Method for rapidly and efficiently screening rubber tree anther explants

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for rapidly and efficiently screening rubber tree anther explants, which comprises the following steps: S11, picking rubber tree rachis, and collecting male flowers and female flowers on the rachis; s12, screening the male flowers and the female flowers by using a 10-mesh inspection sieve, and collecting and screening out the male flowers; s13, screening the male flowers screened in the step S12 by using a 16-mesh inspection sieve, and collecting the male flowers in the sieve; s14, screening the male flowers screened in the step S13 by using a 14-mesh inspection sieve, and collecting the male flowers in the sieve and screening out the male flowers; s15, screening the male flowers screened in the step S14 by using a 12-mesh inspection sieve, and collecting the screened male flowers; s16, the male flowers screened out in the step S14 and the step S15 are soaked and disinfected, the male flowers which do not meet the maturity requirement are removed and then washed and filtered, and the hevea brasiliensis anther explants are obtained. According to the method provided by the invention, the picking time of the anther explants can be greatly shortened, the inoculation quantity and the culture effect are improved, and an important method is provided for rapidly and efficiently obtaining different varieties of anther explants of the rubber trees.

Description

Technical Field

[0001] The present application relates to the technical field of plant tissue culture, and in particular to a method for rapidly and efficiently screening anther explants of rubber trees. Background Art

[0002] Anther culture is the main technical approach for tissue culture of Hevea brasiliensis. In addition to anther culture, it is relatively difficult to cultivate asexual plants using other culture methods such as unpollinated ovules and ovaries. Anthers are the main explant source for obtaining somatic embryonic plants of Hevea brasiliensis. The somatic embryonic plants start from the somatic cells of the anther wall and form complete regenerated plants through the somatic embryogenesis pathway. The somatic embryonic plants obtained by in vitro culture of anthers have juvenile characteristics and autonomously developed root systems. They are called self-rooted juvenile asexual plants of Hevea brasiliensis. Compared with old bud grafted asexual plants, they grow faster, have higher yields, and are more resistant to stress. They are a new generation of superior planting materials for the development of the natural rubber industry. At present, the conventional method for obtaining anther explants of rubber trees is: the flowering side branches are picked back in the morning of the same day, and the pickers hold pointed tweezers and select male flowers of appropriate size on the inflorescence, disinfect them and peel out the stamens for inoculation. However, the male flowers of rubber trees are relatively small, about 2 to 4 mm long, and the rubber flowers are panicles, with male and female flowers in the same order and different flowers. There are male flowers of very similar size and color but different maturity on the same inflorescence. Manual picking is time-consuming and laborious, and the time-consuming picking greatly reduces the daily inoculation time and affects the number of inoculations.

[0003] When manually picking anther explants, a skilled inoculator can inoculate 1,800 seeds per day in 6 hours. Based on the daily inoculation requirements of 3 people, 3 people are required to work together for about 1 to 1.5 hours. During this period, due to the long picking time, the picked male flowers are prone to dehydration and weakened vitality. At the same time, manual picking may also cause damage to the anthers due to improper techniques or uneven force, affecting the vitality of the anthers and the culture effect.

[0004] In summary, manual picking of rubber tree anther explants takes a long time, affecting the number of inoculations, and the process is easily affected by multiple factors, making it difficult to ensure that the anthers picked each time remain consistent in terms of vitality and culture effect. Therefore, a new screening method is needed to overcome the current problem of low efficiency in obtaining rubber tree anther explants. Summary of the invention

[0005] The problem that the present application aims to solve is to provide a method for quickly and efficiently screening anther explants of rubber trees, which can greatly reduce the time for picking anther explants and reduce manpower input, thereby increasing the time available for inoculation every day, improving the number of inoculations and the culture effect, and providing an important method for quickly and efficiently obtaining anther explants of different varieties of rubber trees and inoculating more culture materials during the flowering period of rubber trees each year.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] The present application provides a method for quickly and efficiently screening anther explants of rubber trees, comprising the following steps:

