Method for culturing and somatic embryogenesis of a cytisus cultivar

Abstract

The application discloses a method for culturing and somatic embryogenesis of a cyclamen persicum cell line. The method comprises the following steps: picking healthy unopened flower buds of the cyclamen persicum, performing disinfection treatment, and taking filaments as explants; inoculating the filaments into a callus induction culture medium to perform induction culture, and obtaining yellowish blocky callus; inoculating the yellowish blocky callus into a callus subculture medium to perform subculture, and obtaining loose granulated callus; inoculating the loose granulated callus into a liquid culture medium to perform oscillation culture, and obtaining a cyclamen persicum suspension cell line; inoculating the cyclamen persicum suspension cell line into an embryogenic induction culture medium with filter paper on a surface to perform induction culture, and obtaining cyclamen persicum somatic embryos. The application uses callus to obtain a large number of crescent cells through a suspension liquid culture mode for the first time, and finally induces formation of cyclamen somatic embryos, thereby breaking through the problem that a single cell of a lily is difficult to develop into a somatic embryo.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for culturing double-flowered lily cell lines and somatic embryogenesis. Background Technology

[0002] Lilies (Lilium spp.) are important bulbous monocotyledonous plants, typically used for medicinal, edible, and ornamental purposes. Their vibrant flowers, strong fragrance, and adaptability make them a popular choice for cut flowers and potted plants in the flower market. Currently, common cut lilies include LA, Asiatic lilies, LO, Trumpet lilies, OT, and Oriental lilies, with Asiatic lilies, Oriental lilies, and Trumpet lilies being the three most prevalent types. With the increasing demand for cut flowers in China, traditional lily propagation methods can no longer meet production needs. Somatic embryo propagation offers advantages such as high induction rate, rapid seedling emergence, and large-scale production, quickly increasing the propagation speed of lilies, obtaining large quantities of virus-free plants, and effectively shortening the breeding cycle.

[0003] Plant cell suspension culture refers to a biotechnology that utilizes the rapid proliferation capacity of cells to achieve large-scale culture of plant cells in bioreactors, offering high efficiency and controllability. Suspension cell lines also exhibit characteristics such as consistent origin, uniform genetic background, and no inter-individual differences, demonstrating good cell totipotency. Previous studies have applied suspension culture systems to plants from which secondary metabolites are extracted, such as grapes and yews. Lilies, a typical asexually reproducing plant, typically reproduce through scale cuttings or tissue culture because they lack seeds, but these methods severely limit production scale. Therefore, somatic embryogenesis in suspension cell culture will become one of the most effective methods in plant biotechnology.

[0004] Lily explants can induce embryogenic callus to maintain embryogenicity, thereby differentiating into complete regenerated plants. However, the resulting callus granules are usually hard, and the somatic embryos are not easily dispersed, requiring a longer time to culture into cell lines that reproduce quickly. Currently, there are no patents on mature lily cell suspension systems in the literature. Although there are some reports, such as the establishment of Minjiang lily suspension cell lines and plant regeneration, these remain at the level of scientific data and are still some distance from industrialization technology. Therefore, this invention screens and obtains double-flowered lily materials, and utilizes their callus to induce and culture cell lines to achieve efficient subculturing and somatic embryogenesis, solving a pressing technical problem for the mass propagation and industrialization of lilies. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of poor regeneration capacity and difficulty in suspension culture of lily cells in the prior art, and to provide a method for lily cell line culture and somatic embryogenesis.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for culturing and somatic embryogenesis of a double-flowered lily cell line, comprising the following steps:

[0008] (1) Remove healthy, unopened flower buds of double-flowered lilies, disinfect them, and use the filaments as explants;

[0009] (2) The filaments were inoculated into the callus induction medium for induction culture to obtain pale yellow blocky callus tissue; the formula of the callus induction medium was: MS + 2.0 mg / L Picloram + 0.5 mg / L NAA + 0.2 mg / L TDZ + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8;

[0010] (3) The pale yellow blocky callus tissue was inoculated into the callus tissue subculture medium for subculture to obtain loose granular callus tissue; the formula of the callus tissue subculture medium is: MS + 0.5 mg / L Picloram + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8.

