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

Abstract

The application discloses an in-situ regeneration method of apple rootstock under non-tissue culture condition and belongs to the field of plant biotechnology.The technical problem to be solved by the application is low in-situ regeneration efficiency of apple rootstock under non-tissue culture condition, the regeneration system is imperfect, and a key regeneration induction part is not clear.The in-situ regeneration method of apple rootstock under non-tissue culture condition comprises the steps of cutting the top of an apple rootstock seedling once, removing the top, reserving part of active apical meristem and then culturing to promote the cut surface to regenerate adventitious buds.The method further comprises the steps of cutting off the lateral bud along the cotyledon base, removing the top again, culturing to promote the cut surface to regenerate adventitious buds when the lateral bud appears at the cotyledon base.The method can induce and realize high-efficiency in-situ regeneration of apple rootstock under non-tissue culture condition, and provides a simple and efficient regeneration system reference for breeding and genetic improvement of woody apple rootstock.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology regeneration system exploration, specifically involving an in-situ regeneration method for apple rootstock under non-tissue culture conditions. Background Technology

[0002] Apple (Malus domestica Borkh.) is one of the world's most important cultivated fruit trees. Apple rootstock is a crucial component of the apple industry's development, playing a significant role in enhancing tree resistance, optimizing tree structure, improving fruit quality, and shortening the seedling cycle. Modern bio-breeding technologies, such as transgenics, gene editing, and whole-genome selection, have provided vital support for the genetic improvement and bio-breeding of apple rootstock. Plant regeneration technology is widely used in crop breeding, especially in genetic improvement and transgenic technology. Therefore, an efficient regeneration system for apple rootstock is of great importance. Current research on apple rootstock regeneration technology mainly focuses on asexual propagation and genetic transformation under tissue culture conditions, with little attention paid to in situ regeneration under non-tissue culture conditions. Achieving in situ regeneration of apple rootstock under non-tissue culture conditions is of great significance for exploring in situ genetic transformation systems.

[0003] Plant regeneration refers to the process by which plant cells, under the influence of natural or human factors, are activated by environmental signals and internal regulatory networks, thereby leveraging the totipotency of plant cells to repair damaged tissues or regenerate into a complete plant. Plant regeneration can occur in the natural environment or under human intervention without tissue culture conditions, or it can be achieved through complete artificial induction under tissue culture conditions. Based on 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] Plant organ regeneration under tissue culture conditions refers to the process of inducing the regeneration of new organs or complete plants from isolated plant tissues (such as leaves, stem segments, and roots) in an artificially controlled sterile environment through the regulation of hormones and culture medium formulations. The totipotency of plant somatic cells is the basis for regeneration under tissue culture conditions. Cell totipotency is activated through specific molecular signals, and the dynamic balance of auxin and cytokinin is the core mechanism regulating organ regeneration. A high auxin / cytokinin ratio promotes root organogenesis, while a low ratio induces bud differentiation. In apples, adventitious bud regeneration systems are well-established for some varieties, such as 'Gala', 'RoyalGala', 'M26', and 'Fuji'. In apples, leaf explants are easy to manipulate under tissue culture conditions and readily induce adventitious bud differentiation within a short period. Somatic cell clonal variation is small, and the genetic stability of the recipient plant is well maintained. Utilizing other apple explants, such as shoot tips, stem segments, cotyledons, and callus tissue, has also improved the regeneration efficiency of adventitious buds in some difficult-to-regenerate apple species.

[0006] Research on ex vivo organ regeneration under tissue culture conditions is quite advanced and forms the basis for rapid propagation and genetic transformation of many crops. However, it also faces challenges such as strong explant genotype dependence, high regeneration difficulty, and cumbersome procedures. The leaf regeneration ability of apple plants is significantly influenced by genotype. Even with strict consistency of conditions throughout the genetic transformation process, the regeneration rate and transformation efficiency still depend on the type of scion and rootstock materials. For example, the highly regenerable material 'GL-3' has become the most widely used recipient material in apple genetic transformation due to its high regeneration capacity. While other parts of the apple, such as shoot tips, anthers, cotyledons, protoplasts, and embryos, can improve the adventitious bud regeneration ability of some difficult-to-regenerate materials, this requires the addition of complex ratios of hormones and chemical reagents, and the procedures are complex and demanding.

