CN102487825B - Method for inducing Morinda citrifolia calluses and regenerating plants using root as explant - Google Patents

Abstract

The invention relates to a method for inducing Morinda citrifolia calluses and regenerating plants using a root as an explant, belonging to a culture method of a plant in-vitro expant, especially an in-vitro culture method of a Morinda citrifolia expant. The root of a Morinda citrifolia test-tube plantlet is used as the explant, and in-vitro culture is carried on an MS culture medium with different concentrations of 6-BA to induce calluses. Results show that the culture medium most suitable for callus induction is MS+1.5 mg/L6-BA, most of induced calluses are earth yellow and granulated and has moderate hardness; the culture medium most suitable for callus subculture is also MS+1.5 mg/L6-BA, and the survival rate reaches 91.25%. The differentiation rates of adventitious buds of calluses in MS culture mediums with 1.0 mg/L and 1.5 mg/L 6-BA concentrations are respectively 24.13% and 23.64%; and buds induced on the calluses can grow into robust complete plants.

Description

Induction of noni callus using roots as explants and method for plant regeneration

technical field

The invention belongs to a method for in vitro cultivation of plant explants, in particular a method for in vitro cultivation of noni explants.

Background technique

Noni ( Morinda citrifolia ) is an evergreen small tree or shrub of the Rubiaceae Morinda genus growing in the tropics and subtropics. Noni is a kind of natural fruit, which is very rich in nutrients and contains almost all the nutrients needed by the human body. The distribution area of Noni in the world is very rare. Our country is short of Noni resources. It is only distributed in Hainan, Xisha Islands and Taiwan. In recent years, it has been introduced and cultivated in the tropical area of Yunnan. The establishment of Noni in vitro regeneration system is very important for subsequent genetic engineering operations, but most of the research at home and abroad is still at the stage of introduction and breeding, and there are few reports on in vitro regeneration at home and abroad. "Studies on Rapid Propagation of Noni ( Morinda citrifolia L.) in Vitro" is an article we retrieved from the CNKI database of China National Knowledge Infrastructure. According to this article, the cotyledon and hypocotyl of the embryo after Noni seeds germinated were used as experimental materials. And according to the method it introduced, we repeated the experiment for 3 times but no callus was induced. The research on the callus induction and plant regeneration of Noni will lay the foundation for the rapid in vitro propagation of Noni in an industrialized way and the subsequent research on Noni genetic engineering.

Contents of the invention

The object of the present invention is to take the root of Noni test-tube seedling as explant, carry out in vitro culture on the MS medium that adds different concentrations of 6-BA, carry out Noni rapid propagation in vitro, and provide a kind of Noni callus Methods of induction and plant regeneration.

The object of the present invention is achieved in the following manner, and the steps include:

(1) Induction of callus:

Cut off the root of noni aseptic seedlings, cut into small sections of 0.5cm ~ 1cm as explants, gently insert them into the medium, and make the explants fully contact with the medium, the medium is MS medium ( Add 3% sucrose and 6‰ agar powder) + 1.5mg/L 6-BA, after inoculation for about 15 days, fine granular callus will gradually form, the color is khaki or green, and the hardness is moderate;

(2) Induction of adventitious buds on callus:

Observed at 20 days, adventitious buds gradually differentiated on the callus, cut off the adventitious buds when they grew to about 1cm, and cultured in MS medium (with 3% sucrose and 6‰ agar powder) + 1.0 mg/L 6-BA Or MS medium (with 3% sucrose and 6‰ agar powder added) + 1.5 mg/L 6-BA for 35 to 40 days;

(3) Regeneration of adventitious buds:

When the adventitious buds grown in step (2) grow to 1cm-2cm, cut them off and insert them into MS medium (adding 3% sucrose and 6‰ agar powder) + 1.0 mg/L 6-BA for medium culture about 30 days;

(4) Strong seedlings take root:

