A method for inducing adventitious buds using amaryllis leaf tissue culture

Abstract

The present invention discloses a method for inducing adventitious buds by tissue culture of amaryllis leaves. The present invention selects leaves within a range of less than 10 cm from the bulb, and after explant disinfection, cuts the leaves into 2-3 cm long leaf segments to obtain explants; then inoculates the explants into an induction culture medium supplemented with melatonin, and darkens the culture medium for 10-14 days after inoculation; then alternates between darkness and light; and forms adventitious buds after 30-40 days. One segment of leaves can induce multiple adventitious buds. The leaves of the present invention are easy to obtain, and do not require high disinfection technology. In addition, adventitious buds can be directly induced, and induction and proliferation are carried out simultaneously without going through the callus induction stage, resulting in a fast seedling formation speed. For varieties with few individuals or single seedlings, using leaves to induce adventitious buds can significantly increase the number of materials collected.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a method for inducing adventitious buds (cultivating plants) using amaryllis leaf tissue culture. Background Technology

[0002] Amaryllis (Hippeastrum striatum (Lam.) HE Moore) is a perennial herbaceous plant belonging to the Amaryllidaceae family. Its bulb is nearly spherical; its leaves are bright green and strap-shaped. Amaryllis flowers are vibrant in color and open towards the sun, making them suitable for potted plants to decorate rooms, living rooms, hallways, and corridors. They can also be cultivated in gardens and used as cut flowers.

[0003] The main propagation methods for amaryllis include seed propagation, tissue culture, scale cutting, and bulb propagation. Among these, tissue culture is a highly efficient method that can produce a large number of amaryllis seedlings in a short period of time. Using flower buds as explants to induce callus redifferentiation into seedlings is time-limited, as amaryllis flowers only once a year, and the induction of regeneration takes a long time. Using scales with a bulb disc at the base as explants is challenging because the bulbs grow in the soil and have a large number of microorganisms attached to their surface and inside, making explant sterilization difficult. Furthermore, endophytic bacteria easily develop after a period of tissue culture, leading to a high risk of tissue culture failure. In addition, amaryllis seedlings obtained through hybridization only have one bulb, resulting in a high risk of failure. Often, they need to be cultivated for another year until bulb tillering occurs before tissue culture, delaying the development of new varieties.

[0004] Studies have shown that it is difficult to induce callus tissue from young leaves of cultivated amaryllis, or from the base of leaves of primary tissue culture or first-passage small plants derived from bulbs. Direct induction of callus tissue from amaryllis leaves is challenging and requires multiple subcultures on culture medium before callus tissue can be induced. Furthermore, the process of regenerating plants from somatic embryos takes a long time to induce regeneration. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for inducing adventitious buds using amaryllis leaf tissue culture. This invention selects leaves within 10cm of the bulb, sterilizes the explant, and directly induces adventitious buds, which are then cultivated into seedlings. Leaves are easy to obtain, sterilization techniques are not demanding, and adventitious buds are induced directly, with induction and proliferation occurring simultaneously, bypassing the callus (somatic embryo) induction stage, resulting in rapid seedling formation. For varieties with few individuals or single seedlings, using leaves to induce adventitious buds can significantly increase the quantity of material obtained.

[0006] The technical solution of this invention is: a method for inducing adventitious buds using amaryllis leaf tissue culture, characterized in that...

[0007] 1) Select leaves within 10cm of the bulb, disinfect the explants, and cut the leaves into 2-3cm long segments to obtain the explants;

[0008] 2) Under aseptic conditions, explants are inoculated into induction medium supplemented with melatonin (MT). After inoculation, the plants are treated in the dark for 10-14 days; then, alternating between dark and light, with a light duration of 10-14 hours every 24 hours, a light intensity of 800-1000 lux, and a temperature of 25±2℃; adventitious buds will form after 30-40 days. Multiple adventitious buds can be induced from a single leaf segment.

[0009] Furthermore, adventitious buds are individually cut off and inoculated into a subculture medium. After culturing for 50-80 days under a light intensity of 1000-2000 lux and a temperature of 25±2℃, seedlings are generated. Once the seedlings have grown, they are inoculated into a rooting medium and rooted under a light intensity of 1000-2000 lux and a temperature of 25±2℃.

