A method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources

Abstract

This invention belongs to the field of forest tree breeding and seedling technology, specifically involving a method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources, including the following steps: selecting seedlings grown to have three leaf lobes or clonal seedlings, and observing the seedling population when the top leaf lobes are in a stable growth period; observing the length of the leaflets and whether the distance between the three leaflets is separated in the lower part of the second leaf lobes from the top leaf lobes as preliminary selection criteria, using each plant or clonal line as a unit; selecting those that simultaneously meet the conditions of short and thick leaflets and non-separation of the distance between the three leaflets as drought-resistant germplasm resources; and further selecting the selected germplasm resources by subjecting them to natural drought stress, and determining that plants with only a few wrinkles at the top of the top leaf lobes and no softening and drooping of the petioles are drought-resistant germplasm. This invention enables efficient, low-cost, large-scale, and precise identification of drought resistance in rubber tree germplasm resources, with an identification accuracy rate of over 90%. It can also significantly shorten the drought resistance identification time and accelerate the process of drought-resistant rubber tree breeding.

Description

Technical Field

[0001] This invention belongs to the field of forest tree breeding and seedling technology, specifically relating to a method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources. Background Technology

[0002] Natural rubber is an important strategic resource. Latex is the primary byproduct of rubber trees in the production of natural rubber; it is a white, milky substance. During plant evolution, latex production may be related to its defense against pests and various forms of abiotic stress. Although there are over 2,000 plant species worldwide that produce latex, the rubber tree, with its unique advantages such as tall and robust growth, long productive lifespan, high latex yield, and high natural rubber content (over 30%), is currently the only species used for large-scale industrial production of natural rubber.

[0003] my country lacks wild rubber tree resources, and introduced varieties exhibit poor overall adaptability, posing significant challenges to the cultivation of superior varieties. While large-scale production and planting bases have been established in Hainan, Yunnan, and Guangdong, over 70 years of practice have proven Yunnan to be my country's best rubber-growing region, boasting the highest average yield per unit area, minimal typhoon damage, and relatively low risk. Despite Yunnan's status as my country's best rubber-growing region, several unfavorable factors remain. While wind damage is less severe, seasonal drought is significantly exacerbated in Yunnan's rubber-growing areas. Furthermore, the increasingly severe winter and spring droughts in Yunnan in recent years have intensified, making seasonal drought more pronounced in most rubber-growing areas. The most direct impact of this drought is delayed leaf emergence, inhibited tree growth, increased pests and diseases such as powdery mildew, and delayed tapping, leading to reduced yields and becoming a serious constraint on the rubber industry.

[0004] Therefore, given the complex and ever-changing climate conditions in Yunnan and the increasingly frequent occurrence of natural disasters such as drought, it is particularly important to select rubber tree germplasm with strong drought resistance for the cultivation of superior drought-resistant and water-saving varieties. This will not only ensure high and stable yields, but also play a vital role in conserving limited water resources.

[0005] Currently, research on the drought resistance identification of rubber trees is scarce. Previous studies have used artificial water stress to measure physiological substances in leaves to identify a limited number of varieties or clones. This identification method involves indoor physiological and biochemical index identification, which requires a high level of professional skill from the operators, is cumbersome, requires significant manpower and resources, is costly, and is difficult to scale up quickly. Furthermore, it suffers from poor repeatability. This method fails to achieve the goals of "rapid" and "large-scale" implementation, thus hindering the large-scale screening of drought-resistant rubber tree germplasm resources. Summary of the Invention

[0006] The purpose of this invention is to overcome the limitations of single methods for identifying drought-resistant germplasm resources of rubber trees and the shortcomings of laboratory identification techniques. It provides an efficient and large-scale method for identifying the drought resistance of rubber tree germplasm resources, solving problems such as difficulty in identifying drought resistance of rubber tree germplasm resources, high testing costs, and low efficiency. This method enables accurate, rapid, and large-scale drought resistance identification results, and is not affected by laboratory conditions, thereby accelerating the process of drought-resistant breeding of rubber trees.

[0007] The objective of this invention is achieved through the following technical solution: a method for efficiently and on a large scale screening of drought-resistant rubber tree germplasm resources, comprising the following steps: (1) Cultivation of seedlings and selection of leaf foliage: Seedlings or clonal seedlings with 3 leaf foliages were cultivated by field seedling cultivation method. Seedlings with good growth (good means no visible diseases, pests and damage) with 3 leaf foliages and the top leaf foliage in a stable growth period were selected for observation.

[0008] (2) Selection of leaf trait indicators: Using plants or clones as units, observe the length of the leaflets in the middle of the second leaflet from the top leaf and whether the distance between the three leaflets is separated.

