CN116998405A - Method for improving embryoid seedling rate of eggplant anther culture - Google Patents
Method for improving embryoid seedling rate of eggplant anther culture Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a method for improving the seedling rate of embryoid cultured by eggplant anther, wherein embryoid is singly separated and transferred into embryoid germination medium for seedling; shearing off the well-developed rooting-free seedlings or parasitic embryoid seedlings, transferring the seedlings into a rooting and sprouting culture medium to induce rooting, forming complete plants, and transplanting and hardening the seedlings; rooting the rooting and sprouting culture medium and developing embryoid seedlings of more than 1 branch, and directly transplanting and hardening the seedlings; the cluster seedlings which grow well are torn to the root along the branches, the cluster seedlings are divided into a plurality of plants, the seedlings with roots are directly planted, and the branches without roots are transplanted and subjected to moisture preservation and heat preservation management according to a tissue culture seedling hardening method, and are moved to the field after survival; transplanting field survival multi-branch seedlings, and carrying out selfing seed reserving on branch strips or cutting/grafting preservation on branch strips. The method greatly improves the efficiency of creating new germplasm of the eggplant by using anther culture technology.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to a method for improving the seedling rate of embryoid cultured by eggplant anther.
Background
Eggplant (Solarium melongena L) is cultivated in various provinces in China in Asian tropical zone of original production. Herbs or sub-shrubs can be used for vegetable and fruit, and root, stem, leaf and seed can be used as medicines. At the beginning of the 70 s, the Riana and the like successfully grow seedlings through the callus way by the anther culture of the eggplant, and at present, no matter the anther or pollen (microspore) culture of the eggplant is carried out, a lot of successful reports exist, but the report of breeding new eggplant varieties by utilizing homozygous parents obtained by anther or pollen culture in China is not so much. The reason for this is that the culture of eggplant anther or pollen mainly forms seedlings through callus differentiation, and the frequency of forming seedlings with complete roots and stems is very low; the frequency of generating regenerated plants through embryogenesis and germination approaches is theoretically much higher than that of callus redifferentiation approaches, but the premise of realizing the method is that the frequency of inducing eggplant anthers to generate embryoids is high and stable, and the embryoids are more, so that most researchers cannot do the problem at present, and therefore, a systematic method for promoting embryoids to germinate into seedlings and a report of the final plant yield are not seen.
The subject group establishes a culture procedure of eggplant anther culture in 2019 by inducing plants such as homozygous diploid from eggplant anther through embryogenesis route and continuously optimizing eggplant anther culture conditions in 2014Haploid, diploid and polyploid plants are regenerated by the generation way, and the embryoid generation frequency reaches 0.4-0.6 per bud on average [1] Slightly higher than the range of 0.3-0.35 reported by Liu Duchen [2] . After that, the subject group continuously improves all technical links such as a culture procedure, a culture medium formula, a seedling hardening technology in a transplanting process and the like, and a stable and repeatable eggplant anther culture technology system with the anther embryo emergence rate of more than 30 percent and 1 to tens of embryoids regenerated from each anther is established at present, so that a test material is provided for further researching the germination of embryoids into seedlings.
