CN115589943B - In-vitro regeneration method for tea trees - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to the field of plant tissue culture, and particularly relates to an in-vitro regeneration method of tea trees. The high-efficiency adventitious bud in vitro generation system of the tea tree stem is established by taking the tea tree stem as an initial explant, so as to provide technical support for large-scale breeding, genetic engineering, gene editing, germplasm resource preservation and the like of high-quality seedlings of the tea tree.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to an in-vitro regeneration method of tea trees.
Background
Tea tree (Camellia sinensis (L.) Kuntze) is a perennial woody plant of the genus Camellia in the family Theaceae, is an important cash crop in China, has long cultivation history (Chen Zong. Chinese tea passage [ M ]. Shanghai: shanghai culture Press, 1994:5), and tea is a main product, and tea beverage is one of three non-alcoholic beverages in the world and has large market demand. Along with the continuous enhancement of people's great health consciousness and the increasing of living standard, genetic improvement of tea trees and high-quality seedling production are increasingly paid attention to. The production of the clone tea seedlings in China mainly uses short spike cutting propagation and secondary layering propagation. The cutting of the short spikes can well maintain the original excellent characteristics of the variety, but has the defects of long seedling period, low propagation coefficient, season limitation and the like; the layering propagation mode is simple to operate, root system is achieved, but the layering propagation mode is limited in large-scale production and propagation of tea tree seedlings. Compared with the traditional asexual propagation method, the plant in-vitro regeneration technology has the advantages of short culture period, high proliferation rate, no time and region limitation in seedling production, artificial controllability of culture conditions, time and labor saving, low cost and the like, and has important significance in the aspects of large-scale propagation of high-quality seedlings of tea trees, preservation of germplasm resources, rapid popularization and application of good varieties and the like.
In vitro regeneration studies of tea trees have been reported to obtain adventitious buds or regenerated plants mainly via the organogenesis pathway. Borchetia et al used different explants of tea tree (stem tip, axillary buds and cotyledons) as variables, studied the influence factor of tea tree in vitro rapid propagation, and the result showed that the axillary buds were the safest material for keeping the parent in micropropagation, and the random polymorphism analysis showed that the similarity between the plants cultivated by these three explants and the parent was compared, and the axillary buds were the highest (Borchetia S, das SC, handique PJ, et al high multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.) [ J ]. Scientia Horticulturae, 2009,120 (4): 544-550.). Chen Zexiong the in vitro regeneration research of tea tree by taking axillary buds of tea tree of Yucha No. 1 as explants, the induction rate of adventitious buds reaches 71.1%, the proliferation coefficient reaches 3.37, the rooting rate reaches 55.6% (Chen Zexiong, liu Yiqing, huang Dengyan Yucha No. 1' in vitro culture and plant regeneration research [ J ]. University of southwest (Nature scientific edition), 2009,31 (12): 67-70). Liu Jing et al performed the induction culture of tea tree axillary buds with an induction rate of 92%, but no cluster buds were found, and only single fixed buds were found (Liu Jing. Tea tree with axillary bud stem tissue culture research [ J ]. Shaanxi agricultural science 2020,66 (10): 43-45). Chen Lijie and the like establish a quick-propagation system of the tea stem of the Huaxi ancient tree, the optimal proliferation coefficient is 1.5, the rooting rate is 56.7 percent (Chen Lijie, yang Xia, high-definition, etc. the in vitro propagation technology of the Huaxi ancient tea tree is studied as first report [ J ]. Seed journal of the seed industry, 2019 (08): 10-13). The tissue culture explant differentiation pathway of tea tree is first axillary bud germination, the germinated fixed buds grow well, and then the tissue culture explant differentiation pathway is used for induction of the adventitious buds, so that the induction rate and the propagation coefficient are improved. The explant is damaged to some extent after disinfection treatment, and if the explant is directly used for adventitious bud induction, an ideal effect is hardly achieved (Xie Enjun. Tissue culture and rapid propagation of tea tree No. 1 in Zhonghuang and regeneration system establishment [ D ]. Guizhou university, 2019.). The stem of tea tree is a common report of the initiation of explant-induced bud, but the efficiency is not high, and the propagation coefficient is about 3. At present, no report of a genetic stable and efficient adventitious bud generation system of tea trees is found, and especially the genetic stability of optimal tea tree seedlings is slow to progress, the efficiency is low, and the genetic stability of regenerated buds is difficult to guarantee.