[0009] (3) Characteristics of drought-resistant germplasm in the field: Plants that simultaneously meet the two conditions of short and thick leaflet pillow and non-separation of the three leaflets are initially selected as drought-resistant germplasm resources.

[0010] Preferably, the method for determining the length of the leaf pulvinus in step (3) is as follows: the determination is made based on the actual measured length of the leaf pulvinus. If the length of the leaf pulvinus of the leaf to be tested is less than 0.60cm, it is considered short and thick; otherwise, it is considered long and thin.

[0011] Preferably, the method for determining whether the three leaflets are not separated in step (3) is as follows: determine whether the three leaflets are separated based on their proximity. If the three leaflets are in contact with each other, they are determined to be not separated; otherwise, they are separated.

[0012] Preferably, in step (2), the observation method is field visual inspection and measurement statistics.

[0013] Preferably, a method for efficiently and on a large scale identifying the drought resistance of rubber tree germplasm resources is characterized by further comprising the following steps: Re-identification and screening of drought-resistant plants: The germplasm resources initially selected as drought-resistant in step (3) were subjected to natural drought stress treatment, and drought-resistant plants were screened again according to the degree of leaf damage.

[0014] Preferably, the method of natural drought stress treatment is as follows: the plants initially selected as drought-resistant in step (3) are placed in an artificial climate chamber with an indoor temperature of 28°C, humidity of 30%, light intensity of 1500 lx, and natural drought culture for 8 days.

[0015] Preferably, the method for further screening of drought-resistant plants based on the degree of leaf damage is as follows: Plants with wrinkles appearing in less than or equal to 1 / 3 of the top of the main stem, while more than 80% of the large petioles of the entire plant do not wilt or droop (not drooping means the angle between the large petiole and the main stem is greater than or equal to 80°), are considered drought-resistant germplasm resources. Plants with wrinkles appearing in more than 1 / 3 of the entire stem from the top leaf to the base of the plant, with dense wrinkles at the top of the stem, while more than 80% of the large petioles of the entire plant wilt and droop at an angle less than 80° to the main stem, are considered drought-resistant germplasm resources.

[0016] Compared with existing technologies, this invention provides a method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources, which has the following beneficial effects: (1) Using the drought resistance identification method of the present invention, rubber trees with strong drought resistance can be selected in large quantities quickly, with an accuracy rate of over 90%.

[0017] (2) The present invention is low in cost and easy to operate. It can be identified by visual observation of leaf morphological indicators and can be efficiently and on a large scale.

[0018] (3) The present invention requires a short cycle and is not affected by laboratory testing technology. It can greatly shorten the time for identifying the drought resistance of rubber trees, improve the identification effect, and accelerate the process of selecting and breeding drought-resistant rubber trees. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the three leaf foliages of a rubber tree.

[0020] Figure 2 The morphology is that of a small leaflet pulvinus, where A is a short and thick leaflet pulvinus and B is a slender leaflet pulvinus.

[0021] Figure 3 The diagram shows the separation of the three lobules, where A and B are not separated, and C shows the separation of the three lobules.

[0022] Figure 4 The images show the morphology of stem wrinkles and large petioles after natural drought. In image A, the stem tip is slightly wrinkled, and most of the large petioles have not softened and drooped. In image B, the stem has wrinkles from the tip to the base, and most of the petioles have softened and drooped. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 A method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources includes the following steps: (1) Selection of seedling leaf canopy: 2,500 open pollinated seeds of rubber tree variety GT1 were collected and sown in a conventional sand bed for germination. After about 15 days, seedlings with two large petioles were grown, resulting in a total of 2,100 seedlings. They were transplanted into nutrient bags as individual plants and managed conventionally. After about 7 months of cultivation, when the seedlings had grown to have three leaf canopies and the top leaf was in a stable growth period, the seedling population was observed and identified.

[0025] (2) Observation of leaf trait indicators: On a single plant basis, observe the length of the leaf pulvinus and the separation of the three leaflets in the lower part of the second leaf pulvinus from the top leaf pulvinus as leaf trait indicators. Figure 1 Three schematic diagrams of the Rubber Tree are shown.

[0026] Reference Figure 2 and Figure 3 The specific methods for determining whether the length of the lobule pillow and the distance between the three lobules are separated are as follows: Method for judging the length of leaflet pulvinus: The judgment is based on the actual measured length of the leaflet pulvinus. If the length of the leaflet pulvinus of the leaf foliage to be measured is less than 0.60cm, it is considered short and thick; otherwise, it is considered long and thin. Figure 2 Figure A shows a short and thick small leaf pillow, while Figure B shows a long and thin small leaf pillow; Method for determining whether the lobules are separated: Determine whether the lobules are separated based on their proximity. If all three lobules are in contact, they are considered not separated; otherwise, they are considered separated. Figure 3 Figures A and B show three lobules that are not separated, while Figure C shows three lobules that are separated.