[0008] S11. Picking the inflorescence axis of the rubber tree and collecting the male and female flowers on the inflorescence axis;

[0009] S12. The male and female flowers were screened with a 10-mesh test sieve, and the male flowers were collected;

[0010] S13. The male flowers screened out in S12 were screened with a 16-mesh inspection sieve, and the male flowers in the sieve were collected;

[0011] S14. The male flowers in the sieve of S13 were screened with a 14-mesh inspection sieve, and the male flowers in the sieve and the male flowers screened out were collected;

[0012] S15. The male flowers in the sieve of S14 were screened with a 12-mesh inspection sieve, and the male flowers screened were collected;

[0013] S16. The male flowers screened out in S14 and S15 are immersed and disinfected, and the yellow male flowers that do not meet the maturity requirements are removed, washed and filtered to obtain the rubber tree anther explants.

[0014] In some embodiments, the 10-mesh inspection sieve, the 12-mesh inspection sieve, the 14-mesh inspection sieve, and the 16-mesh inspection sieve are all standard inspection sieves.

[0015] In some examples, the standard test sieve is a 20 cm diameter standard test sieve produced by Xinxiang Dahan Vibration Machinery Co., Ltd.

[0016] In some embodiments, in S16, the immersion disinfection includes the following steps:

[0017] S21. The male flowers are soaked in ethanol and filtered to obtain a preliminary sterilized rubber tree anther explant;

[0018] S22. The preliminarily sterilized rubber tree anther explants are soaked in HgCl2 solution and Tween 80 and then filtered to obtain completely sterilized rubber tree anther explants.

[0019] In some embodiments, in S21, the concentration of ethanol is 75%.

[0020] In some embodiments, in S22, the concentration of the HgCl2 solution is 0.1% to 0.15%.

[0021] In some embodiments, in S22, the amount of Tween 80 is 1 to 5 drops.

[0022] In some embodiments, in S21, the soaking time is 30 to 60 seconds.

[0023] In some embodiments, in S22, the soaking time is 8 to 12 minutes.

[0024] In some embodiments, in S16, the method for removing the yellow male flowers includes: during the soaking disinfection, scooping out the suspended yellow male flowers.

[0025] In some embodiments, in S16, the washing cleaning solution is sterile water, and the number of washing times is 5 times.

[0026] This application has the following beneficial effects:

[0027] (1) The present application successfully completes the screening of rubber tree anther explants by using test sieves of different specifications, and at the same time greatly shortens the selection time of anther explants, thereby avoiding the influence of the culture effect on the selection time of anther explants being too long.

[0028] (2) The rubber tree anther explants screened by the present application can maintain or even have a higher callus induction rate and somatic embryogenesis rate than those selected manually.

[0029] (3) Test sieves are currently mainly used in the fields of food, mining, metallurgy, construction and environmental monitoring, but have not yet been used in the field of plant tissue culture. The present application creatively uses test sieves for the screening of anther explants, which can greatly reduce the difficulty of screening anther explants.

[0030] (4) The method for rapid and efficient screening of rubber tree anther explants provided in the present application can greatly reduce the difficulty of picking rubber tree anther explants, reduce the workload of pickers, save manpower input in obtaining anther explants, and thereby invest more human resources in the work of anther explant inoculation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the present disclosure, the following briefly introduces the drawings required to be used in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams, and are not limitations on the actual process of the method involved in the embodiments of the present disclosure.

[0032] Figure 1The present application provides a step for rapid and efficient screening of anther explants of the rubber tree variety 'Reken 628';

[0033] Figure 2 The present application provides a step for rapid and efficient screening of anther explants of the rubber tree variety 'Yunyan 73-477';

[0034] Figure 3 This application provides a step for rapid and efficient screening of anther explants of the rubber tree variety 'Yunyan 7346'. DETAILED DESCRIPTION

[0035] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0036] Example 1 Preparation of callus induction medium

[0037] The callus induction medium was obtained by taking MS medium as the basic medium, adding KT 1.0 mg / L, 2,4-D 1.0 mg / L, NAA 1.0 mg / L, Picloram 0.5 mg / L, coconut water 50 ml / L, sucrose 70 g / L and agar 6 g / L.

