A method for cultivating new varieties of amaryllis variegated with a mixed regeneration pathway using somatic embryos and callus tissue.

Abstract

This invention discloses a method for cultivating new variegated varieties of Amaryllis using a mixed regeneration pathway of somatic embryos and callus. This invention establishes a highly efficient cultivation method for new variegated varieties of Amaryllis through this mixed regeneration pathway. By optimizing the entire process from young leaf induction, embryogenic callus culture, somatic embryo induction and proliferation, to germination and seedling formation, this method significantly improves the propagation efficiency and success rate of new variegated varieties of Amaryllis, providing a new approach for leaf color mutation breeding of Amaryllis. This invention provides an efficient and stable technical solution for the rapid cultivation of new variegated varieties of Amaryllis.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for cultivating new varieties of amaryllis variegated plants using a mixed regeneration pathway of somatic embryos and callus tissue. Background Technology

[0002] Amaryllis (Hippeastrum spp.) is a perennial herbaceous plant belonging to the Amaryllidaceae family, native to Central and South America. There are approximately 100 native species, and it is widely popular in the market due to its large, brightly colored flowers and high ornamental value. Currently, the commercial propagation of amaryllis mainly relies on traditional methods, including seed propagation, bulb division, and scale cuttings. However, these methods have significant limitations, such as slow propagation speed, low propagation coefficient, and susceptibility to seasonal and environmental conditions. Therefore, developing an efficient and stable propagation technology is of great significance to meeting the needs of large-scale amaryllis production.

[0003] With the upgrading of flower consumption, the market demand for new variegated amaryllis varieties is becoming increasingly urgent. Variegated amaryllis have unique ornamental value, with leaves displaying various colored patterns or stripes, making them more ornamental and artistic compared to common amaryllis. Currently, there are relatively few variegated amaryllis varieties on the market, and their prices are relatively high. Therefore, cultivating more new variegated amaryllis varieties through innovative propagation methods can not only meet market demand but also enhance the economic value and market competitiveness of amaryllis.

[0004] Plant tissue culture is a technique that, under sterile conditions, cultivates plant tissues, organs, or cells on artificially prepared culture media to allow them to grow, differentiate, and form complete plants. This technique boasts advantages such as rapid propagation, high propagation coefficient, and independence from seasonal and environmental limitations, and has been widely applied in fields such as rapid plant propagation, variety improvement, and genetic engineering. While some research has been reported on the tissue culture of amaryllis, it mainly focuses on inducing callus and adventitious bud formation using explants such as scales and leaves. No research has been reported on the use of a mixed regeneration pathway of somatic embryos and callus to cultivate new variegated amaryllis varieties. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings by providing a method for cultivating new varieties of amaryllis variegated with a mixed regeneration pathway using somatic embryos and callus tissue. This invention effectively establishes a stable method for cultivating amaryllis variegated varieties through unique treatment methods, cultivation techniques, and culture media.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for cultivating new variegated varieties of amaryllis using a mixed regeneration pathway of somatic embryos and callus tissue includes the following steps:

[0008] (1) Preparation of tender leaves: On a clean bench, the small bulbs of amaryllis with the leaves and roots cut off are inoculated into the bud induction medium and cultured under light for 30-40 days to obtain tender leaves.

[0009] (2) Embryogenic callus induction: On a clean bench, take the base of the sterile seedling leaf obtained in step (1), cut off both ends and cut into small pieces, with the leaf face up, inoculate it into the embryogenic callus induction medium, culture in the dark for 2-3 days, then culture in the light for 10-15 days, and then subculture once in the same medium; after subculture for 30-45 days, embryogenic culture is obtained.

[0010] (3) Somatic embryo induction and proliferation: The embryonic culture obtained in step (2) is transferred to somatic embryo induction medium A and cultured under low light. Subculture is performed every 20-30 days, and the culture is repeated twice. During the somatic embryo proliferation stage, the concentration of plant growth regulators in the culture medium needs to be adjusted in time to maintain the embryonic state of the culture and reduce the rate of deformed embryos. The specific method is as follows: After 20-30 days of proliferation culture, the culture is transferred to a new proliferation medium B, and then the two media are used alternately, and the medium is changed every 20-30 days.

[0011] (4) Statistical analysis of leaf variation rate of somatic embryo germination and seedling: Under dark culture conditions, the proliferated somatic embryos are inoculated into somatic embryo development medium for somatic embryo germination and growth culture. After the somatic embryos germinate leaves, they are transferred to rooting and seedling strengthening medium for culture until they become new plants.

