A method for in vitro regeneration of Napier grass

Abstract

This invention discloses a method for the in vitro regeneration of *Pennisetum alopecuroides*. The method includes the following steps: 1) using *Pennisetum alopecuroides* seeds as explants, inducing callus formation in an induction medium to obtain embryogenic callus; the induction medium includes 2,4-D, NAA, and 6-BA; 2) differentiating the embryogenic callus in a differentiation medium to obtain callus that differentiates into clustered shoots; the differentiation medium includes GA, NAA, and 6-BA; 3) rooting the clustered shoots in a rooting medium to obtain *Pennisetum alopecuroides* seedlings. This invention's in vitro regeneration method for *Pennisetum alopecuroides* using seeds as explants achieves a high callus formation rate, improves production efficiency, and reduces costs. The in vitro regeneration system established by this invention lays a good foundation for *Pennisetum alopecuroides* variety improvement and biotechnology breeding, and is of great significance for the efficient propagation and commercial production of *Pennisetum alopecuroides*.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for the in vitro regeneration of Pennisetum alopecuroides, and particularly to a method for the in vitro regeneration of Pennisetum alopecuroides using seeds as explants. Background Technology

[0002] *Pennisetum alopecuroides* (L.) is a C4 plant belonging to the genus *Pennisetum* in the family Poaceae. It is an annual or perennial herb, mainly distributed in tropical and subtropical regions, including Northeast, North, East, Central South, and Southwest my country. *Pennisetum alopecuroides* grows erect in clumps, reaching a height of 0.3-1.2 m. The leaves are linear, with cilia on the ligule. The inflorescence is an erect, conical panicle, with the main axis densely covered in soft hairs. The spikelets are linear-lanceolate, mostly solitary, and the caryopsis is oblong. *Pennisetum alopecuroides* grows rapidly, has a large biomass, and is characterized by high protein content, good palatability, and strong resistance to adverse conditions. It is an important energy forage, fodder, and miscellaneous grain crop. Its robust fibrous roots can also be used for environmental greening and soil and water conservation.

[0003] Napier grass primarily reproduces through seeds, but its breeding research in my country is still in its early stages. While modern molecular breeding techniques are relatively mature in major food and forage crops, they have not yet been applied to Napier grass. Therefore, there is an urgent need to establish an efficient and stable Napier grass regeneration system. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient and stable method for the in vitro regeneration of Napier grass.

[0005] Firstly, this invention claims a method for the in vitro regeneration of Napier grass.

[0006] The method for in vitro regeneration of Napier grass claimed in this invention includes the following steps:

[0007] 1) Using Napier grass seeds as explants, the Napier grass seeds were cultured in an induction medium to induce callus tissue and obtain embryogenic callus tissue; the induction medium included 2,4-D, NAA and 6-BA.

[0008] 2) The embryogenic callus is cultured in a differentiation medium to obtain callus that differentiates into clustered shoots; the differentiation medium includes GA, NAA and 6-BA;

[0009] 3) The clustered buds are rooted in a rooting medium to obtain Napier grass seedlings.

[0010] In the above method, step 1) is preceded by a step of disinfecting the seeds of Napier grass.

[0011] In one embodiment of the invention, the disinfection method comprises the steps of disinfecting Napier grass seeds in a 75% ethanol solution and disinfecting them in a 10% sodium hypochlorite solution containing Triton X-100.

[0012] In one specific embodiment of the present invention, the disinfection treatment method comprises the steps of disinfecting the seeds of Napier grass in a 75% ethanol solution for 1 minute (soaking) and disinfecting them in a 10% sodium hypochlorite solution containing Triton X-100 (one drop) for 1.5 hours (on a shaker).