[0011] (4) Loose granular callus tissue was inoculated into liquid culture medium for suspension culture to obtain a suspension cell line; the liquid culture medium was selected from MP1 liquid culture medium and MP2 liquid culture medium; the formula of MP1 liquid culture medium was: MS + 2.0 mg / L Picloram + 0.5 mg / L NAA + 2 g yeast extract + 35 g / L sucrose, pH 5.8; the formula of MP2 liquid culture medium was: MS + 1.0 mg / L Picloram + 0.2 mg / L NAA + 35 g / L sucrose, pH 5.8;

[0012] (5) The suspension cell line was inoculated into an embryogenic induction medium with filter paper on the surface for induction culture to obtain double-petaled lily embryos; the embryogenic induction medium was formulated as follows: MS + 1.5 mg 6-BA / L + 0.15 mg / L NAA + 3.0 mg / L GA + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8.

[0013] The specific steps (1) are as follows: take healthy, unopened flower buds of double-flowered lilies, peel off the outer 1-2 layers of petals with tweezers, then soak the flower buds in 75% alcohol solution for 1-2 minutes, rinse with sterile water 4-5 times, soak in 3% mercuric chloride solution for 10 minutes, rinse with sterile water 5-7 times, air dry at room temperature, peel off the filaments, and use the peeled filaments as explants.

[0014] The specific steps (2) are as follows: the filaments are inoculated into the callus induction culture medium and cultured for 4 to 5 weeks at a temperature of 27°C, a relative humidity of 50%, and in the dark to obtain pale yellow blocky callus tissue.

[0015] The specific steps (3) are as follows: the pale yellow blocky callus tissue is inoculated into the callus tissue subculture medium and cultured at a temperature of 26°C, relative humidity of 50%, and in the dark. Subculture is performed every 15 days, and the subculture is performed 3 to 5 times to obtain loose granular callus tissue.

[0016] Step (4) is as follows: 0.5-0.8g of double-petaled lily subcultured callus tissue is added to a 100mL Erlenmeyer flask containing 30mL of MP1 liquid culture medium. The flask is placed on a shaker and cultured in the dark at 23℃ and 120rpm. Subculture is performed every 4 days. Each time, 2 / 3 of the supernatant is discarded and fresh MP1 liquid culture medium is added to 30mL for continued suspension culture. Subculture is performed 3-4 times under the above conditions. After each subculture, 2 / 3 of the supernatant is discarded and fresh MP2 liquid culture medium is added to 30mL. The suspended cells are then filtered through a 20-mesh sterilized sieve, and only the suspended cells that can pass through the 20-mesh sieve are retained for continued suspension culture. The suspension culture is performed for 20-60 days to complete the establishment of the double-petaled lily cell suspension cell line.

[0017] The specific steps (5) are as follows: the suspended cell line is filtered through a 40-mesh sieve, and the cell clusters remaining on the sieve are inoculated into an embryogenic induction medium with filter paper on the surface. The cells are cultured for 14 to 25 days at a temperature of 26°C, a relative humidity of 50%, and in the dark to obtain a somatic embryo.

[0018] The significant advantages of this invention are:

[0019] The significant advantages of this invention lie in the screening and acquisition of double-flowered lily materials capable of rapidly developing from somatic cells into somatic embryos. It is the first time that optimal genotype materials capable of rapid cell suspension have been obtained in lilies, and the use of filter paper bridges further enhances the probability of somatic embryogenesis. Secondly, a lily embryogenic suspension cell line, characterized by crescent-shaped embryogenic cells, can be established within a short period of 1.0-2.0 months. This breakthrough enables the subculture of lily cell lines, significantly improving the propagation and proliferation capacity of suspension cells, and providing a technological platform for the future application of cell suspension in the large-scale, industrialized propagation of lily seedlings. Attached Figure Description

[0020] Figure 1: Schematic diagram of the lily cell line culture and somatic embryogenesis process of this invention. A, Sterilized flower bud morphology. B, Callus induction state of filaments. C, Secondary subculturing of callus. D, Cutting small callus pieces and inoculating them into liquid culture medium. E, First cell culture state. F, Cell cluster culture state. G, Microscopic observation of cell cluster morphology. H, Cell cluster continuing to grow on filter paper bridge. I, New somatic embryogenesis. J, Somatic embryo induction into small bulbs.

[0021] Figure 2 : Double-petaled lily cell suspension cell line. Detailed Implementation

[0022] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0023] Example 1

[0024] The double-flowered lily variety used in this embodiment is Anouska.