[0012] Preferably, the bud induction medium in step (1) has the following composition: MS + 6-BA 3.0-5.0 mg·L⁻¹ -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0.

[0013] Preferably, the embryogenic callus induction medium in step (2) has the following composition: MS + 2,4-D 1.0-3.0 mg·L⁻¹ -1 +TDZ 0.5-1.5 mg·L -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0.

[0014] Preferably, the somatic embryo induction culture medium A in step (3) has the following composition: MS + NAA 1.0 mg·L⁻¹ -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1+Picloram 2.0-4.0 mg·L -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0.

[0015] Preferably, the proliferation medium B in step (3) has the following composition: MS + NAA 1.0 mg·L⁻¹ -1 +KT 1.0mg·L -1 +TDZ 0.25 mg·L -1 +Picloram 0.5-1.0 mg·L -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0.

[0016] Preferably, the composition of the somatic embryonic development medium in step (4) is: MS + 6-BA 1.0-2.0 mg·L⁻¹ -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0; the rooting and seedling strengthening medium is MS + NAA 1.0-2.0 mg·L⁻¹. -1 +20-40g / L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0.

[0017] Preferably, the light cultivation conditions in steps (1) and (2) are a temperature of 25±2℃, a light intensity of 1500-2500lx, and a light duration of 12-16 hours / day.

[0018] Preferably, the low-light culture conditions in step (3) are a temperature of 25±2℃, a light intensity of 500-600lx, and a light exposure time of 12-16 hours / day.

[0019] Preferably, the cutting into small pieces in step (2) is to cut into small pieces with a length of 0.5-1.0cm and a width of 0.4cm.

[0020] The present invention has the following beneficial effects:

[0021] 1. Improve the breeding efficiency of new amaryllis variegated varieties: This invention establishes an efficient breeding method for new amaryllis variegated varieties through a mixed regeneration pathway of somatic embryos and callus. This method significantly improves the propagation efficiency and success rate of new amaryllis variegated varieties by optimizing the entire process of young leaf induction, embryogenic callus culture, somatic embryo induction and proliferation, and germination and seedling formation.

[0022] 2. Reducing the proportion of deformed embryos and improving plant survival rate: During the somatic embryo proliferation stage, adjusting the concentration of plant growth regulators in the culture medium effectively reduced the incidence of deformed embryos and improved the normal development rate of somatic embryos. Simultaneously, by alternating the use of two different proliferation media, the growth environment of the somatic embryos was dynamically controlled, thereby improving the final plant survival rate.

[0023] 3. Significantly improves variation rate and breeding potential: This invention increases the possibility of variation in amaryllis leaves through embryogenic callus induction and somatic embryogenesis, providing rich genetic resources for the breeding of new varieties.

[0024] 4. Simplified operation process and lower technical threshold: The present invention adopts a standardized culture medium formula and culture conditions, which makes the operation process simpler and suitable for large-scale industrial production.

[0025] In summary, this invention combines high efficiency, stability, and controllability, providing a reliable technical solution for the industrial cultivation of new varieties of amaryllis with variegated leaves, and has significant application value and broad development prospects. Attached Figure Description

[0026] Figure 1 This describes the in vitro regeneration process of amaryllis somatic embryo induction and mosaic variant plants. A: Detailed image of leaf-induced embryogenic callus (cultured for 35 days); B: Overall image of leaf-induced embryogenic callus (cultured for 35 days); C: Embryogenic callus proliferation and somatic embryogenesis (cultured for 60 days); D: Leaf variant buds produced by somatic embryos; E: Variant leaves emerging from somatic embryos; F: Leaf color variation after transplanting amaryllis.

[0027] Figure 2 The figure shows the effect of different concentrations of 2,4-D and TDZ combinations on the induction of embryogenic callus in 'Bangkok Rose' amaryllis. Figures 1, 2, 3, and 4 represent the culture conditions on the corresponding culture media numbers.

[0028] Figure 3 This is about the maintenance and proliferation of embryonic cells of 'Bangkok Rose' amaryllis. The numbers 1-9 in the figure represent the culture status of the corresponding culture medium numbers. Detailed Implementation

[0029] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0030] Example 1: Inducing mutations in leaves of 'Bangkok Rose' (Hippeastrum 'Bangkok Rose')

[0031] 1. Preparation of tender leaves: On a clean bench, inoculate small amaryllis bulbs (with leaves and roots removed) into MS solution with 4.0 mg / L of 6-BA. -1+ 30g / L of sucrose -1 +4.8g / L agar -1 Tender leaves were obtained after culturing in a medium with a pH of 5.8 at a temperature of 25°C, a humidity of 85% in the culture bottle, a light intensity of 2500 lx, a light duration of 16 hours per day and a dark duration of 8 hours per day for 35 days. The regeneration rate of uncontaminated leaves reached 100%.