[0013] In a preferred embodiment of the present invention, the disinfection treatment method includes the following steps: selecting plump and relatively uniform Napier grass seeds and rinsing them twice with clean water to remove dust and impurities from the seed surface; then soaking the washed seeds in a 75% ethanol solution for 1 minute; then disinfecting the soaked seeds in a 10% sodium hypochlorite solution containing one drop of surfactant Triton X-100 on a shaker for 1.5 hours; finally rinsing the disinfected seeds 5-6 times with sterile water until no foam remains, soaking the seeds in an appropriate amount of sterile water, and placing them at 4°C overnight.

[0014] In the above method, in step 1), the induction culture medium further includes maltose.

[0015] In one embodiment of the present invention, the concentration of maltose in the induction medium may be 20-30 g / L, specifically 20 g / L or 30 g / L, preferably 30 g / L.

[0016] In one embodiment of the present invention, the concentration of 2,4-D in the induction medium may be 1-5 mg / L, 1-3 mg / L, or 3-5 mg / L, specifically 1 mg / L, 3 mg / L, or 5 mg / L, preferably 3 mg / L.

[0017] In one embodiment of the present invention, the concentration of NAA in the induction medium may be 0.1-0.5 mg / L, 0.1-0.2 mg / L, or 0.2-0.5 mg / L, specifically 0.1 mg / L, 0.2 mg / L, or 0.5 mg / L, preferably 0.2 mg / L.

[0018] In one embodiment of the present invention, the concentration of 6-BA in the induction medium may be 1-5 mg / L, specifically 1 mg / L or 5 mg / L, preferably 1 mg / L.

[0019] In one specific embodiment of the present invention, the induction medium is obtained by mixing water, MS salt, maltose, 2,4-D, NAA, 6-BA and plant gel, wherein the concentration of MS salt in the induction medium is 4.43 g / L, the concentration of maltose in the induction medium is 30 g / L, the concentration of 2,4-D in the induction medium is 3 mg / L, the concentration of NAA in the induction medium is 0.2 mg / L, the concentration of 6-BA in the induction medium is 1.0 mg / L, and the concentration of plant gel in the induction medium is 4 g / L.

[0020] In one specific embodiment of the present invention, the pH of the induction medium is 5.8.

[0021] In the above method, in step 2), the differentiation medium further includes CuSO4. Further, the differentiation medium also includes maltose and CuSO4.

[0022] In one embodiment of the present invention, the concentration of maltose in the differentiation medium may be 20-30 g / L, specifically 20 g / L or 30 g / L, preferably 20 g / L.

[0023] In one embodiment of the present invention, the concentration of CuSO4 in the differentiation medium may be 0.5-2.5 mg / L, 0.5-1.25 mg / L, or 1.25-2.5 mg / L, specifically 0.5 mg / L, 1.25 mg / L, or 2.5 mg / L, preferably 1.25 mg / L.

[0024] In one embodiment of the present invention, the concentration of GA in the differentiation medium may be 0.1-1 mg / L, 0.1-0.5 mg / L, or 0.5-1 mg / L, specifically 0.1 mg / L, 0.5 mg / L, or 1 mg / L, preferably 0.5 mg / L.

[0025] In one embodiment of the present invention, the concentration of NAA in the differentiation medium may be 0.1-0.5 mg / L, 0.1-0.2 mg / L, or 0.2-0.5 mg / L, specifically 0.1 mg / L, 0.2 mg / L, or 0.5 mg / L, preferably 0.2 mg / L.

[0026] In one embodiment of the present invention, the concentration of 6-BA in the differentiation medium may be 1-5 mg / L, specifically 1 mg / L or 5 mg / L, preferably 1 mg / L.

[0027] In one specific embodiment of the present invention, the differentiation medium is obtained by mixing water, MS salt, maltose, CuSO4, GA, NAA, 6-BA and plant gel, wherein the concentration of MS salt in the differentiation medium is 4.43 g / L, the concentration of maltose in the differentiation medium is 20 g / L, the concentration of CuSO4 in the differentiation medium is 1.25 mg / L, the concentration of GA in the differentiation medium is 0.5 mg / L, the concentration of NAA in the differentiation medium is 0.2 mg / L, the concentration of 6-BA in the differentiation medium is 1.0 mg / L, and the concentration of plant gel in the differentiation medium is 4 g / L.

