In-situ regeneration method of apple rootstock under non-tissue culture condition

Abstract

The invention discloses an in-situ regeneration method of an apple rootstock under a non-tissue culture condition, and belongs to the technical field of plant biology. The invention aims to solve the technical problems of low in-situ regeneration efficiency, imperfect regeneration system and indefinite key regeneration induction part of the apple rootstock under the non-tissue culture condition. The in-situ regeneration method for the apple rootstock under the non-tissue culture condition comprises the following steps: performing primary cutting and topping on epicotyl of an apple rootstock seedling, retaining part of active apical meristem, and culturing to promote the section to regenerate adventitious buds. The method further comprises the step of cutting off lateral buds along the base parts of the cotyledons under the condition that the lateral buds appear at the base parts of the cotyledons and then carrying out culture to promote the sections to regenerate adventitious buds. According to the method disclosed by the invention, non-tissue culture efficient in-situ regeneration of the apple rootstock is realized through induction, and a simple, convenient and efficient regeneration system reference is provided for breeding and genetic improvement of the woody apple rootstock.

Description

Technical Field

[0001] The present invention belongs to the field of exploration of plant biotechnology regeneration systems, and particularly relates to an in-situ regeneration method of apple rootstocks under non-tissue culture conditions. Background Art

[0002] Apple (Malus domestica Borkh.) is one of the most important cultivated fruit trees in the world. Apple rootstocks are an important part of the development of the apple industry, and are of great significance for enhancing the stress resistance of the tree body, optimizing the tree shape structure, improving the fruit quality, and shortening the seedling raising cycle. The emergence of modern biological breeding technologies, such as transgenic, gene editing, and whole genome selection technologies, provides important support for the genetic improvement and biological breeding of apple rootstocks. Plant regeneration technology is widely used in crop breeding, especially in genetic improvement and transgenic technology. Therefore, an efficient regeneration system for apple rootstocks is of great significance. Currently, the research on apple rootstock regeneration technology mainly focuses on asexual propagation and genetic transformation under tissue culture conditions, and little research is involved in in-situ regeneration of the main body under non-tissue culture conditions. Achieving in-situ regeneration of apple rootstocks under non-tissue culture conditions is of great significance for the exploration of in-situ genetic transformation systems.

[0003] Plant regeneration refers to the activation of plant cell totipotency under the action of natural or artificial factors, under the activation of environmental signals and internal regulatory networks, so as to repair damaged tissues or regenerate into complete plants. Plant regeneration can occur in the natural environment or under artificial intervention under non-tissue culture conditions, or can be achieved through complete artificial induction under tissue culture conditions. According to whether the regeneration process depends on in vitro tissue culture conditions, plant regeneration can be divided into the following two types:

[0004] (1) In vitro regeneration of tissues and organs under tissue culture conditions

[0005] In vitro organ regeneration of plants under tissue culture conditions refers to the process of inducing the regeneration of new organs or complete plants from in vitro tissues (such as leaves, stem segments, roots, etc.) of plants through the regulation of hormones and culture medium formulations in an artificially controlled aseptic environment. The totipotency of plant somatic cells is the basis for regeneration under tissue culture conditions. The totipotency of cells is activated by specific molecular signals, and the dynamic balance of auxin and cytokinin is the core mechanism for regulating organ regeneration. Among them, a high auxin / cytokinin ratio promotes root organogenesis, while a low ratio induces bud differentiation. In apples, the adventitious bud regeneration systems of some apples have been mature, such as 'Gala', 'Royal Gala', 'M26', 'Fuji', etc. In apples, leaf explants are easy to operate under tissue culture conditions and can be induced to differentiate into adventitious buds in a relatively short period. The somaclonal variation is small, and it can better maintain the genetic stability of the recipient plant. By using other explants of apples, such as shoot tips, stem segments, cotyledons, and callus, etc., the regeneration efficiency of adventitious buds of some difficult-to-regenerate apple species has also been improved.

[0006] The research on in vitro organ regeneration under tissue culture conditions is relatively in-depth and is the basis for the rapid propagation and genetic transformation of many crops. However, there are also problems such as strong genotype dependence of explants, great difficulty in regeneration, and cumbersome operation procedures. The leaf regeneration ability of apple plants is greatly affected by genotypes. Even if the conditions are strictly ensured to be consistent throughout the entire process of genetic transformation, the regeneration rate and transformation efficiency still depend on the material types of scions and rootstocks. For example, the high-regeneration material 'GL-3' found has become the most widely used recipient material in current apple genetic transformation due to its high regeneration ability. Although other parts of apples, such as shoot tips, anthers, cotyledons, protoplasts, embryos, etc., can improve the adventitious bud regeneration ability of some difficult-to-regenerate materials, it is necessary to add hormones and chemical reagents with complex ratios, and the operation process is complex and the requirements are more stringent.

