A method for efficient regeneration of Saxifraga macrostegia Hance

Abstract

The present invention relates to a method for efficient regeneration of Saxifraga bronchialis, comprising the following steps: (1) material treatment; (2) induction of callus and adventitious buds; (3) proliferation of adventitious buds; (4) in-vitro rooting culture; (5) acclimatization and transplantation. The present invention uses the leaves of tissue-cultured seedlings of Saxifraga bronchialis as explants, establishes an efficient regeneration system for the leaves of Saxifraga bronchialis, determines suitable callus induction media, proliferation media, rooting media and suitable substrates for transplanting tissue-cultured seedlings. The rooted cluster buds grow well after transplantation, providing new ideas for the seedling breeding, genetic breeding of Saxifraga bronchialis and the development and utilization of plants of the genus Saxifraga.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and more specifically, it relates to a method for efficient regeneration of Saxifraga fortunei Hook.f. var. fortunei. Background Art

[0002] Plants of the genus Saxifraga Tourn. ex L. are widely distributed globally, with 440 - 500 species, being the largest genus in the family Saxifragaceae. They are distributed in both the north and south of China, mainly in the southwest, Qinghai, Gansu and other places, and mostly grow on alpine rocks or in rock crevices at an altitude of 3000m. Currently, there are more than 1600 internationally registered Saxifraga varieties, while there are only 3 in China, leaving a large research space in the breeding, development and utilization of Saxifraga varieties. Saxifraga plants like shady and humid environments, are tolerant of barrenness, cold and drought, and have strong adaptability. Saxifraga has bright flower colors, small flower shapes, unique leaf shapes, and spots on the back of the leaves, with high ornamental value. It has been applied in landscaping, and can be used as potted plants, vertical greening and garden landscaping decorations, etc.

[0003] In addition, because Saxifraga plants contain various secondary metabolites such as polyphenols, flavonoids, organic acids, etc., Saxifraga plants also have more medicinal values, with effects such as anti - inflammation, anti - virus, liver protection, anti - cancer, antioxidant, etc. However, current research on Saxifraga plants mainly focuses on drug effects and pharmacology, and there are few research reports on breeding techniques, and there are few reports on tissue culture research, and a mature and perfect regeneration system has not been established.

[0004] Currently, Saxifraga is generally propagated by sowing and dividing plants using stolons. The propagation coefficient is small, which limits its development and utilization methods. Studying the propagation methods of Saxifraga plants has important practical significance for the rational utilization of its germplasm resources. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for efficient regeneration of Saxifraga fortunei Hook.f. var. fortunei. Using the leaves of tissue - cultured seedlings of Saxifraga fortunei Hook.f. var. fortunei as explants, an efficient regeneration system for the leaves of Saxifraga fortunei Hook.f. var. fortunei is established, and the appropriate callus induction medium, proliferation medium, rooting medium and suitable substrate for transplanting tissue - cultured seedlings are determined. The rooted cluster buds grow well after transplantation, providing new ideas for the seedling breeding, genetic breeding of Saxifraga fortunei Hook.f. var. fortunei and the development and utilization of Saxifraga plants.

[0006] The above - mentioned technical purpose of the present invention is achieved through the following technical solutions:

[0007] A method for efficient regeneration of Saxifraga fortunei Hook.f. var. fortunei, the steps are as follows:

[0008] (1) Material treatment: Select the leaves of Saxifraga bronchialis tissue culture seedlings with a size of about 0.5 cm * 0.5 cm as explants;

[0009] (2) Induction of callus and adventitious buds: Inoculate the explants onto the callus induction medium for culture to induce adventitious buds. Among them, the callus induction medium includes 1 / 2 MS medium and the first growth regulator group;

[0010] The first growth regulator group is 6-BA and NAA; or the first growth regulator group is KT and NAA; or the first growth regulator group is TDZ and NAA;

[0011] (3) Proliferation of adventitious buds: Inoculate the adventitious buds obtained in step (2) into the proliferation medium to proliferate the adventitious buds. Among them, the proliferation medium includes 1 / 2 MS medium and the second growth regulator group;

[0012] The second growth regulator group is 6-BA and NAA; or the second growth regulator group is KT and NAA; or the second growth regulator group is TDZ and NAA;

[0013] (4) In-vitro rooting culture: Divide the proliferated adventitious buds obtained in step (3) into cluster buds and transfer them to the rooting medium for culture to obtain rooted cluster buds. Among them, the rooting medium includes 1 / 2 MS medium and IBA;

[0014] (5) Acclimatization and transplantation: Acclimatize the proliferated non-rooted buds and then transplant them into the substrate to cultivate and form new plants.