[0028] In one specific embodiment of the present invention, the pH of the differentiation culture medium is 5.8.

[0029] In the above method, step 1) further includes the step of subculturing embryonic callus (embryonic callus with proliferative and differentiation capabilities) in a subculture medium.

[0030] The subculture medium includes proline, 2,4-D, NAA, and 6-BA. Further, the subculture medium includes maltose, proline, 2,4-D, NAA, and 6-BA.

[0031] In one embodiment of the present invention, the concentration of maltose in the subculture medium may be 20-30 g / L, specifically 20 g / L or 30 g / L, preferably 30 g / L.

[0032] In one embodiment of the present invention, the concentration of proline in the subculture medium may be 0.5-2 mg / L, 0.5-1 mg / L, or 1-2 mg / L, specifically 0.5 mg / L, 1 mg / L, or 2 mg / L, preferably 1 mg / L.

[0033] In one embodiment of the present invention, the concentration of 2,4-D in the subculture medium may be 1-5 mg / L, 1-3 mg / L, or 3-5 mg / L, specifically 1 mg / L, 3 mg / L, or 5 mg / L, preferably 3 mg / L.

[0034] In one embodiment of the present invention, the concentration of NAA in the subculture medium may be 0.1-0.5 mg / L, 0.1-0.2 mg / L, or 0.2-0.5 mg / L, specifically 0.1 mg / L, 0.2 mg / L, or 0.5 mg / L, preferably 0.2 mg / L.

[0035] In one embodiment of the present invention, the concentration of 6-BA in the subculture medium may be 1-5 mg / L, specifically 1 mg / L or 5 mg / L, preferably 1 mg / L.

[0036] In one specific embodiment of the present invention, the subculture medium is obtained by mixing water, MS salt, maltose, proline, 2,4-D, NAA, 6-BA and plant gel, wherein the concentration of MS salt in the subculture medium is 4.43 g / L, the concentration of maltose in the subculture medium is 30 g / L, the concentration of proline in the subculture medium is 1 g / L, the concentration of 2,4-D in the subculture medium is 3 mg / L, the concentration of NAA in the subculture medium is 0.2 mg / L, the concentration of 6-BA in the subculture medium is 1.0 mg / L, and the concentration of plant gel in the subculture medium is 4 g / L.

[0037] In one specific embodiment of the present invention, the pH of the subculture medium is 5.8.

[0038] In the above method, the rooting culture medium includes maltose.

[0039] In one embodiment of the present invention, the concentration of maltose in the rooting culture medium can be 15-30 g / L, specifically 15 g / L or 30 g / L, preferably 15 g / L.

[0040] In one specific embodiment of the present invention, the rooting medium is obtained by mixing water, MS salt, maltose and plant gel, wherein the concentration of MS salt in the rooting medium is 2.215 g / L, the concentration of maltose in the rooting medium is 15 g / L, and the concentration of plant gel in the rooting medium is 4 g / L.

[0041] In one specific embodiment of the present invention, the pH of the rooting medium is 5.8.

[0042] In the above method, in step 1), the conditions for induction culture can be 25±2℃ and dark culture for 20-40 days, specifically 25℃ and dark culture for 30 days.

[0043] In step 2), the differentiation culture conditions are 25℃±2℃, 16h light / 8h darkness for 30-60 days, specifically 25℃, 16h light / 8h darkness for 40 days.

[0044] In step 3), the rooting culture conditions are 25℃±2℃, 16h light / 8h darkness for 15-30 days, specifically 25℃, 16h light / 8h darkness for 15 days.

[0045] The conditions for the subculture can be 25±2℃, dark culture.

[0046] During the subculture process, the proliferated callus tissue was divided into 2*2*2mm sections every 30 days. 3 The small pieces were placed in a new subculture medium and the culture continued.

[0047] The number of subcultures can be 2-3 times.

[0048] Step 3 above also includes hardening off the seedlings and transplanting.

