the Hyg concentration was further adjusted to 5 mg / L, which basically eliminated the browning phenomenon. At the same time, the addition of 3 mM AMP to culture medium F significantly promoted the differentiation rate of resistant buds. Under 25°C light culture (16 hours light / 8 hours dark), the resistant buds grew into regenerated seedlings after about 20-30 days ( Figure 3 ), grow to about 3-4 cm resistant seedlings. Medium G, change every 2 weeks.

[0078] The formula of the above-mentioned medium F is as follows: 4.4 g / L MS, 2 mg / L NAA, 3 mg / L 6-BA, 8 mg / L Hyg, 200 mg / L Cef, 3 mM adenosine monophosphate (AMP), 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0079] The formula of the above-mentioned culture medium G is as follows: 4.4 g / L MS, 2 mg / L NAA, 3 mg / L 6-BA, 5 mg / L Hyg, 200 mg / L Cef, 30 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0080] Example 5 Rooting and transplanting of cold-resistant Pennisetum seedlings The resistant seedlings of about 3-4 cm in size in Example 4 were transferred to culture medium H for resistance rooting culture and then transplanted. The specific implementation process is divided into two steps: (1) The resistant seedlings were transferred to culture medium H. In order to quickly induce rooting of the resistant seedlings, the Hyg selection pressure was removed from the culture medium H, which significantly improved the rooting efficiency of the resistant seedlings. Under 25°C light culture (16 hours light / 8 hours dark), the resistant seedlings took root after about 14-20 days ( Figure 4). (2) After hardening the resistant rooted seedlings obtained above for 2-3 days in a sterile environment, transplant them into 1 / 2 Hogland nutrient solution and culture them for 3-4 weeks ( Figure 5 ), and the obtained hydroponic resistant seedlings were subjected to subsequent molecular identification of transgenic positive plants.

[0081] The formula of the above-mentioned culture medium H is as follows: 2.2 g / L MS, 0.5 mg / L NAA, 0.5 mg / L IBA, 200 mg / L Cef, 25 g / L sucrose, 7 g / L agar, and pH 5.8-6.0.

[0082] Example 6 Screening and molecular identification of transgenic cold-tolerant Pennisetum plants Leaves from the cold-tolerant Pennisetum transgenic plants described in Example 5, as well as corresponding wild-type (WT) leaves, were excised and DNA was extracted from the leaves according to the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit instructions. DNA concentrations were measured using a Quawell 5000 nucleic acid and protein analyzer. Using these as templates, primers P1 and P2 were used to identify transgenic plants. The results showed that only 45 cold-tolerant Pennisetum strains amplified the 289-bp hyg fragment, while the wild-type strain did not amplify the target fragment ( Figure 6 ), confirming the successful integration of the hyg gene into the cold-tolerant Pennisetum genome. Further statistical analysis revealed that the genetic conversion rates for the cold-tolerant Pennisetum varieties Changsui, Liren, Liqiu, and No. 3 were 11.67%, 13.33%, 5.83%, and 6.67%, respectively (Table 1).

[0083] Table 1 Genetic conversion rate of Pennisetum with different cold resistance

[0084] The above results show that the Agrobacterium-mediated genetic transformation method established in the present invention is suitable for the genetic transformation of cold-resistant Pennisetum with different genotypes, provides technical support for the utilization of its excellent stress-resistant genes and molecular breeding, and has broad application prospects.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A genetic transformation method for cold-tolerant Pennisetum alopecuroides, characterized in that, The method comprises the following steps: Using the seeds or young spikes of Pennisetum americanum with cold tolerance as explants, inducing callus from the explants to obtain embryogenic callus as the transformation receptor; Infecting the transformation receptor with recombinant Agrobacterium containing the vector of the target gene, and then successively performing co-culture, resistant callus screening culture, resistant bud point differentiation culture, resistant cluster bud differentiation culture and resistant rooting culture to obtain genetically transformed plants; Among them, medium C is used for the co-culture, and medium C contains a basal medium, a carbon source, a coagulant, and 6-BA, 2,4-D, KT and acetosyringone; Medium F is used for the resistant bud point differentiation culture, and medium F contains a basal medium, a carbon source, a coagulant, and NAA, 6-BA, an antibiotic and adenosine monophosphate; Medium G is used for the resistant cluster bud differentiation culture, and medium G contains a basal medium, a carbon source, a coagulant, and NAA, 6-BA and an antibiotic.