[0015] In one embodiment, in step (2), the first growth regulator group is 1 - 3 mg·L -1 of 6-BA and 0.2 mg·L -1 of NAA; or the first growth regulator group is 1 - 4 mg·L -1 of KT and 0.2 mg·L -1 of NAA; or the first growth regulator group is 0.05 - 0.4 mg·L -1 of TDZ and 0.2 mg·L -1 of NAA.

[0016] In one embodiment, in step (2), the culture temperature is 25 ± 1 °C. After culturing the explants in the dark for 15 d, transfer them to the light for continued culture until adventitious buds are induced. Among them, when culturing under light conditions, the light intensity is 27 - 36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0017] In one embodiment, in step (3), the second plant growth regulator group is 6-BA at 0.5-1 mg·L -1 and NAA at 0.2 mg·L -1 ; or the second plant growth regulator group is KT at 1-2 mg·L -1 and NAA at 0.2 mg·L -1 ; or the second plant growth regulator group is TDZ at 0.1-0.2 mg·L -1 and NAA at 0.2 mg·L -1 .

[0018] In one embodiment, in step (3), the proliferation culture of adventitious buds is carried out under light conditions, the light intensity is 27-36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0019] In one embodiment, in step (4), the concentration of IBA is 0-0.5 mg·L -1 .

[0020] In one embodiment, in step (4), the in-vial rooting culture is carried out in the dark.

[0021] In one embodiment, in step (5), the substrate is a mixture formed by peat soil, perlite and vermiculite, wherein the volume ratio of peat soil, perlite and vermiculite is 2:1:1.

[0022] In one embodiment, in step (5), the substrate is a mixture formed by perlite + vermiculite, wherein the volume ratio of perlite and vermiculite is 1:2.

[0023] In one embodiment, in step (5), the culture conditions are: the culture temperature is 25±1 °C, the light intensity is 27-36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0024] In summary, the present invention has the following beneficial effects:

[0025] The present invention uses the leaves of Saxifraga okamotoi tissue culture seedlings as explants, establishes an efficient regeneration system for Saxifraga okamotoi leaves, determines suitable callus induction media, proliferation media, rooting media and suitable substrates for transplanting tissue culture seedlings. The rooted cluster buds grow well after transplantation, providing new ideas for the seedling breeding, genetic breeding of Saxifraga okamotoi and the development and utilization of Saxifraga plants. Description of the Drawings

[0026] Figure 1 They are tissue-cultured seedlings of Saxifraga macrophylla var. pallida with petiole regeneration;

[0027] Figure 2 It is a schematic diagram of inoculating the leaves of tissue-cultured seedlings as explants onto the induction medium;

[0028] Figure 3 It is a schematic diagram of a small amount of callus and adventitious buds induced from the leaves;

[0029] Figure 4 It is a schematic diagram of the leaves gradually callusing and differentiating into more adventitious buds;

[0030] Figure 5 It is a schematic diagram of more buds proliferated from adventitious buds;

[0031] Figure 6 It is a schematic diagram of the growth state after the cluster buds take root;

[0032] Figure 7 It is a schematic diagram of the growth state of the root system

[0033] Figure 8 It is a schematic diagram of transplanting tissue-cultured seedlings. Specific implementation method

[0034] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0035] It should be noted that the orientation terms such as "upper" and "lower" involved in this article are relative to the perspective of the accompanying drawings, and are only for convenience of description, and cannot be construed as a limitation to the technical solution.

[0036] The present invention provides a method for efficient regeneration of Saxifraga macrophylla var. pallida, and the specific steps are as follows:

[0037] Select tissue-cultured seedlings of Saxifraga macrophylla var. pallida with good rooting, strong plant growth and normal leaf development. As Figure 1 shown.

[0038] (1) Material treatment: Select leaves of tissue-cultured seedlings of Saxifraga macrophylla var. pallida with a size of about 0.5 cm * 0.5 cm as explants, as Figure 2 shown.

[0039] (2) Induction of callus and adventitious buds: Inoculate the explants onto the callus induction medium for culture to induce adventitious buds. Among them, the callus induction medium includes 1 / 2 MS medium and the first growth regulator group.

[0040] In step (2), the first growth regulator group can be a combination of multiple growth regulators. For example: the first growth regulator group is 1 - 3 mg·L -1of 6-BA (6-benzyladenine) and 0.2 mg·L -1 of NAA (naphthaleneacetic acid); or the first growth regulator group is 1 - 4 mg·L -1 of KT (kinetin) and 0.2 mg·L -1 of NAA; or the first growth regulator group is 0.05 - 0.4 mg·L -1 of TDZ (thidiazuron) and 0.2 mg·L -1 of NAA.