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

[0008] In situ plant regeneration refers to the process by which plants, after being damaged by external stimuli, re-differentiate cells at the wound site through their own regulatory mechanisms without relying on exogenous tissue culture, thereby repairing damaged tissue or regenerating new individuals. In situ regeneration under non-tissue culture conditions typically requires minimal external intervention, relying primarily on the plant's own regenerative capacity. Examples include the formation of new branches from axillary buds or adventitious buds after the removal of the apical meristem; the formation of adventitious roots after root damage; and the healing of wounds after pruning. Types of in situ regeneration in woody plants include: regeneration of the growing point after apical meristem removal, regeneration of secondary vascular tissue after girdling, and tissue healing and regeneration after mechanical damage. Under current controlled conditions, the most widely utilized method is in situ regeneration of the apical meristem growing point. When the stem or root tip of a plant is partially removed, surrounding cells can initiate meristem regeneration through various signaling mechanisms.

[0009] In situ regeneration of plants under non-tissue culture conditions has broad application prospects, but there are still many problems, especially in woody plants such as fruit trees. How to efficiently induce and realize in situ regeneration and genetic transformation under non-tissue culture conditions is a current research hotspot and difficulty. In situ regeneration systems related to apple rootstocks are still rarely reported. Summary of the Invention

[0010] This invention aims to address the problems of low in situ regeneration efficiency, imperfect regeneration systems, and unclear optimal in situ regeneration induction sites for apple rootstocks under non-tissue culture conditions. Although in vitro regeneration technology under tissue culture conditions is relatively mature, and some materials have established relatively stable adventitious bud regeneration systems, these systems generally suffer from 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; however, research on in situ regeneration technology for non-tissue culture conditions is limited. Achieving efficient in situ regeneration of apple rootstocks without tissue culture under natural or artificial environments is a prerequisite for establishing in situ genetic transformation systems and improving breeding efficiency and application value.

[0011] This invention provides an in-situ regeneration method for apple rootstock under non-tissue culture conditions, which includes a step of cutting off the hypocotyl of an apple rootstock seedling, retaining some active apical meristem, and then culturing the cut surface to promote the regeneration of adventitious buds.

[0012] The above method also includes a step of cutting off the lateral buds along the base of the cotyledons when lateral buds appear, removing the tops again, and then cultivating them to promote the regeneration of adventitious buds from the cut surface of the cotyledon base.

[0013] In the above method, the apple rootstock can be Pinyi sweet tea or Malus baccata.

[0014] In the above method, when the apple rootstock is Pingyi sweet tea and Malus blakeana, the length of the retained epicotyl is 0.3-0.5cm after the first cutting and topping.

[0015] In the above method, the cultivation conditions are a temperature of 20-25℃ 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] This invention establishes an in-situ regeneration technology system for apple rootstocks under non-tissue culture conditions, breaking through the limitations of traditional regeneration methods that mainly rely on tissue culture. It solves problems such as cumbersome tissue culture operations, strong dependence on exogenous hormones, and genotype limitations, 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 topping of the epicotyl" and "two-time topping" system proposed in this invention can effectively improve the in-situ regeneration rate of adventitious buds in apple rootstocks. This invention has conducted in-depth research on key factors affecting regeneration efficiency, including the cutting location (epicosome, cotyledon base, hypocotyl, young root), and the specific cutting position. Through the screening and utilization of these factors, the controllability and repeatability of in-situ regeneration are significantly improved.