Disclosure of Invention
In view of the above, the invention takes the tea plant stem as an initial explant, develops research on the in-vitro regeneration influence factor of the genetic stability of the tea plant stem, establishes an in-vitro generation system of the efficient adventitious buds of the tea plant stem, and aims to provide technical support for large-scale breeding, genetic engineering, gene editing, germplasm resource preservation and the like of high-quality seedlings of the tea plant.
The invention provides an in-vitro regeneration method of tea trees, which comprises the following steps:
(1) Preparation of the explant material: taking a stem segment with a stem node as an explant;
(2) And (3) fixed bud induction: inoculating the stem segments cut in the step (1) into a fixed bud induction culture medium, wherein the fixed bud induction culture medium minimal medium takes an MS culture medium as a minimal medium, and 1-4 mg/L6-BA is added;
(3) Induction and proliferation of adventitious buds: inoculating the stem segments with the fixed buds in the step (2) into an adventitious bud induction culture medium, wherein the basic culture medium of the adventitious bud induction culture medium is an MS culture medium, and 1-3 mg/L6-BA, 0.1-0.3mg/L NAA, 0.05-0.15mg/L KT and 0.5-1.5mg/L proline are added;
(4) Adventitious bud elongation: inoculating the adventitious bud induced in the step (3) into an adventitious bud elongation culture medium, wherein a basic culture medium of the adventitious bud elongation culture medium is an MS culture medium, and 0.2-1mg/L of 6-BA and 0.02-0.1mg/L of NAA are added;
(5) Adventitious root induction: selecting the strong elongated sprouts in the step (4), inoculating the individual plants into a rooting medium, wherein the basic medium is 1/2MS medium, and adding 1-4mg/L IBA.
Preferably, the method further comprises the steps of:
(6) Hardening seedlings: selecting tea seedlings with developed root systems in the step (5), removing the sealing film, adding a small amount of distilled water into a culture flask, and hardening the seedlings at room temperature for 2-4 d.
More preferably, the method further comprises the steps of:
(7) Transplanting: taking out the culture medium of the roots of the tea seedlings after hardening off in the step (6), transplanting the culture medium into a plastic basin containing mixed soil, and placing the culture medium in a greenhouse for growth.
In a specific embodiment, 25-35g/L of sucrose and 5-9g/L of agar powder are added into the basic culture medium in the steps (2) to (5), and the pH value of the culture medium is regulated to be 5.8+/-0.1.
In another embodiment, the culture conditions of steps (2) to (5) are illumination intensity of 1500-2500lx, illumination per day of 14-18 hours, and culture temperature of 24+ -2 ℃.
In the preferred mode, in the step (1), the specific operation is to cut the half lignified stem section of the current year with partial leaves, the length of the stem section is 0.5-2cm, the leaves are cut off at the far end, leaves close to the petiole part are reserved, the petiole part is taken as a stem section node, the upper end and the lower end are cut flatly, and the stem section explant with one stem node is cut.
In a preferred embodiment, in step (2), the culture is continued for 3-5 weeks, and 2-3mg/L6-BA is added to the medium.
In a preferred embodiment, in step (3), the culture is continued for 3-5 weeks, and 2 mg/L6-BA, 0.2mg/L NAA, 0.1mg/L KT and 1mg/L proline are added to the medium.
In a preferred embodiment, in step (4), the culture period is 3-5 weeks, and 0.8mg/L6-BA, 0.08mg/LNAA is added to the medium.
In a preferred embodiment, in the step (7), the volume ratio of the nutrient soil to the perlite in the mixed soil is 3:1.