[0007] (2) In situ regeneration under non-tissue culture conditions

[0008] In-situ regeneration of plants refers to the process by which, after being stimulated by external damage, plants activate the redifferentiation of cells at the wound site through an autonomous regulatory mechanism without relying on exogenous tissue culture, and then repair the damaged tissue or regenerate new individuals. In-situ regeneration under non-tissue culture conditions usually requires little external intervention and mainly relies on its own regeneration ability. For example, after the apical meristem is removed, axillary buds or adventitious buds form new branches; after the root system is damaged, adventitious roots are formed; the wound site heals after pruning, etc. The types of in-situ regeneration in woody plants include: growth point regeneration after apical meristem excision, regeneration of secondary vascular tissue after girdling treatment, tissue healing regeneration after mechanical damage, etc. Under current artificially controlled conditions, the most widely used is the in-situ regeneration of the apical meristem growth point. When the shoot tip or root tip of a plant is partially excised, surrounding cells can initiate meristem regeneration mediated by various signals.

[0009] In-situ regeneration of plants under non-tissue culture conditions has broad application prospects, but there are still many problems at present. Especially in woody plants such as fruit trees, how to efficiently induce and achieve in-situ regeneration and genetic transformation under non-tissue culture conditions is a current research hotspot and difficulty. There are still few reports on the in-situ regeneration system related to apple rootstocks. Summary of the Invention

[0010] The present invention aims to solve problems such as low efficiency of in-situ regeneration of apple rootstocks under non-tissue culture conditions, imperfect regeneration system, and unclear optimal in-situ regeneration induction site. Although in-vitro regeneration technology under tissue culture conditions has been relatively mature, relatively stable adventitious bud regeneration systems have been established for some materials, but these systems generally have problems such as cumbersome operation, strong genotype dependence, high dependence on exogenous hormones, and low transformation efficiency. Existing research mainly focuses on in-vitro organ regeneration under tissue culture conditions, while there is less research on in-situ regeneration technology of the main body under non-tissue culture conditions. Inducing efficient in-situ regeneration of apple rootstocks without tissue culture in natural or artificial environments is a prerequisite for establishing an in-situ genetic transformation system and improving breeding efficiency and application value.

[0011] The present invention provides a method for in-situ regeneration of apple rootstocks under non-tissue culture conditions, which includes the step of making a single cut at the hypocotyl of apple rootstock seedlings, retaining part of the active apical meristem, and then culturing to promote the regeneration of adventitious buds on the cut surface.

[0012] In the above method, when lateral buds appear at the base of the cotyledons, the lateral buds are excised along the base of the cotyledons, and then decapitated again and cultured to promote the regeneration of adventitious buds on the cut surface of the cotyledon base.

[0013] In the above method, the apple rootstock can be Malus hupehensis Rehd. or Malus robusta (Carr.) Rehd.

[0014] In the above method, when the apple rootstock is Malus hupehensis (Pamp.) Rehd. and Malus robusta (Carr.) Rehd., the length of the epicotyl retained after the first cutting and topping is 0.3 - 0.5 cm.

[0015] In the above method, the culture conditions are a temperature of 20 - 25 °C and a relative humidity of 70 - 80%.

[0016] The present invention also provides the application of the above method in the regeneration and propagation of apple rootstocks.

[0017] The present invention also provides the application of the above method in apple cultivation.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention has established an in-situ regeneration technology system for apple rootstocks under non-tissue culture conditions, breaking through the limitations of the traditional regeneration mainly relying on tissue culture methods, and solving problems such as cumbersome tissue culture operations, strong dependence on exogenous hormones, and genotype restrictions, providing a simple and efficient regeneration system reference for the breeding and genetic improvement of woody apple rootstocks.

[0020] 1) Significantly improved regeneration efficiency: The "one-time epicotyl topping" and "secondary topping" systems proposed by the present invention can effectively improve the in-situ regeneration rate of adventitious buds of apple rootstocks. The present invention has deeply studied the key factors affecting the regeneration efficiency, including the cutting site (epicotyl, cotyledon base, hypocotyl, young root), specific cutting position, etc. Through the screening and utilization of these factors, the controllability and repeatability of in-situ regeneration have been significantly improved.

