CN118451941A - A method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources - Google Patents

Abstract

The invention belongs to the technical field of forest genetic breeding, and particularly relates to a method for rapidly screening cold-resistant rubber tree germplasm resources in a large scale, which comprises the following steps: 1. selecting seedlings to grow to 3-4 She Pengdai seedlings or clone, and observing the seedling group when the canopy leaves are in a stable growth period; 2. taking a plant or clone as a unit, observing the petiole length, the leaf base included angle and the first-stage lateral pulse distribution regularity of the lower leaf in the second She Peng from the top She Peng downwards of the seedling as leaf character indexes; 3. plants meeting more than two indexes of ① small petiole length smaller than similar groups, ② large leaf basal included angle and ③ first-level lateral pulse distribution irregularity are classified as plants with strong cold resistance. The invention can select the germplasm of the rubber tree with strong cold resistance in batches with high efficiency and low cost, has the discrimination accuracy rate of more than 90 percent, can greatly shorten the cold resistance identification time and accelerate the cold resistance selection breeding process of the rubber tree.

Description

A method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources

Technical Field

The invention belongs to the technical field of forest tree genetic breeding, and in particular relates to a method for rapid and large-scale screening of rubber tree germplasm resources with strong cold resistance.

Background Art

The rubber tree (Hevea brasiliensis) is a perennial tropical tree species native to the Amazon River Basin in South America. It is currently the only source of natural rubber that can be industrialized on a large scale. The average annual temperature suitable for the growth of rubber trees is 20-30℃. When the temperature is below 10℃, they will suffer varying degrees of cold damage. China's rubber planting area is located on the northern edge of the tropics and is a non-traditional rubber planting area. Low temperature is the main reason restricting the development of the rubber industry in this area. The Yunnan rubber planting area is located at a latitude that is more northerly and at a higher altitude. There is insufficient heat in winter, and cold damage often occurs, causing great losses to the rubber tree industry. Therefore, it is particularly important to carry out the identification of cold-resistant germplasm of rubber trees.

At present, the main methods for identifying cold-resistant germplasm are: cold-resistant sentinel nursery comparison identification method, field natural low temperature identification method and indoor physiological and biochemical index identification. These methods have obvious disadvantages, are time-consuming and labor-intensive, and have low efficiency. For example, the sentinel nursery and natural low temperature identification methods have high requirements for the environmental climate of the selected test site, and both need to wait until winter to carry out identification, which is subject to strong time constraints; at the same time, affected by the weather, either no extreme low temperature is encountered, no cold damage occurs, or the cold damage is severe, all plants have been harmed, resulting in identification failure. The identification of indoor physiological and biochemical indicators requires a high level of professional and technical skills of the operator, the steps are cumbersome, and a large amount of manpower and material resources are required. The measurement cost is high, and it is difficult to quickly achieve large-scale testing, and there are also problems such as poor repeatability. These methods are difficult to achieve the goals of "rapidity" and "scale".

Summary of the invention

The object of the present invention is to overcome the deficiencies in the prior art and provide a method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources, so as to solve the problems of long cycle, large climate influence, high determination cost and low efficiency in cold-resistant identification of rubber tree germplasm resources, so that the cold-resistant identification result is accurate and fast, and is not restricted by season, thereby accelerating the cold-resistant breeding process of rubber trees.

The object of the present invention is to achieve the following technical scheme: a method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources, comprising the following steps:

S1: Selection of seedling leaf canopy: Select seedlings grown from bagged seedlings or clones with 3-4 leaf canopies, and observe the seedling population when the top canopy leaves are in the stable growth period;

S2: Observation of leaf trait indexes: Taking the plant or clone as the unit, the petiole length, leaf base angle and the regularity of the primary lateral vein distribution of the leaves in the lower part of the second leaf canopy from the top leaf canopy of the seedlings were observed as leaf trait indexes;

S3: Classification of plants with strong cold resistance in the field: Plants that meet two or more of the following leaf trait indicators: ① The length of the petiole is significantly smaller than that of the same group, ② The angle of the leaf base is large, and ③ The distribution of the primary lateral veins is irregular are classified as plants with strong cold resistance.

Further, in step S3, the method for determining whether the petiole length is significantly smaller than that of the same group is as follows:

Through internal comparison within the group, if the petiole length of the lower leaf in the second leaf canopy below the top leaf canopy is less than 25% of the petiole length of plants in the same group, it is judged that the petiole length is significantly smaller than that of the same group, otherwise it is the general petiole length;

The method for determining that the leaf base angle is large is as follows:

The judgment is made based on the angle between the main vein in the middle of the leaf base and the leaf edge. If the leaf base angle of the lower leaf in the second leaf canopy from the top leaf canopy of the plant to be judged is greater than 30°, it is judged that the leaf base angle is large; otherwise, the leaf base angle is small.