2. The genetic transformation method of Pennisetum setaceum var. rubrum according to claim 1, characterized in that, In the medium C, the concentrations of 6-BA, 2,4-D, KT and acetosyringone are as follows: 0.05 - 0.15 mg / L 6-BA, 2.0 - 4.0 mg / L 2,4-D, 0.5 - 1.5 mg / L KT, 80 - 100 μM acetosyringone; And / or, in the medium F, the concentrations of NAA, 6-BA and adenosine monophosphate are as follows: 1.5 - 2.3 mg / L NAA, 1.5 - 3.2 mg / L 6-BA, 1.5 - 3.5 mM adenosine monophosphate; And / or, in the medium G, the concentrations of NAA and 6-BA are as follows: 1.3 - 2.1 mg / L NAA, 1.6 - 3.4 mg / L 6-BA.

3. The genetic transformation method of Pennisetum setaceum cv. ‘Rubrum’ according to claim 1 or 2, characterized in that, The antibiotic is hygromycin and / or cephalosporin; among them, the antibiotics in medium F include 5 - 8 mg / L hygromycin and 200 - 300 mg / L cephalosporin; the antibiotics in medium G include 3 - 5 mg / L hygromycin and 200 - 300 mg / L cephalosporin.

4. The genetic transformation method of cold-resistant Pennisetum alopecuroides according to any one of claims 1 to 3, characterized in that, Medium C contains the following components: 4.2 - 4.6 g / L MS, 0.05 - 0.15 mg / L 6-BA, 2.0 - 4.0 mg / L 2,4-D, 0.5 - 1.5 mg / L KT, 80 - 100 μM acetosyringone, 25 - 35 g / L sucrose, 6 - 10 g / L agar; And / or, medium F contains the following components: 4.2 - 4.6 g / L MS, 1.5 - 2.3 mg / L NAA, 1.5 - 3.2 mg / L 6-BA, 5 - 8 mg / L hygromycin, 200 - 300 mg / L cephalosporin, 1.5 - 3 mM adenosine monophosphate, 25 - 35 g / L sucrose, 6 - 10 g / L agar; And / or, the medium G contains the following components: 4.2 - 4.6 g / L MS, 1.3 - 2.1 mg / L NAA, 1.6 - 3.4 mg / L 6 - BA, 3 - 5 mg / L hygromycin, 200 - 300 mg / L cefamycin, 25 - 35 g / L sucrose, 6 - 10 g / L agar.

5. The genetic transformation method of cold-resistant Pennisetum alopecuroides according to any one of claims 1 to 4, characterized in that, The resistant callus screening culture includes sequentially using medium D and medium E for resistant callus screening; wherein, both medium D and medium E contain a basal medium, a carbon source, a solidifying agent, as well as 6 - BA, 2,4 - D, KT, an antibiotic, and ascorbic acid; In the medium D, the concentrations of 6 - BA, 2,4 - D, KT, the antibiotic, and ascorbic acid are: 0.08 - 0.21 mg / L 6 - BA, 2.2 - 3.6 mg / L 2,4 - D, 0.6 - 1.8 mg / L KT, 15 - 20 mg / L hygromycin, 200 - 300 mg / L cefamycin, 160 - 220 mg / L ascorbic acid; In the medium E, the concentrations of 6 - BA, 2,4 - D, KT, the antibiotic, and ascorbic acid are: 0.06 - 0.18 mg / L 6 - BA, 1.2 - 2.4 mg / L 2,4 - D, 0.6 - 1.8 mg / L KT, 8 - 10 mg / L hygromycin, 200 - 300 mg / L cefamycin, 150 - 210 mg / L ascorbic acid; Preferably, the medium D contains the following components: 4.2 - 4.6 g / L MS, 0.08 - 0.21 mg / L 6 - BA, 2.2 - 3.6 mg / L 2,4 - D, 0.6 - 1.8 mg / L KT, 15 - 20 mg / L hygromycin, 200 - 300 mg / L cefamycin, 160 - 220 mg / L ascorbic acid, 25 - 35 g / L sucrose, 5 - 10 g / L agar; The medium E contains the following components: 4.2 - 4.6 g / L MS, 0.06 - 0.18 mg / L 6 - BA, 1.2 - 2.4 mg / L 2,4 - D, 0.6 - 1.8 mg / L KT, 8 - 10 mg / L hygromycin, 200 - 300 mg / L cefamycin, 150 - 210 mg / L ascorbic acid, 25 - 35 g / L sucrose, 6 - 10 g / L agar.