[0049] In one specific embodiment of the present invention, the seedling hardening and transplanting method includes the following steps: taking out the Napier grass seedlings, washing off the culture medium from the roots, first transplanting them into the culture soil (black soil: vermiculite: humus = 1:1:1) for cultivation (22℃, 16h light / 8h darkness for 2 days), and then moving them to a greenhouse for further cultivation to obtain Napier grass plants.

[0050] Secondly, this invention claims protection for complete culture media.

[0051] The complete culture media claimed by this invention include the above-mentioned induction culture medium and / or the above-mentioned differentiation culture medium and / or the above-mentioned subculture culture medium and / or the above-mentioned rooting culture medium.

[0052] In one specific embodiment of the present invention, the complete culture medium further includes the above-mentioned culture soil (black soil: vermiculite: humus = 1:1:1).

[0053] The complete culture medium can be used for the regeneration culture of Napier grass.

[0054] Thirdly, the present invention claims protection for any of the applications described in a1)-a5):

[0055] a1) Application of the above methods in in vitro regeneration culture of Pennisetum arvense;

[0056] a2) Application of the above-mentioned complete culture medium in the in vitro regeneration culture of Napier grass;

[0057] a3) Application of the above-mentioned complete culture medium in the preparation of products from in vitro regeneration culture of Napier grass;

[0058] a4) Application of the above-mentioned induction culture medium in improving the healing rate of Pennisetum alopecuroides;

[0059] a5) Application of the above-mentioned induction culture medium in the preparation of products that improve the healing rate of Napier grass.

[0060] In any of the methods or applications described above, the Napier grass can be any Napier grass germplasm resource, variety or strain, specifically late-maturing Napier grass, American Napier grass PI535955, purple Napier grass, long-spike Napier grass or Ningza No. 3 Napier grass.

[0061] This invention establishes an in vitro regeneration method for *Pennisetum alopecuroides* using seeds as explants. By optimizing the formulation of the induction medium in this regeneration method, a high cure rate was achieved, improving production efficiency and reducing costs. The in vitro regeneration system established in this invention lays a solid foundation for *Pennisetum alopecuroides* variety improvement and biotechnology breeding, and is of great significance for the efficient propagation and commercial production of *Pennisetum alopecuroides*. Attached Figure Description

[0062] Figure 1 The callus induction of late-maturing Napier grass seeds under different hormone ratios.

[0063] Figure 2 Phenotypic images are shown in the in vitro regeneration method of *Pennisetum oleraceum* using seeds of *Pennisetum oleraceum* PI535955 as explants. A shows the morphology of *Pennisetum oleraceum* PI535955 seeds after 15 days of induction culture on induction medium; B shows callus tissue divided into small pieces and transferred to subculture medium for subculture; C shows callus tissue differentiated into shoot clusters after 30 days of light culture on differentiation medium; D shows shoot clusters growing into roots after 15 days of rooting medium; E shows regenerated seedlings transferred to nutrient soil for continued growth.

[0064] Figure 3 Phenotypic images are shown in the in vitro regeneration method of *Pennisetum purpureum* using seeds as explants. A shows the morphology of *Pennisetum purpureum* seeds after 15 days of induction culture; B shows callus tissue divided into small pieces and transferred to subculture medium for subculture; C shows callus tissue differentiated into shoot clusters after 30 days of light exposure on differentiation medium; D shows shoot clusters developing roots after 15 days of rooting medium growth; E shows regenerated seedlings transferred to nutrient soil for continued growth.

[0065] Figure 4 Phenotypic diagrams are shown in the in vitro regeneration method of *Pennisetum longicornis* using seeds as explants. A shows the morphology of *Pennisetum longicornis* seeds after 15 days of induction culture; B shows callus tissue divided into small pieces and transferred to subculture medium for subculture; C shows callus tissue differentiated into shoot clusters after 30 days of light exposure on differentiation medium; D shows shoot clusters developing roots after 15 days of rooting medium growth; E shows regenerated seedlings transferred to nutrient soil for continued growth.

