Method for rapidly identifying dragon fruit seed seedling high branch change grafting

Abstract

The invention belongs to the technical field of dragon fruit breeding, and particularly relates to a dragon fruit seed seedling high branch change grafting rapid identification method. According to the method for rapidly identifying the dragon fruit seed seedling high branch change grafting, an auxiliary containing glycerol and 6-BA is attached to the surface of a scion cut, the moisture of the cut surface can be maintained, moisture evaporation is reduced, and then wound healing is accelerated; in addition, a wedge grafting mode is combined for grafting, and the survival rate of grafted seedlings is increased due to the fact that the number of sections of wedge-shaped notches is small; in addition, topping and cable-stayed pulling are carried out in the grafting culture process, so that not only is the aging of branches promoted, but also the flowering and fruiting are accelerated, the original 3-5-year identification process of the dragon fruit seedlings is shortened to 5-6 months, and the breeding process of the dragon fruits is greatly accelerated. Therefore, the technical scheme provided by the invention has better production practicability, and provides scientific guidance for dragon fruit seedling breeding and new hybrid variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of dragon fruit breeding technology, specifically involving a method for rapid identification of dragon fruit seedlings through high-branch grafting. Background Technology

[0002] pitaya( Hylocereus undatus Native to South America, this fruit has high nutritional value and has become an important emerging tropical specialty fruit in my country. By the end of 2017, the domestic planting area exceeded 40,000 hectares. 2 The annual output value exceeds 5 billion yuan. With the rapid development of the dragon fruit industry, existing dragon fruit varieties can hardly meet the current needs of the industry's rapid growth. Therefore, it is urgent to breed some superior dragon fruit varieties.

[0003] There are three common methods for propagating dragon fruit seedlings: seedling propagation, cutting propagation, and grafting propagation. Seedling propagation uses dragon fruit seeds and has the longest growth cycle, so it is rarely used nowadays. Cutting propagation is suitable for fast-growing red-skinned, red-fleshed dragon fruit, but not for slow-growing varieties like the "bird's nest" variety. Grafting is a commonly used method, including flat grafting, wedge grafting, approach grafting, insert grafting, and overgrafting. However, these methods are either cumbersome and have low survival rates, or are time-consuming and labor-intensive, resulting in low efficiency, or require a large number of scions and have high requirements for both rootstock and scion, which limits the application of dragon fruit grafting and reduces grafting efficiency and survival rate. Therefore, dragon fruit breeding has a long cycle, high cost, and slow yield, resulting in low willingness and enthusiasm in the industry to engage in this work.

[0004] To accelerate the dragon fruit breeding process, existing technologies often shorten the juvenile stage of dragon fruit seedlings, promoting earlier flowering and fruiting of hybrid offspring to quickly identify the phenotypic characteristics of the hybrids, thereby achieving the goal of accelerating breeding. For example, Chinese patent CN 108617498 B discloses a method for efficient hybrid pollination of dragon fruit, which greatly improves the number of hybrid fruits, single fruit weight, seed purity, fruit development, seed germination rate, and seedling uniformity by optimizing steps such as bagging of pistils and flower buds before flowering and pollination during nighttime flowering; another example is Chinese patent CN208549383. A seedling tray and device for assisting dragon fruit hybridization breeding have been developed. The device allows for humidity adjustment within the seedling tray as needed. Water in the seedling holes is drawn in through capillary action via the water absorption holes, effectively preventing seedlings from rotting due to excessive water absorption. It also supports seedlings in the early stages of growth, preventing lodging and facilitating large-scale seedling production and management. Furthermore, Chinese patent CN107980617A discloses a method for rapidly identifying hybrid dragon fruit offspring with a shortened juvenile period. A method for rapidly identifying dragon fruit hybrid offspring includes: a "young scion on an older scion" grafting process to accelerate seedling growth; after successful grafting, two rounds of forced pinching of the scion's tender shoots to encourage the emergence of third-generation shoots, promoting their transition out of juvenile stage; and once these shoots reach 20-30cm in length, a "long scion on a fruiting branch" grafting process to accelerate flowering and fruiting. This method shortens the generation cycle of dragon fruit hybrid breeding from the original 24-36 months to 8-10 months. While existing technologies can be widely applied to dragon fruit hybrid breeding and new variety propagation, shortening breeding and seedling production timelines, there is still room for improvement in shortening dragon fruit seedling propagation and rapidly developing new hybrid varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapid identification of dragon fruit seedlings through high-branch grafting, which can not only improve the survival rate of dragon fruit propagation, but also accelerate dragon fruit breeding.