[0010] The induction medium consisted of: MS + 6-BA 3.5-4.5 mg / L + NAA 1.8-2.2 mg / L + sucrose 28-32 g / L + agar 5-8 g / L + MT10. -8 -10 -9 mol / L, preferably MS + 6-BA 4.0 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + agar 6 g / L + MT10 -9 mol / L. Subculture medium is the same as induction medium.

[0011] The rooting medium is: 1 / 2 MS + IBA (indolebutyric acid) 0.90-1.10 mg / L + NAA 0.09-0.11 mg / L + sucrose 28-32 g / L + agar 5-8 g / L, preferably: 1 / 2 MS + IBA (indolebutyric acid) 1.00 mg / L + NAA 0.10 mg / L + sucrose 30 g / L + agar 6 g / L.

[0012] Preferably, the disinfection in step 1) is performed by sequentially using 75% alcohol and 0.1% mercuric chloride with added Tween 80; specifically: the first disinfection with 75% alcohol lasts 30-60 seconds, followed by rinsing with sterile water once; the second disinfection with 0.1% mercuric chloride (with 2-3 drops of Tween 80) lasts 5-10 minutes, followed by rinsing with sterile water with continuous shaking 3 times, each time for 2-3 minutes; after disinfection, the surface of the explant is dried with sterile paper.

[0013] Preferably, in step 1), leaves are selected within a range of 5-10 cm from the bulb.

[0014] The technical effects of this invention are:

[0015] 1. This invention selects leaves within 10cm of the bulb, making the explant material easy to obtain, not limited by time, and with low requirements for disinfection. Furthermore, during the breeding process, for a single excellent individual selected, the plant can be propagated simultaneously using traditional scale cuttings and leaf tissue culture methods, thus expanding the range of propagation materials.

[0016] 2. Through the improvement of tissue culture formula, adventitious buds can be directly induced from leaves and then cultivated into seedlings without going through the stage of inducing callus tissue (somatic embryo), thus improving the efficiency of tissue culture propagation. Attached Figure Description

[0017] Figure 1 Inducing adventitious buds from amaryllis leaf tissue culture;

[0018] Figure 2 Leaves differentiate from adventitious buds;

[0019] Figure 3 To allow adventitious buds to differentiate into seedlings;

[0020] Figure 4 To allow adventitious buds to grow into seedlings. Detailed Implementation

[0021] The effects are illustrated below with reference to specific examples.

[0022] Example 1:

[0023] 1. Explants: Leaves within 20cm of the bulb were used as explants, divided into four groups according to their distance from the bulb: ≤5cm leaves, >5cm and ≤10cm leaves, >10cm and ≤15cm leaves, and >15cm and ≤20cm leaves. The explants were disinfected for the first time with 75% alcohol for 45 seconds, followed by rinsing once with sterile water. A second disinfection was performed with 0.1% mercuric chloride (with 2 drops of Tween 80) for 8 minutes, followed by rinsing three times with sterile water, shaking continuously, for 2.5 minutes each time. After disinfection, the surface moisture of the explants was blotted dry with sterile paper. The leaves were cut into 2-3cm long segments in one go, with clean cuts. Under sterile conditions, the explants were inoculated into the induction medium. The survival rates of different explants are shown in Table 1. Table 1 shows that the survival rate of leaves within 5-20cm of the bulb was over 85%.

[0024] Table 1 Survival rates of different explants

[0025]

[0026] 2. Under aseptic conditions, explants were inoculated onto induction medium supplemented with melatonin (MT). The basal medium consisted of MS medium containing 4.0 mg / L 6-BA (6-benzylaminopurine) + 2.0 mg / L NAA (naphthaleneacetic acid) + 30 g / L sucrose + 6 g / L agar. Melatonin (MT) was added to each medium. -9 -10 -5 mol / L, with no MT added as a control. After inoculation, the plants were treated in the dark for 12 days; then alternated between dark and light, with 12 hours of light every 24 hours, a light intensity of 1000 lux, and a temperature of 25±2℃. Adventitious buds formed after 30-40 days. Multiple adventitious buds can be induced from a single leaf segment. Leaves <10cm in length near the bulb can directly induce adventitious buds, such as... Figure 1 As shown, adventitious buds differentiate into leaves as follows Figure 2 As shown, adventitious buds differentiate into seedlings as follows Figure 3 As shown, adventitious buds grow into seedlings as follows Figure 4 As shown.