[0041] In step (2), the total culture time is 30 d - 40 d, the culture temperature is 25 ± 1°C. The explants are cultured in the dark for 15 d and then transferred to light for continued culture for 15 d - 25 d until more adventitious buds are induced. Preferably, when cultured under light conditions, the light intensity is 27 - 36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0042] In step (2), the 1 / 2MS medium refers to the medium prepared by halving the macroelements of MS while keeping the others unchanged. The same applies hereinafter.

[0043] (3) Proliferation of adventitious buds: The adventitious buds obtained in step (2) are inoculated into the proliferation medium to proliferate the adventitious buds. Among them, the proliferation medium includes 1 / 2MS medium and the second growth regulator group.

[0044] In step (3), the second growth regulator group can be a combination of multiple growth regulators. For example: the second growth regulator group is 0.5 - 1 mg·L -1 of 6-BA and 0.2 mg·L -1 of NAA; or the second growth regulator group is 1 - 2 mg·L -1 of KT and 0.2 mg·L -1 of NAA; or the second growth regulator group is 0.1 - 0.2 mg·L -1 of TDZ and 0.2 mg·L -1 of NAA.

[0045] In step (3), the culture time is 30 d - 40 d. The proliferation culture of adventitious buds is carried out under light conditions. The light intensity is 27 - 36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0046] (4) Rooting culture in vitro: Divide the proliferated adventitious buds obtained in step (3) into cluster buds and transfer them to a rooting medium for culture to obtain rooted cluster buds. Among them, the rooting medium comprises 1 / 2 MS medium and IBA (indolebutyric acid).

[0047] In step (4), preferably, the concentration of IBA is 0 - 0.5 mg·L -1 .

[0048] In step (4), the culture time is 30d - 40d, and the rooting culture in vitro is carried out in the dark.

[0049] (5) Hardening-off and transplanting: Harden off the proliferated rootless buds and transplant them into the substrate to culture and form new plants.

[0050] In step (5), the substrate is a mixture formed by peat soil, perlite and vermiculite. Among them, the volume ratio of peat soil, perlite and vermiculite is 2:1:1.

[0051] In step (5), the substrate is a mixture formed by perlite + vermiculite. Among them, the volume ratio of perlite and vermiculite is 1:2.

[0052] In step (5), the culture conditions are: the culture temperature is 25 ± 1 °C, the light intensity is 27 - 36 μmol·m -2 ·s - -1, and the light time is 12 h·d -1 .

[0053] The technical solution of the present invention is illustrated by the following examples.

[0054] Callus induction rate (%) = number of leaves with callus / number of inoculated leaves × 100.

[0055] Adventitious bud induction rate (%) = number of leaves with differentiated buds / number of inoculated leaves × 100.

[0056] Cluster bud induction rate % = number of leaves with differentiated cluster buds / number of inoculated leaves.

[0057] Bud proliferation coefficient (%) = total number of buds after proliferation / number of inoculated buds × 100.

[0058] Seedling survival rate (%) = number of survived seedlings / number of inoculated seedlings × 100.

[0059] Cluster bud rooting rate (%) = number of rooted cluster buds / number of inoculated cluster buds × 100.

[0060] Number of rooting branches = total number of rooting branches / number of inoculated cluster buds.

[0061] Transplanting survival rate (%) = number of survived seedlings / number of transplanted seedlings × 100.

[0062] The above data was sorted and statistically analyzed using Excel software, and the significance analysis of differences was performed using the STST variance analysis software by Wang Shaohua of Nanjing Agricultural University.

[0063] Example 1

[0064] This example is about the induction effects of different first growth regulator groups on callus and adventitious buds.

[0065] Using leaves as explants and 1 / 2MS medium as the basic medium, different combinations of plant growth regulators were set, namely 6-benzyladenine (6-BA) (0.5, 1, 2, 3, and 4 mg·L -1 ) combined with naphthaleneacetic acid (NAA 0.2 mg·L -1 ), kinetin (KT) (0.5, 1, 2, 3, and 4 mg·L -1 ) combined with NAA 0.2 mg·L -1 , thidiazuron (TDZ) (0.05, 0.1, 0.2, 0.3, and 0.4 mg·L -1 ) combined with NAA 0.2 mg·L -1 ), a total of 15 callus and adventitious bud induction media. After 30 days, the induction rates of callus and adventitious buds were statistically analyzed. At least 30 leaves were inoculated for each treatment, and the experiment was repeated 3 times.