[0006] This invention provides a method for rapid identification of dragon fruit seedlings through high-branch grafting, comprising the following steps: After applying the adjuvant to the cut surface of the scion, a pretreated scion is obtained; The pretreated scion is wedge-grafted onto the rootstock for grafting culture to obtain the grafted plant; The grafting cultivation includes: pinching off the top and slanting pull; The adjuvants include glycerol and 6-BA.

[0007] Preferably, the scion comprises: dragon fruit seedlings aged 6-8 weeks.

[0008] Preferably, the additive uses water as a solvent and includes: 0.2% glycerol by volume and 50 mg / L of 6-BA.

[0009] Preferably, the treatment method for the scion incision includes: Remove the cotyledons and roots of the dragon fruit seedlings, and cut the base of the scion into a wedge shape to form a scion incision.

[0010] Preferably, the first pinching is performed on the 30th-35th day after grafting; The time interval between two consecutive pinching operations is 30-35 days; the pinching operation is performed 2-3 times.

[0011] Preferably, the inclined pulling time is 30 days.

[0012] Preferably, the temperature when applying the additive is 25-32℃.

[0013] Preferably, the rootstock includes: 1.5-2.5-year-old Dalbergia odorifera rootstock.

[0014] Preferably, the treatment method for the rootstock includes: After cutting the branches into 20-25cm sections, remove the pulp from the bottom of the branches, leaving 3-5cm of the xylem and removing the areoles from the branches, then air dry them in the shade.

[0015] This invention provides the application of the method described in the above technical solution in improving the survival rate of dragon fruit propagation and / or accelerating dragon fruit breeding.

[0016] Beneficial effects: This invention provides a method for rapid identification of dragon fruit seedlings through high-branch grafting, comprising the following steps: applying an adjuvant to the cut surface of the scion to obtain a pretreated scion; wedging the pretreated scion onto the rootstock for grafting culture to obtain a grafted plant; the grafting culture includes: pinching off the top and oblique pulling; the adjuvant includes: glycerol and 6-BA.

[0017] This invention involves applying an adjuvant containing glycerol and 6-BA to the cut surface of the scion, which helps maintain the moisture of the cut surface, reduces water evaporation, and thus accelerates wound healing. In addition, the grafting method combined with wedge grafting improves the survival rate of grafted seedlings due to the smaller cut surface of the wedge-shaped incision. Furthermore, the pinching and oblique pulling during the grafting cultivation process not only promotes the aging of branches but also accelerates flowering and fruiting. This reduces the original 3-5 year process of identifying dragon fruit seedlings to 5-6 months, greatly accelerating the dragon fruit breeding process. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This invention demonstrates the grafting method provided by the present invention. Figure 2 Flowcharts illustrating different grafting methods for dragon fruit provided by this invention; Figure 3 This invention provides a method for grafting dragon fruit seedlings using a wedge grafting technique. Figure 4 Dragon fruit seedlings at different developmental stages provided by this invention; Figure 5 The effects of different topping methods provided by this invention on promoting branch maturation; Figure 6 A statistical chart showing how different topping methods provided by this invention promote the growth of dragon fruit seedling scions. Detailed Implementation

[0020] When applying the adjuvant to the cut surface of the scion as described in this invention, there are no special requirements for the application method, as long as the adjuvant completely submerges the cut surface of the scion and the cut surface is completely coated with the adjuvant.