From the robust plants cultivated in step (3), cut the shoot tips or other budding stem segments with a length of about 1 cm, and inoculate them in MS medium (with 3% sucrose and 6‰ agar powder) + 0.3 mg/L NAA Induce rooting, cultivate for 30 to 50 days, and obtain complete plants;

(5) Transplanting:

Move the culture bottle with a well-developed Noni root system to a greenhouse with a shading rate of 75%. After 7 days of hardening, remove the Noni test-tube seedlings from the culture bottle, wash off the agar, cut off the leaves near the root, and transplant them into the seedling tray. , spray water in an appropriate amount to keep the leaves and soil moist, and manage normally under natural light;

The pH value of the above MS medium was 5.8; the medium was sterilized at 121°C for 20 min under a pressure of 1.1 kg/cm ² ; the callus induction culture experiments were all carried out at 25±2°C and 2000 lx light intensity, 12 hours of light every day.

The method is further to transfer the callus granules from step (1) cultured and induced for about 28 days to MS (additional 3% sucrose and 6‰ agar powder)+1.5mg/L 6-BA medium for callus Subculture, and then follow the above steps (2) to (5) to operate.

Effect and significance that the present invention has are:

When most plants induce callus in vitro, they usually need to add various growth regulators such as NAA, IAA, and 2,4-day. Because the root of the noni test-tube plantlet is used as the explant in the present invention, the root itself contains more growth hormone, which provides the biological basis for the normal growth of the individual plant for the explant cultivated in vitro. Therefore, only adding 6-BA as a growth regulator in the MS medium of the present invention can also induce root callus and obtain more callus with better quality. Moreover, among the three concentration gradients of 6-BA, the high concentration of 6-BA was more conducive to the induction of noni root callus. Especially when the concentration of 6-BA is 1.5 mg/L, the induction rate of root callus reaches 95.65%, and the time is short, and it only takes about 15 days for callus to start to occur.

In the callus germination test, the callus was cultured in a medium with a higher concentration of 6-BA, and the germination rate was also higher, indicating that a high concentration of 6-BA was beneficial to the differentiation of shoots; The quality of callus induced by the medium with higher concentration of 6-BA is better, therefore, it is beneficial to the differentiation of shoots in the induction of germination. In addition, when the callus was continuously cultured in the original medium without replacing the fresh medium, the callus could also differentiate into small shoots in about 40 days, but the germination rate was low.

The present invention obtains the complete plant of Noni from the induction of the callus of root, the induction of adventitious bud, strong seedling rooting to transplanting, and the whole process is about 4 months, and the embryo cotyledon and cotyledon after the germination of the currently known Noni seed The time required by the noni in vitro rapid propagation method with the hypocotyl as the experimental material is basically the same, or even shorter than the current method.

Therefore, the present invention achieves the purpose of laying a foundation for the in vitro rapid propagation of noni in an industrialized manner and the subsequent research on noni genetic engineering.

Description of drawings

Fig. 1 is a schematic diagram of the growth of callus formed from the root of the present invention.

Fig. 2 is a schematic diagram of the subculture growth of the callus of the present invention.

Fig. 3 is a schematic diagram of the growth of shoots grown on the callus of the present invention.

Fig. 4 is a schematic diagram of the growth of a complete plant of Noni of the present invention.

The present invention will be further described below in conjunction with the accompanying drawings. The present invention includes but is not limited to the following examples. the

Detailed ways

(1) The inventive method

The Noni used was taken from the tissue culture room of the Southwest Forestry University School of Landscape Architecture, and the roots of healthy and sterile Noni seedlings were used as explants.

In the following methods, the medium for comparison is MS+0.5 mg/L 6-BA; MS+1.0 mg/L 6-BA; MS+1.5 mg/L 6-BA. Among them, the pH value of MS medium is 5.8, and 3% sucrose and 0.6% agar are added. The medium was sterilized at 121°C for 20 min at a pressure of 1.1 kg/cm ² . The callus induction culture experiments were carried out at 25±2°C and 2000 lx light intensity, and the light was 12 hours a day. The medicines are domestic analytical grade, and the experimental water is ultrapure water.

