AU2020104372A4

AU2020104372A4 - An effective seedling hardening matrix for promoting corn callus seedling growth

Info

Publication number: AU2020104372A4
Application number: AU2020104372A
Authority: AU
Inventors: Langlang Ma; Yaou Shen
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-03-18
Anticipated expiration: 2028-12-29

Abstract

An effective seedling hardening matrix for promoting corn callus seedling growth, the invention provides high rooting efficiency, good rooting quality, good seedling hardening effect and high transplanting survival rate. 1. When the tissue culture seedlings grow three main roots, the surface of the culture medium is covered with a layer of hardening agent. 2. After 7 days' hardening, the seedlings are transplanted into hardening matrix, which is formed by mixing garden soil and nutrient soil in proportion. 3. First put 2/3 of the substrate into the seedling cup, put the seedlings into the seedling cup, and then bury the roots of the seedlings with the substrate. 4. The seedlings planted in the seedling cup should be covered with a plastic cup cover with holes for 7 days. 5. After removing the cup cover, cultivate in the greenhouse for 10 days, and move outside the greenhouse to harden seedlings for 3 days and transplant to the field. 6. After planting in the field, cover the ground with plastic film, and cover it with sunshade net for 7 days. 7. When some seedlings grow in culture bottles, they will have the phenomenon of heart leaf rolling. For hardening seedlings, the heart leaf of the heart rolling seedlings will be peeled off to improve the survival rate.

Description

AN EFFECTIVE SEEDLING HARDENING MATRIX FOR PROMOTING CORN CALLUS SEEDLING GROWTH TECHNICAL FIELD

The invention relates to a method with high rooting efficiency, good rooting quality, good seedling hardening effect and high transplanting survival rate.

BACKGROUND

The rapid development of genetically modified technology has brought huge benefits to agricultural production. This technology has achieved directional breeding and improved breeding efficiency. Since the late 1980s, scientists began to conduct maize transgenic research and have achieved some important results. There are already some commercially planted genetically modified corn varieties, such as high-lysine genetically modified corn, corn borer resistant genetically modified corn, herbicide resistant genetically modified corn, etc. Therefore, transgenic breeding has become an important means of modern molecular breeding. The general process of transgenic breeding can be divided into three stages: molecular construction, tissue culture regeneration, and field breeding. Tissue culture regeneration stage refers to the transformation of the vector containing the target gene into Agrobacterium, which then infects callus, obtains resistant callus after resistance screening, and then obtains regeneration seedlings through differentiation culture, refines seedlings and transplants, and finally plants in field. The more regenerated seedlings planted in the field, the more positive seedlings obtained, and later field breeding will be more effective. Therefore, in the process of transgenic breeding, tissue culture regeneration is an extremely important and indispensable link. At present, the most widely used maize transgenic acceptor material is embryogenic callus obtained by induction and culture of maize immature embryos. The tissue culture technology based on this has some defects: 1. The rooting effect of the currently used rooting and strong seedling medium is not good. Many regenerated seedlings still have no roots after being cultured on the rooting and strengthening seedling medium for 50 days, resulting in death; 2. In the process of transplanting test-tube seedlings, due to the lack of effective control methods, the death loss was serious, resulting in few transgenic plants, even none, which made transgenic breeding very difficult. 3. In the existing seedling training method, a layer of sterilized distilled water is added to the surface of the culture medium in the culture bottle and even if a thick layer of water is added, it cannot avoid the growth of mold and the death of the seedlings, and thick water the layer guides the seedlings to anaerobic respiration, which also accelerates the seedling's death. 4. The soil for planting seedlings also has some problems: if the ratio of nutrient soil is too high, it is easy to burn the seedlings and cause the seedlings to die; if the ratio of nutrient soil is too low, the soil is compacted, and the seedlings are not easy to survive. 5. The seedlings that have just been planted in the flowerpot are severely dehydrated and wilted, resulting in death; 6. The seedlings that grow well in the flowerpot have a high mortality rate after being planted in the field.

SUMMARY

The invention provides a corn transgenic tissue culture method with high rooting efficiency, good rooting quality, good seedling hardening effect and high transplanting survival rate.