[0027] The adventitious shoot induction rate and proliferation coefficient of different explants and different culture media (with different MT contents) are shown in Table 2.

[0028] Table 2: Adventitious shoot induction rate and proliferation coefficient of different explants and different culture media (with different MT contents)

[0029]

[0030]

[0031] Table 2 shows that leaves within 10cm of the bulb exhibit better adventitious bud induction rate and proliferation coefficient, and higher melatonin (MT) content. -9 The effect is best at mol / L. Leaves that are more than 10 cm away from the bulb are difficult to induce adventitious buds, and only a few leaves can produce a few adventitious buds.

[0032] 3. Cut off individual adventitious buds and inoculate them onto subculture medium (MS + 6-BA 4.0 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + agar 6 g / L + MT 10). -9 Seedlings were generated after culturing in (mol / L) at 1000-2000 lux of light and 25±2℃ for 50-80 days.

[0033] 4. After the seedlings have grown, they are inoculated into rooting medium (1 / 2 MS + IBA 1.00 mg / L + NAA 0.10 mg / L + sucrose 30 g / L + agar 6 g / L). Under light conditions of 1000-2000 lux and temperature of 25±2℃, the rooting rate can reach 100%.

Claims

1. A method for inducing adventitious buds using amaryllis leaf tissue culture, characterized in that, 1) Select leaves within 10cm of the bulb, disinfect the explants, and cut the leaves into 2-3cm long segments to obtain the explants; 2) Under aseptic conditions, explants were inoculated into induction medium supplemented with melatonin MT and treated in the dark for 10-14 days after inoculation; then, alternating between darkness and light, with a light duration of 10-14 hours every 24 hours, a light intensity of 800-1000 lux, and a temperature of 25±2℃; adventitious buds formed after 30-40 days. The induction medium was: MS + 6-BA 3.5-4.5 mg / L + NAA 1.8-2.2 mg / L + sucrose 28-32 g / L + agar 5-8 g / L + MT10 -8 -10 -9 mol / L.

2. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 1, characterized in that, Individual adventitious buds are cut off and inoculated into a subculture medium. After culturing for 50-80 days under a light intensity of 1000-2000 lux and a temperature of 25±2℃, seedlings are generated. Once the seedlings have grown, they are inoculated into a rooting medium and rooted under a light intensity of 1000-2000 lux and a temperature of 25±2℃.

3. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 1, characterized in that, The induction medium was: MS + 6-BA 4.0 mg / L + NAA 2.0 mg / L + sucrose 30 g / L + agar 6 g / L + MT10 -9 mol / L.

4. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 2, characterized in that, The subculture medium is the same as the induction medium.

5. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 2, characterized in that, The rooting medium is: 1 / 2 MS + IBA 0.90-1.10 mg / L + NAA 0.09-0.11 mg / L + sucrose 28-32 g / L + agar 5-8 g / L.

6. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 1, characterized in that, The disinfection in step 1) is carried out by sequentially using 75% alcohol and 0.1% mercuric chloride with added Tween 80.

7. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 6, characterized in that, The disinfection in step 1) is as follows: disinfect for the first time with 75% alcohol for 30-60 seconds, then rinse once with sterile water; disinfect for the second time with 0.1% mercuric chloride (containing Tween 80) for 5-10 minutes, then rinse three times with sterile water, shaking continuously for 2-3 minutes each time; after disinfection, use sterile paper to absorb the moisture on the surface of the explant.

8. The method for inducing adventitious buds using amaryllis leaf tissue culture as described in claim 1, characterized in that, Step 1) Select leaves within a range of 5-10cm from the bulb.

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