[1] Liu Duchen, fang Chao, li Jianyue, et al eggplant anther culture induced embryoid seedling [ J ]. Southwest agricultural journal 2008,21 (6): 1643-1646
[2] Wang Chang, cui Qunxiang, zhu Haijia, etc. the influence of the culture procedure and the medium composition on the anther culture of eggplants, preferably plants [ J ]. Vegetables, 2019, (4): 15-21
Disclosure of Invention
The invention aims to: aiming at the defects of small quantity of embryoid seedlings generated by each bud and low transplanting seedling rate in the prior art, the method for improving the seedling rate of embryoid cultured by the anther of the eggplant is provided, and the efficiency of germplasm creation and new variety breeding of the eggplant is improved.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for improving the seedling rate of embryoid cultured by eggplant anther, comprising the following steps:
(1) The embryoid is singly separated and transferred into embryoid germination culture medium for seedling formation;
(2) Secondary seedling formation of embryoid: shearing off the well-developed rooting-free seedlings or parasitic embryoid seedlings, transferring the seedlings into a rooting and sprouting culture medium to induce rooting, forming complete plants, and transplanting and hardening the seedlings;
(3) And (3) directly transplanting the rooting embryoid seedlings with strong development: rooting the rooting and sprouting culture medium and developing embryoid seedlings of more than 1 branch, and directly transplanting and hardening the seedlings;
(4) Transplanting survival cluster seedlings to separate plants: the cluster seedlings which grow well are torn to the root along the branches, the cluster seedlings are divided into a plurality of plants, the seedlings with roots are directly planted, and the branches without roots are transplanted and subjected to moisture preservation and heat preservation management according to a tissue culture seedling hardening method, and are moved to the field after survival;
(5) Transplanting field survival multi-branch seedlings, and carrying out selfing seed reserving on branch strips or cutting/grafting preservation on branch strips.
Specifically, in the step (1), the embryoid bodies are separated and transferred within 10d after appearance of the embryoid bodies are observed by naked eyes, wherein the method is that the single independent embryoid bodies are directly transferred, and the embryoid bodies which are closely together are separated into the single independent embryoid bodies by a blade or a dissecting needle and then transferred, and all anthers are transferred.
Specifically, in the step (1), the period and the method for separating the single embryoid bodies are as follows: and (3) after anther inoculation, generating green and white embryoid which is visible to the naked eye and is not healed, wherein embryoid which are close together, have obvious size difference and inconsistent growth direction are separated under aseptic condition on an ultra-clean workbench by using a dissecting needle and a blade.
Specifically, in the step (1), the embryoid germination medium is a B5 or NLN solid medium containing a small amount of auxin and/or mitogen substances, and the concentration of the auxin substances is higher than that of the mitogen substances; preferably, the concentration of the auxin substances is 0.2mg/L, and the concentration of the mitogen substances is 0.1mg/L. The culture conditions are that the culture is carried out in an illumination incubator for about 10 days, the culture temperature is 25 ℃, the photoperiod is 12-16 h, the dark period is 8-12 h, and the illumination intensity is 1000-1500 lx.
Specifically, in the step (2), the rooting and sprouting medium is a B5/NLN solid medium with or without 0.1-0.2mg/L auxin.
Specifically, the transplanting method in the step (2) is as follows: taking out the seedling suitable for transplanting from the culture flask, and immersing the root in the carbendazim solution for 5-30 min, preferably 10-30 min; taking out, cleaning with water, gently sucking to dry root drop, soaking in NAA or IBA solution for 1s-1min, then planting in conventional seedling substrate, watering thoroughly without water accumulation, covering with high transparency film, and placing in illumination incubator for 2 weeks. The film is tightly covered around to prevent water loss from the upper part of the seedling, the film is not opened in the first 1 week, a part of the film is uncovered after 1 week, the humidity of the seedling is reduced, the leaf color of the transplanted seedling is deepened and the leaf becomes thicker after 1 week, the covered film is uncovered, and normal water and fertilizer management is carried out according to the growth condition of the survival seedling.
Specifically, the seedling substrate is a commercial vegetable seedling substrate or 100% vermiculite, the thickness is not less than 10cm, and the seedling substrate is thoroughly poured but does not accumulate water; the root and stem parts of the seedlings are buried in the matrix, the leaves are reserved at the upper part of the matrix, the row spacing of the plants is about 4cm, and a small amount of water is sprayed around the plants after the plants are planted, so that the roots are tightly contacted with the matrix.
Specifically, the carbendazim solution is obtained by diluting carbendazim wettable powder with the active ingredient content of 50wt% with water according to the mass ratio of 1:500-800; the NAA or IBA solution is 100-1000 ppm.
Specifically, in the step (3), the embryoid Miao Miaogao is more than 2cm and the transplanting and hardening seedlings are directly carried out.
