CN107173234A - A kind of method of the direct Rapid Rooting of tissue-cultured seedling - Google Patents

Abstract

The invention provides a method for direct and rapid rooting of tissue culture seedlings. The above rooting method induces the rooting of the tissue cultured seedlings by obliquely cutting the stem base of the tissue cultured seedlings, evenly smearing the jelly substance containing rooting hormone on the slope of the stem base, and then inserting the tissue cultured seedlings into the matrix for cultivation. The above method of the present invention not only has a high rooting rate and a high survival rate of later transplanting, but also is easy to operate, does not need to be cultivated on a rooting medium, does not need to prepare a large amount of rooting medium, greatly reduces costs, and does not need to change the medium or follow-up. During the transplanting process, the culture medium at the roots of the tissue culture seedlings is removed, which saves time and effort. Said method of the present invention also optimizes the kind and consumption of rooting hormone, and the method after optimization is particularly suitable for the rooting of woody plants, especially chestnut and cherry, and the rooting rate of chestnut and cherry is from 55% and 70% of traditional rooting method , increased to 81-99%.

Description

A kind of method of direct rapid rooting of tissue culture seedling

technical field

The invention relates to the technical field of plant tissue culture, in particular to a method for direct and rapid rooting of tissue culture seedlings.

Background technique

Plant tissue culture seedlings are complete plants cultivated under sterile and suitable artificial conditions using explants based on the totipotency of plant cells. Rooting is to let the tissue culture seedlings grow roots. At present, the common rooting method of plant tissue culture seedlings at home and abroad is the rooting medium rooting method, that is, on the basis of 1/2MS and WPM basal medium, add plant hormones IBA or NAA with different concentrations, and then the tissue culture seedlings Place it on the rooting medium for culture, and induce the tissue culture shoots to take root. The medium rooting method has been generally applied on the rooting of plant tissue culture seedlings, and in addition, no other innovative rooting methods have been seen.

There are many defects in traditional rooting medium rooting method:

(1) It takes a lot of time and energy: the rooting of tissue cultured seedlings on the medium first needs to prepare a large amount of rooting medium. In order to speed up the rooting speed, fresh medium needs to be replaced every two weeks, and attention should be paid to the replacement of the medium. Remove the old medium remaining on the seedling roots before they can be connected to fresh medium. When transplanting to the hole plate in the later stage, it is also necessary to clean up all the remaining medium on the seedling roots, which is time-consuming and laborious.

(2) High experimental cost: the consumption of reagents used in the preparation of the culture medium is large, and the experimental cost is high.

(3) Material waste, low rooting rate: in the process of changing the medium and transplanting the plug, it is inevitable that the root system of the plant will be damaged, resulting in a waste of experimental materials. The rooting rate of Chinese chestnut is 55.33%, and the rooting rate of cherry is 55.33%. Only 70-80%.

(4) Due to the loss of training seedlings, the transplanting survival rate is low: when the rooted seedlings growing well on the rooting medium are transplanted to the hole trays, they need to experience environmental changes from the aseptic medium to the soil with bacteria matrix. It is a great test, and it is easy to lose rooted seedlings and reduce the survival rate. As far as chestnuts are concerned, the survival rate of transplanted to plug trays is only 20-30%.

In view of this, the present invention is proposed.

Contents of the invention

The first object of the present invention is to provide a method for direct and rapid rooting of tissue cultured seedlings. The rooting method has the advantages of simple operation, low cost, high rooting rate and high survival rate after transplanting.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

A method for direct rapid rooting of tissue cultured seedlings, said method comprising the following steps: (1) obliquely cutting the base of the stem of the tissue cultured plant to expose the slope of the base of the stem; Smear it on the slope of the base of the stem and the stem; (3) insert the tissue culture seedling into the substrate and cultivate it until it takes root.

The rooting method of the present invention is obviously better than the traditional rooting method of tissue cultured seedlings: first, according to the embodiments of the present invention description, the rooting rate of the tissue cultured seedlings of the method of the present invention is obviously higher than the rooting method of traditional rooting medium . Secondly, since the tissue-cultured seedlings have been separated from the sterile environment during the rooting process and are in contact with the substrate, the tissue-cultured seedlings of the present invention can adapt to the external environment more easily, and the survival rate of subsequent transplants is also significantly higher than that of traditional in-plants. Rooting method for rooting in a sterile environment. Furthermore, the tissue cultured seedlings of the above method of the present invention do not need to be cultivated in the rooting medium, which saves a large amount of rooting medium on the one hand and greatly reduces the cost of rooting; The medium is replaced during the process, which saves the cumbersome steps of cleaning up the old medium at the roots of the tissue cultured seedlings, which not only saves time and effort, but also avoids secondary damage to the roots of the tissue cultured seedlings.

