Method for inducing adventitious buds through hippeastrum leaf tissue culture

Abstract

The invention discloses a method for inducing adventitious buds by using hippeastrum hippeastrum leaf tissue culture. The method comprises the following steps: selecting a leaf which is less than 10cm away from a bulb, sterilizing an explant, and cutting the leaf into leaf sections with the length of 2-3cm to obtain the explant; then inoculating the explant into an induction culture medium added with melatonin, and carrying out dark treatment for 10-14 days after inoculation; darkness and illumination are alternated; adventitious buds are formed after 30-40 days. A plurality of adventitious buds can be induced by a section of leaf. According to the method, leaves are easy to obtain, the requirement for the disinfection technology is not high, adventitious buds are directly induced, induction and proliferation are conducted at the same time, the callus induction stage is omitted, and the seedling forming speed is high. For seedling individuals with rare variety individuals or single plants, adventitious buds are induced by using leaves, so that the material taking quantity can be obviously increased.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to a method for inducing adventitious buds (cultivating to form plants) by using amaryllis leaf tissue culture. Background Art

[0002] Hippeastrum striatum (Lam.) HE Moore, a perennial herbaceous plant of the Amaryllis family, has a nearly spherical bulb and bright green ribbon-shaped leaves. Hippeastrum has bright flowers and blooms towards the sun. It is suitable for potted plants to decorate rooms, living rooms, aisles and corridors. It can be used for garden cultivation or as fresh cut flowers.

[0003] The main propagation methods of Amaryllis include sowing propagation, tissue culture, scale cutting method and bulb propagation method. Among them, tissue culture can obtain a large number of Amaryllis seedlings in a short time, which is a highly efficient propagation method. Using flower buds as explants, inducing callus to differentiate into seedlings, the material collection is limited by time, and Amaryllis blooms once a year; and the induction regeneration time is long. Using scales with bulb disks at the base as explants, because the bulbs grow in the soil, a large number of microorganisms are attached to the surface and body, the explants are difficult to disinfect, and endophytes are prone to be produced after a period of tissue culture, resulting in a high risk of tissue culture failure. In addition, for Amaryllis seedlings obtained through hybridization, there is only one bulb, and the risk of failure is high. It is often necessary to cultivate for another year and wait for the bulbs to tiller before tissue culture, which delays the cultivation process of new varieties.

[0004] Studies have shown that it is difficult to induce callus from young leaves of cultivated Amaryllis, or the base of leaves of primary tissue culture or first-generation subcultured plantlets from bulbs. It is difficult to directly induce callus from Amaryllis leaves, and callus can only be induced after multiple subcultures on the culture medium. Moreover, it takes a long time to induce regeneration to form plants through somatic embryo regeneration. Summary of the invention

[0005] In view of the above problems, the present invention provides 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, sterilizes the explants, directly induces adventitious buds, and then cultivates them into seedlings. The leaves are easy to obtain, the disinfection technology requirements are not high, and the adventitious buds are directly induced, and the induction and proliferation are carried out simultaneously without going through the callus (somatic embryo) induction stage, and the seedling speed is fast. For varieties with rare individuals or single seedlings, the number of materials can be significantly increased by inducing adventitious buds using leaves.

[0006] The technical solution of the present invention is: a method for inducing adventitious buds by tissue culture of Amaryllis leaves, characterized in that:

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

[0008] 2) Under sterile conditions, the explants were inoculated into an induction medium supplemented with melatonin (MT), and kept in the dark for 10-14 days after inoculation; then the dark and light conditions were alternating, with a light duration of 10-14 hours per 24 hours, a light intensity of 800-1000 lux, and a temperature of 25±2°C; adventitious buds were formed after 30-40 days. Multiple adventitious buds can be induced from one leaf segment.

[0009] Furthermore, the adventitious buds are individually cut off, inoculated into a subculture medium, and cultured for 50-80 days at a light intensity of 1000-2000 lux and a temperature of 25±2°C to generate seedlings; after the seedlings grow, they are inoculated into a rooting medium and rooted at a light intensity of 1000-2000 lux and a temperature of 25±2°C.

[0010] Among them, the induction medium is: MS+6-BA3.5-4.5mg / L+NAA1.8-2.2mg / L+sucrose 28-32g / L+agar 5-8g / L+MT10 -8 -10 -9 mol / L, preferably MS+6-BA4.0mg / L+NAA2.0mg / L+sucrose30g / L+agar6g / L+MT10 -9 mol / L. Subculture medium is the same as induction medium.

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

[0012] Preferably, the disinfection in step 1) is: disinfection is carried out using 75% alcohol and 0.1% mercuric chloride with Tween 80 added in sequence; specifically: disinfection with 75% alcohol for the first time, 30-60s, and rinse with sterile water once; disinfection with 0.1% mercuric chloride (with Tween 80, 2-3 drops) for the second time, 5-10min, and rinse with sterile water for 3 times with continuous shaking, each time for 2-3min; after disinfection, the moisture on the surface of the explant is absorbed with sterile paper.

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

[0014] The technical effects of the present invention are:

[0015] 1. The present invention selects leaves that are less than 10 cm away from the bulb, so that the explant material is easy to obtain, is not limited by time, and has low disinfection difficulty requirements. In addition, during the breeding process, for a single excellent individual selected for breeding, the traditional scale cutting and leaf tissue culture methods can be used to simultaneously propagate the plant, thereby expanding the range of propagation materials.

[0016] 2. Through the improvement of tissue culture formula, leaves can directly induce adventitious buds and then cultivate them into seedlings without going through the stage of inducing callus tissue (somatic embryos), thereby improving the efficiency of tissue culture propagation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To induce adventitious buds from Amaryllis leaf tissue culture;

[0018] Figure 2 Differentiate leaves from adventitious buds;

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

[0020] Figure 4 For adventitious buds to grow into seedlings. DETAILED DESCRIPTION

[0021] The effects are described below with reference to embodiments.

