Method for obtaining distant hybrid new germplasm of intergeneric hybrid potato based on grafting mongolian guide technology
By using grafting techniques, and through grafting and self-pollination of wolfberry and potato, the barriers to distant breeding of potatoes were overcome, and new potato germplasm resistant to late blight was obtained, thus expanding breeding resources.
Patent Information
- Application Number
- CN202311773374.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-21
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2043-12-21
AI Technical Summary
Existing technologies have failed to effectively overcome the barriers to sexual breeding among distant relatives of potatoes, making it difficult to obtain intergeneric hybrid offspring through grafting techniques.
Using grafting techniques, robust wolfberry shoots were selected as rootstocks and diploid potato seedlings as scions. Through cleft grafting and post-grafting management, combined with pollination and self-pollination, distant hybrid germplasm was obtained.
The transfer of the disease-resistant gene from wolfberry to potato plants was achieved, resulting in distant hybrid offspring resistant to late blight and expanding potato resistance breeding resources.
Smart Images
Figure CN117581714B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of potato breeding, and particularly relates to a method for obtaining distant hybrid new germplasm of intergeneric hybrid potato based on grafting and guiding technology. BACKGROUND
[0002] Potato is originally from the Andes in South America, and the genetic basis of potato cultivars worldwide is narrow, and inbreeding degeneration is serious, and resistance breeding is limited by resources. Wolfberry is also a Solanaceae plant, and will not cause late blight, and is drought-tolerant and saline-alkali-tolerant, and is a pioneer tree species for afforestation in saline-alkali land. Grafting is a commonly used technical means in agricultural and forestry production, and plays an important role in improving production efficiency, improving quality, preserving germplasm resources and new mutant genes, etc. At present, potato late blight is one of the important diseases threatening potato production, and causes serious losses to the industry every year. Studies have shown that the genome between rootstock and scion will exchange in grafting, and the vascular bundle and intercellular filaments may provide conditions for gene horizontal transfer. Stegemann and Rock used two transgenic tobacco lines to graft each other, and the nuclear genes and plastid genes of the two transgenic tobacco lines carried different markers and reporter genes, respectively, and the detection of the reporter genes showed that the plastid genome would be short-distance transferred at the grafting interface. If the disease-resistant genes of wolfberry can be transferred to potato plants through grafting means, new germplasm resources or intermediate materials are obtained, which will provide a new idea for the research on potato resistance to late blight. Ohta (1991) of Japan has been engaged in grafting hybridization experiments of peppers for many years, and not only obtained grafting hybrids, but also further studied the grafting hybrids from the gene level, and summarized some characteristics of Mendelian traits in genetic variation after grafting, and found that the mechanism of inducing these variations is similar to the transformation mechanism of higher organisms. Stegemann and Bock (2006) of Germany grafted two transgenic tobacco lines, and the results showed that the marker genes can be frequently exchanged between cells at the grafting site, indicating that there is horizontal gene transfer between rootstock and scion.
[0003] At present, there is no report on grafting and guiding technology with potato as the female parent and wolfberry as the male parent to overcome the sexual breeding obstacles of distant hybrid plants of potato, and to obtain intergeneric hybrid offspring. SUMMARY
[0004] The technical problem to be solved by the present application is to provide a method for obtaining distant hybrid new germplasm of intergeneric hybrid potato based on grafting and guiding technology, to overcome the sexual breeding obstacles of distant hybrid plants of potato, and to obtain intergeneric hybrid offspring.