In some embodiments, the rooting hormone is IBA; preferably, the mass fraction of the IBA in the jelly-like substance is 0.25-0.35%; or, the mass fraction of the IBA in the jelly-like substance is 0.28-0.33% %; or, the mass fraction of the IBA in the colloidal substance is 0.30～0.32%; more preferably, the mass fraction of the IBA in the colloidal substance is 0.25%; or, the IBA in the colloidal substance The mass fraction of the IBA is 0.31%; or, the mass fraction of the IBA in the colloidal substance is 0.35%.

The present invention also optimizes the type and dosage of the rooting hormone, and finally finds that the best rooting hormone suitable for the rooting method of the present invention is IBA, the preferred working concentration of IBA is 0.25-0.35%, and the best working concentration is 0.31%. According to Examples 1 to 10 of the description of the application, within this range, the rooting rate of the method of the present invention is as high as 95 to 99%, which is not only significantly better than the traditional rooting method, but also better than using other rooting hormones or other rooting hormones. The jelly-like substance of concentrated IBA smears the rooting method of the stem slope.

In some embodiments, glycerin is also included in the jelly-like substance.

In some embodiments, the tissue cultured seedlings are woody plant tissue cultures; preferably, the woody plant tissue cultures are chestnut tissue cultures or cherry tissue cultures.

Chestnut (Castanea mollissima) is a plant of the genus Chestnut in the family Fagaceae. It has a well-developed root system and strong adaptability. It is an important ecological and economic forest tree species in my country. However, there are few reports on chestnut tissue culture and it is often accompanied by low rooting rate and limited by the number of subcultures. Xu Chen et al. (2013) used WPM basal medium to soak the plant stems in 2 mg/L IBA for 4 minutes before rooting in the establishment of chestnut tissue culture system, and the rooting rate reached 55.33% after 30 days.

Cherry (Cerasus pseudocerasus), a deciduous tree of Rosaceae Pseudocerasus, is an excellent small species of characteristic fruit with important economic value. Bao Jiuling et al. (2016) published a paper on tissue culture and rapid propagation of Guizhou big cherry, using 1/2MS basic medium, plus the hormone IBA with an optimum concentration of 0.5 mg/L, and the rooting rate reached 75% after 30 days -80%.

This shows that the rooting rate of existing Chinese chestnut and cherry is low.

And according to embodiment 1～10 of specification sheet of the present application, comparative example 1～14 and experiment example 1～2, it can be known that the above-mentioned rooting method of the present invention can promote the rooting rate of Chinese chestnut and cherry tissue culture seedling to 90～99%, after transplanting The survival rate is increased to 100%, which is obviously higher than the rooting method of existing traditional chestnuts and cherries.

In some embodiments, the tissue cultured plantlets are tissue cultured plantlets whose plant height is higher than 3 cm, diameter is larger than 2 mm, and has not yet taken root.

In some embodiments, the matrix in step (3) has a hole, and the tissue culture plantlet is inserted into the hole of the matrix, and the matrix is extruded so that the tissue culture plantlet and the matrix are seamlessly fitted, and the inner wall of the hole is coated with There is the gel-like substance.

The above method of the present invention has a hole on the substrate, inserts the tissue cultured seedling into the hole of the substrate, makes the operation of inserting the tissue cultured seedling into the substrate softer, and can avoid the loss of tissue cultured seedlings in the substrate when directly inserted into the substrate. Solid particles cause mechanical damage to tissue culture seedlings. At the same time, apply jelly-like substances on the inner wall of the hole, squeeze the matrix, and make the matrix and the tissue culture seedlings fit seamlessly, which can make the matrix and the tissue culture seedlings bond more closely, and reduce the pores between the matrix and the tissue culture seedlings , exhaust air, avoid the growth of mold, etc.