[0021] 2) Low genotype dependence and strong adaptability: This method is applicable to multiple apple rootstock materials, such as Pingyi sweet tea and Malus blakeana. Different materials showed good regeneration ability under non-tissue culture conditions, overcoming the technical bottleneck of genotype dependence on regeneration ability in previous tissue culture systems.

[0022] 3) Simple operation, no sterile conditions required: Compared with traditional tissue culture technology, this invention does not rely on a complex sterile culture system and exogenous hormone induction. Regeneration can be induced simply by artificially creating wounds and controlling environmental conditions (humidity, temperature), which greatly simplifies the operation process and reduces costs. Attached Figure Description

[0023] Figure 1 This shows the in-situ regeneration of different parts of the Malus octagonalis seedling in Example 1 of the present invention. Figure 1 A represents the statistical results of the number of seedlings regenerated from the cut surface after wounding treatment at different parts of the seedlings; Figure 1 B and Figure 1 C represents the regeneration of the cut surface after the epicotyl is cut; Figure 1 D and Figure 1E represents the regeneration status of the wound cut surface after the hypocotyl is scratched; Figure 1 F and Figure 1 G represents the regeneration status at the wound site after the young root is scratched.

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

[0025] Figure 3 This is a photograph of the adventitious bud regeneration process of the epicotyl incision in Pingyi sweet tea in Embodiment 2 of the present invention. Figure 3 A and Figure 3 B represents the regeneration of adventitious buds in group A after retaining an epicotyl length of 0-0.2cm. The arrows indicate the lateral buds released from the base of the cotyledons. Figure 3 C and Figure 3 D represents the regeneration of adventitious buds in group B after retaining an epicotyl length of 0.3-0.5 cm. The arrows indicate the regeneration of adventitious buds on the epicotyl wound surface. Scale bar = 5 mm.

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

[0027] Figure 5 This diagram illustrates the efficient in-situ regeneration process of the supercolumn after secondary decapitation according to the present invention.

[0028] Figure 6 This is the observation result of the in-situ growth point of the adventitious buds at the base of the cotyledons after the second topping of Pingyi sweet tea in Example 3 of the present invention. Figure 6 A is a schematic diagram of cross-sectional sampling, and the direction indicated by the arrow is the sampling direction. Figure 6 B is a schematic diagram of longitudinal section sampling, and the direction indicated by the arrow is the sampling direction. Figure 6 C is a paraffin section observed in cross-section, and the arrow points to the growth point of the bud primordium. Figure 6 D is a longitudinal section of paraffin section for observation, and the arrow points to the growth point of the bud primordium. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The apple rootstock Pingyi sweet tea seedlings in the following examples were obtained by commercially purchased seeds that were stratified at 4°C for about 30 days before germination and sowing.

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

[0033] Example 1: Exploration of the optimal non-tissue culture in situ regeneration site of apple rootstock

[0034] Apple rootstock seedlings consist of four parts from top to bottom: epicotyl, cotyledons, hypocotyl, and young roots. To explore the differences in in-situ regeneration methods and efficiency among these parts, three-leaf-aged seedlings of the apple rootstock *Malus octagonale*, which exhibit strong regeneration capabilities, were selected. Wounding treatment and regeneration induction were performed on each of the four different parts, as detailed below:

[0035] (1) Epicotyl: Remove the epicotyl and retain a certain length so that it can regenerate on the cut surface. Place it in a high humidity and normal light condition for culture, and cover it with a transparent lid to maintain humidity (temperature 20-25℃, relative humidity 70-80%). Observe whether there is callus formation and adventitious bud differentiation on the cut surface.