[0066] Figure 5Phenotypic images of the in vitro regeneration method of *Pennisetum purpureus* using Ningza 3 seeds as explants are shown. A shows the morphology of Ningza 3 *Pennisetum purpureus* seeds after 15 days of induction culture; B shows callus tissue divided into small pieces and transferred to subculture medium for subculture; C shows callus tissue differentiated into shoot clusters after 30 days of light exposure on differentiation medium; D shows the morphology of shoot clusters after 30 days of growth on rooting medium; E shows regenerated seedlings transferred to nutrient soil for continued growth. Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0069] The late-maturing Napier grass in the following examples is described in the literature “Zou Jingyuan, Cui Yu, Zeng Xianhuang, et al. Effects of 6 kinds of forage grass on the physicochemical properties of red soil in Nanxiong, Guangdong [J]. Forestry and Environmental Science, 2024, 40(01):91-98.”

[0070] The American foxtail grass PI535955 described in the following examples is described in the literature “Sun M, Yan H, Zhang A, et al. Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets. Plant Biotechnol J. 2023 Nov; 21(11):2348-2357.”

[0071] The purple-lighted pampas grass in the following examples is described in the literature "Miao Shan, Su Xiaojing, Chen Bo, et al. Study on salt tolerance of three kinds of pampas grass [J]. Journal of Beijing Vocational College of Agriculture, 2019, 33(02):28-33."

[0072] The long-spike Napier grass in the following examples is described in the literature “Miao Shan, Xia Zhenping, Li Zhiqiang. Effects of NaCl stress on the growth and physiological characteristics of three Napier grass species [J]. Heilongjiang Agricultural Sciences, 2019, (06): 132-136.”

[0073] The Ningza No. 3 Napier grass in the following examples is described in the literature "Xiang Zhen, Fan Jigui, Zhou Mingtang, et al. High-yield cultivation techniques of Ningza No. 3 Napier grass [J]. Agricultural Science and Technology Communication, 2002, (12): 24."

[0074] The MS salt used in the following examples is a Phytotech product, catalog number M519.

[0075] Example 1: A method for in vitro regeneration of Pennisetum arvense using seeds as explants

[0076] I. Seed disinfection

[0077] Select a number of plump, relatively uniform-sized Napier grass seeds and place them in a 50mL centrifuge tube. Disinfect the seeds according to the following steps: First, rinse twice with clean water to remove dust and impurities from the seed surface. Then, soak the washed seeds in a 75% ethanol solution for 1 minute. Next, disinfect the soaked seeds in a 10% sodium hypochlorite solution (with one drop of surfactant Triton X-100) on a shaker for 1.5 hours. Finally, rinse the disinfected seeds 5-6 times with sterile water until no foam remains (all operations from this step onwards must be performed in a laminar flow hood). After disinfection, soak the seeds in an appropriate amount of sterile water and incubate overnight at 4°C.

[0078] II. Induction of Embryonic Callus

[0079] Discard the sterile water, pour the seeds onto three layers of sterile filter paper to absorb excess moisture, then place the seeds on the induction medium and incubate in the dark at 25°C. Check the callus every 2-3 days; if contamination occurs, replace the medium immediately. After 30 days of culture, callus tissue is obtained, at which point the callus rate is calculated (callus rate = number of seeds that induced callus tissue / total number of seeds placed in the induction medium × 100%).

[0080] The above-mentioned induction medium was prepared by mixing water, MS salt, maltose, 2,4-D, NAA, 6-BA and plant gel, and adjusting the pH to 5.8 with 1M NaOH. The concentrations of MS salt, maltose, 2,4-D, NAA, 6-BA and plant gel in the induction medium were 4.43 g / L, 30 g / L, 3 mg / L, 0.2 mg / L, 1.0 mg / L, and 4 g / L, respectively.