[0021] In this invention, there are no special requirements for the methods of pinching off the top and pulling at an angle; techniques known in the art can be used. For example, pinching off the top involves cutting off 1-2 cm of the branch tip to remove apical dominance and promote the development of lateral buds; pulling at an angle involves fixing the top of the dragon fruit branch with a rope, bending it at least 90 degrees to ensure that the top faces the ground, and fixing the other end of the rope to the ground to maintain the apical dominance, thereby altering the transport of auxin and promoting flowering.

[0022] In this invention, unless otherwise specified, there are no special requirements for the specific methods of joining, such as flat joint, wedge joint, contact joint, insertion joint, sleeve joint, and budding, and any method known in the art can be used.

[0023] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1: Grafting method and screening of adjuvants for hybrid dragon fruit seedlings Materials and methods 1. Dragon fruit seedling propagation For rootstock, select branches of the common variety Da Hong that are about 2 years old. Remove weak, diseased, and underdeveloped branches, and cut the branches into 20-25cm sections. Scrape off the pulp from the bottom of the branches, leaving 3-5cm of the xylem for rooting. Cut off the areoles on the branches and let them air dry before use.

[0025] For specific methods, please refer to the relevant content in CN206932730U, "A Rapid Budding Cut for Dragon Fruit" (Lu Shuhua et al., Guangxi Institute of Botany, Chinese Academy of Sciences, Guangxi Zhuang Autonomous Region).

[0026] 2. Dragon fruit seedlings Using seeds from the self-pollinated fruit of the common dragon fruit variety, Big Red, as material, the peel and pulp were removed, and the seeds were sown in seedling pots. The soil was kept warm and moist to promote seed germination. Seedlings at different growth stages (i.e., seedlings at different times after seed germination) were used as scions for grafting to carry out related experiments.

[0027] 3. Grafting method Dragon fruit propagation methods include flat grafting, wedge grafting, approach grafting, insert grafting, loop grafting, and bud grafting. Specific grafting methods are as follows: Figure 1 As shown.

[0028] The main problems with approach grafting are: the rootstock and scion are not tightly joined, the scion is prone to falling off, and the survival rate is not high; the main problems with cleft grafting are: slow scion preparation, low efficiency, and low survival rate; the main problems with overlay grafting are: the xylem of the rootstock directly connects to the fleshy part of the scion; there is less sap; the contact is not tight, resulting in a low survival rate, and the results are as follows. Figure 2 As shown (in) Figure 2In the diagram, Figures A-F represent grafting, with Figure A showing the completed grafting process; Figure B showing the completed grafting process; Figure C showing the survival rate of the grafted seedling; Figure D showing the initial state of pith healing between the rootstock and scion; Figure E showing the anatomical diagram of pith lignification healing after the grafted seedling has survived; Figure F showing relatively smooth and complete pith lignification healing; Figures GL represent cleft grafting, with Figure G showing the pretreatment of the scion before cleft grafting, where the fleshy stem is scraped off, leaving 3-5 cm of lignified pith; Figure H showing the insertion of the scion into the rootstock. Partial; Figure I shows the completed grafted seedling; Figure J shows the binding and fixing method; Figure K shows the abnormal healing after the grafted seedling survives; Figure L shows the anatomical diagram of the healing of the abnormal lignified pith; Figure MU shows the approach graft, where Figure M shows the pretreatment of the rootstock grafting surface, requiring the rootstock grafting surface to be smooth and slightly larger than the scion; Figure N shows the scion cutting diagram; Figure O shows the other side of the scion, requiring 2-3 buds; Figure P shows the scion, requiring a smooth cut surface without xylem pith; Figure Q shows the scion and rootstock in contact, and the contact... After grafting, the graft is secured by binding; Figure R shows grafting failure due to the scion drying out after grafting; Figure S shows grafting failure due to loose adhesion; Figure T shows grafting failure due to the scion not sprouting; Figure U shows grafting failure due to a narrow healing surface; Figures V and W show groove grafting, with Figure V showing grafting failure due to a side groove graft; Figure W shows grafting failure due to excessive callus tissue formation in a side groove graft; Figure X shows grafting failure due to the scion drying out in a top groove graft; Figures Y-A4 show budding, with Figure Y showing the rootstock before budding. Wood pretreatment involves cutting off the buds with an L-shaped stainless steel knife to create a right-angle grafting surface; Figure Z shows a dragon fruit budding knife, which can be used for pretreatment of rootstock and scion; Figure A1 shows a scion in excellent condition, ideally with growing buds; Figure A2 shows a scion that is too small and cannot fully fit the grafting surface of the rootstock, resulting in a low survival rate; Figure A3 shows a budded scion without young buds, leading to slow growth of the grafted seedling; Figure A4 shows a relatively successful budding, with vigorous scion growth and a completely and smoothly healed graft union.