The method for determining the irregular distribution of the first-level lateral veins is as follows:

The judgment is made based on the parallelism of the lateral vein arrangement, whether it extends to the leaf edge, and whether there is any crossing. If most of the lateral veins in the lower leaves of the second leaf canopy from the top leaf canopy of the plant to be judged have poor parallelism between each pair, some lateral veins are far away from the leaf edge, and the leaf veins cross, then it is judged to be irregular, otherwise it is regular.

Furthermore, in step S2, the observation method is field visual inspection.

Furthermore, the above-mentioned method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources also includes the following steps:

Re-screening of cold-resistant plants: The cold-resistant plants classified in step S3 are subjected to artificial low-temperature treatment, and then restored to growth after the treatment, and the cold-resistant plants are screened again according to the degree of leaf damage.

Furthermore, the method of artificial low temperature treatment is: placing the cold-resistant plants classified in step S3 into an artificial low temperature incubator, setting the temperature to 3°C, the humidity to 75%, the light intensity to 1500lx, and the incubation time to 4 days.

Furthermore, the method for recovering growth after the treatment is: placing the plants treated with artificial low temperature outdoors at normal temperature and allowing them to recover growth naturally for 3 days.

Furthermore, the leaves are seedling canopy leaves.

Furthermore, the method for re-screening plants with strong cold resistance according to the degree of leaf damage is as follows: observe the canopy leaves of the plant seedlings after artificial low temperature treatment, and screen the leaves that are intact or have a small amount of water stains in the middle or a small amount of water stains on the edge near the tip as germplasm with strong cold resistance; and the leaves that only have a small amount of normal green near the main vein in the middle or the entire leaf has become water-stained as germplasm with weak cold resistance.

Further, the above-mentioned method for rapid and large-scale screening of cold-resistant rubber tree germplasm resources is characterized in that it also includes the following steps:

Re-screening of plants with strong cold resistance: The plants with strong cold resistance obtained by classification in step S3 are screened again by the conductivity method.

The beneficial effects of the present invention are:

1. The cold resistance identification method of the present invention can quickly select rubber tree germplasms with strong cold resistance in batches, and the identification accuracy rate is as high as more than 90%.

2. The present invention has low cost and is easy to operate. It can be identified only by visual observation of leaf trait indicators, and has high identification efficiency.

3. The required cycle of the present invention is short and is not restricted by season and environment. It can greatly shorten the cold resistance identification time and accelerate the cold resistance breeding process of rubber trees.

4. The cold resistance identification method of the present invention is combined with the indoor low temperature identification method, which can not only further improve the identification accuracy, but also because most of the rubber trees obtained by the cold resistance identification method of the present invention have cold resistance, so it is possible to avoid the damage of the non-cold-resistant rubber trees by the low temperature in the indoor low temperature identification method, and the normal growth of the non-cold-resistant rubber trees is not affected.

BRIEF DESCRIPTION OF THE DRAWINGS

The upper column of Figure 1 shows leaves with generally long petioles, and the lower column shows leaves with short petioles;

Figure 2 is a comparison of leaf base angles of plants with strong cold resistance and plants with weak cold resistance;

The upper column of Figure 3 shows leaves with irregular primary lateral vein distribution, and the lower column shows leaves with regular bilateral lateral vein distribution;

Figure 4 shows the degree of damage to the leaves of different rubber trees after artificial low temperature treatment and recovery growth. From left to right, they are the leaf morphological phenotypes from strong to weak cold resistance.

DETAILED DESCRIPTION

The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

Example 1

Step 1: Selection of seedling leaf canopies: Collect 3,000 open-pollinated seeds of rubber tree variety GT1, sow and germinate them on a conventional sand bed, and grow 2,600 seedlings with a seedling height of 18 to 22 cm in about one month. Transplant them into nutrient bags as individual plants, manage them conventionally, and cultivate them for 6 to 8 months. When the seedlings grow to have 3 to 4 leaf canopies and the top canopy leaves are in a stable growth period, observe and identify the seedling population.

Step 2: Observation of leaf trait indicators: Taking the plant or clone as a unit, observe the petiole length, leaf base angle and regularity of primary lateral vein distribution of the lower leaves in the second leaf canopy from the top leaf canopy of the seedling as leaf trait indicators.