[0005] The technical problem to be solved by the present application is solved by the following technical scheme:
[0006] A method for obtaining distant hybrid new germplasm of intergeneric hybrid potato based on grafting and guiding technology, comprising the following steps:
[0007] (1) Rootstock cultivation and selection: select the non-lignified tender branches of the upper part of the healthy and disease-free Lycium barbarum as the rootstock;
[0008] (2) Cultivation and selection of scion: diploid original cultivated species Solanum tuberosum 2-3 unfolded leaf seedlings as scion;
[0009] (3) Grafting: cut the Solanum tuberosum seedling from the base with a blade, remove all leaves, then use the blade to cut the scion into a wedge-shaped symmetrical bevel, the size of the wedge is consistent with the size of the rootstock cut, keep the wedge clean and tidy, graft in a way of split grafting, insert the scion into the rootstock cut, align the two cuts, then use the safety film to fix the rootstock and the scion;
[0010] (4) Post-grafting management: after grafting, enter post-grafting management;
[0011] (5) Obtaining of distant hybrid seed: select the Solanum tuberosum flower buds of grafted seedlings for pollination, obtain self-pollinated Solanum tuberosum seeds, then germinate the self-pollinated seeds to produce seedling tubers, graft the seedling tubers, pollinate and obtain distant hybrid seeds, and harvest F1 generation of asexually cloned line original seed after the distant hybrid seeds are expanded.
[0012] Preferably, the step (5) specifically comprises the following steps:
[0013] (51) Select the Solanum tuberosum flower buds of grafted seedlings, remove the stamens after removing the petals;
[0014] (52) Pollination, bagging, and labeling: collect Lycium barbarum pollen, pollinate the Solanum tuberosum / Lycium barbarum grafted plants every morning;
[0015] (53) Ovary swelling: the ovary of the primary grafted Solanum tuberosum after pollination by Lycium barbarum pollen is rarely swollen, and the shedding time is delayed by 5-7 days compared with the same control; collect self-pollinated Solanum tuberosum seeds with good distant graft compatibility and vigorous growth of scion, for standby use;
[0016] (54) Germination of self-pollinated seeds and production of seedling tubers: after pretreatment of the scion hybrid seeds obtained last year, mix with fine sand, sow, and harvest seedling tubers in autumn;
[0017] (55) Re-grafting of seedling tubers: sow the seedling tubers obtained in step (54), and after germination, the offspring of the same parent are called sister lines, which are grafted with Lycium barbarum again, and the method is the same as step (3) above;
[0018] (56) Pollination of sister line scion: collect Lycium barbarum pollen, pollinate the Solanum tuberosum / Lycium barbarum grafted plants every morning;
[0019] (57) Ovary swelling of sister line scion: 7-10 days after pollination, the ovary can swell;
[0020] (58) Sterile culture of the hybrid seed: the hybrid seed is sowed on 1 / 2MS medium in a tissue culture room, and the asexual propagation stems are planted in a greenhouse with vermiculite, and the F1 generation asexual clone original seed is harvested.
[0021] Preferably, the pollination method of step (52) and step (56) is that the pollen of the male parent wolfberry is collected and applied to the stigma of the emasculated flower, and then the flower is bagged and labeled with the hybridization date and the parent and child variety names.
[0022] Preferably, step (54) is specifically:
[0023] The hybrid seed obtained last year is soaked in 1000 times gibberellin liquid for 48 hours;
[0024] The seed is mixed with fine sand and evenly spread on the plug tray, and then covered with a layer of soil;
[0025] After 40 days of sowing, the seedlings are transplanted into 10cm*10cm nutrient pots, and normal fertilization and water management are carried out, and the seedling tubers are harvested in autumn.
[0026] Preferably, step (55) is specifically: after the potato and wolfberry are grafted, a series of seedling tubers produced by self-pollination of the same variety are called sister lines, and the sister lines are sowed, and then grafted with wolfberry after germination, and the method is the same as step (3) above.
[0027] Preferably, the stock selected in step (1) is treated as follows:
[0028] The top bud of the wolfberry is removed, and the split grafting method is used between the first fully expanded leaf and the second fully expanded leaf at the top of the stem, and a double-edged blade is vertically cut downward from the top of the stock, and the depth of the cutting edge is about 1.0-1.5cm.
[0029] Preferably, in step (3), the blade and hands are disinfected with 75% alcohol before grafting, and the blade and hands are disinfected with alcohol after completing each group of grafting, and the next operation is carried out after the alcohol is completely volatilized.