In some embodiments, the matrix includes peat soil, rice husk charcoal, river sand, perlite, vermiculite or rock wool.

In some embodiments, during the cultivation process of step (3), the temperature is kept constant at 20-25°C, the humidity is kept constant at 80-100%, and the light time is 14-18 hours/day; preferably, the temperature is kept constant at 25 ℃, the humidity is constant at 100%, and the light time is 16 hours/day.

In some embodiments, water is sprayed once every 6-8 days during the culturing in step (3).

In some embodiments, the method further includes transplanting the obtained tissue cultured plantlets together with the substrate into plug trays or land. The above method of the present invention transplants the tissue cultured seedlings together with the matrix, which can reduce the damage to the roots of the tissue cultured seedlings during the transplanting process.

Compared with prior art, the beneficial effect of the present invention is:

1), the rooting rate of the rooting method of the present invention and the survival rate after transplanting are higher;

2), the method of the present invention is easy to operate and does not need to be cultivated on the rooting medium. On the one hand, there is no need to prepare a large amount of rooting medium, which greatly reduces the cost. On the other hand, there is no need to change the medium or the subsequent transplanting process. Remove the culture medium at the roots of tissue culture seedlings, saving time and effort;

3), the method of the present invention optimizes the kind and consumption of rooting hormone, the method after optimization is particularly suitable for the rooting of woody plants, especially chestnut and cherry, the rooting rate of chestnut and cherry is from 55% of traditional rooting method And 75 ~ 80%, raised to 95 ~ 99%.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the front view of the Chinese chestnut seedling growth situation described in embodiment 1 after rooting culture 4 weeks;

Fig. 2 is the top view of the Chinese chestnut seedling growth situation described in embodiment 1 after rooting culture 4 weeks;

Fig. 3 is the front view of the cherry seedling growth situation described in embodiment 6 after rooting culture 4 weeks;

Fig. 4 is the top view of the growth situation of the cherry seedling described in Example 6 after rooting and culturing for 4 weeks.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example 1

Make chestnut tissue culture seedling take root fast as follows:

1. Take chestnut tissue-cultured seedlings grown in a culture bottle, grow well, have a plant height higher than 3 cm, a diameter larger than 2 mm, and have not taken root, and place them on filter paper; Cut obliquely with the knife, exposing the beveled stem.

2. Take the jelly substance with a medicine spoon, and evenly apply it on the slope of the stem base and the stem, so that there is a jelly substance around the entire stem.

3. Use a fruit pick to pierce a cylindrical hole with a diameter of about 3mm and a depth of 2/3 of the matrix block in the center of the matrix block that has absorbed enough water, and use a fruit pick to dip the jelly-like substance and spread it on the inner wall of the cylindrical hole as evenly as possible.

4. Insert the tissue-cultured seedlings whose stems are coated with jelly-like substance directly into the matrix block coated with jelly-like substance, and knead the matrix block with hands to make the seedling and the matrix seamlessly fit.

5. Spray an appropriate amount of water with a watering can, culture conditions: 25°C, 16 hours of light per day, 8 hours of dark cultivation in a culture room, spray water once a week; after 4 weeks, transplant the rooted tissue culture seedlings together with the substrate into the hole tray .

The jelly-like substance in steps 2 and 3 was a hydrogel containing 0.31% (w/w) IBA.

Example 2

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.25% (w/w).

Example 3

With reference to the method described in Example 1, the tissue-cultured seedlings took root rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.28% (w/w).

Example 4

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.33% (w/w).

Example 5

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.35% (w/w).

Experimental example 6

Make cherry tissue culture seedling take root fast as follows:

1. Take the cherry tissue culture seedlings that grow in the culture bottle, grow well, have a plant height higher than 3cm, a diameter greater than 2mm, and have not taken root on the filter paper; on the filter paper, use a scalpel to cut the base of the tissue culture seedling stem Cut obliquely with the knife, exposing the beveled stem.

2. Take the jelly substance with a medicine spoon, and evenly apply it on the slope of the stem base and the stem, so that there is a jelly substance around the entire stem.

3. Use a fruit pick to pierce a cylindrical hole with a diameter of about 3mm and a depth of 2/3 of the matrix block in the center of the matrix block that has absorbed enough water, and use a fruit pick to dip the jelly-like substance and spread it on the inner wall of the cylindrical hole as evenly as possible.