[0036] (2) Hypocotyl: The hypocotyl is located between the base of the cotyledons and the young roots. Its tissue is relatively tender and rich in primary vascular tissue. The experiment involved making transverse cuts at different locations on the hypocotyl to observe its regeneration ability. Simultaneously, during the induction of hypocotyl regeneration, the epicotyl and lateral buds at the base of the cotyledons of the seedlings were periodically removed. The seedlings were cultured under high humidity and normal light conditions, and covered with a transparent lid to maintain humidity (temperature 20-25℃, relative humidity 70-80%). The study observed whether this stimulated callus formation and the differentiation of adventitious roots and buds.

[0037] (3) Cotyledon part: Make longitudinal cuts on the cotyledon part, cut a part along the midrib of the cotyledon or remove a part of the cotyledon directly, at a temperature of 20-25℃ and a relative humidity of 70-80%, and observe the regeneration of callus and adventitious buds.

[0038] (4) Young Roots: Healthy and stable seedlings were selected for the experiment. First, the young roots were treated with different methods of wounding, including root tip removal (cutting off the root tip 1-2 mm), transverse cutting (cutting horizontally 2-5 mm from the root tip), and puncture wounding (for thicker roots). Then, the treated seedlings were planted back into the substrate in the seedling trays. The seedlings were grown in a high-humidity, suitable-temperature, and normal-light environment (temperature 20-25℃, relative humidity 70-80%). Simultaneously, during the cultivation period, the epicotyl and lateral buds released from the base of the cotyledons were repeatedly removed.

[0039] Three replicates were set up, with 100 seedlings treated for each excision method at each site in each replicate.

[0040] After being cultured normally for about 10 days 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 wound treatment at different sites was compared.

[0041] The percentage of seedlings that regenerate adventitious buds in situ = (Number of seedlings that regenerate adventitious buds on the cut surface / Total number of seedlings) × 100% — Formula 1

[0042] The results showed that the highest proportion of seedlings regenerating adventitious buds in situ was from the epicotyl (6.46%), while the regeneration of adventitious buds in situ was poor in the hypocotyl and young root regions, at 2.38% and 1.36% respectively. Furthermore, no regeneration was observed in the cotyledon section. Figure 1 The most suitable part of apple rootstock for in-situ regeneration without tissue culture is the epicotyl.

[0043] Example 2: Exploring the optimal retention length for in-situ regeneration of hypocotyls in apple rootstocks

[0044] According to Example 1, it was found that the most suitable non-tissue culture in situ regeneration site for apple rootstock is the epicotyl of the seedling. In order to further obtain the optimal cutting length to be retained during epicotyl in situ regeneration, an experiment was conducted using apple rootstock Pingyi sweet tea.

[0045] Mature and plump apple rootstock seeds of Pingyi sweet tea were selected and subjected to low-temperature stratification treatment (stratification for 30 days under 4℃ humid conditions) to break dormancy, after which the seeds began to sprout. The sprouting seeds were then placed at room temperature for 2-3 days to promote germination. Once the hypocotyl began to elongate, they were sown in seed trays with moist vermiculite substrate and placed in a humid environment at normal room temperature (20-25℃) for germination and growth. Approximately 2-3 days after sowing, the epicotyl elongated, and the cotyledons began to emerge from the soil, followed by cotyledon unfolding and epicotyl elongation.

[0046] After the cotyledons unfold and the epicotyl emerges and elongates, use a scalpel to remove the first true leaf and the portion above it, retaining the cotyledons and leaving part of the epicotyl. See the flowchart. Figure 2 The retained upper axis is available in two lengths:

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

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

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

[0050] Cover the seedlings with plastic wrap to maintain humidity (temperature 20-25℃, relative humidity 70-80%), observe the bud regeneration of the cut surfaces, and after 10 days, calculate the proportion of seedlings with in situ regenerated adventitious buds according to Formula 1, and calculate the lateral bud release rate according to Formula 2.