(1) Induction of callus by different concentrations of 6-BA

Cut off the roots of noni sterile seedlings, cut them into small pieces of 0.5-1cm, and gently insert them into the medium to ensure full contact with the medium. The time of callus formation was recorded, the induction rate of callus was calculated, and the color and texture of callus were observed. Wherein, induction rate=number of explants producing callus ÷ number of inoculated non-contaminated explants.

After 15 days of inoculation, fine granular callus formed successively, and after 30 days, the callus became larger, and the color was khaki and light green. The test results showed (Table 1) that when the concentration of 6-BA was 0.5 mg/L, the callus induction rate was only 75.51%, the appearance color of the callus was khaki, and the particles were loose; when the concentration of 6-BA When the concentration of 6-BA was 1.0 mg/L, the induction rate of callus was 85.11%, and the appearance color of callus was mostly khaki, and a small part was light green; The induction rate was the highest, reaching 95.65%. The appearance color of the callus was mostly light green, and the texture was tight and tight (Fig. 1).

Table 1. Effects of Different Concentrations of 6-BA on Root Callus Induction

6-BA (mg/L) Number of inoculations (pieces) The number of wounds produced (pieces) Induction rate (%) color and texture 0.5 49 37 75.51 Earthy yellow, loose, granular 1 47 40 85.11 Most of them are khaki, a small amount of light green, loose and granular 1.5 46 44 95.65 Most of them are khaki, some of them are light green, dense and granular

(2) Induction of adventitious buds on callus

After the callus is generated in step (1), part of the callus is sequentially transferred to the same fresh medium as the three mediums in step (1) for continuous culture, and the germination rate is counted.

As shown in Figure 3, after about 20 days observation, small buds grew successively on the callus. After 35 days, the small shoots grow to about 1 cm. The results showed (Table 3) that the callus had the lowest germination rate of 10.9% in the culture medium with 6-BA concentration of 0.5 mg/L; In the medium, the germination rates were 24.13% and 23.64%, respectively, and the germination rates were higher.

table 3. Effects of Different Concentrations of 6-BA on Callus Bud Growth after Replacement of Fresh Medium

6-BA (mg/L) number of wounds Number of sprouts (pieces) Germination rate(%) 0.5 55 6 10.9 1 58 14 24.13 1.5 55 13 23.64

(3) Regeneration of adventitious buds

When the adventitious buds grown in step (2) grew to 1-2 cm, they were cut off and cultured in the bud regeneration medium of MS+1.0 mg/L 6-BA, and the growth of buds was observed.

The number of inoculated buds was 20. After the buds were inserted into the medium for 7 days, the average height was 2.5 cm; after 15 days, the buds grew significantly taller, and new leaves grew, with an average height of 3.6 cm; The color is reddish, and the average height is 4.2cm; after 30 days, the plants grow robustly, with an average height of 5.1cm.

(4) Strong seedlings take root

From the robust plants cultivated in step (3), cut the shoot tip or other budding stem segments with a length of about 1 cm, inoculate them in the rooting medium of MS+0.3 mg/L NAA, and observe the rooting situation.

After 30 days, adventitious roots appeared near the incision of the shoot tip, and the roots were slender and multi-branched, light yellow, forming a dense root group and forming a complete plant (Fig. 4); after 50 days, near the incision of other noni bud stem segments Adventitious roots begin to form. Loose callus is easily formed at the incision of the stem segment, and part of the root system occurs from the callus. During adventitious rooting, the plant grows more slowly. After 50 days, the average height of the plants was only 3.2 cm. From the experimental results, it can be seen that the shoot tip has the shortest time for adventitious roots and the strongest growth.