[0021] 2) Low genotype dependence and strong adaptability: This method is applicable to multiple apple rootstock materials, such as Malus hupehensis (Pamp.) Rehd. and Malus robusta (Carr.) Rehd. Different materials all show good regeneration ability under non-tissue culture conditions, overcoming the technical bottleneck of regeneration ability depending on genotype in the previous tissue culture system.

[0022] 3) Simple operation and no need for sterile conditions: Compared with the traditional tissue culture technology, the present invention does not rely on a complex sterile culture system and exogenous hormone induction, and can induce regeneration only through artificial wounding and environmental condition control (humidity, temperature), greatly simplifying the operation process and reducing costs. Description of the Drawings

[0023] Figure 1 It shows the in-situ regeneration situation of the cut surfaces of different parts of the Malus robusta (Carr.) Rehd. seedlings in Example 1 of the present invention. Figure 1 A shows the statistical situation of the number of regenerated cut surfaces after wounding treatment of different parts of the seedlings; Figure 1 B in Figure 1 and C in Figure 1 show the regeneration situation of the cut surface after cutting the epicotyl; Figure 1E shows the regeneration of the wound section after the hypocotyl is scratched; Figure 1 F and Figure 1 G show the regeneration of the wound after the young root is scratched.

[0024] Figure 2 This is a diagram of the in-situ regeneration process after the first decapitation of the epicotyl of the present invention.

[0025] Figure 3 This is a photo of the adventitious bud regeneration process at the epicotyl incision of Malus hupehensis Rehd. in Example 2 of the present invention. Figure 3 A and Figure 3 B show the adventitious bud regeneration after retaining the epicotyl length of 0 - 0.2 cm in Group A, and the arrow indicates the lateral bud released from the base of the cotyledon; Figure 3 C and Figure 3 D show the adventitious bud regeneration after retaining the epicotyl length of 0.3 - 0.5 cm in Group B, and the arrow indicates the adventitious bud regenerated from the wound surface of the epicotyl. Scale bar = 5 mm.

[0026] Figure 4 This is the statistical result of the adventitious bud regeneration after retaining different lengths of the epicotyl in Example 2 of the present invention.

[0027] Figure 5 This is a diagram of the efficient in-situ regeneration process after the second decapitation of the epicotyl of the present invention.

[0028] Figure 6 This is the observation result of the in-situ growth point of adventitious buds at the base of the cotyledon after the second decapitation of Malus hupehensis Rehd. in Example 3 of the present invention. Figure 6 A shows a schematic diagram of cross-section sampling, and the arrow indicates the sampling direction. Figure 6 B shows a schematic diagram of longitudinal-section sampling, and the arrow indicates the sampling direction. Figure 6 C shows the observation of paraffin sections of the cross-section, and the arrow indicates the growth point of the bud primordium. Figure 6 D shows the observation of paraffin sections of the longitudinal-section, and the arrow indicates the growth point of the bud primordium. Detailed implementation methods

[0029] The present invention will be further described in detail below in combination with the detailed implementation methods. The examples given are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for further improvement by those of ordinary skill in the art and do not constitute any limitation to the present invention in any way.

[0030] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0031] The apple rootstock Pingyi sweet tea seedlings in the following examples are commercially purchased seeds that were germinated and sown after being stratified at 4°C for about 30 days.

[0032] The apple rootstock Malus multiflora seedlings in the following examples are obtained by germination and sowing after commercially purchased seeds were stratified at 4°C for about 40 days.

[0033] Example 1: Exploration of the Optimal Non-tissue Culture In-situ Regeneration Site of Apple Stock

[0034] Apple rootstock seedlings have four parts from top to bottom, namely epicotyl, cotyledon, hypocotyl and young root. In order to explore the differences in in situ regeneration mode and regeneration efficiency of each part, three-leaf seedlings with strong regeneration ability of apple rootstock Malus octagonalis were selected, and wounding treatment and regeneration induction were performed on four different parts. The specific parts are as follows:

[0035] (1) Epicotyl: Cut the epicotyl and keep a certain length to allow it to regenerate on the cut surface. Cultivate under high humidity and normal light conditions and cover with a transparent lid to maintain humidity (temperature 20-25°C, relative humidity 70-80%). Observe the cut surface for callus formation and differentiation of adventitious buds.