[0051] Lateral bud release rate = (Number of seedlings sprouting from lateral buds / Total number of seedlings) × 100% (Formula 2)

[0052] When the length of the retained epicotyl is different, the regeneration of the buds shows significant differences, such as... Figure 3 and Figure 4 As shown, when the epicotyl length is short (0-0.2 cm), the cut surface turns brown and cannot regenerate adventitious buds. 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 from the cotyledon base is about 17.6%. This indicates that the optimal length of the epicotyl to be retained after cutting for in situ regeneration of apple rootstock epicotyl is 0.3-0.5 cm.

[0053] Example 3: In-situ regeneration of cotyledon base in apple rootstock (secondary topping)

[0054] Based on the exploratory experiment in Example 2 on the optimal retention length of the hypocotyl for in-situ regeneration of apple rootstock, it was found that after one topping of the hypocotyl, in addition to in-situ regeneration occurring at the cut surface, a certain proportion of lateral buds were also released at the base of the cotyledons. To explore the in-situ regeneration system at the base of the cotyledons in apple rootstock, approximately 10 days after the first topping of the hypocotyl, seedlings with lateral buds released at the base of the cotyledons from groups A and B in Example 2 were taken. Two released lateral buds were removed along the base of the cotyledons using a scalpel. The complete flowchart from the first removal is shown below. Figure 5 This involves a second cutting to remove the top. After cutting, the tube is placed under a moisturizing plastic cover (temperature 20-25℃, relative humidity 70-80%).

[0055] The results showed that adventitious buds would regenerate in situ at the base of the cotyledons after the removal of lateral buds. To investigate the regeneration characteristics of adventitious buds in apple rootstock after a second topping treatment at the base of the cotyledons, this study observed the growth points of bud primordia using sampling and paraffin sectioning techniques. Figure 6 As shown in Figures A and B, the cross-section and longitudinal section diagrams of the sampling site are shown respectively, and the red arrows indicate the specific sampling direction.

[0056] The formation of adventitious bud primordia and their tissue characteristics can be clearly observed through paraffin sectioning. (Observation in cross-section...) Figure 6 In section C), the area indicated by the arrow shows small cell size, dense arrangement, thin cell walls, and large nuclei, exhibiting typical meristem characteristics, indicating that new shoot primordia have formed in this area. In the longitudinal section ( Figure 6In area D), a type of structure with active cell division is also visible, further verifying the in-situ regeneration trend of adventitious buds after deapering treatment. The section results show that after a second deapering treatment at the base of the cotyledons, apple rootstock can stimulate the activation of potential meristematic tissue and induce the in-situ formation of adventitious buds (the same phenomenon is also observed in apple rootstock Malus baccata).

[0057] Ten days after the second topping (removal of released lateral buds), the efficiency of in-situ regeneration of adventitious buds from the base of the cotyledons was calculated according to Formula 3:

[0058] Cotyledon base adventitious bud regeneration rate = (Total number of regenerated buds at the base of cotyledons / Total number of seedlings) × 100% (Formula 3)

[0059] The statistical results are shown in Table 1, with a regeneration rate of 186.1%.

[0060] Table 1. Regeneration of adventitious buds after removing basal lateral buds from the apple rootstock, Pingyi sweet tea (secondary topping).

[0061] 108 201 186.1％

[0062] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for in-situ regeneration of apple rootstock under non-tissue culture conditions, characterized in that, The method includes a step of cutting off the top of the hypocotyl of apple rootstock seedlings, retaining some active apical meristems, and then culturing the cut surface to promote the regeneration of adventitious buds. The length of the retained hypocotyl is 0.3-0.5 cm. The method further includes a step of cutting off the lateral buds along the base of the cotyledons when lateral buds appear, removing the tops again, and then culturing the cut surface to promote the regeneration of adventitious buds; the culturing conditions are a temperature of 20-25℃ and a relative humidity of 70-80%; The apple rootstock is either Pinyi sweet tea or Malus baccata.

2. The application of the method of claim 1 in the regeneration and propagation of apple rootstocks.

3. The application of the method of claim 1 in apple cultivation.

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