4. Insert the tissue-cultured seedlings whose stems are coated with jelly-like substance directly into the matrix block coated with jelly-like substance, and knead the matrix block with hands to make the seedling and the matrix seamlessly fit.

5. Spray an appropriate amount of water with a watering can, culture conditions: 25°C, 16 hours of light per day, 8 hours of dark cultivation in a culture room, spray water once a week; after 4 weeks, transplant the rooted tissue culture seedlings together with the substrate into the hole tray .

The jelly-like substance in steps 2 and 3 was a hydrogel containing 0.31% (w/w) IBA.

Experimental example 7

With reference to the method described in Example 6, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.25% (w/w).

Experimental example 8

With reference to the method described in Example 6, the tissue-cultured seedlings were rooted rapidly, the only difference being that the IBA content in the jelly-like substance of step 3 and step 4 was 0.28% (w/w).

Experimental example 9

With reference to the method described in Example 6, the tissue culture seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.33% (w/w).

Experiment 10

With reference to the method described in Example 6, the tissue cultured plantlets took root rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.35% (w/w).

Comparative example 1

Rooting culture is carried out in the following manner: the base of the Chinese chestnut tissue culture seedlings whose plant height is higher than 3cm and has not taken root is cut off under aseptic conditions, and the oblique section is exposed, so that the wound is exposed to the IBA solution with a concentration of 2mg/L. After soaking in medium for 4 minutes, the tissue culture seedlings were inoculated into WPM medium without IBA and cultured for 4 weeks, and the tissue culture seedlings were transplanted into plug trays after 4 weeks. The culture conditions are: temperature 25° C., humidity 100%, light intensity 2500 lx, 16 hours of light per day and 8 hours of dark cultivation.

Comparative example 2

With reference to the method described in Example 1, the tissue culture shoots were rooted rapidly, the only difference being that the rooting hormone contained in the gelatinous substance in steps 3 and 4 was IAA with a mass fraction of 0.3%.

Comparative example 3

Refer to the method described in Example 1 to make the tissue cultured shoots take root quickly, the only difference being that the rooting hormone contained in the jelly-like substance in steps 3 and 4 is NAA with a mass fraction of 0.3%.

Comparative example 4

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.1% (w/w).

Comparative example 5

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.2% (w/w).

Comparative example 6

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.4% (w/w).

Comparative example 7

With reference to the method described in Example 1, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.5% (w/w).

Comparative example 8

Carry out rooting culture in the following manner: transfer the cherry tissue cultured seedlings with a plant height higher than 3 cm and not yet rooted under sterile conditions to the rooting medium for 4 weeks, and transplant the tissue cultured seedlings to plugs after 4 weeks middle. Wherein, the formulation of rooting medium is: MS medium containing 1.0mg/L IBA and 2g/L sucrose. The culture conditions are: temperature 25° C., humidity 100%, light intensity 2500 lx, 16 hours of light per day and 8 hours of dark cultivation.

Comparative example 9

Refer to the method described in Example 6 to make the tissue culture shoots take root quickly, the only difference being that the rooting hormone contained in the jelly-like substance in steps 3 and 4 is IAA with a mass fraction of 0.3%.

Comparative example 10

Refer to the method described in Example 6 to make the tissue cultured seedlings take root quickly, the only difference being that the rooting hormone contained in the jelly-like substance in steps 3 and 4 is 0.3% NAA by mass fraction.

Comparative example 11

With reference to the method described in Example 6, the tissue-cultured seedlings were rooted rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.1% (w/w).

Comparative example 12

With reference to the method described in Example 6, the tissue-cultured seedlings were rooted rapidly, the only difference being that the IBA content in the jelly-like substance of step 3 and step 4 was 0.2% (w/w).

Comparative example 13

With reference to the method described in Example 6, the tissue-cultured seedlings were rooted rapidly, the only difference being that the IBA content in the jelly-like substance of step 3 and step 4 was 0.4% (w/w).

Comparative example 14

With reference to the method described in Example 6, the tissue-cultured seedlings took root rapidly, the only difference being that the content of IBA in the jelly-like substance of step 3 and step 4 was 0.5% (w/w).