Specifically, in the step (4), the method for dividing plants comprises the following steps: after the plant is separated, the plants with developed root systems are directly transplanted into a matrix, and the plants are placed in an incubator to be subjected to heat preservation and moisture preservation for 1 week, and then the surviving plants can be transplanted into the field; and transplanting the plants without roots at the bottoms after the division according to a method for transplanting the tissue culture seedlings out of the bottles.
Specifically, in the step (5), the branch selfing method comprises the following steps: the flowers on each branch are subjected to homoflower selfing, or the flowers on the same branch are subjected to pollination selfing, and the flowers on different branches cannot be mutually pollinated; if the selfing fruits on all branches cannot be obtained at the same time due to excessive branches, batch selfing is needed, namely, after the mature harvesting of the fruits on the branches of the early-stage selfing fruit setting, the selfing and seed reserving are carried out on the flower buds on the other branches.
Preferably, the same-flower selfing mode is adopted, and the single-flower selfing process is as follows: the method comprises the steps of (1) carrying out on sunny days with the temperature not exceeding 32 ℃, clamping the front ends of petals of large flower buds which are opened on each branch by using grafting clips on the 2 nd day or the 3 rd day, paying attention to clamping petals but not clamping the petals on the column heads, finding out flowers with the petals developed and the top ends fixed together by the clips, using the front ends of small milk powder stirrers to be close to flower handles, turning on a power switch, electrifying and vibrating the flowers for about 10s, and marking the flowers in a hanging mode; after the petals shrink, the petals fall off together with the grafting clip, and the fruits grow into calyx, which indicates that the selfing is successful.
The beneficial effects are that:
by applying the method, the independent embryoids generated spontaneously or separately are transferred into a rooting and sprouting culture medium, the sprouting rate of the embryoids can reach 40% -60%, and several to tens or even hundreds of embryoids can be separated from each anther; transplanting the rooting strong embryoid seedlings to a survival rate of 93% or more; counting Miao Zhan 38.5.5 percent (15/39) of the multiple branches in 39 transplanted survival seedlings, dividing and transplanting after the survival multiple branches seedlings (seedling clusters) grow for 1 month, obtaining 2-9 plants from 1 survival seedling, and changing the plant yield to be more than 1.69 (66/39) times of the original plant yield; about 1/3 of the plants transplanted to the field develop into multi-terminal bud plants, and the plant parts need to dredge redundant terminal buds, so that 1 to a plurality of remained terminal buds develop into branches and bloom, the branches are selfed into 1 to a plurality of selfed lines, or 1 to a plurality of asexual lines are separately propagated, and the plants without the thinning die prematurely and the offspring cannot be obtained. In this way, the germination rate of embryoid bodies exceeds 32 plants/bud on average, and 1 (0.6 plants/bud of Wangchang et al at maximum) has not been reported. The method greatly improves the efficiency of creating new germplasm of the eggplant by using anther culture technology.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 shows a suitable for the anther and embryoid body of eggplant with no switch over and over. A. Anthers and embryoids suitable for switching: arrows show anthers that produce embryoids, red boxes indicate that embryoids are different in size and direction; B. overdue, uninverted anthers and embryoids: arrows show embryoid callus formation.
FIG. 2 embryoid and anther classification switch. A. The embryoid 1 which needs to be separated and transferred; B. the embryoid body 2 which needs to be separated and transferred; C. the small embryoid on embryoid 2 needs to be peeled off as early as possible; D. a small embryoid that can be transferred with anther; E. embryoid bodies suitable for anther stripping and transferring into germination medium; the anthers that did not produce embryoid bodies were all transferred to germination medium.
FIG. 3 requires embryoid seedlings that induce secondary rooting into seedlings. a embryoid seedlings with undeveloped root systems; b, embryoid seedlings with vigorous growth vigor are too little in branches and too many in seedlings; and c, the root system of the middle embryoid is parasitic in the stem, and the arrow indicates.