[0038] Example 2 Preparation of somatic embryogenic medium

[0039] The somatic embryogenesis medium is obtained by using modified MS medium (the concentration of inorganic salts of macroelements is 80% of that of MS) ​​as the basic medium, adding 6-BA 1.0 mg / L, KT 2.0 mg / L, NAA 0.2 mg / L, GA 30.5 mg / L, ABA 0.8 mg / L, sucrose 70 g / L and agar 6 g / L.

[0040] Example 3 Rapid and efficient screening of anther explants of 'Reken 628' and callus induction and somatic embryogenesis

[0041] (1) Rapid and efficient screening of anther explants of 'Reken 628' (e.g. Figure 1 shown):

[0042] S1. Pick the inflorescence axis of 'Reken 628' in the morning, and rub the male and female flowers on the inflorescence axis with your palms;

[0043] S2. The male and female flowers were screened with a 10-mesh 2mm pore size test sieve, and the twigs, female flowers and male flowers that had opened were discarded and the male flowers that were screened were collected;

[0044] S3. The male flowers sieved out in S2 were screened with a 16-mesh 1.25 mm pore size test sieve, the smaller male flowers sieved out were discarded, and the male flowers remaining in the sieve were collected;

[0045] S4. The male flowers remaining in the sieve in S3 were screened with a 14-mesh test sieve with a pore size of 1.43 mm, and the male flowers remaining in the sieve and the male flowers sieved out of the 14-mesh sieve were collected;

[0046] S5. The male flowers remaining in the sieve in S4 were screened with a 12-mesh test sieve with a pore size of 1.6 mm, and the male flowers sieved out of the 12-mesh sieve and the male flowers remaining in the sieve were collected;

[0047] S6. The 14-mesh male flowers, 12-mesh male flowers and 12-mesh male flowers were soaked in 75% ethanol for 30 seconds and then filtered to obtain a preliminary sterilized rubber tree anther explant;

[0048] S7. The preliminary sterilized rubber tree anther explants were soaked in a 0.1% HgCl2 solution and 1 drop of Tween 80 for 12 min, during which the suspended yellow male flowers were removed and discarded, and filtered after the soaking to obtain completely sterilized rubber tree anther explants;

[0049] S8. Wash the completely sterilized rubber tree anther explants with sterile water for 5 times and then filter them to obtain the rubber tree anther explants.

[0050] (2) Callus induction and somatic embryogenesis

[0051] The stamens of 300 sterilized male flowers with 14 mesh, 300 sterilized male flowers with 12 mesh, 300 sterilized male flowers with 12 mesh retained, and 300 sterilized male flowers manually picked were peeled off with a dissecting needle and inoculated onto callus induction medium respectively. Callus were obtained after culturing in a dark room at 26°C for 50 days, and the callus induction rate was observed and counted.

[0052] 200 calli obtained from 14-mesh, 12-mesh, 12-mesh retained and manually selected anther explants after 50 days of culture were inoculated on somatic embryogenesis medium and cultured in a dark room at 24°C for 50 days to induce somatic embryogenesis, and the somatic embryogenesis rate was observed and counted.

[0053] The callus induction rate and somatic embryogenesis rate of anther explants of 'Reken 628' are shown in Table 1:

[0054] Table 1 Screening of anther explants and induction effect of 'Reken 628'

[0055]

[0056] As can be seen from the results in Table 1, the anther explants of 'Reken 628' screened by the present application can maintain or even have a higher anther callus induction rate and somatic embryogenesis rate of rubber trees. Among them, the callus induction rate of the male flowers screened through 14 meshes is higher than that of the male flowers screened through 12 meshes after the stamens are stripped and induced and cultured, but the somatic embryogenesis rate and anther callus size of the male flowers screened through 12 meshes are greater than those screened through 14 meshes, and the length and diameter of the male flowers screened through 12 meshes are larger, making it easier to strip the anthers. The explants left in the sieve of 12 meshes failed to induce callus and somatic embryos after inoculation, indicating that the present invention can quickly and efficiently obtain the best explants suitable for this variety. The present application uses male flowers screened through 14 meshes or 12 meshes as anther explants of 'Reken 628'.