[0081] III. Callus Subgeneration

[0082] In a clean bench, using sterile forceps and a scalpel, redundant parts of the callus's primary embryo and spongy tissue are removed to locate the hard "nucleus," which is the embryogenic callus capable of proliferation and differentiation. This nucleus is then placed in a subculture medium for subculture, maintaining the same dark incubation conditions at 25°C. Every 30 days, the proliferated callus is divided into 2*2*2mm sections. 3 The small pieces were placed in a new subculture medium and the culture continued.

[0083] The subculture medium was prepared by mixing water, MS salt, maltose, proline, 2,4-D, NAA, 6-BA, and plant gel, and adjusting the pH to 5.8 with 1M NaOH. The concentrations of MS salt, maltose, proline, 2,4-D, NAA, 6-BA, and plant gel in the subculture medium were 4.43 g / L, 30 g / L, 1 g / L, 3 mg / L, 0.2 mg / L, 1.0 mg / L, and 4 g / L.

[0084] IV. Differentiation of callus tissue

[0085] Callus tissues that have undergone 2-3 subcultures are inoculated into differentiation medium for differentiation culture under the following conditions: 25℃, 16h light / 8h dark. After about 10 days of culture, pale green buds will appear on the surface of the callus tissues. Continue culture until green shoots form. After about 30 days of culture, the shoot clusters have basically emerged. At this point, the differentiation rate is calculated (differentiation rate = number of shoots emerging from the callus tissue / total number of callus tissues placed in the differentiation medium × 100%).

[0086] The differentiation medium was prepared by mixing water, MS salt, maltose, CuSO4, GA, NAA, 6-BA, and plant gel, and adjusting the pH to 5.8 with 1M NaOH. The concentrations of MS salt, maltose, CuSO4, GA, NAA, 6-BA, and plant gel in the differentiation medium were 4.43 g / L, 20 g / L, 1.25 mg / L, 0.5 mg / L, 0.2 mg / L, 1.0 mg / L, and 4 g / L.

[0087] V. Rooting Culture

[0088] The differentiated shoots were transferred to rooting medium and cultured under the following conditions: 25°C in a culture room with 16 hours of light and 8 hours of darkness. After about 15 days of culture, sterile seedlings with roots (regenerated seedlings) were obtained. At this time, the rooting rate was calculated (rooting rate = number of shoots with roots / total number of shoots placed in the rooting medium × 100%).

[0089] The above-mentioned rooting medium was prepared by mixing water, MS salt, maltose and plant gel, and adjusting the pH to 5.8 with 1M NaOH. The concentration of MS salt in the rooting medium was 2.215 g / L, the concentration of maltose in the rooting medium was 15 g / L, and the concentration of plant gel in the rooting medium was 4 g / L.

[0090] VI. Seedling hardening and transplanting

[0091] The regenerated seedlings were removed with tweezers, the culture medium was washed off the roots, and they were transplanted into the culture soil (black soil: vermiculite: humus = 1:1:1). They were then cultured in an artificial climate incubator at 22℃ (photoperiod of 16h light / 8h darkness) for 2 days. Finally, they were moved to a greenhouse for further cultivation to obtain the Napier grass plants.

[0092] Example 2: Optimization of Induction Culture Medium

[0093] I. Seed disinfection

[0094] Late-maturing Napier grass seeds were used as test materials and were disinfected according to the method in step one of implementation 1.

[0095] II. Induction of Embryonic Callus

[0096] Discard the sterile water, pour the seeds onto three layers of sterile filter paper to absorb excess moisture, then place the seeds on the induction medium and incubate in the dark at 25°C. Check the callus every 2-3 days; if contamination occurs, replace the medium immediately. After 30 days of culture, callus tissue is obtained, at which point the callus rate is calculated (callus rate = number of seeds that induced callus tissue / total number of seeds placed in the induction medium × 100%).