The transgenic corn tissue culture method for realizing the purpose of the invention comprises the following steps.

1. Divide the maize tissue culture seedlings which grow to 3cm-4cm on the differentiation medium in the culture bottle into individual plants, peel off the callus from the roots, and move them to the rooting and strengthening seedling culture medium for rooting. The components of the rooting and strengthening seedling culture medium are improved MS culture medium, and the hormones used are rooting powder ABT and chlormequat uniconazole, with the concentrations of 1mg/L and 0.25mg/L respectively, and the antibiotic used is cefotaxime with a concentration of 200 mg/L.

2. The tissue culture seedlings grow at least three main roots, each of which is about 15cm long. When capillary roots grow on the main roots, open the cover to harden the seedlings, and cover the surface of the culture medium with a layer of 1 2mm hardening agent, so as to avoid the death of seedlings due to the cultivation of basal fungi. A total of 7 days were spent on seedling training. In the first 3 days, the seedlings were trained with a cover, which gave them a gradual process to adapt to the external environment, so as to avoid withering. In the last 4 days, the cover was removed to train seedlings, so that the seedlings could adapt to the external environment completely. 3. After 7 days' hardening, the seedlings will become strong and green, and they will be transplanted into hardening matrix, which is made of garden soil and nutrient soil in a ratio of 8:2. The proportion of garden soil is too high, which is easy to harden, airtight and absorbent, which is not conducive to the survival and growth of seedlings; If the proportion of nutrient soil is too high, there will be seedling burning phenomenon, which will lead to seedling death; It is more appropriate to mix garden soil and nutrient soil in the ratio of 8:2, which not only solves the problem of hardening of garden soil, but also avoids the phenomenon of burning seedlings with nutrient soil, and can also provide certain nutrient substrate for seedlings. Moreover, this mixed substrate is loose, breathable, moisture-retaining and beneficial to the survival and growth of seedlings. The matrix for hardening seedlings does not need to be sterilized, so as to prevent the nutrient components in the matrix from being damaged by high temperature. After planting seedlings, dilute the stock solution of hardening agent in step 2 with tap water to an aqueous solution with a working concentration of 400mg/L, and pour the matrix thoroughly. 4. First, put 2/3 of the hardening matrix into the seedling cup, and gently pull out the seedlings from the culture medium with tweezers. Avoid washing the roots with water to reduce the damage to the roots. If the roots are stained with large pieces of culture medium, they can be gently clipped off with tweezers. Then put the seedlings in the middle of the seedling cup, spread the roots as far as possible, bury the roots of the seedlings with the matrix, and press them gently.

5. The seedlings planted in the seedling cup should be covered with a plastic cup cover with holes for about 7 days, and can be taken away when the seedlings grow new leaves. The cup cover can keep water and moisture, and prevent the seedlings just planted in the flowerpot from withering. The seedlings planted in flower pots should be given sufficient light, which is conducive to the survival of the seedlings. 6. After removing the cup cover, it will be cultivated in the greenhouse for about 10 days. When the seedlings grow to about 20cm high, they will be moved outside the greenhouse for 3 days and then transplanted to the field. 7. After planting in the field, cover the ground with plastic film and shade it for 7 days to prevent the sun from dying. When the seedlings are restored to strong green, the sunshade net can be removed. 8. When some test-tube plantlets grow in culture bottles, due to problems such as lack of space or water, they will roll up. When the seedlings are hardened or planted in a seedling cup, the heart of the rolled seedlings will still survive. If you don't peel them off, these seedlings will die. Every transgenic seedling obtained is very precious, so try to make every one survive. The rooting and seedling strengthening medium in step 1 is characterized in that the medium is an improved MS medium, and the formula of the improved MS medium is shown in the following table:

Note: PH is 6.0.

The preparation method of the hardening agent in step 2 is as follows: 100 nystatin tablets per bottle are ground into powder by mortar and put in bottles for later use. Before use, the powdery nystatin was dissolved in sterile distilled water to prepare mg/ml stock solution of the hardening agent, which was left for more than 2 hours to dissolve. When hardening seedlings, 10ml of 40mg/ml nystatin stock solution was added to each liter of sterile distilled water to prepare a hardening agent, which was added to the surface of the culture medium for hardening seedlings.