[0032] 2. Embryogenic callus induction: On a clean bench, take the base of the leaves of the sterile seedlings obtained in step 1, cut off both ends, and cut them into small pieces 0.6 cm long and 0.4 cm wide. With the leaf face up, inoculate them onto MS + 2,4-D 1.0 mg·L⁻¹. -1 +TDZ 0.5 mg·L -1 + 30g / L of sucrose -1 +4.8g / L agar -1 In an embryogenic callus induction medium with a pH of 5.8, after culturing in the dark for 2 days, followed by light culture (under the same conditions as step 1) for 40 days, relatively firm, light yellow or light green embryogenic callus tissue can be observed near the small cut of the leaf, with an induction rate of up to 92.6%.

[0033] Table 1. Effects of different concentrations of 2,4-D and TDZ combinations on callus induction in 'Bangkok Rose' amaryllis embryogenesis.

[0034]

[0035]

[0036] Table 1 shows the effects of different concentrations of 2,4-D and TDZ combinations on callus induction in 'Bangkok Rose' amaryllis. Figure 2 .

[0037] 3. Maintenance and proliferation of embryonic callus:

[0038] The embryogenic culture obtained in step 2 was transferred to somatic embryo induction medium A (MS + NAA 1.0 mg·L⁻¹). -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1 +Picloram 3.0 mg·L -1 + 30g / L of sucrose -1 +4.8g / L agar -1In a low-light culture medium (pH 5.8), at 25℃ and 85% humidity, with a light intensity of 500-600 lx, a daily light duration of 16 hours and a dark duration of 8 hours, subcultured every 10 days for a total of 2 subcultures, embryogenic callus can be induced and proliferated to form 15-40 somatic embryos. During the somatic embryo proliferation stage, it is necessary to adjust the concentration of plant growth regulators in the culture medium in a timely manner to maintain the embryogenic state of the culture and reduce the rate of deformed embryos. The specific method is as follows: after 20 days of proliferation culture, the culture is transferred to a new proliferation medium B (MS + NAA 1.0 mg·L⁻¹). -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1 +Picloram 1.0 mg·L -1 + 30g / L of sucrose -1 +4.8g / L agar -1 The culture medium was prepared in a pH value of 5.8, and then the two culture media were used alternately, with the medium being changed every 20 days.

[0039] Table 2. Effects of different concentrations of Picloram or 2,4-D on the maintenance and proliferation of embryogenic callus in 'Bangkok Rose' amaryllis.

[0040]

[0041]

[0042] The other components of the culture medium in Table 2 are MS + NAA 1.0 mg·L⁻¹. -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1

[0043] Table 2 shows the effects of different concentrations of Picloram or 2,4-D on the maintenance and proliferation of embryonic cells in 'Bangkok Rose' amaryllis. Figure 3 .

[0044] 4. Statistical analysis of leaf variation rate during somatic embryo germination and seedling emergence:

[0045] After 12 months of subculture, the variation rate of somatic embryo germination was observed. Under dark culture conditions, the callus tissue blocks formed by the treatment groups 2, 3, 4, and 6, which showed better proliferation performance as shown in Table 2, were subcultured in MS medium supplemented with 6-BA 2.0 mg·L⁻¹. -1 + 30g / L of sucrose -1 +4.8g / L agar -1Somatic embryos were cultured in a medium with a pH of 5.8. After about 10-15 days of culture, each transplanting unit could form 10-30 mature somatic embryos. These were then placed in a tissue culture room under light conditions (temperature 25℃, humidity 85%, light intensity 1500 lx, 16 hours of light per day, 8 hours of darkness per day) for another 30 days. It was observed that most of the somatic embryos had sprouted leaves. They were then transferred to a rooting and seedling strengthening medium (MS + NAA 1.0 mg·L⁻¹). -1 + 30g / L of sucrose -1 +4.8g / L agar -1 On a medium (pH 5.8), the rooting rate was 100%. After rooting and hardening off, the seedlings were transplanted into a greenhouse, and leaf color changes were observed. The seedlings were cultured on MS medium (MS + NAA 1.0 mg·L⁻¹). -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1 +Picloram 3.0 mg·L -1 + 30g / L of sucrose -1 +4.8g / L agar -1 The variation at pH 5.8 reached 91.67%, and the mosaic rate was 50.33%.