[0057] Example 4 Rapid and efficient screening of anther explants of 'Yunyan 73-477' and callus induction and somatic embryogenesis (1) Rapid and efficient screening of anther explants of 'Yunyan 73-477' (eg Figure 2 shown):

[0058] S1. Pick the inflorescence axis of 'Yunyan 73-477' in the morning, and rub the male and female flowers on the inflorescence axis with your palms;

[0059] S2. The male and female flowers were screened with a 10-mesh 2mm pore size test sieve, and the twigs, female flowers and male flowers that had opened were discarded and the male flowers that were screened were collected;

[0060] S3. The male flowers sieved out in S2 were screened with a 16-mesh 1.25 mm pore size test sieve, the smaller male flowers sieved out were discarded, and the male flowers remaining in the sieve were collected;

[0061] S4. The male flowers remaining in the sieve in S3 were screened with a 14-mesh test sieve with a pore size of 1.43 mm, and the male flowers remaining in the sieve and the male flowers sieved out of the 14-mesh sieve were collected;

[0062] S5. The stamens left in the sieve in S4 were screened with a 12-mesh 1.6 mm pore size test sieve, and the 12-mesh male flowers and the 12-mesh male flowers left in the sieve were collected;

[0063] S6. The 14-mesh male flowers, 12-mesh male flowers and 12-mesh male flowers were soaked in 75% ethanol for 45 seconds and then filtered to obtain a preliminary sterilized rubber tree anther explant;

[0064] S7. The preliminary sterilized rubber tree anther explants were soaked in a 0.13% HgCl2 solution and 3 drops of Tween 80 for 10 min, during which the suspended yellow male flowers were removed and discarded, and after the soaking was completed, the anther explants were filtered to obtain completely sterilized rubber tree anther explants;

[0065] S8. Wash the completely sterilized rubber tree anther explants with sterile water for 5 times and then filter them to obtain the rubber tree anther explants.

[0066] (2) Callus induction and somatic embryogenesis

[0067] The stamens of 300 sterilized male flowers with 14 mesh, 300 sterilized male flowers with 12 mesh, 300 sterilized male flowers with 12 mesh retained, and 300 sterilized male flowers manually picked were peeled off with a dissecting needle and inoculated onto callus induction medium respectively. Callus were obtained after culturing in a dark room at 26°C for 50 days, and the callus induction rate was observed and counted.

[0068] 200 calli obtained from 14-mesh, 12-mesh, 12-mesh retained and manually selected anther explants after 50 days of culture were inoculated on somatic embryogenesis medium and cultured in a dark room at 24°C for 50 days to induce somatic embryogenesis, and the somatic embryogenesis rate was observed and counted.

[0069] The callus induction rate and somatic embryogenesis rate of anther explants of 'Yunyan 73-477' are shown in Table 2:

[0070] Table 2 Screening of anther explants and induction effect of 'Yunyan 73-477'

[0071]

[0072]

[0073] As shown in Table 2, the anther explants of 'Yunyan 73-477' screened by the present application can maintain or even have a higher callus induction rate and somatic embryogenesis rate of rubber tree anthers. Among them, there is no significant difference in callus induction rate between the male flowers screened out by 14 mesh and 12 mesh, but the callus obtained by the anther explants screened out by 12 mesh has a somatic embryogenesis rate greater than that of 14 mesh after differentiation culture, and the male flowers screened out by 12 mesh have a larger length and diameter, which is easier to strip the anthers. Therefore, the present application preferably uses the male flowers screened out by 12 mesh as the anther explants of 'Yunyan 73-477'. In addition, the explants left in the sieve of 12 mesh failed to induce callus and somatic embryos after inoculation, indicating that the present invention can quickly and efficiently obtain the best explants suitable for this variety.