[0036] (2) Hypocotyl position: The hypocotyl is located between the base of the cotyledon and the young root. Its tissue is relatively tender and rich in primary vascular tissue. The experiment observes its regeneration ability by making cross-sections and incisions at different positions of the hypocotyl. At the same time, during the induction of hypocotyl regeneration, the lateral buds at the epicotyl and cotyledon base of the seedlings are regularly removed. Cultivate under high humidity and normal light conditions and cover with a transparent lid to maintain humidity (temperature 20-25°C, relative humidity 70-80%) to observe whether it can stimulate the formation of callus tissue and the differentiation of adventitious roots and adventitious buds.

[0037] (3) Cotyledon: Cut the cotyledon longitudinally, cut a part of it longitudinally along the midrib of the cotyledon or directly remove a part of the cotyledon, at a temperature of 20-25°C and a relative humidity of 70-80%, and observe the callus and regeneration of adventitious buds.

[0038] (4) Young root position: Select healthy and stable seedlings for the experiment. First, the young roots are wounded in different ways, including root tip removal (cutting off 1-2 mm of the root tip), cross-section (cutting horizontally 2-5 mm from the root tip), and puncture (thicker roots). Subsequently, the treated seedlings are planted back into the plug tray matrix. The seedling growth environment is a high humidity, suitable temperature, and normal light environment (temperature 20-25°C, relative humidity 70-80%). At the same time, the lateral buds released from the epicotyl and cotyledon base are always excised multiple times during the culture period.

[0039] Set 3 replicates, and for each replicate, 100 seedlings are treated with each excision method for each part.

[0040] After normal cultivation for about 10 d in a highly humid and warm environment, the regeneration of the wound site was statistically analyzed according to Formula 1, and the proportion of seedlings that regenerated adventitious buds in situ after wounding treatment of different parts was compared.

[0041] The proportion of seedlings that regenerated adventitious buds in situ = the number of seedlings with adventitious buds regenerated from the cut surface / the total number of seedlings × 100% —— Formula 1

[0042] The results showed that the proportion of seedlings that regenerated adventitious buds in situ at the epicotyl part was the highest (6.46%), the regeneration of adventitious buds in situ at the hypocotyl and young root parts was relatively poor, being 2.38% and 1.36% respectively, while the cut surface of the cotyledon part could not regenerate ( Figure 1 ), that is, the most suitable part for non-tissue culture in situ regeneration of apple rootstocks is the epicotyl.

[0043] Example 2: Exploration of the optimal remaining length for in situ regeneration of apple rootstock epicotyl

[0044] According to Example 1, it was found that the most suitable part for non-tissue culture in situ regeneration of apple rootstocks is the epicotyl part of the seedlings. In order to further obtain the optimal cutting length retained during in situ regeneration of the epicotyl, Malus hupehensis Rehd. was used for experimental exploration.

[0045] Select mature and plump Malus hupehensis Rehd. seeds. After breaking dormancy by cold stratification treatment (stratify at 4°C under moist conditions for 30 days), the seeds began to show white tips. The seeds with white tips were placed at room temperature for 2 - 3 days for germination acceleration. After the hypocotyl began to elongate, they were sown in a plug tray filled with moist vermiculite substrate and placed in a moisturizing environment at a conventional room temperature (20 - 25°C) for germination and growth. About 2 - 3 days after sowing, the epicotyl of the seeds elongated, the cotyledons began to emerge successively, and then the cotyledons unfolded and the epicotyl elongated.

[0046] After the cotyledons unfolded and the epicotyl emerged and elongated, use a surgical blade to cut off the part above the first true leaf, retain the cotyledons, and leave part of the epicotyl. The flow chart is shown in Figure 2 . Set two lengths for the retained epicotyl:

[0047] Group A: The length of the retained epicotyl is 0 - 0.2 cm.

[0048] Group B: The length of the retained epicotyl is 0.3 - 0.5 cm.

[0049] Set 3 replicates, with 100 plants in each replicate for each group.

[0050] Cover with plastic wrap and keep moist (temperature 20-25°C, relative humidity 70-80%), observe the bud regeneration of the cut surface, and after 10 days, calculate the proportion of seedlings that regenerate adventitious buds in situ on the cut surface according to formula 1, and calculate the lateral bud release rate according to formula 2:

[0051] Lateral bud release rate = number of seedlings from lateral buds / total number of seedlings × 100% Formula 2

[0052] When the length of the retained epicotyl was different, the regeneration of the buds was significantly different, such as Figure 3 and Figure 4 When the epicotyl length is short (0-0.2 cm), the cut surface turns brown and adventitious buds cannot be regenerated. When the epicotyl length is long (0.3-0.5 cm), the proportion of seedlings that regenerate adventitious buds in situ from the cut surface is about 85%, and the efficiency of lateral bud release at the base of the cotyledons is about 17.6%, indicating that the optimal length of the epicotyl retained after cutting in situ regeneration of apple rootstock epicotyl is 0.3-0.5 cm.