[0029] Therefore, three grafting methods—flat grafting, wedge grafting, and approach grafting—were selected to graft dragon fruit seedlings to compare the grafting success rates of different methods.

[0030] Specific experimental procedure: Two-month-old dragon fruit seedlings were used as materials and grafted onto *Pterocarya stenoptera* rootstock using three methods: flat grafting, wedge grafting, and approach grafting. Each treatment had at least 15 scions, and the grafts were repeated once. After two weeks of cultivation in the same environment, the effect of grafting method on survival rate was statistically analyzed (correlation and significance analysis were performed using Excel). The results are shown in Table 1 and [Table data would be inserted here]. Figure 3 AF in Figure 3 In the diagram, A shows a 2-month-old dragon fruit seedling; B shows the seedling with its cotyledons and roots removed to prepare the scion; C shows the base of the scion cut into a wedge shape; D shows each ridge of the red rootstock with a wedge-shaped cut; E shows the scion being inserted into the wedge-shaped cut of the rootstock; and F shows the graft union secured with tape.

[0031] Table 1. Survival rate statistics for three grafting methods

[0032] Based on the data in Table 1, it was found that the survival rates of both flat grafting and approach grafting methods were low. The main reasons were the tenderness of the scions, the excessively large incisions, and the tendency for the scions to dry out easily due to water loss. Two weeks later, the survival rates were 15.07±2.57% and 28.30±3.30%, respectively. Figure 3 It can be seen that compared with flat grafting and approach grafting, the grafting survival rate of wedge grafting is as high as 47.01±5.88%. The possible reason is that the wedge-shaped cut has less surface area, which can effectively reduce water evaporation and allow more time for the cut surface between the graft and the rootstock to heal.

[0033] A comparison of different grafting methods revealed that while wedge grafting had the highest survival rate, it was still insufficient for the requirements of hybrid population analysis, with only about 50% survival. The resulting populations were prone to losing important traits, leading to population deviations and affecting subsequent analysis. Improving the survival rate is a crucial aspect of grafting technique optimization. Reducing moisture evaporation from the cut surface while accelerating the healing of the two cut surfaces is essential. Therefore, further improvements in survival rate can be achieved through the use of adjuvants.

[0034] 4. Composition and usage of adjuvants (protective and accelerator agents) The main function of adjuvants (protective and accelerators) is to accelerate the healing process between the scion and rootstock, thereby increasing the graft survival rate. Before joining the scion and rootstock cuts, the scion cut is dipped in the adjuvant before joining to promote healing. Different hormones are combined with different solvents in the adjuvants (protective and accelerators), and the specific composition of the adjuvants is shown in Table 2 below.

[0035] Table 2 Composition and concentration of different additives

[0036] Note: When preparing the excipients listed in Table 2, if the excipient contains 6-BA, it should first be dissolved in anhydrous ethanol. For example, dissolve 50 mg of 6-BA in 2-3 mL of anhydrous ethanol, and then bring the volume up to a final volume with sterile water.