[0097] The aforementioned induction medium was prepared by mixing water, MS salt, maltose, 2,4-D, NAA, 6-BA, and plant gel, and adjusting the pH to 5.8 with 1M NaOH. The concentrations of MS salt, maltose, and plant gel in the induction medium were 4.43 g / L, 30 g / L, and 4 g / L, respectively. Nine induction media (numbered A1, A2, A3, B1, B1, B3, C1, C2, and C3) were prepared according to different ratios of 2,4-D, NAA, and 6-BA hormones, as shown in Table 1.

[0098] The results showed that the hormone combination shown in B1 (3 mg / L 2,4-D + 0.2 mg / L NAA + 1.0 mg / L 6-BA) had the highest cure rate (Table 1). Figure 1 Furthermore, it was found that when the hormone ratio was inappropriate, the length of the primary coleoptile was longer, which was not conducive to callus induction. Therefore, the optimal induction medium formula is as follows (solvent is water): 4.43 g / L MS + 30 g / L maltose + 3 mg / L 2,4-D + 0.2 mg / L NAA + 1.0 mg / L 6-BA + 4 g / L plant gel.

[0099] Table 1. Seed emergence rate of late-maturing Napier grass under different hormone ratios.

[0100]

[0101] Example 3: A method for in vitro regeneration of *Pennisetum purpureus* using seeds of *Pennisetum purpureus* PI535955 as explants.

[0102] The seeds of American foxtail grass PI535955 were used as test materials to carry out in vitro regeneration of foxtail grass according to the method in Example 1, and the healing rate, differentiation rate and rooting rate were statistically analyzed.

[0103] Phenotypic diagrams of the in vitro regeneration method of *Pennisetum oleraceum* established using seeds of *Pennisetum oleraceum* PI535955 as explants are shown below. Figure 2 As shown.

[0104] The statistical results of the callus rate, differentiation rate and rooting rate of the in vitro regeneration method of *Pennisetum oleraceum* established using seeds of *Pennisetum oleraceum* PI535955 as explants are as follows: callus rate was 91.3%, differentiation rate was 66.7% and rooting rate was 100%.

[0105] Example 4: A method for in vitro regeneration of *Pennisetum purpureum* using seeds as explants.

[0106] Using seeds of *Pennisetum purpureum* as the test material, *Pennisetum purpureum* was regenerated in vitro according to the method in Example 1, and the healing rate, differentiation rate, and rooting rate were statistically analyzed.

[0107] Phenotypic diagrams of the in vitro regeneration method of *Pennisetum purpureus* using seeds as explants are shown below. Figure 3 As shown.

[0108] The statistical results of the callus rate, differentiation rate and rooting rate of the in vitro regeneration method of Pennisetum purpureum using seeds as explants are as follows: the callus rate was 92.0%, the differentiation rate was 41.7% and the rooting rate was 100%.

[0109] Example 5: A method for in vitro regeneration of Pennisetum longicornis using seeds as explants.

[0110] Using long-spike foxtail grass seeds as test material, in vitro regeneration of foxtail grass was carried out according to the method in Example 1, and the healing rate, differentiation rate and rooting rate were statistically analyzed.

[0111] Phenotypic diagrams of the in vitro regeneration method of *Pennisetum longicornis* using seeds as explants are shown below. Figure 4 As shown.

[0112] The statistical results of the callus rate, differentiation rate and rooting rate of the in vitro regeneration method of Pennisetum longicornis using seeds as explants are as follows: the callus rate was 95.4%, the differentiation rate was 6.1% and the rooting rate was 100%.

[0113] Example 6: A method for in vitro regeneration of *Pennisetum affine* using seeds of Ningza No. 3 as explants.

[0114] Using Ningza No. 3 Pennisetum seeds as test material, Pennisetum in vitro regeneration was carried out according to the method in Example 1, and the healing rate, differentiation rate and rooting rate were statistically analyzed.

[0115] Phenotypic diagrams of the in vitro regeneration method of *Pennisetum affine* established using seeds of Ningza 3 as explants are shown below. Figure 5 As shown.