The seedling hardening matrix in step 3 is characterized in that it is formed by mixing garden soil and nutrient soil according to the ratio of 8:2, and the seedling hardening matrix does not need to be sterilized, so as to prevent the nutrient components in the matrix from being damaged by high temperature. After planting seedlings, it is diluted into an aqueous solution with a working concentration of 400mg/L with tap water, and the matrix can be poured thoroughly.

In the step 5, in order to prevent the seedlings just planted in the seedling cup from dying of water loss, the seedlings are covered with a plastic cup cover with holes. The plastic cup cover is made of disposable milk tea cup, and three holes are punched in the bottom of the cup for ventilation.

In the step 6, after the cup cover is removed, the seedlings are cultured in the greenhouse for about 10 days, and when the seedlings grow to a height of about 20cm, the seedlings are moved outside the greenhouse for hardening for 3 days before being transplanted to the field.

In the step 7, after planting in the field, the ground surface is covered with plastic film, and the sun screen is used for 7 days to prevent the sun from dying.

In step 8, when some test-tube seedlings grow in the culture bottle, due to problems such as insufficient space or water shortage, the phenomenon of core rolling will occur. When the seedlings are hardened or planted in a seedling cup, the heart of the rolled seedlings will still survive.

In the step 1, the thickness of the rooting and strengthening seedling culture medium is 3cm, which is enough to provide nutrients needed by the tissue culture seedlings for 50 days, so there is no need to change the culture medium during rooting, thus reducing the damage to the root system of the tissue culture seedlings.

In the step 5, the tissue culture seedlings just planted into the seedling raising cup should break the conventional operation of avoiding light, and sufficient light should be given to promote the survival of the seedlings.

The efficient tissue culture method for transgenic corn has the following beneficial effects.

1. According to the rooting and seedling strengthening culture medium provided by the invention, the rooting rate reaches 100%, the adventitious roots are long and thick, and the capillary roots are developed, so that the transplanting survival rate is effectively improved. It solves the problems of low rooting rate and poor rooting quality of maize tissue culture seedlings, and has significant scientific research value. 2. According to the seedling hardening agent provided by the invention, after the seedlings are hardened for 7 days, there is still no long bacteria, which is in sharp contrast with the previous hardening method that the long bacteria rate is over 80%, and the seedling hardening agent is strong and green, and the survival rate of hardening seedlings reaches 100% at this stage. 3. The seedling hardening matrix provided by the invention not only solves the problem of hardening of garden soil, but also avoids the phenomenon of burning seedlings by nutrient soil, and can also provide certain nutrient matrix for seedlings; and the mixed matrix is loose, breathable, moisture-retaining and moisture-preserving, which is beneficial to the survival and growth of seedlings, and remarkably improves the transplanting survival rate at this stage. This seedling hardening matrix does not need sterilization, which greatly reduces the workload and equipment consumption. 4. The tissue culture seedlings planted in the seedling cup will be seriously dehydrated and wither due to the change of culture environment, and the mortality rate is very high. After being covered with plastic cups with holes, the transplanted tissue culture seedlings will not die because of water loss. After 7 days, the seedlings grow new leaves, which fully adapt to the greenhouse environment and grow well. 5. After planting in the field, covering the ground with plastic film and covering it with sun-shading net for 7 days can prevent the sun from dying, and significantly improve the field survival rate of tissue culture seedlings. 6. When the seedlings are hardened or planted in a seedling cup, the heart leaf of the rolled seedlings is peeled off, and the tissue culture seedlings will grow new leaves and still survive with remarkable effect

BRIEF DESCRIPTION OF THE FIGURES

No figures

DESCRIPTION OF THE INVENTION

The specific implementation steps of the present invention are as follows.