[0053] Example 3: In situ regeneration of the cotyledon base of apple stock (secondary decapitation)

[0054] According to the exploratory test of the optimal epicotyl retention length for in situ regeneration of epicotyls of apple stock in Example 2, it was found that after the epicotyl was toppled once, in addition to the in situ regeneration occurring on the cut surface, a certain proportion of lateral buds were released at the base of the cotyledons. In order to explore the in situ regeneration system at the base of the cotyledons of apple stock, about 10 days after the epicotyl was toppled once, the seedlings with lateral buds released at the base of the cotyledons of groups A and B in Example 2 were taken, and the two released lateral buds were cut along the base of the cotyledons using a surgical blade. The complete flow chart starting from the first cutting is shown in Figure 5 After cutting, place the plants under a moisturizing plastic cover (temperature 20-25°C, relative humidity 70-80%).

[0055] The results showed that after the lateral buds were removed, adventitious buds would regenerate in situ at the base of the cotyledons. In order to explore the regeneration characteristics of adventitious buds after the cotyledon base of the apple rootstock was removed for the second time, this study observed the growth point of the bud primordium through sampling and paraffin section technology. Figure 6 As shown in A and B, the cross-section and longitudinal section diagrams of the sampling site are shown respectively, and the red arrow indicates the specific sampling direction.

[0056] Through the results of paraffin section, the formation of adventitious bud primordium and its tissue characteristics can be clearly observed. Figure 6 In C), the area indicated by the arrow shows small cells, dense arrangement, thin cell walls, and large nuclei, showing typical meristem characteristics, indicating that new bud primordia have been formed in this area. Figure 6In D), a structure in which a certain type of cell is actively dividing can also be seen in the indicated area, further verifying the in-situ regeneration trend of adventitious buds after decapitation. The section results show that after the apple rootstock is decapitated a second time at the cotyledon base, it can stimulate the activation of potential meristems and induce the in-situ formation of adventitious buds (the same phenomenon also occurs in the apple rootstock Malus robusta Rehd.).

[0057] Ten days after the second decapitation, that is, removing the released lateral buds, the efficiency of in-situ regeneration of adventitious buds at the cotyledon base was statistically analyzed according to Formula 3:

[0058] Adventitious bud regeneration rate at the cotyledon base = total number of regenerated buds at the cotyledon base / total number of seedlings × 100% Formula 3

[0059] The statistical results are shown in Table 1, and the regeneration rate reached 186.1%.

[0060] Table 1 Regeneration of adventitious buds after removing the lateral buds at the cotyledon base of Malus hupehensis (Pamp.) Rehd. (second decapitation)

[0061] Total number of seedlings Number of adventitious bud regeneration Adventitious bud regeneration rate 108 201 186.1％

[0062] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. An in-situ regeneration method of apple rootstock under non-tissue culture conditions, characterized in that The method includes a step of making a single cut to remove the top of the epicotyl of the apple rootstock seedling, retaining a part of the active apical meristem, and then culturing to promote the regeneration of adventitious buds on the cut surface.

2. The method according to claim 1, wherein: The method further includes a step of, when lateral buds appear at the base of the cotyledons, cutting off the lateral buds along the base of the cotyledons to remove the top again, and then culturing to promote the regeneration of adventitious buds on the cut surface.

3. The method according to claim 1 or 2, characterized in that: The apple rootstock is Malus hupehensis (Pamp.) Rehd.

4. The method according to claim 3, wherein: For the single cut to remove the top of the epicotyl, the length of the retained epicotyl is 0.3 - 0.5 cm.

5. The method according to claim 1 or 2, characterized in that: The apple rootstock is Malus robusta (Carr.) Rehd.

6. The method according to any one of claims 1-5, characterized in that: The conditions for the culture are a temperature of 20 - 25°C and a relative humidity of 70 - 80%.

7. Application of the method according to any one of claims 1 - 6 in the regeneration and propagation of apple rootstocks.

8. Application of the method according to any one of claims 1 - 6 in apple cultivation.

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