[0074] Example 5 Rapid and efficient screening of anther explants of 'Yunyan 7346' and callus induction and somatic embryogenesis

[0075] (1) Rapid and efficient screening of anther explants of 'Yunyan 7346' (e.g. Figure 3 shown):

[0076] S1. Pick the inflorescence axis of 'Yunyan 7346' in the morning and rub the male and female flowers on the inflorescence axis with your palms;

[0077] S2. The male and female flowers were screened with a 10-mesh 2mm pore size test sieve, and the twigs, female flowers and male flowers that had opened were discarded and the male flowers that were screened were collected;

[0078] S3. The male flowers sieved out in S2 were screened with a 16-mesh 1.25 mm pore size test sieve, the smaller male flowers sieved out were discarded, and the male flowers remaining in the sieve were collected;

[0079] S4. The male flowers remaining in the sieve in S3 were screened with a 14-mesh test sieve with a pore size of 1.43 mm, and the male flowers remaining in the sieve and the male flowers sieved out of the 14-mesh sieve were collected;

[0080] S5. The male flowers remaining in the sieve in S4 were screened with a 12-mesh 1.6 mm pore size test sieve, and the 12-mesh male flowers were collected;

[0081] S6. The 14-mesh male flowers and the 12-mesh male flowers were soaked in 75% ethanol for 60 seconds and then filtered to obtain a preliminary sterilized rubber tree anther explant;

[0082] S7. The preliminarily sterilized rubber tree anther explants were soaked in a 0.15% HgCl2 solution and 5 drops of Tween 80 for 8 min, and filtered after the soaking to obtain completely sterilized rubber tree anther explants;

[0083] S8. The completely sterilized rubber tree anther explants are washed 5 times with sterile water, and the male flowers suspended in the 12-mesh filter are fished out, that is, the 12-mesh filter is suspended, and filtered separately to obtain the rubber tree anther explants.

[0084] (2) Callus induction and somatic embryogenesis

[0085] The stamens of 300 sterilized male flowers with 14 mesh, 300 sterilized male flowers with 12 mesh, 300 sterilized male flowers with 12 mesh retained, and 300 sterilized male flowers manually picked were peeled off with a dissecting needle and inoculated onto callus induction medium respectively. Callus were obtained after culturing in a dark room at 26°C for 50 days, and the callus induction rate was observed and counted.

[0086] 200 calli obtained from 14-mesh, 12-mesh, 12-mesh suspension and manually selected anther explants after 50 days of culture were inoculated on somatic embryogenesis medium and cultured in a dark room at 24°C for 50 days to induce somatic embryogenesis, and the somatic embryogenesis rate was observed and counted.

[0087] The callus induction rate and somatic embryogenesis rate of anther explants of 'Yunyan 7346' are shown in Table 3:

[0088] Table 3 Screening of anther explants and induction effect of 'Yunyan 7346'

[0089]

[0090] As shown in Table 3, the anther explants of 'Yunyan 7346' screened by the present application can maintain or even have a higher anther callus induction rate and somatic embryogenesis rate of rubber trees. Because the size of the male flowers of the 'Yunyan 7346' variety is not much different, there are male flowers with the same diameter but significantly different maturity in the male flowers screened out of 12 meshes. After the male flowers screened out of 12 meshes are disinfected and suspended male flowers are used for cultivation and comparison, it is found that the suspended explants of 12 meshes fail to induce callus and somatic embryos after inoculation, indicating that the present invention can quickly and efficiently obtain the best explants suitable for this variety. In addition, the male flowers screened out of 14 meshes have significantly higher callus induction rates and somatic embryogenesis rates than the male flowers screened out of 12 meshes. Therefore, the present application preferably uses the male flowers screened out of 14 meshes as the anther explants of 'Yunyan 7346'.