[0025] S1: Pick healthy, unopened buds of double-flowered lilies, peel off the outer 1-2 layers of petals with tweezers, then disinfect the buds and separate the stamens and petals. Collect robust bulbs of double-flowered lilies, remove the outermost scales that are in contact with the soil, and select the clean, disease-free middle-layer scales for disinfection. The disinfection process is as follows: first, soak in a 75% alcohol solution for 1-2 minutes, rinse 4-5 times with sterile water, then soak in a 3% mercuric chloride solution for 10 minutes, rinse 5-7 times with sterile water, and air dry at room temperature.

[0026] S2: Using the petals, filaments, and scales of the double-flowered lily obtained in step S1 as explants, inoculate them into callus induction medium for induction. Culture under conditions of 27°C, 50% relative humidity, and darkness. The callus induction medium is MPNT medium (MS + 2.0 mg / L Picloram + 0.5 mg / L NAA + 0.2 mg / L LTDZ + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5–5.8) or MP medium (MS + 0.5 mg / L Picloram + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5–5.8). The callus formation from various explants was observed. The results showed that induction using double-flowered lily filaments in MPNT medium resulted in the formation of pale yellow, blocky callus after 4-5 weeks of culture, with a callus induction rate of 92% and a browning rate of 2%. Induction using double-flowered lily petals in MPNT medium resulted in the formation of blocky callus after 4 weeks of culture, but the callus induction rate was less than 30%. Induction using double-flowered lily scales in MPNT medium resulted in the formation of blocky callus after 2 months of culture, but the callus induction rate was only 65%, and the callus surface hardened. Induction using double-flowered lily filaments in MP medium resulted in the formation of blocky callus after 4 weeks of culture, but the callus induction rate was only 45%, and the callus surface hardened.

[0027] S3: The pale yellow, blocky callus tissue induced in step S2 by inoculating double-petaled lily filaments into MPNT medium is cut into small cubes of 2-5 mm in size using a sterile scalpel. These cubes are then inoculated into subculture medium for subculture. The subculture conditions are: temperature 26℃, relative humidity 50%, and darkness. The subculture cycle is 15 days. The subculture medium is either MP medium (MS + 0.5 mg / L citronella + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8) or MD medium (MS + 0.5 mg / L 2,4-D + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8). The state of the callus obtained by subculture was observed. The results showed that after 2 to 3 subcultures in MP medium, the callus grew vigorously, was granular and loose, and the callus proliferation rate was 85%. After 4 subcultures in MD medium, the callus proliferation rate was 65%, but the callus was watery and poorly granulated.

[0028] S4: Take 0.5-0.8g of the loose, granular callus obtained from the pale yellow, blocky callus in step S3, subculture it in MP medium 2-3 times, and add it to a 100mL Erlenmeyer flask containing 30mL of MP1 liquid medium. Place the flask on a shaker and culture in the dark at 23℃ and 120rpm. Subculture every 4 days, discarding 2 / 3 of the supernatant each time and adding fresh MP1 liquid medium to 30mL. Continue subculturing for 3-4 times. From the 4th to 5th subculture, discard 2 / 3 of the supernatant each time and add fresh MP2 liquid medium to 30mL. Filter the suspended cells through a 20-mesh sterile sieve, retaining only the cells that can pass through the 20-mesh sieve for further suspension culture. After 20 days of suspension culture, microscopic examination reveals that most of the culture consists of uniformly sized round, oval, or crescent-shaped single cells or small cell clusters with uniform growth and good dispersion. Figure 2 This completes the establishment of a double-flowered lily cell suspension line, with a cell growth rate of 6 times / 24h. The MP1 liquid culture medium formulation is: MS + 2.0 mg / L icloram + 0.5 mg / L NAA + 2 g yeast extract + 35 g / L sucrose, pH 5.8; the MP2 liquid culture medium formulation is: MS + 1.0 mg / L icloram + 0.2 mg / L NAA + 35 g / L sucrose, pH 5.8.

[0029] S5: Filter the suspension cell culture obtained from step S4 after 20 days of suspension culture through a 40-mesh sieve. The cell clusters remaining on the sieve are then evenly inoculated onto embryogenic induction medium (MS + 1.5 mg 6-BA / L + 0.15 mg / L NAA + 3.0 mg / L GA + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5-5.8) lined with filter paper or onto embryogenic induction medium without filter paper. Culture conditions are 26℃, 50% relative humidity, and darkness. Somatic embryogenesis is observed. Results show that a large number of somatic embryos (85%) appear after 14-25 days of culture on embryogenic induction medium with filter paper; however, no normal somatic embryos are obtained after 45 days of culture on embryogenic induction medium without filter paper, only large clumps of callus tissue form.