The inventor searches the finally obtained high-efficiency tea tree in-vitro regeneration method through research and can ensure genetic stability. The choice of the explant is that the semi-lignified stem with the axillary buds has special consideration, for example, when the old branches of tea trees are used as the explant, the stem lignification degree is high, the buds are dormant buds, the axillary buds germination rate is low, and the browning death degree is high; the stem node explant has higher bud proliferation rate than the stem tip because the stem tip is tender and is easy to damage during disinfection to cause browning and necrosis of the plant body. Therefore, the method selects a semi-lignified twig induction way with axillary buds of tea trees to obtain regenerated plants; the MS medium was selected from the various basal media to give the best results.
Aiming at the differentiation path of the tissue culture explant of the woody plant of the tea tree, the axillary bud germination path is selected. Because the stem sections after the axillary buds germinate grow strongly, if the axillary buds are used for induction of the adventitious buds, the induction rate and the propagation coefficient of the adventitious buds are high. And the explant has a certain damage after disinfection treatment, and if the explant is directly used for adventitious bud induction, an ideal effect is difficult to achieve. Therefore, the invention first selects for the generation of fixed buds for explants. According to the invention, the research shows that when the concentration of 6-BA is 2-3mg/L, more callus is generated at the bottom of the stem segment, and part of fixed buds grow out and the growth vigor is better in view of the comprehensive fixed bud induction rate, chassis expansion rate and stem segment growth condition.
In the growth stage of the adventitious bud, firstly, research shows that after the adventitious bud grows out, the callus at the basal part needs to further absorb nutrition, and two substances KT and proline are added to supplement each other, so that the state of the callus at the basal part is better, and the induction and proliferation of the adventitious bud are facilitated. Meanwhile, the hormone proportion screening experiment shows that the hormone concentration is 2 mg/L6-BA and 0.2mg/L NAA are the best combination, the formed absorption chassis state is best, and the adventitious bud induction and proliferation effects are best.
The adventitious bud induction and elongation are divided into two stages, and researches show that when 6-BA and NAA are respectively 0.4-1mg/L and 0.06-0.1mg/L, the adventitious buds have the effect of elongation.
In the rooting stage, the research table is that a 1/2MS culture medium is adopted to facilitate adventitious root induction of the bud seedlings; and the IBA with different concentrations can induce adventitious roots, and along with the increase of the IBA concentration, the rooting rate of the sprouts is in a trend of rising first and then falling, so that the adventitious roots are screened out to be thicker, lateral roots are more, and the plant grows healthily.
And (3) carrying out genetic stability identification on the in-vitro regenerated plant obtained by the invention. And selecting an in-vitro regenerated plant of the same stem segment of the robust tea tree, and taking a parent material of the in-vitro regenerated plant as a control, wherein the detection results of RAPD and ISSR molecules show that polymorphism bands are not detected between the parent and the regenerated plant, and the regenerated plant has no variation of DNA level.
Drawings
FIG. 1 shows selected explants of the present invention.
Figure 2 WPM medium induced shoot induced growth status of stem segment cultures.
FIG. 3 DCR medium induced shoot induced growth status of stem segment cultures.
FIG. 4 N6 culture medium induced shoot induced growth status of stem segment cultures.
FIG. 5 MS culture medium induced shoot induced growth status of stem segment cultures.
FIG. 6 shoot culture induced fixed bud growth conditions.
Adventitious bud proliferation phase II in the example of fig. 7.
The adventitious bud in the example of FIG. 8 is elongated.
Adventitious root induction in the embodiment of fig. 9.
The surviving plants were transplanted in the example of fig. 10.
FIG. 11 shows an amplified RAPD pattern of a regenerated plant obtained according to the present invention. Wherein a is primer S132; b, primer S133; m, DNA Marker;1-18: an isolated regenerated plant group of the same tea plant stem segment; 1-6 of 'Shucha early' generation regeneration plants; 7-12: 'Shucha early' second generation regenerated plants; 13 to 18: 'Shucha early' third generation regeneration plants; CK, female parent material.