(5) Transplanting

 Transplant the culture bottle with well-developed Noni root system into a greenhouse with a shading rate of 75% for 7 days to harden the seedlings. Remove the noni test-tube seedlings from the culture bottle, wash off the agar, cut off the leaves near the roots, transplant them into the seedling tray, and spray water appropriately to keep the leaves and soil moist. Manage normally under natural light.

In the first 3 to 4 days when the Noni test tube seedlings were transplanted into the seedling tray, the seedling leaves were easy to become soft and wilted, and water was often sprayed to keep the leaf surface moist; after 5 days, the seedling leaves gradually hardened and the color darkened; 7 After 15 days, the seedlings can keep standing upright, you can stop spraying the leaves, just keep the soil moist; after 15 days, the plants begin to grow taller and grow new leaves. Statistics after 30 days showed that the survival rate was 81.25%.

Example 2:

In this example, except that the callus induced in step (1) of Example 1 was correspondingly transferred to the fresh medium with the same three medium formulations as in step (1) for subculture, the rest were carried out according to the example Steps (2) to (5) of 1 are carried out, that is, the adventitious buds are differentiated, and then the adventitious buds grow into a complete plant.

The results of subculture are shown in Table 2. On the culture medium with the concentration of 0.5 mg/L of 6-BA, the callus was khaki, with a small part of browning and death, and the survival rate was 81.82%; in the medium with the concentration of 1.0 mg/L of 6-BA On the medium, most of the calli were khaki, a few were light green and loose, and the survival rate was 89.66%; while on the medium with the concentration of 1.5 mg/L of 6-BA, most of the calli were khaki to yellow, some are yellow-green to light green, relatively dense (see Figure 2), and the survival rate reaches 91.25%. Transferring the callus to MS supplemented with 6-BA concentrations of 0.5 mg/L, 1.0 mg/L, and 1.5 mg/L for subculture had good subculture effects, and the 6-BA concentration was 1.5 mg/L medium had the best callus subculture effect and survival rate.

Table 2. Effects of Different Concentrations of 6-BA on Subculture Growth of Callus

Claims (1)

1. the method for induction and plant regeneration of the Noni callus taking root as explant, the steps include: (1) Induction of callus: Cut off the root of the noni sterile seedling, cut it into small sections of 0.5cm ~ 1cm as explants, gently insert them into the medium, and make the explants fully contact with the medium, the medium is MS medium + 3% sucrose + 6‰ agar powder + 1.5mg/L 6-BA, about 15 days after inoculation, fine granular callus will gradually form, the color is khaki or green, and the hardness is moderate; (2) Induction of adventitious buds on callus: Observed at 20 days, adventitious buds gradually differentiated from the callus, cut off the adventitious buds when they grew to 1 cm, and cultured in MS medium + 3% sucrose + 6‰ agar powder + 1.0 mg/L 6-BA or MS Base + 3% sucrose + 6‰ agar powder + 1.5 mg/L 6-BA for 35 to 40 days; (3) Regeneration of adventitious buds: When the adventitious buds grow to 1cm-2cm, cut them off, insert them into MS medium + 3% sucrose + 6‰ agar powder + 1.0 mg/L 6-BA and cultivate them for about 30 days; (4) Strong seedlings take root: From the robust plants cultivated in step (3), cut the shoot tips or other budding stem segments with a length of 1 cm, and inoculate them in MS medium + 3% sucrose + 6‰ agar powder + 0.3 mg/L NAA to induce rooting, Cultivate for 30-50 days to obtain complete plants; (5) Transplanting: Move the culture bottle with a well-developed Noni root system to a greenhouse with a shading rate of 75%. After 7 days of hardening, remove the Noni test-tube seedlings from the culture bottle, wash off the agar, cut off the leaves near the root, and transplant them into the seedling tray. , spray water in an appropriate amount to keep the leaves and soil moist, and manage normally under natural light; The pH value of the above MS medium was 5.8; the medium was sterilized at 121°C for 20 min under a pressure of 1.1 kg/cm ² ; the induction culture of callus was carried out at 25±2°C and 2000 lx light intensity, every day Light for 12h.

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