FIG. 4 embryoid sprouting that can be transplanted directly. A. Normal seedlings; B. multiple embryos are used for seedling combination, and after survival, the seedlings are required to be separated; C. multiple seedlings with terminal buds being symbiotic, after survival, branch strip selfing and seed reserving or asexual propagation (cutting/grafting) are needed.
FIG. 5 diploid, clumped seedlings are planted in separate plants. 1 embryoid-developing cluster strain containing 9 branches; B. 6 complete plants separated from the cluster plants; C. 3 root-free seedlings separated from the cluster plants are planted after being treated by carbendazim and auxin; and D.9 separate plants are diploid, bloom and fruit.
FIG. 6 haploid clumped seedlings were planted in separate plants. A.1 cluster plant with embryoid development; a1, dividing the plant into 1, and making root system vigorous; a2, dividing the plant into 2 without roots; a3, dividing the plant into 3, and making root system vigorous; b1, A1 plants bloom; b2, the root-free seedlings of the A2 survive by rooting; b3, survival of A3 and new leaf growth.
FIG. 7 is a multi-terminal bud plant and its fruit developed from a transplanted, viable embryoid body. A. A shoot-co-growing multiple terminal bud plant; B. 6 branches are developed from the multi-terminal bud plant; c-d. the arrow in fruit a on two branches shows the multiple terminal buds (the arrow in b.c.d shows the fruit on three branches).
Fig. 8 is a process of single flower selfing. A. Flower buds suitable for flower clamping and isolation; B. flowers suitable for supplementary pollination are clamped on the 2 nd day; C. supplementary pollination selfing by a milk powder stirrer; D. pollen is fully stained on the stigma after pollination; E. and after selfing, the flower clamping isolation and temporary flower clamping handle marking are carried out, and then the plastic hanging label is replaced for marking.
Detailed Description
The invention will be better understood from the following examples.
The test was carried out at the gardening laboratory center of the national academy of sciences of the science and technology at 10 th 2018-7 th 2023. The embryoid is the offspring of the commercial variety 'new black dragon' and its polymeric hybridization, 1 diploid plant is produced by anther culture in 2018, the seedling population is established after seed sowing and is collected from the plant, and the embryoid is induced by anther culture. The 50% wp carbendazim powder is produced by Shanghai Yue Ling chemical industry Co., ltd, the content of active ingredient is 50wt%, each liter of tap water contains 1.25 g powder, and the suspension is prepared by diluting 800 times; 1-Naphthylacetic acid (NAA, molecular weight 186.21) produced by Shanghai chemical reagent company of China medical group, the powder was dissolved with a small amount of 0.5N NaOH and diluted to 1000ppm with ultra pure water.
(1) Embryoid isolation and germination culture: anthers of embryoid bodies which are visible to the naked eye are generated on an embryoid induction culture medium, each embryoid body is independent of the other embryoid bodies, root hair and embryo do not appear, before callus does not appear, the anthers of single independent large embryoid bodies are peeled off and transferred into an embryoid germination culture medium, the embryoid bodies are transferred into the embryoid germination culture medium after being separated into single embryoid bodies along the outline by a blade, and the small embryoid bodies are transferred into the embryoid germination culture medium together with the anthers, and all the anthers without embryoid bodies are transferred into the embryoid germination culture medium (figures 1 and 2);
(2) Secondary rooting and seedling formation of embryoid seedlings: the seedlings with the length exceeding 2cm are cut off, inoculated into a rooting culture medium and cultured for about 20 days to induce rooting to form complete plants.