FIG. 12 shows ISSR patterns amplified from regenerated plants obtained by the present invention. Wherein, a is primer UBC854; b, primer UBC880; m, DNA Marker;1-18: an isolated regenerated plant group of the same tea plant stem segment; 1-6 of 'Shucha early' generation regeneration plants; 7-12: 'Shucha early' second generation regenerated plants; 13 to 18: 'Shucha early' third generation regeneration plants; CK, female parent material.
Detailed Description
The invention will be further illustrated by the following detailed description in order to provide a better understanding of the invention, but without limiting the invention.
1. Preparation of explant material
(1) Selecting healthy and strong early tea leaf-loosening aseptic seedlings, cutting the half lignified stem segments with partial leaves in an ultra clean bench, cutting the distal ends of the leaves, reserving the leaves close to the petioles, taking the petioles as stem segment nodes, cutting the upper and lower ends of the petioles flatly, and cutting into stem segment explants with one stem node (figure 1).
2. Determination of minimal Medium
(2) The cut stem sections were inoculated onto 4 kinds of minimal medium such as MS, WPM, DCR, N6 to which 1 mg/L6-BA and 0.1mg/L NAA were added, and the growth conditions of the cultures were examined. The results show (Table 1) that the different media have a large difference in the effect of the induction of the stem segments. The growth vigor is weaker in the WPM culture medium, the bud stem section is thinner, and the leaf blade is reddish (figure 2); the buds growing on the DCR medium were thin and thin, and the leaves were yellowish (FIG. 3); the petioles were slightly reddish and the buds were thin and weak in N6 medium (fig. 4); the stem sections grew well in the MS medium, the sprouts were tender, the leaves were shiny, and the whole culture was viable (FIG. 5).
TABLE 1 Effect of different basal media on shoot induction
3. Fixed bud induction
(3) Inoculating the stem segment in the step (1) into a fixed bud induction culture medium, wherein a basic culture medium is an MS culture medium, and adding 0-4 mg/L6-BA. The results show (Table 2) that with the increase of the concentration of 6-BA, the induction rate of the fixed buds is in a trend of increasing and then decreasing, and the induction rate of the fixed buds is highest in 3mg/L6-BA and reaches 84.44%; the chassis expansion rate gradually increased with increasing 6-BA concentration, up to 88.89%, but part of the chassis cracked. From the viewpoints of the induction rate of fixed buds, the expansion rate of the chassis and the growth condition of the stem segments, more callus is generated at the bottom of the stem segments when the concentration of 6-BA is 2-3mg/L, and part of fixed buds grow out and grow better (figure 6). The main reason that the induction rate and the propagation coefficient of the adventitious buds are relatively high is that the adventitious buds are closely related to two factors of the induction of the adventitious buds and the absorption of the chassis, the healthy and strong adventitious buds grow into a photosynthesis main body under a certain culture medium formula, the basal part of the stem is enlarged, the absorption of the chassis is good, the activity of the whole culture is good, and the fertility is strong.
TABLE 2 Effect of different concentrations of 6-BA on shoot initiation at the stem
4. Adventitious bud induction and proliferation
(4) Inoculating the stem segment with the fixed bud in the step (3) into an MS culture medium with 0-3 mg/L6-BA, 0-0.3 mg/L NAA, 0.1mg/LKT and 1mg/L proline, and carrying out proliferation induction on the stem segment adventitious bud. The results showed (Table 3) that after 4 weeks of culture, adventitious buds were induced successively, and the induction rate of adventitious buds and the average adventitious bud number increased and then decreased with increasing 6-BA and NAA concentrations; when neither 6-BA nor NAA was added, the adventitious bud induction amount was small; when 6-BA is 1mg/L and NAA is 0.1mg/L, only 1 adventitious bud is formed on average, and the leaf surface is thin and curled; when 2mg/L of 6-BA and 0.2mg/L of NAA are added, the induction effect of adventitious buds is optimal, the induction rate is 88.89%, the average number of adventitious buds is 7.88, the buds are green, and the leaves are stretched (figure 7); when 6-BA and NAA were 3mg/L and 0.3mg/L, respectively, the adventitious bud induction rate was 71.11%, the number of adventitious buds was 3-4 on average, and a large amount of callus appeared at the bottom, and the adventitious bud leaves were yellow. Thus, the preferred formulation for shoot adventitious bud induction is MS+2 mg/L6-BA+0.2 mg/L NAA.