[0037] Detailed experimental procedure: Wedge grafting was used. Before grafting, the cut end of the scion was dipped in one of the adjuvants listed in Table 1. Each adjuvant was used as one treatment, with a control group (scions grafted directly without adjuvant) as a blank control. At least 15 scions were used for each treatment, and the experiment was repeated once. After two weeks of cultivation in the same environment, the average survival rate of the grafted seedlings was calculated. The results are shown in Tables 3 and 4. Figure 3 GI in Figure 3In the diagram, graph G represents the result after treatment with adjuvant number 2 in Table 2; graph H represents the result after treatment with adjuvant number 1 in Table 2; and graph I represents the result after treatment with adjuvant number 3 in Table 2.

[0038] Table 3. Statistical analysis of the effects of different adjuvant treatments on grafting survival rate of dragon fruit seedlings.

[0039] Based on the data in Table 3, both 6-BA and NAA improved the survival rate, with 6-BA showing a stronger effect than NAA. NAA tends to produce aerial roots and multiple buds, which is not conducive to subsequent experiments. Adding lanolin and glycerin can improve the survival rate, but adding glycerin is more effective. Lanolin has too strong a water-retention capacity, which can easily lead to bud wilting. Overall, adjuvant No. 3 showed the best effect, with a survival rate as high as 85.62±4.62%, which is 38.56% higher than the 47.06±5.88% of the control group (without adjuvant), showing the most significant effect, promoting faster wound healing, and faster bud sprouting.

[0040] Example 2: The impact of different sizes of dragon fruit hybrid seedlings on the low success rate of high-branch grafting. The experimental results of Example 1 showed that pretreatment with the wedge grafting agent effectively improved the survival rate of grafted seedlings. However, the grafted seedlings selected in Example 1 were all two-month-old seedlings. To accelerate the breeding and identification process, it is worthwhile to further explore whether smaller seedlings can also be successfully grafted. Therefore, the following experiment was conducted: Morphology of dragon fruit seedlings at different developmental stages is shown in the figure. Figure 4 (exist Figure 4 In the diagram, Figure A shows seedlings one week after germination, Figure B shows seedlings two weeks after germination, Figure C shows seedlings three weeks after germination, Figure D shows seedlings four weeks after germination, Figure E shows seedlings six weeks after germination, and Figure F shows seedlings eight weeks after germination.

[0041] Combination Figure 4 Observations revealed that in the third week after germination, the growth of the fleshy stem between the two cotyledons could be observed. As the growth time increased, the fleshy stem continued to elongate and thicken, eventually becoming a scion.

[0042] Dragon fruit seedlings were selected at four, six, and eight weeks after germination, respectively. The cotyledons and roots of the seedlings were removed to serve as scions, and the base of the scions was cut into a wedge shape. The adjuvant numbered 3 in Table 2 was selected, and the cut ends of the scions in each treatment were dipped in the adjuvant (ensuring that the adjuvant completely submerged the cut ends). Wedge-shaped incisions were made on each ridge of the red rootstock, and the adjuvant-dipped scions were inserted into the wedge-shaped incisions of the rootstock. The connection between the rootstock and the scion was fixed with tape. After two weeks, the survival rate and growth of the grafted seedlings in each treatment were observed. Each treatment had no less than 15 scions and was repeated once. The results are shown in Table 4.

[0043] Table 4. Survival rate of dragon fruit seedlings grafted at different growth stages

[0044] The statistics in Table 4 show that the survival rates of dragon fruit seedlings after grafting at 4, 6, and 8 weeks of germination were 50.34±8.35%, 83.77±1.62%, and 86.30±4.33%, respectively, with the survival rate increasing with the growth time of the seedlings. Further observation of the scion phenotype in the later stages of grafting revealed that 4-week-old scions were more tender, prone to wilting and drying, had smaller growing points, making grafting more difficult and less suitable for securing with tape, which could easily compress the scion and cause secondary damage. Considering the need for further analysis of the hybrid population, a survival rate that is too low is unsuitable for further experiments; therefore, seedlings at 6 weeks of germination are recommended as more suitable scions.