[0030] The above technical scheme of the present application has the following beneficial effects:
[0031] The starting point of the present application is that the genetic basis of potato cultivars is narrow worldwide, inbreeding degeneration is serious, resistance breeding is limited by resources, and at present, potato late blight is one of the important diseases threatening potato production. On this basis, the present application is created based on the reasons that wolfberry will not occur late blight, is drought and saline-alkali tolerant, and is also a solanaceous plant. The present application effectively reduces the incompatibility of wolfberry and potato, rapidly realizes the distant hybridization of wolfberry and potato, and for the first time obtains the offspring of distant hybridization, thereby providing a material basis for widening the resistance breeding resources and distant sexual hybridization of potato. BRIEF DESCRIPTION OF DRAWINGS
[0032] Figure 1 It is a morphological feature diagram of potato and wolfberry grafted plants. Among them: A external conditions, B initial stage, C healing, D scion metamorphic stem, E growth potential, F flowering period, G ovary swelling by wolfberry pollen, H mature period of scion self-pollination and seed setting.
[0033] Figure 2 It is a diagram of ovary swelling of potato / wolfberry grafted plants by wolfberry pollen for 20 days.
[0034] Figure 3 It is the offspring obtained in the ovary of potato / wolfberry grafted plants x wolfberry.
[0035] Figure 4 It is the production of microtubers, wherein a: the tissue culture expanded bottle seedlings are transplanted into the vermiculite coconut husk substrate; b: F1 microtubers are obtained. DETAILED DESCRIPTION
[0036] The specific embodiments of the present application are described in detail below in order to further understand the present application.
[0037] In the following examples, all experimental methods used are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.
[0038] A method for obtaining intergeneric hybrid potato new germplasm based on grafting Mong guide technology, comprising the following steps:
[0039] (1) Cultivation and selection of rootstocks:
[0040] The un-woody tender branches of the upper part of the wolfberry growing in a healthy and disease-free manner are selected as rootstocks.
[0041] (2) Cultivation and selection of scions:
[0042] The diploid original cultivar potato 2-3 unfolded leaf seedlings are used as scions.
[0043] (3) Grafting:
[0044] Before grafting, use 75% alcohol to disinfect the knife and hands. After finishing a group of grafting, disinfect the knife and hands with alcohol, and then proceed to the next step after the alcohol completely evaporates. The following is specific:
[0045] (31) Preparation of the stock:
[0046] Remove the top bud of the wolfberry, and use the wedge grafting method to cut the stem between the first fully expanded leaf and the second fully expanded leaf from the top of the stock vertically downward with a double-edged knife. The depth of the cut is about 1.0-1.5 cm.
[0047] (32) Selection of the scion variety:
[0048] Select potato varieties with high graft compatibility with wolfberry. The growth of grafted potato plants is significantly weaker than that of ungrafted potato plants. After grafting, the height of the scion is significantly lower than that of the control (Table 1), with a decrease of 47.1%-52.8%. The number of stems and nodes is not significantly different from the control, but the average internode length is significantly shorter, with an average decrease of 49.7%. The average number of branches also significantly decreases.
[0049] Table 1 Comparison of growth potential of different scion potato varieties and non-grafted potato plants at flowering stage
[0050]
[0051] Note: In the table, the same column with different lowercase letters indicates significant differences at the 0.05 level in multiple range test analysis. The values in the table represent mean ± standard error.
[0052] (33) Grafting:
[0053] Cut the potato seedling from the base with a knife, remove all leaves, then cut the scion into a symmetrical wedge-shaped surface with a knife. The size of the wedge surface is consistent with the size of the stock cut, and the wedge surface is kept clean and neat. Graft in a wedge grafting manner, insert the scion into the stock cut, align the two cuts, then use the safety film to fix the stock and scion.
[0054] (4) Post-grafting management:
[0055] After grafting, cover the grafting site with a plastic bag, then proceed to the incubation and moisture preservation (see Figure 1 Fig. A and Fig. B).
[0056] Spray 1 g / L of carbendazim every day to prevent the growth of pathogens.
[0057] After 7 days, regularly ventilate and expose to sunlight, and gradually extend the ventilation time as the grafting site grows.
[0058] 20-25 days later, when the scion potato grows 2-3 leaves, the shading and warming measures can be completely removed, and the routine management is carried out, and attention should be paid to removing the new sprouts of the grafted part on the Lycium barbarum rootstock. At this time, in order to provide sufficient nutrients and prevent the breeding of diseases and pests, full nutrient solution should be irrigated regularly, and fungicides and insecticides should be sprayed.