TABLE 3 Effect of plant growth regulators on adventitious bud proliferation induction
5. Elongation of adventitious bud
(5) Transferring the adventitious buds induced in the step (4) into an adventitious bud elongation culture medium, wherein a basic culture medium is an MS culture medium, adding 0.2-1 mg/L6-BA and 0.02-0.1mg/L NAA, and counting the elongation of the adventitious buds after culturing for 4 weeks. The results showed that the different hormone combinations had the effect of adventitious bud elongation, but the adventitious bud elongation induced by the different hormone combinations was different (Table 4), and the adventitious bud elongation tended to increase and decrease with increasing growth regulator. When the concentration of 6-BA was 0.8mg/L, NAA and 0.08mg/L, the elongation effect of adventitious buds was best, and the elongation was the highest, up to 70% (FIG. 8).
TABLE 4 Effect of plant growth regulators on adventitious bud elongation
6. Adventitious root induction
Robust elongation sprouts were selected and transferred to 1-4mg/LIBA MS and 1/2MS medium for adventitious root induction. The results showed that 7d began with slightly enlarged stem base, 15d primordia initiated, 20d adventitious root formation, and the rate of adventitious root induction in 1/2MS medium after 6 weeks was generally higher than in MS medium (Table 5). Therefore, the 1/2MS culture medium is beneficial to adventitious root induction of the bud seedling. The IBA with different concentrations can induce adventitious roots, the rooting rate of the sprouts is in a trend of rising and then falling along with the rising of the IBA concentration, when the IBA is 3mg/L, the rooting rate of the sprouts is highest and is 85.56%, the induced adventitious roots are thicker, lateral roots are more, and the plants grow healthily (figure 9).
TABLE 5 Effect of different concentrations of basal salts and IBA on adventitious root induction
7. Hardening and transplanting
Selecting bottle seedlings with developed root systems, removing a sealing film, adding a small amount of distilled water into a culture bottle, hardening the seedlings at room temperature for 3 days, taking out a culture medium for cleaning the root parts of the tea seedlings, transplanting the tea seedlings into a plastic basin containing mixed soil (the volume ratio of nutrient soil to perlite is 3:1), placing the plastic basin in a greenhouse, and watering in time to keep the soil moist, wherein the transplanting survival rate of the root seedlings is counted after 4 weeks, and reaches 86.67% (figure 10).
8. Genetic stability identification
Selecting the continuous third generation in vitro regeneration plants of the same stem segment of the obtained healthy and strong Shucha early, and carrying out RAPD and ISSR molecular marker genetic stability identification by taking the parent material of the Shucha early as a control. The RAPD and ISSR molecular markers co-amplified 161 bands, an average of 6.4 bands per primer (Table 6 and Table 7).
TABLE 6 RAPD primers and amplified spectra thereof
TABLE 7 ISSR primers and amplified spectra thereof
The result shows that the 15 RAPD primers amplify 113 clear bands with the size of 250-5000 bp. The number of amplified bands of a single RAPD primer varies from 4 to 12, the RAPD amplification pattern of primer S132 is shown in FIG. 11 a, and the RAPD amplification pattern of primer S133 is shown in FIG. 11 b. The 10 ISSR primers amplify 48 distinct bands with the size of 300-3000 bp. The number of bands amplified by a single ISSR primer is between 2 and 6, the ISSR amplification pattern of primer UBC854 is shown in FIG. 12 a, and the ISSR amplification pattern of UBC880 is shown in FIG. 12 b.