6. The genetic transformation method of Pennisetum setaceum resistant to cold according to any one of claims 1 to 5, characterized in that, The resistant rooting culture uses medium H, and the medium H contains a basal medium, a carbon source, a solidifying agent, as well as NAA, IBA, and an antibiotic; Among them, the concentrations of NAA, IBA, and the antibiotic are 0.3 - 0.6 mg / L NAA, 0.4 - 0.7 mg / L IBA, 200 - 300 mg / L cefamycin; Preferably, the medium H comprises the following components: 2.0 - 2.4 g / L of MS, 0.3 - 0.6 mg / L of NAA, 0.4 - 0.7 mg / L of IBA, 200 - 300 mg / L of cefamycin, 20 - 30 g / L of sucrose, and 6 - 10 g / L of agar.

7. The genetic transformation method of cold-resistant Pennisetum alopecuroides according to any one of claims 1 to 6, characterized in that, The callus induction uses medium A, which comprises a basal medium, a carbon source, a solidifying agent, and 6 - BA, 2,4 - D, and KT; Among them, the concentrations of 6 - BA, 2,4 - D, and KT are as follows: 0.05 - 0.15 mg / L of 6 - BA, 2.0 - 4.0 mg / L of 2,4 - D, 0.5 - 1.5 mg / L of KT; Preferably, the medium A comprises the following components: 4.2 - 4.6 g / L of MS, 0.05 - 0.15 mg / L of 6 - BA, 2.0 - 4.0 mg / L of 2,4 - D, 0.5 - 1.5 mg / L of KT, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; Preferably, the embryogenic callus is used as a transformation receptor after sub - culturing 1 - 2 times or directly as a transformation receptor; Among them, the sub - culturing uses medium B, which comprises a basal medium, a carbon source, a solidifying agent, and 6 - BA, 2,4 - D, and KT; the concentrations of 6 - BA, 2,4 - D, and KT are as follows: 0.06 - 0.18 mg / L of 6 - BA, 1.2 - 2.2 mg / L of 2,4 - D, 0.8 - 1.5 mg / L of KT; Preferably, the medium B comprises the following components: 4.2 - 4.6 g / L of MS, 0.06 - 0.18 mg / L of 6 - BA, 1.2 - 2.2 mg / L of 2,4 - D, 0.8 - 1.5 mg / L of KT, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar.

8. The genetic transformation method of Pennisetum setaceum resistant to cold according to any one of claims 1 to 7, characterized in that, The callus induction is carried out in the dark at 23 - 26 °C for 40 - 50 d; And / or, the co - culturing is carried out in the dark at 23 - 26 °C for 4 - 6 d; And / or, the resistant callus induction and screening culture is carried out in medium D in the dark at 23 - 26 °C for 13 - 15 d, and then in medium E in the dark at 23 - 26 °C for 20 - 30 d; And / or, the differentiation of resistant bud points is carried out under light at 23 - 26 °C for 20 - 30 d; And / or, the differentiation of resistant multiple shoots is carried out under light at 23 - 26 °C for 20 - 30 d; And / or, the resistant rooting culture is carried out under light at 23 - 26 °C for 14 - 20 d; Preferably, the cold - tolerant Pennisetum is Pennisetum longissimum, Pennisetum orientale, Pennisetum 'Liqiu' or Pennisetum 'No. 3'.