1. According to the formula of the rooting and strong seedling culture medium provided by the present invention, the rooting and strong seedling culture medium is prepared, autoclaved at 121°C for 15 minutes, and poured into a sterile culture bottle on a clean bench. The thickness of the culture medium is 3 cm. Steam to dry and use after l day.Divide the corn tissue culture seedlings that grow to 3cm-4cm on the culture flask differentiation medium into individual plants, peel off the callus of the roots, and transfer to the rooting and strong seedling medium prepared in step 1 for rooting.

2. Cultivate for about 30 days. Note that the medium does not need to be replaced during this period. The tissue cultured seedlings should grow at least 3 taproots, each of which is about 15cm long. When capillary roots grow on the taproots, open the lid and cover the culture medium with a layer of1-2mm seedling strengthening agent. The seedlings were tempered for 7 days. The seedlings were tempered with a cover in the first 3 days to give the seedlings a gradual process of adapting to the external environment to prevent the seedlings from wilting. In the next 4 days, the cover was removed and the seedlings were tempered to make the seedlings fully adapt to the external environment.

3. After 7 days'hardening, the seedlings will become strong and green, and they will be transplanted into hardening matrix, which is made of garden soil and nutrient soil in a ratio of 8:2. The matrix for hardening seedlings does not need to be sterilized, so as to prevent the nutrient components in the matrix from being damaged by high temperature. After planting seedlings, dilute the stock solution of hardening agent in step 2 with tap water to an aqueous solution with a working concentration of 400mg/L, and pour the matrix thoroughly.

4. After 7 days'hardening, the seedlings will become strong and green, and they will be transplanted into hardening matrix, which is made of garden soil and nutrient soil in a ratio of 8:2. The matrix for hardening seedlings does not need to be sterilized, so as to prevent the nutrient components in the matrix from being damaged by high temperature. After planting seedlings, dilute the stock solution of hardening agent in step 2 with tap water to an aqueous solution with a working concentration of 400mg/L, and pour the matrix thoroughly.

5. The seedlings planted in the seedling cup should be covered with a plastic cup cover with holes for about 7 days, and can be taken away when the seedlings grow new leaves. The seedlings planted in flowerpots should be given sufficient light, which is conducive to the survival of the seedlings.

6. After the cup cover is removed, the seedlings are cultured in the greenhouse for about 10 days. When the seedlings grow to a height of about 20cm, they are moved outside the greenhouse to practice seedlings for 3 days, and then they can be transplanted to the field.

7. After planting in the field, cover the ground with plastic film and shade it for 7 days to prevent the sun from dying. When the seedlings are restored to strong green, the sunshade net can be removed.

8. When some test-tube plantlets grow in culture bottles, due to problems such as lack of space or water, they will roll up. When the seedlings are hardened or planted in a seedling cup, the heart of the rolled seedlings will still survive.

The above are only specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or replacements that are not thought of through creative work shall be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An effective seedling hardening matrix for promoting corn callus seedling growth with high rooting efficiency, effective seedling hardening method, good seedling hardening substrate and transplanting technology with high survival rate wherein the matrix comprises self-made seedling hardening agent, and the formula is as follows: 100 nystatin tablets per bottle, which grind into powder by mortar and put in bottles for prepare; dissolve powdery nystatin with sterile distilled water before use, prepare 40mg/ml stock solution of hardening agent, and let it dissolve after standing for more than 2 hours; when hardening seedlings, 10ml of 40mg/ml nystatin stock solution was added to every litre of sterile distilled water, and then the hardening agent was prepared, which was added on the surface of culture medium for hardening seedlings.

2. The effective seedling hardening matrix according to claim 1 is characterized in that it is formed by mixing garden soil and nutrient soil according to the ratio of 8:2, and the seedling hardening matrix does not need to be sterilized, so as to prevent the nutrient components in the matrix from being destroyed by high temperature. After planting seedlings, it can be diluted into an aqueous solution with a working concentration of 400mg/L with tap water, and the matrix can be poured thoroughly.

3. In the effective seedling hardening method as claimed in claim 1, some test tube seedlings will roll up due to lack of space or water when growing in the culture bottle. When hardening seedlings or planting them in the seedling cup, peeling off the heart of the rolled-up seedlings will significantly improve the survival rate.