[0059] The potato and the Lycium barbarum are grafted into survival 10-15 days after grafting, and then enter the vigorous growth stage. The stolon stems of the scion cannot enter the soil to form tubers, resulting in that the nutrients cannot be transported to the storage organs (underground tubers), causing the stem at the grafting interface to swell, and some stolon stems will develop into sprouts (see D figure of Figure 1 ).
[0060] The scion grows well but the internodes are shortened, and the growth potential is weakened (see E figure of Figure 1 ).
[0061] (5) Obtaining of distant hybrid germplasm:
[0062] The potato scion with strong affinity can normally bloom in the later growth stage (see F figure of Figure 1 ).
[0063] (51) Selection of potato flower buds, and the flower buds 1-2 days before flowering are removed after the petals are removed;
[0064] (52) Pollination, bagging, and labeling: collect Lycium barbarum pollen, and pollinate the potato / Lycium barbarum grafted plants every morning. Collect the pollen of the male Lycium barbarum and smear it on the stigma of the emasculated flower, and then bag and label, indicating the hybridization date, the male and female variety names;
[0065] (53) Ovary swelling: the ovary of the primary grafted potato after being pollinated by Lycium barbarum pollen is rarely swollen, and the shedding time is delayed by 5-7 days compared with the same control (the potato variety not grafted is pollinated by Lycium barbarum pollen). Collect the seeds of the potato variety with good distant grafting affinity and vigorous scion growth that can self-pollinate (see H figure of Figure 1 ).
[0066] (54) Germination of self-pollinated seeds and production of seedling tubers: the scion hybrid seeds obtained last year are soaked in 1000 times gibberellin liquid for 48 hours. The seeds are mixed with fine sand and evenly spread on the plug tray, and then covered with a layer of soil. After 40 days of sowing, transplant the seedlings into 10 cm x 10 cm nutrient pots, and normally manage the fertilization and watering. Harvest the seedling tubers in autumn.
[0067] (55) Re-grafting of seedling tubers: after the potato is grafted with Lycium barbarum, a series of seedling tubers produced from the self-pollinated seeds of the same variety are called sister lines. The sister lines are sowed, and then grafted with Lycium barbarum after germination, and the method is the same as step (3) above.
[0068] (56) Pollination of sister system scion: Collect pollen of wolfberry, and pollinate potato / wolfberry grafted plant every morning. Collect pollen of male wolfberry, and smear on the stigma of emasculated flower. Put bag on the flower, and label the date of cross and the name of male and female varieties.
[0069] (57) Sister system scion ovary swelling: 7-10 days after pollination, ovary can swell (see Figure 2 ).
[0070] (58) Aseptic culture of distant hybrid seed: sow distant hybrid seed on 1 / 2MS medium (see Figure 3 ) in tissue culture room, and propagate the tissue. Plant the asexual propagation stem segment in greenhouse vermiculite, and harvest F1 generation asexual clone line original stock (see Figure 4 ).
[0071] Although the present application has been disclosed with reference to the examples as above, it is not intended to limit the present application, and any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, and the scope of protection of the present application is defined by the claims and their equivalents.