1-3, the specific method for determining the petiole length, leaf base angle and the regularity of the primary lateral vein distribution of the leaf blade is as follows:

Method for judging petiole length: through internal comparison within the group, if the petiole length of the lower leaf in the second leaf canopy from the top leaf canopy is less than 25% of the petiole length of plants in the same group, it is judged that the petiole length is significantly smaller than that of the same group, otherwise it is the normal petiole length;

Method for judging the angle of leaf base: judge according to the angle between the main vein in the middle of the leaf base and the leaf edge. If the leaf base angle of the lower leaf in the second leaf canopy from the top leaf canopy of the plant to be judged is greater than 30°, it is judged that the leaf base angle is large; otherwise, the leaf base angle is small.

The method for judging the regularity of the distribution of the primary lateral veins on leaves is as follows: the regularity is judged based on the parallelism of the lateral vein arrangement, whether it extends to the leaf edge, and whether there is any crossing. If most of the lateral veins in the lower leaves of the second leaf canopy from the top leaf canopy of the plant to be judged have poor parallelism between each pair, some lateral veins are far away from the leaf edge, and the veins cross, then it is judged to be irregular, otherwise it is regular.

Step 3: Classification of plants with strong cold resistance in the field: Plants that meet two or more of the following leaf trait indicators: ① The length of the petiole is significantly smaller than that of the same group, ② The angle of the leaf base is large, and ③ The distribution of the primary lateral veins is irregular are classified as plants with strong cold resistance.

The classification results are shown in Table 1:

Table 1 GT1 rubber tree field preliminary screening results

Example 2

On the basis of Example 1, Example 2 further includes the following steps:

Step 4: Artificial low temperature treatment: Place the cold-resistant seedlings selected in step 3 into an artificial low-temperature incubator, set the temperature to 3°C, humidity to 75%, light intensity to 1500lx, and incubate for 4 days.

Step 5: Resume growth after treatment: Take out the seedlings after low-temperature culture, place them at normal temperature outdoors, and make sure to water them in the nutrient bags to keep them moist. When they resume growth naturally for 3 days, their cold resistance can be identified.

Step 6: Screening of plants with strong cold resistance: Observe and record the growth of the canopy leaves of the seedlings in step 5, and quickly identify the cold resistance in batches based on the different degrees of damage to the leaves. The specific identification method for the different degrees of damage to the leaves is: the leaves that are intact or have a small amount of water stains in the middle or a small amount of water stains on the edge near the tip are identified as germplasms with strong cold resistance, while the leaves that only have a small amount of normal green near the main vein in the middle or the entire leaf has become water-stained are identified as germplasms with weak cold resistance (refer to Figure 4).

The screening and classification results are shown in Table 2:

Table 2 Results of rapid and large-scale screening of cold-resistant rubber tree germplasm resources by GT1 rubber tree

As can be seen from Table 2, the present invention can quickly and massively screen out 86 plants with strong cold resistance from 2600 rubber trees only by the method of field initial screening, and after re-screening and verification by the indoor identification method, it is confirmed that 79 germplasm resources with strong cold resistance can be obtained. Therefore, the screening method of the present invention is low in cost, easy to operate, high in accuracy and efficiency, and greatly accelerates the cold-resistant breeding process of rubber trees.

Example 3

Step 1: Selection of leaf canopies of seedlings: Open-pollinated seed seedlings of rubber tree variety GT1 are used to cultivate bagged seedling rootstocks, and 160 branches of Wickham germplasm that year are used as scions. After bud grafting, routine management is carried out and cultivation is carried out for 6 to 8 months. When the seedlings grow to have 3 to 4 leaf canopies and the top canopy leaves are in the stable growth period, the seedling population is observed and identified.

Steps 2 to 6 are the same as in Examples 1 and 2, and the screening and classification results are shown in Table 3 below:

Table 3 Results of rapid and large-scale screening of cold-resistant rubber tree germplasm resources by GT1/Weikehan rubber tree

As can be seen from Table 3, the present invention can quickly and massively screen out 20 plants with strong cold resistance from 160 rubber trees only by the method of field initial screening, and screen and verify again by the indoor identification method, confirming that 18 germplasm resources with strong cold resistance can be obtained. Therefore, the screening method of the present invention is low in cost, easy to operate, high in accuracy and efficiency, and greatly accelerates the cold-resistant breeding process of rubber trees.

Example 4

The plants with strong cold resistance classified by the field preliminary screening in the above-mentioned Examples 1 and 3 were taken, and the relative conductivity was identified by a conductivity meter. The smaller the conductivity, the stronger the cold resistance, and the larger the conductivity, the weaker the cold resistance. The conductivity of the budding seedlings of the rubber tree variety GT1 was used as a control.

The experimental results are as follows:

From the above experimental results, it can be seen that the method for screening the cold resistance of rubber tree germplasm resources provided by the present invention has a judgment accuracy rate of more than 90%. Therefore, the present invention is more convenient and fast than the prior art, and has the characteristics of high accuracy.