[0116] The statistical results of the callus rate, differentiation rate and rooting rate of the in vitro regeneration method of Pennisetum 'Ningza 3' seeds established as explants are as follows: the callus rate was 96.2%, the differentiation rate was 18.7%, and the rooting rate was 100%.

[0117] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for in vitro regeneration of Pennisetum, comprising the following steps: 1) taking the Pennisetum species seed as an explant, culturing the Pennisetum species seed in an induction medium to induce callus, and obtaining embryogenic callus; the induction medium is obtained by uniformly mixing water, MS salt, maltose, 2,4-D, NAA, 6-BA and plant gel, wherein, the concentration of the MS salt in the induction medium is 4.43 g / L, the concentration of the maltose in the induction medium is 30 g / L, the concentration of the 2,4-D in the induction medium is 3 mg / L, the concentration of the NAA in the induction medium is 0.2 mg / L, the concentration of the 6-BA in the induction medium is 1.0 mg / L, and the concentration of the phytagel in the induction medium is 4 g / L; 2) culturing the embryogenic callus in a differentiation medium to obtain callus with differentiated shoots; the differentiation medium is obtained by mixing water, MS salt, maltose, CuSO4, GA, NAA, 6-BA and phytagel, wherein the concentration of the MS salt in the differentiation medium is 4.43 g / L, the concentration of the maltose in the differentiation medium is 20 g / L, the concentration of the CuSO4 in the differentiation medium is 1.25 mg / L, the concentration of the GA in the differentiation medium is 0.5 mg / L, the concentration of the NAA in the differentiation medium is 0.2 mg / L, the concentration of the 6-BA in the differentiation medium is 1.0 mg / L, and the concentration of the phytagel in the differentiation medium is 4 g / L; 3) culturing the shoots in a rooting medium to obtain Pennisetum seedlings; the rooting medium is obtained by mixing water, MS salt, maltose and phytagel, wherein the concentration of the MS salt in the rooting medium is 2.215 g / L, the concentration of the maltose in the rooting medium is 15 g / L, and the concentration of the phytagel in the rooting medium is 4 g / L; the step 1) further comprises a step of subculturing the embryogenic callus in a subculture medium; the subculture medium is obtained by mixing water, MS salt, maltose, proline, 2,4-D, NAA, 6-BA and phytagel, wherein the concentration of the MS salt in the subculture medium is 4.43 g / L, the concentration of the maltose in the subculture medium is 30 g / L, the concentration of the proline in the subculture medium is 1 g / L, the concentration of the 2,4-D in the subculture medium is 3 mg / L, the concentration of the NAA in the subculture medium is 0.2 mg / L, the concentration of the 6-BA in the subculture medium is 1.0 mg / L, and the concentration of the phytagel in the subculture medium is 4 g / L; the Pennisetum is late-maturing Pennisetum, American Pennisetum PI535955, purple Pennisetum, long-awned Pennisetum or Ningza No. 3 Pennisetum; the step 1) further comprises a step of sterilizing the Pennisetum seeds; and the induction culture is performed at 25±2 ℃ in the dark for 20-40 days; in the step 2), the differentiation culture is performed at 25±2 ℃ with 16 h light and 8 h dark for 30-60 days. In the step 3), the rooting culture condition is 25℃±2℃, light 16h / dark 8h, and the culture time is 15-30 days.

2. A kit comprising the induction medium, the differentiation medium, the subculture medium and the rooting medium according to any one of claim 1.

3. Use of any one of a1)-a5) as follows: a1) Use of the method according to any one of claim 1 in the in vitro culture of Pennisetum alopecuroides; a2) Use of the kit according to claim 2 in the in vitro culture of Pennisetum alopecuroides; a3) Use of the kit according to claim 2 in the preparation of a product for the in vitro culture of Pennisetum alopecuroides; a4) Use of the induction medium according to any one of claim 1 in the improvement of the callus rate of Pennisetum alopecuroides; a5) Use of the induction medium according to any one of claim 1 in the preparation of a product for the improvement of the callus rate of Pennisetum alopecuroides.

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