[0046] Table 3. Effects of different Picloram concentrations on leaf variation rates in embryogenic callus tissue of 'Bangkok Rose' 'Amaryllis' 'Bangkok Rose'.

[0047]

[0048] The other components of the culture medium in Table 3 are MS + NAA 1.0 mg·L⁻¹. -1 +KT 1.0mg·L -1 +TDZ 0.25mg·L -1 .

[0049] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cultivating new varieties of amaryllis variegated with a mixed regeneration pathway using somatic embryos and callus tissue, characterized in that, Includes the following steps: (1) Preparation of tender leaves: On a clean bench, small bulbs of amaryllis with the leaves and roots removed were inoculated into a bud induction medium and cultured under light for 30-40 days to obtain tender leaves; the composition of the bud induction medium was: MS + 6-BA 3.0-5.0 mg·L -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5 g·L -1 The pH value is 5.4-6.0; (2) Embryogenic callus induction: On a clean bench, take the base of the sterile seedling leaf obtained in step (1), cut off both ends and cut into small pieces, with the leaf face up, and inoculate it into the embryogenic callus induction medium. Incubate in the dark for 2-3 days, then incubate under light for 10-15 days, and then subculture once in the same medium; after subculture for 30-45 days, obtain embryogenic culture; the composition of the embryogenic callus induction medium is: MS + 2,4-D 1.0-3.0 mg·L -1 + TDZ 0.5-1.5 mg·L -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5 g·L -1 The pH value is 5.4-6.0; (3) Somatic embryo induction and proliferation: The embryogenic culture obtained in step (2) is transferred to somatic embryo induction medium A and cultured under low light. Subculture is performed every 20-30 days, and the culture is repeated twice. During the somatic embryo proliferation stage, the concentration of plant growth regulators in the culture medium needs to be adjusted in time to maintain the embryogenic state of the culture and reduce the rate of deformed embryos. The specific method is as follows: After 20-30 days of proliferation culture, the culture is transferred to a new proliferation medium B, and then the two media are used alternately, and the medium is changed every 20-30 days. The composition of somatic embryo induction medium A is: MS + NAA 1.0 mg·L -1 + KT 1.0 mg·L -1 + TDZ 0.25 mg·L -1 + Picloram 2.0-4.0 mg·L -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5 g·L -1 The pH value was 5.4-6.0; the composition of proliferation medium B was: MS + NAA 1.0 mg·L⁻¹ -1 + KT 1.0 mg·L -1 + TDZ 0.25 mg·L -1 + Picloram 0.5-1.0 mg·L -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5g·L -1 The pH value is 5.4-6.0; (4) Statistical analysis of leaf variation rate during somatic embryo germination and seedling formation: Under dark culture conditions, the proliferated somatic embryos were inoculated into somatic embryo development medium for somatic embryo germination and growth culture. After the somatic embryos germinated leaves, they were transferred to rooting and seedling strengthening medium for culture until they became new plants. The composition of the somatic embryo development medium was: MS + 6-BA 1.0-2.0 mg·L. -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5 g·L -1 The pH value is 5.4-6.0; the rooting and seedling strengthening medium is MS + NAA 1.0-2.0 mg·L⁻¹. -1 +20-40 g·L of sucrose -1 +Agar 4.5-5.5 g·L -1 The pH value is 5.4-6.

0.

2. The method for cultivating new varieties of amaryllis variegated foliage using a mixed regeneration pathway of somatic embryos and callus tissue according to claim 1, characterized in that, The light cultivation conditions for steps (1) and (2) are a temperature of 25±2℃, a light intensity of 1500-2500lx, and a light duration of 12-16 hours / day.

3. The method for cultivating new varieties of amaryllis variegated foliage using a mixed regeneration pathway of somatic embryos and callus tissue according to claim 1, characterized in that, The low-light culture conditions in step (3) are a temperature of 25±2℃, a light intensity of 500-600lx, and a light exposure time of 12-16 hours / day.

4. The method for cultivating new varieties of amaryllis variegated plants using a mixed regeneration pathway of somatic embryos and callus tissue according to claim 1, characterized in that, The step (2) of cutting into small pieces refers to cutting into small pieces that are 0.5-1.0 cm long and 0.4 cm wide.

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