[0091] Experimental Example 1: Time comparison between fast and efficient screening and manual picking

[0092] Assuming that each skilled inoculator inoculates 1,800 seeds per day, and the daily inoculation requirement of three people, 5,400 seeds are needed per day. Let one skilled picker perform the method of rapid and efficient screening of 'Reken 628' in Example 3 to screen 5,400 14-mesh anther explants of 'Reken 628', which takes about 10 minutes; let three skilled pickers screen 5,400 anther explants of 'Reken 628' by manual picking, which takes 1 hour to 1.5 hours. It can be seen that the time taken for rapid and efficient screening is much less than manual picking.

[0093] Experimental Example 2 Comparison of daily inoculum yield between rapid and efficient screening and manual picking

[0094] Calculated based on 7 hours per working day, field picking takes about 1 hour every morning, so the daily inoculation time using rapid and efficient screening is about 6 hours, and the daily inoculation time using manual picking is about 5 hours. According to the difficulty of picking male flowers, a skilled inoculator can inoculate 50 to 60 seeds in 10 minutes. If 300 seeds are inoculated in 1 hour, when rapid and efficient screening is used, each person can inoculate 1,800 seeds per day, and 3 people can inoculate about 5,400 seeds in total; when manual picking is used, each person can inoculate 1,500 seeds per day, and 3 people can inoculate about 4,500 seeds in total. It can be seen that the daily inoculation amount using rapid and efficient screening is much higher than that using manual picking.

[0095] In summary, the method provided by the present application for quickly and efficiently screening anther explants of rubber trees can greatly shorten the time and manpower investment for obtaining anther explants, reduce the difficulty of screening anther explants, increase the daily inoculation time, and improve the number of anther explants that can be inoculated daily. At the same time, it can avoid the influence of the culture effect of anther explants due to the long selection time. Moreover, compared with manual picking, the anther explants screened by the present application can maintain or even have a higher callus induction rate and somatic embryogenesis rate.

[0096] The above is only a preferred embodiment of the present disclosure. It should be understood that the present disclosure 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 disclosure, and should be within the scope of protection of the claims attached to the present disclosure.

Claims

1. A method for quickly and efficiently screening rubber tree anther explants, characterized in that, The following steps are involved: S11. Picking the inflorescence axis of the rubber tree and collecting the male and female flowers on the inflorescence axis; S12. The male and female flowers were screened with a 10-mesh test sieve, and the male flowers were collected; S13. The male flowers screened out in S12 were screened with a 16-mesh inspection sieve, and the male flowers in the sieve were collected; S14. The male flowers in the sieve of S13 were screened with a 14-mesh inspection sieve, and the male flowers in the sieve and the male flowers screened out were collected; S15. The male flowers in the sieve of S14 were screened with a 12-mesh inspection sieve, and the male flowers screened were collected; S16. The male flowers screened out in S14 and S15 are immersed and disinfected, and the yellow male flowers that do not meet the maturity requirements are removed, washed and filtered to obtain the rubber tree anther explants.

2. The method according to claim 1, characterized in that The 10-mesh test sieve, the 12-mesh test sieve, the 14-mesh test sieve and the 16-mesh test sieve are all standard test sieves.

3. The method according to claim 1, characterized in that In S16, the immersion disinfection comprises the following steps: S21. The male flowers are soaked in ethanol and filtered to obtain a preliminary sterilized rubber tree anther explant; S22. The preliminarily sterilized rubber tree anther explants are soaked in HgCl2 solution and Tween 80 and then filtered to obtain completely sterilized rubber tree anther explants.

4. The method according to claim 3, characterized in that In S21, the concentration of ethanol is 75%.

5. The method according to claim 3, characterized in that: In S22, the concentration of the HgCl2 solution is 0.1% to 0.15%.

6. The method according to claim 3, characterized in that In S22, the amount of Tween 80 used is 1 to 5 drops.

7. The method according to claim 3, characterized in that In S21, the soaking time is 30 to 60 seconds.

8. The method according to claim 3, characterized in that In S22, the soaking time is 8 to 12 minutes.

9. The method according to claim 1, characterized in that: In S16, the method for removing the yellow male flowers includes: during the soaking disinfection, fishing out the suspended yellow male flowers.

10. The method according to claim 1, characterized in that In S16, the washing solution is sterile water, and the number of washing times is 5 times.

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