[0027] (3) Classification of drought-resistant plants in the field: Plants that simultaneously meet the two conditions of leaf trait index ① short and thick leaflet pillow and ② non-separation of the three leaflets are initially selected as drought-resistant germplasm resources. Finally, 96 drought-resistant plants were obtained in the field (Table 1).

[0028] The classification results are shown in Table 1 below.

[0029] Table 1. Initial field screening results of offspring seedlings of rubber tree variety GT1 (4) Natural drought stress treatment: The drought-resistant plants (96 plants) identified in step (3) were placed in an artificial climate chamber with an indoor temperature of 28°C, humidity of 30%, light intensity of 1500 lx, and natural drought culture for 8 days.

[0030] (5) Further screening of drought-resistant plants: Observe the seedlings after natural drought in the artificial climate chamber in step (4), and record the changes in the petioles and stems of the seedlings in step (4). The drought resistance is quickly determined in batches based on whether the petioles wilt and droop and the extent of wrinkles on the stems. The specific judgment method is as follows: If less than 1 / 3 of the top of the main stem has wrinkles, and more than 80% of the petioles of the entire plant do not wilt and droop, it is judged as a germplasm resource with strong drought resistance; If wrinkles appear on the entire stem from the top leaf to the base of the plant, and the wrinkles are dense at the top of the stem, and more than 80% of the petioles of the entire plant wilt and droop at an angle of less than 80° to the main stem, it is judged as a germplasm resource with weak drought resistance (refer to...). Figure 4 ), Figure 4 Figure A in the middle shows a rubber tree with strong drought resistance. Figure 4 Figure A shows that the angle between the large petiole and the main stem is greater than or equal to 80°, and the wrinkles on the main stem are less than or equal to 1 / 3 of the main stem; Figure B shows a rubber tree with weak drought resistance. Figure 4 As can be seen in B, the angle between the large petiole and the main stem is less than 80°, and the wrinkles on the main stem are greater than 1 / 3 of the main stem.

[0031] The results of the further filtering and classification are shown in Table 2 below.

[0032] Table 2 Results of rapid large-scale screening of drought-resistant germplasm resources from progeny seedlings of rubber tree variety GT1 As shown in Table 2, this invention can rapidly and on a large scale screen 96 drought-resistant plants from 2100 rubber trees using only a field screening method. Further screening and verification using an artificial climate indoor natural drought stress identification method confirmed that 89 of the initially selected 96 plants exhibited strong drought resistance. Therefore, the screening method of this invention is low-cost, easy to operate, accurate, and efficient, significantly accelerating the identification and breeding process of drought-resistant rubber tree germplasm.

[0033] Example 2 A method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources includes the following steps: (1) Selection of seedling leaf canopy: The open pollinated seedlings of rubber tree variety GT1 were used to cultivate bagged seedlings as rootstocks. 140 branches of Wei Kehan ​​germplasm that sprouted in the current year were used as scions. After bud grafting, asexual seedlings were obtained. After routine management and cultivation for 7 months, when the seedlings grew to have 3 leaf canopies and the top canopy leaves were in a stable growth period, the seedling population was observed and identified.

[0034] (2) Observation of leaf trait indicators: On a single plant basis, observe the length of the leaf pulvinus and the separation of the three leaflets in the lower part of the second leaf pulvinus from the top leaf pulvinus as leaf trait indicators. Figure 1 Three schematic diagrams of the Rubber Tree are shown.

[0035] Reference Figure 2 and Figure 3 The specific methods for determining whether the length of the lobule pillow and the distance between the three lobules are separated are as follows: Method for judging the length of leaf pulvinus: The length of the leaf pulvinus is judged based on the actual measured length. If the length of the leaf pulvinus of the test leaf stalk is less than 0.60cm, it is short and thick; otherwise, it is slender and long. Method for determining whether the three lobules are separated: Determine whether the three lobules are separated based on their proximity. If the three lobules are in contact with each other, they are considered not separated; otherwise, they are considered separated.

[0036] (3) Classification of drought-resistant plants in the field: Plants that simultaneously meet the two conditions of leaf trait index ① short and thick leaflet pillow and ② non-separation of the three leaflets are initially selected as drought-resistant germplasm resources. Finally, 33 samples were screened and the classification results are shown in Table 3 below.

[0037] (4) Natural drought stress treatment: The drought-resistant plants (33 samples) identified in step (3) were placed in an artificial climate chamber with an indoor temperature of 28°C, humidity of 30%, light intensity of 1500 lx, and natural drought culture for 8 days.