From the RAPD and ISSR molecular detection results, no polymorphism band is detected between the female parent and the regenerated plant, which indicates that the regenerated plant has no variation in DNA level, and the genetic consistency and stability of the Shucha early regenerated plant and the female parent material are verified.
Claims (10)
1. A method for in vitro regeneration of tea trees, comprising the steps of:
(1) Preparation of the explant material: taking a stem segment with a stem node as an explant;
(2) And (3) fixed bud induction: inoculating the stem segments cut in the step (1) into a fixed bud induction culture medium, wherein the fixed bud induction culture medium takes an MS culture medium as a basic culture medium, and 2-3mg/L6-BA is added into the basic culture medium;
(3) Induction and proliferation of adventitious buds: inoculating the stem segments with the fixed buds in the step (2) into an adventitious bud induction culture medium, wherein the basic culture medium of the adventitious bud induction culture medium is an MS culture medium, and adding 2-3mg/L6-BA, 0.2-0.3 mg/L NAA, 0.1mg/L KT and 1mg/L proline;
(4) Adventitious bud elongation: inoculating the adventitious bud induced in the step (3) into an adventitious bud elongation culture medium, wherein a basic culture medium of the adventitious bud elongation culture medium is an MS culture medium, and 0.6-1 mg/L6-BA and 0.06-0.1mg/L NAA are added;
(5) Adventitious root induction: selecting the strong elongated sprouts in the step (4), inoculating the individual plants into a rooting medium, wherein the basic medium is 1/2MS medium, and adding 2-4 mg/L IBA;
further comprises a seedling hardening step to obtain regenerated tea seedlings.
2. The method for in vitro regeneration of tea trees according to claim 1, wherein the step of hardening off is as follows:
(6) Hardening seedlings: selecting tea seedlings with developed root systems in the step (5), removing the sealing film, adding a small amount of distilled water into a culture flask, and hardening the seedlings at room temperature for 2-4 d.
3. The method for in vitro regeneration of tea trees of claim 2, further comprising the steps of:
(7) Transplanting: taking out the culture medium of the roots of the tea seedlings after hardening off in the step (6), transplanting the culture medium into a plastic basin containing mixed soil, and placing the culture medium in a greenhouse for growth.
4. A method for the ex vivo regeneration of tea trees according to any one of claims 1 to 3, wherein the minimal medium in steps (2) to (5) is supplemented with sucrose 25-35g/L, agar powder 5-9g/L and the pH of the medium is adjusted to 5.8±0.1.
5. A method for the ex vivo regeneration of tea trees according to any one of claims 1 to 3, wherein the culture conditions in steps (2) to (5) are light intensity 1500-2500lx, light 14-18h per day and culture temperature 24±2 ℃.
6. The method for in vitro regeneration of tea trees according to claim 5, wherein in step (1), the specific operation is to cut the half lignified stem segments of the current year with partial leaves, the length of the stem segments is 0.5-2cm, the leaves are cut off at the distal end, the leaves close to the petiole part are reserved, the petiole part is taken as a stem segment node, the upper end and the lower end are cut flatly, and the stem segment explants with one stem node are cut.
7. The method for in vitro regeneration of tea trees of claim 5, wherein in step (2), the incubation period is 3 to 5 weeks.
8. The method for in vitro regeneration of tea trees according to claim 5, wherein in step (3), the culture period is 3 to 5 weeks, and 2 mg/L6-BA, 0.2mg/L NAA, 0.1mg/L KT and 1mg/L proline are added to the culture medium.
9. The method for in vitro regeneration of tea trees according to claim 5, wherein in step (4), the culture period is 3 to 5 weeks, and 0.8mg/L6-BA and 0.08mg/L NAA are added to the culture medium.
10. A method for the ex vivo regeneration of tea trees as claimed in claim 3 wherein in step (7) the nutrient soil to perlite volume ratio in the mix is 3:1.
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