(3) And (3) transplanting and hardening seedlings of the complete plants: the whole seedling produced directly on germination medium (figure 4), and the healthy seedling produced by secondary root are taken out from bottle, agar is washed off, put into 800 times carbendazim solution to soak for 10min, the carbendazim solution is washed off, water is absorbed, root is immersed into 1000ppm NAA solution for several seconds, planted into wet and water-free matrix, plastic box is tightly covered with plastic film, then the whole plastic box is put into illumination incubator, temperature of illumination incubator is set to 25 ℃, light-dark period is 12h/12h, and illumination intensity is adjusted to 1500lx. The plastic film is opened after 1 week without opening the film, water is properly poured according to the dry and wet condition of the substrate, the film is covered again, a gap with the width of about 1cm is reserved between the film and the top end of the incubator, the gap width is gradually increased on the basis of ensuring that plants do not wilt, and the leaves of the plants obviously grow after 2 weeks generally, and the film is removed. And finally transplanting the surviving cutting seedlings into a big basin or a field, and managing according to normal cultivation. The survival rate is more than 93% by adopting the method (table 1).
(4) Planting cluster seedlings in a dividing way: the rate of the cluster plants in the survival seedlings reaches 38.5% (table 2), the survival cluster plants are torn down and outwards along the joint parts, each plant is provided with roots as much as possible, the developed root-divided plants are directly planted in a basin, watered and placed in an incubator, and the plants with undeveloped roots or without roots are treated with reference to the step 3 (fig. 5 and 6). The culture condition after planting is the same as that of the step 3.
(5) Selfing or cutting multi-branch seedling branches: the survival of plants with a plurality of elongated branches is realized, the selfing of the same flower pollination is carried out according to the branches or the pollination selfing is carried out among flowers on the same branches, then the seed fruits are collected according to different branches, and different selfing lines are respectively built; for plants with more branches and cannot bear fruits at the same time, the plants can be self-crossed in batches, and finally, all the branches are built into self-crossed lines (figure 7); for branches which cannot fruiting seeds, a cutting/grafting method is adopted to build a clone.
The selfing process of single flower pollination is as follows: the method comprises the steps of (1) carrying out on sunny days with the temperature not exceeding 32 ℃, clamping the front ends of petals of large flower buds which are opened on each branch by using a grafting clamp on the 2 nd day or the 3 rd day, taking care of clamping the petals but not clamping the petals on the column head, finding out flowers with the petals developed and the top ends fixed together by the clamps, closing the front ends of small milk powder stirrers to flower handles, turning on a power switch, electrifying and vibrating the flowers for about 10s, and marking (figure 8); after the petals shrink, the petals fall off together with the grafting clip, and the fruits grow into calyx, which indicates that the selfing is successful.
TABLE 1 eggplant embryoid seedling process and first transplanting seedling rate
TABLE 2 investigation of survival of surviving 39 seedlings by dividing propagation
On embryoid induction medium, 6% -50% of anthers produce embryoids, the average of which exceeds 20%, and the embryoid induction rate reaches 1.25 anthers per bud calculated by 5 anthers per bud; the average number of larger embryoid generated by each anther exceeds 41, the first germination rate is 37.3%, the secondary seedling reproduction coefficient is 1.69, and the average number of regenerated plants generated by each bud exceeds 32.
The invention provides a method for improving the seedling rate of embryoid cultured by eggplant anther, and a method for realizing the technical scheme, wherein the method and the way are a plurality of preferred embodiments of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A method for improving the seedling rate of embryoid cultured by eggplant anther, which is characterized by comprising the following steps:
(1) The embryoid is singly separated and transferred into embryoid germination culture medium for seedling formation;
(2) Secondary seedling formation of embryoid: shearing off the well-developed rooting-free seedlings or parasitic embryoid seedlings, transferring the seedlings into a rooting and sprouting culture medium to induce rooting, forming complete plants, and transplanting and hardening the seedlings;
(3) And (3) directly transplanting the rooting embryoid seedlings with strong development: rooting the rooting and sprouting culture medium and developing embryoid seedlings of more than 1 branch, and directly transplanting and hardening the seedlings;
(4) Transplanting survival cluster seedlings to separate plants: the cluster seedlings which grow well are torn to the root along the branches, the cluster seedlings are divided into a plurality of plants, the seedlings with roots are directly planted, and the branches without roots are transplanted and subjected to moisture preservation and heat preservation management according to a tissue culture seedling hardening method, and are moved to the field after survival;
(5) Transplanting field survival multi-branch seedlings, and carrying out selfing seed reserving on branch strips or cutting/grafting preservation on branch strips.