[0038] (5) Screening of drought-resistant plants: Observe the seedlings after natural drought in the artificial climate chamber in step (4), and record the changes in the petioles and stems of the seedlings in step (4). The drought resistance is quickly determined in batches by whether the petioles wilt and droop and the extent of wrinkles on the stems. The specific judgment method is as follows: If wrinkles appear in less than 1 / 3 of the stem near the top leaf, and more than 80% of the petioles of the entire plant do not wilt and droop, it is judged as a germplasm resource with strong drought resistance; If wrinkles appear on the entire stem from the top leaf to the base of the plant, and the wrinkles are dense at the top of the stem, and more than 80% of the petioles of the entire plant wilt and droop at an angle of less than 80° to the main stem, it is judged as a germplasm resource with weak drought resistance (refer to...). Figure 4 The classification results are shown in Table 3 below.

[0039] Table 3 Results of rapid large-scale screening of drought-resistant rubber tree germplasm resources using the Wei Kehan ​​clone. As shown in Table 3, this invention can rapidly and on a large scale screen 33 drought-resistant plants from 140 rubber tree germplasm resources using only a field screening method. Further screening and verification using an artificial climate indoor natural drought stress identification method confirmed that 30 of the 33 initially screened germplasm resources exhibited strong drought resistance. Therefore, the screening method of this invention is low-cost, easy to operate, accurate, and efficient, significantly accelerating the process of drought-resistant rubber tree breeding.

[0040] Example 3 The drought-resistant plants selected after natural drought stress treatment in steps (4) and (5) of Examples 1 and 2 were verified by measuring the relative conductivity of the plants. The smaller the conductivity, the stronger the drought resistance, and the larger the conductivity, the weaker the drought resistance. The budded clonal seedlings of the drought-resistant rubber tree variety GT1 were used as the control.

[0041] The experimental results are as follows: The experimental results above show that the method for screening rubber tree germplasm resources with strong drought resistance provided by this invention has an accuracy rate of over 90%. Therefore, this invention is more convenient, faster, and more accurate than existing technologies.

[0042] The above description is merely a partial embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources, characterized in that: Includes the following steps: (1) Cultivation of seedlings and selection of leaf canopy: The rubber tree seeds were cultivated to grow into seedlings or clonal seedlings with 3 leaf canopies. Seedling groups with 3 leaf canopies and no visible diseases, pests and damage were selected for observation. (2) Selection of leaf trait indicators: On a plant or clone basis, observe the length of the leaflet pulvinus and the distance between the three leaflets in the middle of the second leaflet pulvinus from the top leaflet of the seedling. (3) Characteristics of drought-resistant germplasm in the field: Plants that simultaneously meet the two conditions of ① short and thick leaflet pillow and ② non-separation of the three leaflets are initially selected as drought-resistant germplasm resources. In step (3), the method for determining the length of the leaf pulvinus is as follows: the determination is based on the actual measured length of the leaf pulvinus. If the length of the leaf pulvinus of the leaf to be tested is less than 0.60cm, it is short and thick; otherwise, it is slender. In step (3), the method for determining whether the three lobes are not separated is as follows: determine whether the three lobes are separated based on their proximity. If the three lobes are in contact with each other, they are determined to be not separated; otherwise, they are separated.

2. The method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources according to claim 1, characterized in that: In step (2), the observation method is field visual inspection and measurement statistics.

3. The method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources according to any one of claims 1-2, characterized in that: It also includes the following steps: Re-identification and screening of drought-resistant plants: The germplasm resources initially selected as drought-resistant in step (3) were subjected to natural drought stress treatment, and drought-resistant plants were screened again according to the degree of leaf damage.

4. The method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources according to claim 3, characterized in that: The method of natural drought stress treatment is as follows: the drought-resistant plants identified in step (3) are placed in an artificial climate chamber with an indoor temperature of 28°C, humidity of 30%, light intensity of 1500 lx, and natural drought culture for 8 days.

5. The method for efficient and large-scale screening of drought-resistant rubber tree germplasm resources according to claim 3, characterized in that: The method for further screening drought-resistant plants based on the degree of leaf damage is as follows: Plants with wrinkles on less than 1 / 3 of the top of the main stem, and where more than 80% of the large petioles of the entire plant do not wilt or droop, are considered drought-resistant germplasm resources. Plants with wrinkles on more than 1 / 3 of the stem from the top leaf to the base of the plant, especially with denser wrinkles at the top, and where more than 80% of the large petioles wilt and droop at an angle of less than 80° to the main stem, are considered drought-resistant germplasm resources.

Images