9. Culture medium group for genetic transformation of Pennisetum setaceum var. rubrum, characterized in that, The medium group includes medium A for callus induction, medium C for co - culturing after recombinant Agrobacterium infection, medium D and medium E for resistant callus screening culture, medium F for resistant bud point differentiation culture, medium G for resistant multiple shoot differentiation culture, and medium H for resistant rooting culture; Among them, the culture medium A contains the following components: 4.2 - 4.6 g / L of MS, 0.05 - 0.15 mg / L of 6-BA, 2.0 - 4.0 mg / L of 2,4-D, 0.5 - 1.5 mg / L of KT, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; The culture medium C contains the following components: 4.2 - 4.6 g / L of MS, 0.05 - 0.15 mg / L of 6-BA, 2.0 - 4.0 mg / L of 2,4-D, 0.5 - 1.5 mg / L of KT, 80 - 100 μM of acetosyringone, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; The culture medium D contains the following components: 4.2 - 4.6 g / L of MS, 0.08 - 0.21 mg / L of 6-BA, 2.2 - 3.6 mg / L of 2,4-D, 0.6 - 1.8 mg / L of KT, 15 - 20 mg / L of hygromycin, 200 - 300 mg / L of cefamycin, 160 - 220 mg / L of ascorbic acid, 25 - 35 g / L of sucrose, and 5 - 10 g / L of agar; The culture medium E contains the following components: 4.2 - 4.6 g / L of MS, 0.06 - 0.18 mg / L of 6-BA, 1.2 - 2.4 mg / L of 2,4-D, 0.6 - 1.8 mg / L of KT, 8 - 10 mg / L of hygromycin, 200 - 300 mg / L of cefamycin, 150 - 210 mg / L of ascorbic acid, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; The culture medium F contains the following components: 4.2 - 4.6 g / L of MS, 1.5 - 2.3 mg / L of NAA, 1.5 - 3.2 mg / L of 6-BA, 5 - 8 mg / L of hygromycin, 200 - 300 mg / L of cefamycin, 1.5 - 3.5 mM of adenosine monophosphate, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; The culture medium G contains the following components: 4.2 - 4.6 g / L of MS, 1.3 - 2.1 mg / L of NAA, 1.6 - 3.4 mg / L of 6-BA, 3 - 5 mg / L of hygromycin, 200 - 300 mg / L of cefamycin, 25 - 35 g / L of sucrose, and 6 - 10 g / L of agar; The culture medium H contains the following components: 2.0 - 2.4 g / L of MS, 0.3 - 0.6 mg / L of NAA, 0.4 - 0.7 mg / L of IBA, 200 - 300 mg / L of cefamycin, 20 - 30 g / L of sucrose, and 6 - 10 g / L of agar; Preferably, the culture medium group further includes a culture medium B for the subculture of embryogenic callus, and the culture medium B contains the following components: 4.2-4.6 g / L MS, 0.06-0.18 mg / L 6-BA, 1.2-2.2 mg / L 2,4-D, 0.8-1.5 mg / L KT, 25-35 g / L sucrose, and 6-10 g / L agar.

10. Use of the genetic transformation method of cold-tolerant Pennisetum according to any one of claims 1 to 8 or the culture medium group for genetic transformation of cold-tolerant Pennisetum according to claim 9 in gene function identification, gene editing, genetic breeding, variety improvement or transgenic plant construction of cold-tolerant Pennisetum; Preferably, the cold-tolerant Pennisetum is Pennisetum longissimum, Pennisetum orientale, Pennisetum 'Liqiu' or Pennisetum americanum × P. purpureum cv. 3.

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