[0022] Embodiment 1:

[0023] 1. Explants: Leaves within 20 cm from the bulb are used as explants, and are divided into four groups according to the distance from the bulb, namely: ≤5 cm leaves, >5 cm and ≤10 cm leaves, >10 cm and ≤15 cm leaves, >15 cm and ≤20 cm leaves. Disinfect with 75% alcohol for 45 seconds for the first time, and rinse with sterile water once; disinfect with 0.1% mercuric chloride (with Tween 80, 2 drops) for 8 minutes for the second time, and rinse with sterile water for 3 times with continuous shaking, 2.5 minutes each time. After disinfection, use sterile paper to absorb the moisture on the surface of the explant, cut the leaves into 2-3 cm long leaf segments, and complete the cutting in one time with a smooth blade. Inoculate the explants into the induction medium under sterile conditions. The survival rates of different explants are shown in Table 1. It can be seen from Table 1 that the survival rate of leaves within 5-20 cm from the bulb is above 85%.

[0024] Table 1 Survival rate of different explants

[0025]

[0026] 2. Inoculate the explants into the induction medium under sterile conditions, add melatonin (MT) to the medium, and use MS + 6-BA (6-benzylaminopurine) 4.0 mg / L + NAA (naphthaleneacetic acid) 2.0 mg / L + sucrose 30 g / L + agar 6 g / L as the basic medium, add melatonin (MT) 10 -9 -10 -5 mol / L, with no MT added as the control. After inoculation, dark treatment was carried out for 12 days; then dark and light were alternated, with 12 hours of light per 24 hours, light intensity of 1000 lux, and temperature of 25±2℃. Adventitious buds were formed after 30-40 days. One leaf segment can induce multiple adventitious buds. Leaves close to the bulb with a length of <10 cm were directly induced to produce adventitious buds. Figure 1 As shown, adventitious buds differentiate into leaves as Figure 2 As shown, adventitious buds differentiate into seedlings. Figure 3 As shown, adventitious buds grow into seedlings as Figure 4 shown.

[0027] The adventitious bud 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 bud induction rate and proliferation coefficient of different explants and different culture media (different MT content)

[0029]

[0030]

[0031] From Table 2, we can see that the leaves within 10 cm from the bulb have better adventitious bud induction rate and proliferation coefficient, and melatonin (MT) 10 -9 The effect is best when the distance from the bulb to the leaves is more than 10 cm. It is difficult to induce adventitious buds, and only a few leaves can produce a few adventitious buds.

[0032] 3. Cut off the adventitious buds individually and inoculate them into the subculture medium (MS+6-BA4.0mg / L+NAA2.0mg / L+sucrose 30g / L+agar 6g / L+MT 10 -9 mol / L); light intensity 1000-2000 lux, temperature 25±2°C, seedlings were generated after 50-80 days of cultivation;

[0033] 4. After the seedlings grow, inoculate them into rooting medium (1 / 2MS + IBA 1.00 mg / L + NAA 0.10 mg / L + sucrose 30 g / L + agar 6 g / L), with light 1000-2000 lux and temperature 25±2℃, the rooting rate can reach 100%.

Claims

1. A method for inducing adventitious buds by tissue culture of Amaryllis leaves, characterized in that: 1) Select leaves within 10 cm of the bulb, sterilize the explants, and cut the leaves into 2-3 cm long segments to obtain explants; 2) Under sterile conditions, the explants were inoculated into an induction medium supplemented with melatonin MT, and darkened for 10-14 days after inoculation; then darkness and light were alternated, with a light duration of 10-14 h per 24 hours, a light intensity of 800-1000 lux, and a temperature of 25±2°C; adventitious buds were formed after 30-40 days.

2. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 1, characterized in that: The adventitious buds are cut off individually, inoculated into the subculture medium, and cultured for 50-80 days at a light intensity of 1000-2000 lux and a temperature of 25±2°C to generate seedlings; after the seedlings grow, they are inoculated into the rooting medium and rooted at a light intensity of 1000-2000 lux and a temperature of 25±2°C.

3. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 1 or 2, characterized in that: The induction medium is: MS+6-BA3.5-4.5mg / L+NAA1.8-2.2mg / L+sucrose 28-32g / L+agar 5-8g / L+MT10 -8 -10 -9 mol / L.

4. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 3, characterized in that: The induction medium is: MS+6-BA4.0mg / L+NAA2.0mg / L+sucrose 30g / L+agar 6g / L+MT10 -9 mol / L.

5. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 2, characterized in that: The subculture medium is the same as the induction medium.

6. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 2, characterized in that: The rooting medium is: 1 / 2MS+IBA 0.90-1.10 mg / L+NAA 0.09-0.11 mg / L+sucrose 28-32 g / L+agar 5-8 g / L.

7. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 1, characterized in that: The disinfection in step 1) is as follows: disinfection is performed using 75% alcohol and 0.1% mercuric chloride added with Tween 80 in sequence.

8. A method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 7, characterized in that: The disinfection in step 1) is as follows: disinfect with 75% alcohol for the first time, 30-60 seconds, and rinse with sterile water once; disinfect with 0.1% mercuric chloride containing Tween 80 for the second time, 5-10 minutes, and rinse with sterile water for 3 times with continuous shaking, each time for 2-3 minutes; after disinfection, dry the moisture on the surface of the explant with sterile paper.

9. The method for inducing adventitious buds by tissue culture of Amaryllis leaves as claimed in claim 1, characterized in that: In the step 1), leaves within a range of 5-10 cm from the bulb are selected.

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