Claims

1. A method for culturing double-flowered lily cell lines and somatic embryogenesis, characterized in that: Includes the following steps: (1) Remove healthy, unopened flower buds of double-flowered lilies, disinfect them, and use the filaments as explants; (2) The filaments were inoculated into the callus induction medium for induction culture to obtain pale yellow blocky callus tissue; the formula of the callus induction medium was: MS + 2.0 mg / L Picloram + 0.5 mg / L NAA + 0.2 mg / L TDZ + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5~5.8; (3) The pale yellow blocky callus was inoculated into the callus subculture medium for subculture to obtain loose granular callus; the formula of the callus subculture medium is: MS + 0.5 mg / L Picloram + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5~5.8; (4) Loose granular callus tissue was inoculated into liquid culture medium for suspension culture to obtain a suspension cell line; the liquid culture medium was selected from MP1 liquid culture medium and MP2 liquid culture medium; the formula of MP1 liquid culture medium was: MS + 2.0 mg / L Picloram + 0.5 mg / L NAA + 2 g yeast extract + 35 g / L sucrose, pH 5.8; the formula of MP2 liquid culture medium was: MS + 1.0 mg / L Picloram + 0.2 mg / L NAA + 35 g / L sucrose, pH 5.8; (5) The suspension cell line was inoculated into an embryogenic induction medium with filter paper on the surface for induction culture to obtain double-petaled lily embryos; the embryogenic induction medium was formulated as follows: MS + 1.5 mg / L 6-BA + 0.15 mg / L NAA + 3.0 mg / L GA + 30 g / L sucrose + 3.0 g / L plant gel, pH 5.5~5.

8.

2. The method according to claim 1, characterized in that: The specific steps (1) are as follows: take healthy, unopened flower buds of double-flowered lilies, peel off the outer 1-2 layers of petals with tweezers, then soak the flower buds in 75% alcohol solution for 1-2 minutes, rinse with sterile water 4-5 times, soak in 3% mercuric chloride solution for 10 minutes, rinse with sterile water 5-7 times, air dry at room temperature, peel off the filaments, and use the peeled filaments as explants.

3. The method according to claim 1, characterized in that: The specific steps (2) are as follows: the filaments are inoculated into the callus induction culture medium and cultured for 4-5 weeks at a temperature of 27°C, a relative humidity of 50%, and in the dark to obtain pale yellow blocky callus tissue.

4. The method according to claim 1, characterized in that: The specific steps (3) are as follows: the pale yellow blocky callus tissue is inoculated into the callus tissue subculture medium and cultured at a temperature of 26°C, relative humidity of 50% and in the dark. Subculture is performed every 15 days and the subculture is performed 3 to 5 times to obtain loose granular callus tissue.

5. The method according to claim 1, characterized in that: Step (4) specifically involves adding 0.5-0.8g of double-flowered lily callus tissue to a 100mL Erlenmeyer flask containing 30mL of MP1 liquid culture medium. The flask is then placed on a shaker at 23℃ and 120rpm in the dark for suspension culture. Subculture is performed every 4 days, and each time 2 / 3 of the total volume of the culture medium is discarded. The supernatant was added to 30 mL of fresh MP1 liquid culture medium and the cells were cultured in suspension. The above conditions were repeated 3-4 times. After each subculture, 2 / 3 of the supernatant was discarded and fresh MP2 liquid culture medium was added to 30 mL. The cells were then filtered through a sterile 20-mesh sieve, and only the cells that could pass through the 20-mesh sieve were retained for further suspension culture. The suspension culture was carried out for 20-60 days to complete the establishment of the double-flowered lily cell suspension cell line.

6. The method according to claim 1, characterized in that: The specific steps (5) are as follows: the suspended cell line is filtered through a 40-mesh sieve, and the cell clusters remaining on the sieve are inoculated into an embryogenic induction medium with filter paper on the surface. The cells are cultured for 14 to 25 days at a temperature of 26°C, a relative humidity of 50%, and in the dark to obtain a somatic embryo.

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