The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims (9)

1. A method for rapidly screening cold-resistant rubber tree germplasm resources in a large scale is characterized by comprising the following steps:

s1: selecting nursery stock She Peng: selecting seedlings to grow to 3-4 She Pengdai seedlings or clone, and observing the seedling group when the canopy leaves are in a stable growth period;

S2: and (3) observing leaf property indexes: taking a plant or clone as a unit, observing the petiole length, the leaf base included angle and the leaf primary side pulse distribution regularity of the lower leaf in the second She Peng from the top She Peng downwards of the seedling as leaf character indexes;

S3: classification of plants with strong cold resistance in fields: plants meeting the leaf character index ①, wherein the length of the petiole is obviously smaller than that of similar groups, the included angle of the basal part of ② leaves is large, and the distribution of ③ primary side veins is irregular are classified as plants with strong cold resistance.

2. The method for rapidly and massively screening cold-resistant rubber tree germplasm resources according to claim 1, wherein in the step S3, the method for judging that the length of the ① petioles is obviously smaller than that of similar groups is as follows:

comparing the inner parts of the groups, wherein the length of the petioles of the lower leaves in the second She Peng with the top She Peng downwards is more than 25% of the length of the petioles of plants in the same group, and judging that the lengths of the petioles are obviously smaller than those of the plants in the same group, otherwise, judging that the lengths of the petioles are the lengths of general petioles;

The method for judging the large included angle of the base part of the ② leaves comprises the following steps:

Judging according to the angle between the main vein in the middle of the leaf base and the edge of the leaf, and judging that the leaf base included angle is large if the leaf base included angle of the lower leaf in the second She Peng of the plant to be judged from the top She Peng downwards is larger than 30 degrees; otherwise, the included angle of the basal part of the leaf is small;

The ③ -level lateral pulse distribution irregularity judging method comprises the following steps of:

Judging according to the parallelism of the side pulses, whether the side pulses extend to the edge of the leaf or not and whether the side pulses are intersected or not, if the parallelism of most of the side pulses of the lower leaf in the second She Peng of the plant to be judged from the top She Peng to the bottom is poor, and if the parallelism of most of the side pulses is poor, the side pulses are far from the edge of the leaf and the side pulses are intersected or not, judging that the plant to be judged is irregular or otherwise, judging that the plant to be judged is regular.

3. The method for rapidly and massively screening cold-resistant rubber tree germplasm resources according to claim 1, wherein in step S2, the observation method is visual field inspection.

4. A method for rapid large-scale screening of cold-resistant rubber tree germplasm resources according to any one of claims 1-3, further comprising the steps of:

Screening the plants with strong cold resistance again: and (3) recovering the plant with strong cold resistance obtained in the step (S3) through artificial low-temperature treatment and growth after treatment, and screening the plant with strong cold resistance again according to the damage degree of the leaf.

5. The method for rapidly and massively screening cold-resistant rubber tree germplasm resources according to claim 4, wherein the manual low-temperature treatment method is as follows: and (3) placing the plants with strong cold resistance obtained in the step (S3) in an artificial low-temperature incubator, wherein the temperature is set to be 3 ℃, the humidity is set to be 75%, the light intensity is 1500lx, and the culture time is 4 days.

6. The method for rapidly and massively screening cold-resistant rubber tree germplasm resources according to claim 4, wherein the method for recovering growth after treatment is as follows: and (3) placing the plant subjected to the artificial low-temperature treatment outdoors at normal temperature, and naturally recovering the plant to grow for 3 days.

7. The method for rapid large-scale screening of cold-resistant rubber tree germplasm resources according to claim 4, wherein said leaves are nursery stock canopy leaves.

8. The method for rapidly and massively screening cold-resistant rubber tree germplasm resources according to claim 4, wherein the method for rescreening cold-resistant plants according to the damaged degree of leaves is as follows: observing the plant seedling canopy leaves subjected to manual low-temperature treatment, and screening the leaves into germplasm with strong cold resistance, wherein the leaves are intact or have a small amount of punctiform or blocky water stains in the middle or have a small amount of water stains at the edges near the leaf tips; and only a small amount of normal green exists near the middle main vein of the leaf, or the whole leaf is changed into water stain to be screened as a germplasm with weak cold resistance.

9. A method for rapid large-scale screening of cold-resistant rubber tree germplasm resources according to any one of claims 1-3, further comprising the steps of:

Screening the plants with strong cold resistance again: and (3) screening the plants with strong cold resistance obtained in the step (S3) through a conductivity method again.