2. The method for improving the seedling rate of embryoid bodies cultured by eggplant anthers according to claim 1, wherein in the step (1), separation and switching are carried out within 10d after the occurrence of embryoid bodies are observed by naked eyes, wherein single independent embryoid bodies are directly switched, embryoid bodies which are closely together are separated into single independent embryoid bodies by a blade or a dissecting needle and then switched, and all anthers are switched; the period and the method for separating the single embryoid are as follows: and (3) after anther inoculation, generating green and white embryoid which is visible to the naked eye and is not healed, wherein embryoid which are close together, have obvious size difference and inconsistent growth direction are separated under aseptic condition on an ultra-clean workbench by using a dissecting needle and a blade.
3. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 1, wherein in the step (1), the embryoid body germination medium is a B5 or NLN solid medium containing a small amount of auxin and/or a mitogen, and the concentration of the auxin is higher than that of the mitogen; preferably, the concentration of the auxin substances is 0.2mg/L, and the concentration of the mitogen substances is 0.1mg/L.
4. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 1, wherein in the step (2), the rooting and sprouting medium is a B5/NLN solid medium with or without 0.1-0.2mg/L auxin.
5. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 1, wherein the transplanting method in the step (2) is as follows: taking out the seedling suitable for transplanting from the culture flask, and immersing the root in the carbendazim solution for 5-30 min; taking out, cleaning with water, gently sucking to dry root drop, soaking in NAA or IBA solution for 1s-1min, then planting in conventional seedling substrate, watering thoroughly without water accumulation, covering with high transparency film, and placing in illumination incubator for 2 weeks.
6. The method for improving the seedling rate of embryoid cultured by eggplant anther according to claim 5, wherein the carbendazim solution is obtained by diluting carbendazim wettable powder with the active ingredient content of 50wt% with water according to the mass ratio of 1:500-800; the NAA or IBA solution is 100-1000 ppm.
7. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 1, wherein in the step (3), the embryoid bodies Miao Miaogao are directly transplanted and acclimatized over 2 cm.
8. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 1, wherein in the step (4), the method for dividing plants is as follows: after the plant is separated, the plants with developed root systems are directly transplanted into a matrix, and the plants are placed in an incubator to be subjected to heat preservation and moisture preservation for 1 week, and then the surviving plants can be transplanted into the field; and transplanting the plants without roots at the bottoms after the division according to a method for transplanting the tissue culture seedlings out of the bottles.
9. The method for increasing embryoid growth rate in anther culture of eggplant according to claim 1, wherein in the step (5), the method for selfing each branch comprises: the flowers on each branch are subjected to homoflower selfing, or the flowers on the same branch are subjected to pollination selfing, and the flowers on different branches cannot be mutually pollinated; if the selfing fruits on all branches cannot be obtained at the same time due to excessive branches, batch selfing is needed, namely, after the mature harvesting of the fruits on the branches of the early-stage selfing fruit setting, the selfing and seed reserving are carried out on the flower buds on the other branches.
10. The method for improving the seedling rate of embryoid bodies cultured by eggplant anther according to claim 9, wherein the co-flowering selfing process is as follows: the method comprises the steps of carrying out on sunny days with the temperature not exceeding 32 ℃, clamping the front ends of petals of large flower buds which are opened on each branch on the 2 nd day or the 3 rd day by using grafting clips, finding out flowers with the petals developed and the top ends fixed together by the clips, closing the front ends of stirrers to flower handles, turning on a power switch, electrifying and vibrating the flowers for about 10s, and removing the petals together with the grafting clips after the petals shrink, wherein the fruits grow out of calyx, so that the selfing is successful.
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