Claims
1. A method for obtaining distant hybrid new germplasm of intergeneric hybrid potato based on grafting Mongolian guide technology, characterized in that, The method comprises the following steps: (1) Cultivation and selection of stock: selecting the non-lignified tender branches of the upper part of the healthy and disease-free Lycium barbarum as the stock; (2) Cultivation and selection of scion: selecting the 2-3 unfolded leaf seedlings of the diploid original cultivated species Solanum tuberosum as the scion; (3) Grafting: cutting the Solanum tuberosum seedling from the base with a blade, removing all leaves, then cutting the scion into a wedge-shaped symmetrical bevel, the size of the wedge surface being consistent with the size of the stock incision, keeping the wedge surface clean and tidy, grafting in the way of wedge grafting, inserting the scion into the stock incision, aligning the incisions of the two, then fixing the stock and the scion with fresh-keeping film; (4) Post-grafting management: after grafting, entering post-grafting management; (5) Obtaining of distant hybrid germplasm: selecting the Solanum tuberosum flower buds of the grafted seedlings for pollination, obtaining self-crossed seed of the Solanum tuberosum variety, then germinating the self-crossed seeds, producing true seed tubers, grafting the true seed tubers, pollinating and obtaining distant hybrid seeds, after the tissue propagation of the distant hybrid seeds, harvesting the F1 generation of the original clone original seed; The step (5) specifically comprises the following steps: (51) Selecting the Solanum tuberosum flower buds of the grafted seedlings, removing the stamens after removing the petals; (52) Pollination, bagging and labeling: collecting the pollen of Lycium barbarum, pollinating the Solanum tuberosum / Lycium barbarum grafted plants every morning; (53) Ovary swelling: after the first generation of grafted Solanum tuberosum is pollinated by Lycium barbarum pollen, the ovary is less swollen, and the shedding time is delayed by 5-7 days compared with the same control; collecting the self-crossed seeds of Solanum tuberosum with good distant graft compatibility and vigorous growth of the scion, which can self-cross and bear fruit, for standby use; (54) Germination of self-crossed seeds and production of true seed tubers: after the scion hybrid seeds obtained last year are pretreated, they are mixed with fine sand, sown, and the true seed tubers are harvested in autumn; (55) True seed tuber grafting again: the true seed tubers obtained in step (54) are sown, and after germination, the offspring of the same parent are called sister lines, which are grafted with Lycium barbarum again by the method of step (3); (56) Pollination of sister line scions: collecting the pollen of Lycium barbarum, pollinating the Solanum tuberosum / Lycium barbarum grafted plants every morning; (57) Ovary swelling of sister line scions: after pollination, the ovary can swell 7-10 days later; (58) Aseptic culture of distant hybrid seeds: the distant hybrid seeds are sown on 1 / 2MS medium in a tissue culture room for tissue propagation, and the vegetative propagation stems are planted in a greenhouse with vermiculite to harvest the F1 generation of the original clone original seed.
2. The method as claimed in claim 1 for obtaining distant hybrid novel germplasm of intergeneric hybrid potato based on grafting Mongolian guide technology, characterized in that, The pollination method of step (52) and step (56) is: collecting the pollen of the male parent Lycium barbarum and smearing it on the stigma of the emasculated flower, then bagging and labeling, indicating the hybridization date and the variety names of the male parent and female parent.
3. The method as claimed in claim 1 for obtaining distant hybrid novel germplasm of intergeneric hybrid potato based on grafting Mongolian guide technology, characterized in that, Step (54) specifically comprises: The scion hybrid seeds obtained last year are soaked in 1,000 times gibberellin liquid for 48 hours; The seeds are mixed with fine sand and evenly spread on the plug tray, and then covered with a layer of soil; After 40 days of sowing, the seedlings are transplanted into 10cm*10cm nutrient pots, and normal fertilization and water management are carried out, and the true seed tubers are harvested in autumn.
4. The method as claimed in claim 1 for obtaining distant hybrid novel germplasm of intergeneric hybrid potato based on grafting Mongolian guide technology, characterized in that, Step (55) specifically comprises: after the Solanum tuberosum and Lycium barbarum are grafted, a series of true seed tubers produced by self-crossing the seeds of the same variety are called sister lines, which are sown, and after germination, they are grafted with Lycium barbarum again by the method of step (3).
5. The method as claimed in claim 1, wherein the method of obtaining distant hybrid novel germplasm of intergeneric hybrid potato based on grafting Mongolian guide technology comprises: The stock selected in step (1) is treated as follows: Remove the top bud of the wolfberry, and use a double-edged blade to cut the stem between the first and second fully expanded leaves from the top of the stock vertically downwards, with a depth of 1.0-1.5 cm.
6. The method of obtaining distant hybrid germplasm of intergeneric cross of potato based on grafting of MGD techniques as claimed in claim 1 wherein, In step (3), before grafting, the blade and hands are disinfected with 75% alcohol. After completing each group of grafting, the blade and hands are disinfected with alcohol, and the next step is performed after the alcohol completely evaporates.