[0045] Example 3: Pinch off the top to promote aging and accelerate flowering of grafted seedlings Based on the results of Example 2, seedlings that had germinated for 6 weeks were selected, with the cotyledons and roots removed, and the base of the scion was cut into a wedge shape. Adjuvant number 3 in Table 2 was selected, and the cut end of the scion was dipped in the adjuvant (ensuring the adjuvant completely submerged the cut end). Wedge-shaped incisions were made on each ridge of the *Cephalotaxus fortunei* rootstock. The adjuvant-dipped scion was then inserted into the wedge-shaped incisions on the rootstock, and the connection between the rootstock and scion was secured with tape. After one month (30 days) of cultivation at 20-35℃, the first topping was performed; the second topping was performed after two months; and the third topping was performed after three months. One month after the final topping, the effects of only one topping, two toppings, and three toppings on promoting branch growth and maturation were statistically analyzed. The results are shown in Table 5. Figure 5 (exist Figure 5 In the diagram, A shows the growth of branches after primary topping; B shows the growth of branches after secondary topping; C shows the growth of branches after tertiary topping; D shows deformed seedlings; and E shows normal growth. Figure 6 .

[0046] Table 5. Effects of different topping times on the growth rate of dragon fruit branches.

[0047] Note: "***" indicates that there is a significant difference between the groups. p <0.01) By observing the growth process and combining the data in Table 5, it was found that pinching promotes branch growth. The more times the branches are pinched, the more significant the growth promotion. Pinching twice and three times significantly promotes branch growth longer than pinching once, and pinching three times significantly promotes growth longer than pinching twice. Furthermore, the rate of maturation and aging also increases with branch length. Scions exceeding 50cm in length can be forcibly bent to induce flowering; therefore, pinching two or three times is sufficient to induce flowering.

[0048] One month after the grafted seedlings that had been pinched three times were bent at the top towards the ground using the oblique pulling method, the proportion of flower buds was counted. According to preliminary statistics, about 80% of the grafted seedlings that had been pinched three times produced flower buds after one month of bending at the top towards the ground using the oblique pulling method, which is sufficient for subsequent identification steps. Grafted seedlings that were not subjected to oblique pulling treatment needed to grow to about one meter in length and droop naturally before they could be stimulated to bloom, which required an additional 2-3 months for the entire process.

[0049] In summary, this method can reduce the dragon fruit seedling identification process, which originally took 3-5 years, to 5-6 months, greatly accelerating the dragon fruit breeding process.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid identification of dragon fruit seedlings through high-branch grafting, characterized in that, Includes the following steps: After applying the adjuvant to the cut surface of the scion, a pretreated scion is obtained; The pretreated scion is wedge-grafted onto the rootstock for grafting culture to obtain the grafted plant; The grafting cultivation includes: pinching off the top and slanting pull; The adjuvants include glycerol and 6-BA.

2. The method according to claim 1, characterized in that, The scions include: dragon fruit seedlings that are 6-8 weeks old.

3. The method according to claim 1, characterized in that, The additive uses water as a solvent and includes: 0.2% glycerol by volume and 50 mg / L of 6-BA.

4. The method according to claim 1, characterized in that, The treatment methods for the scion incision include: Remove the cotyledons and roots of the dragon fruit seedlings, and cut the base of the scion into a wedge shape to form a scion incision.

5. The method according to claim 1, characterized in that, The first pinching should be performed on the 30th-35th day after grafting; The time interval between two consecutive pinching operations is 30-35 days; the pinching operation is performed 2-3 times.

6. The method according to claim 1, characterized in that, The time for the inclined plane was 30 days.

7. The method according to claim 1, characterized in that, The temperature for applying the additive is 25-32℃.

8. The method according to claim 1, characterized in that, The rootstocks include: 1.5-2.5 year old Dalbergia odorifera rootstocks.

9. The method according to claim 1 or 8, characterized in that, The treatment methods for the rootstock include: After cutting the branches into 20-25cm sections, remove the pulp from the bottom of the branches, leaving 3-5cm of the xylem and removing the areoles from the branches, then air dry them in the shade.

10. The application of the method according to any one of claims 1-9 in improving the survival rate of dragon fruit propagation and / or accelerating the breeding of dragon fruit.

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