Experimental example 1

The rooting rate and survival rate of the methods described in Examples 1-5 and Comparative Examples 1-7 were counted, and the results are shown in Table 1. Comparing Experimental Examples 1 to 5, Comparative Examples 2 to 7 with Comparative Example 1, it can be seen that the method of the present invention increases the rooting rate of Chinese chestnut tissue culture seedlings from 55% of the traditional rooting method to 85% to 97%. The survival rate of the seedlings after transplanting is increased from 34% to 90-100%. And experimental example 1～5, comparative example 4～7 are compared with comparative example 2～3 as can be known, when selecting to smear on the kind of rooting hormone contained in the jelly-like substance on the stem base slope and the stem of the tissue cultured seedling, The rooting efficiency of chestnut tissue culture seedlings promoted by IBA was significantly higher than that of IAA and NAA. When comparing Examples 1-5 with Comparative Examples 4-7, it can be found that when the mass fraction of IBA is in the range of 0.25-0.35, the rooting effect of Chinese chestnut tissue culture seedlings is the best.

Rooting rate and survival rate of Chinese chestnut tissue culture seedling in table 1

Experimental example 2

The rooting rate and survival rate of the methods described in Examples 6-10 and Comparative Examples 8-14 were counted, and the results are shown in Table 2. Comparing Experimental Examples 6 to 10, Comparative Examples 9 to 14 with Comparative Example 8, it can be known that the method of the present invention increases the rooting rate of cherry tissue culture seedlings from 70% of the traditional rooting method to 81% to 99%. The survival rate of the seedlings after transplanting is increased from 57% to 90-100%. And experimental example 6～10, comparative example 11～14 are compared with comparative example 9～10 and can know, when selecting to smear on the kind of the rooting hormone contained in the jelly-like substance on the stem base slope and the stem of the tissue culture seedling, The rooting efficiency of cherry tissue culture seedlings promoted by IBA was significantly higher than that of IAA and NAA. When comparing Examples 6-10 with Comparative Examples 11-14, it can be found that when the mass fraction of IBA is in the range of 0.25-0.35, the rooting effect of the cherry tissue culture seedlings is the best.

The rooting rate and survival rate of table 2 Chinese chestnut tissue culture seedling

It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.

Claims (10)

1. a method for tissue culture seedlings to directly take root quickly, is characterized in that, described method comprises the following steps: (1) obliquely cut the stem base of the tissue culture seedling to expose the stem base slope; (2) equably smear the jelly substance containing rooting hormone on the stem base slope and the stem; (3) Insert the tissue cultured seedlings into the substrate and cultivate them until they take root. 2. The method according to claim 1, characterized in that, the rooting hormone is IBA, preferably, the mass fraction of the IBA in the colloidal substance is 0.25～0.35%; more preferably, the IBA is in The mass fraction in colloidal substance is 0.31%. 3. The method according to claim 2, characterized in that, glycerin is also included in the jelly-like substance. 4. The method according to claim 1, wherein the tissue-cultured seedlings are woody plant tissue-cultured seedlings; preferably, the woody plant tissue-cultured seedlings are chestnut tissue-cultured seedlings or cherry tissue-cultured seedlings. 5. The method according to claim 4, characterized in that, the tissue cultured plantlets are tissue cultured plantlets whose plant height is higher than 3cm, diameter is greater than 2mm, and has not yet taken root. 6. The method according to any one of claims 1 to 5, characterized in that, the matrix in step (3) has a hole, and the tissue cultured seedling is inserted into the hole of the matrix, and the matrix is extruded, so that the tissue cultured plantlet It fits seamlessly with the matrix, and the inner wall of the hole is also coated with the gel-like substance. 7. The method according to any one of claims 1 to 5, characterized in that, the substrate comprises multiple types of peat soil, rice husk charcoal, river sand, perlite, vermiculite or rock wool. 8. The method according to any one of claims 1 to 5, characterized in that, during the cultivation process of step (3), the temperature is constant at 20 to 25° C., the humidity is constant at 80 to 100%, and the illumination time is 14 ~18 hours/day; preferably, the temperature is constant at 25° C., the humidity is constant at 100%, and the light time is 16 hours/day. 9. The method according to any one of claims 1-5, characterized in that water is sprayed every 6-8 days during the cultivation process in step (3). 10. The method according to any one of claims 1-5, characterized in that the method further comprises transplanting the obtained tissue-cultured seedlings together with the matrix into plug trays or land.