[0066] The effects of the combination of 6-BA and NAA on the induction of callus and adventitious buds from the leaves of Saxifraga bronchialis L. var. przewalskii Engl. are as follows:

[0067] After the leaves of the tissue-cultured seedlings of Saxifraga bronchialis L. var. przewalskii Engl. were inoculated onto the callus induction medium, after about 20 days, it was observed that some leaves curled up, callus was produced at the leaf cut, and at the same time, a small amount of adventitious buds appeared ( Figure 3 as shown). After continuous culture, it was observed that the callus gradually increased, and buds also continuously appeared. Some leaves gradually became callused, producing more adventitious buds ( Figure 4 as shown).

[0068] It can be seen from the data in Table 1 below that on the 5 media with the combination of 6-BA and NAA, the callus induction rate of the leaves was 86.48 - 100%, the adventitious bud induction rate was 56.20 - 97.78%, and the cluster bud induction rate was 45.02 - 91.11%. The differences among the three were significant, showing a trend of first increasing and then decreasing with the increase of the 6-BA concentration.

[0069] Through comprehensive analysis, it can be seen that when the NAA concentration is constant, the 6-BA concentration of 1 - 3 mg·L -1 is more suitable for the induction of callus, adventitious buds, and cluster buds from the leaves of tissue-cultured seedlings. Among them, 6-BA 2 mg·L -1 +NAA 0.2 mg·L-1 The combined medium had a relatively high induction rate. The callus induction rate reached 100%, the adventitious bud induction rate reached 97.78%, and the cluster bud induction rate reached 91.11%.

[0070] Table 1 Effects of combinations of 6-BA and NAA at different concentrations on leaf callus and adventitious buds

[0071]

[0072] Different lowercase letters after the data in the same column indicate statistical differences (P < 0.05), and the same applies to the following table. The number of cluster buds is 3 or more.

[0073] The effects of the combination of KT and NAA on the induction of leaf callus and adventitious buds of Saxifraga bronchialis were as follows:

[0074] As can be seen from Table 2 below, on the 5 media of the combination of KT and NAA for the leaves of tissue-cultured seedlings of Saxifraga bronchialis, the callus induction rate was between 81.72% and 100%, the adventitious bud induction rate was between 66.26% and 96.85%, and the cluster bud induction rate was between 54.73% and 87.04%. The differences among the three were obvious, showing a trend of first increasing and then slightly decreasing with the increase in the KT concentration.

[0075] Comprehensive analysis showed that when the NAA concentration was constant, the KT concentration of 1 - 4 mg·L -1 was more suitable for the induction of leaf callus, adventitious buds and cluster buds of tissue-cultured seedlings. Among them, the combination of KT 1 mg·L -1 and NAA 0.2 mg·L -1 had the highest leaf induction rate on the medium. The callus induction rate reached 100%, the adventitious bud induction rate reached 96.85%, and the cluster bud induction rate reached 87.04%.

[0076] Table 2 Effects of combinations of KT and NAA at different concentrations on leaf callus and adventitious bud induction

[0077]

[0078] The effects of the combination of TDZ and NAA on the induction of leaf callus and adventitious buds of Saxifraga bronchialis were as follows:

[0079] As can be seen from Table 3 below, on the 5 media of the combination of TDZ and NAA for the leaves of tissue-cultured seedlings of Saxifraga bronchialis, the callus induction rate had no obvious difference and could reach 100%, but there were differences in adventitious bud induction and cluster bud induction. The adventitious bud induction rate and the cluster bud induction rate showed an increasing trend with the increase in the TDZ concentration. Among them, the adventitious bud induction rate ranged from 86.88% to 96.67%, and the cluster bud induction rate was between 77.06% and 94.44%. When the NAA concentration was constant, the TDZ concentration was 0.05 - 0.4 mg·L-1 is beneficial to the induction of leaf callus, adventitious bud induction and cluster bud induction. With 0.4 mg·L -1 + NAA 0.2 mg·L -1 The induction rate of the combined medium is relatively high. The adventitious bud induction rate reaches 96.67%, and the cluster bud induction rate reaches 94.44%.

[0080] Table 3 Effects of different concentrations of TDZ and NAA combinations on leaf callus and adventitious bud induction

[0081]

[0082] Example 2

[0083] This example is about the effects of different second growth regulator groups on adventitious bud proliferation.

[0084] The adventitious buds induced from leaves were inoculated into 6 - BA (0.5 and 1 mg·L -1 ) and NAA 0.2 mg·L -1 combinations, KT (1 and 2 mg·L -1 ) and NAA 0.2 mg·L -1 combinations, TDZ (0.1 and 0.2 mg·L -1 ) and NAA 0.2 mg·L -1 combinations, on a total of 6 bud proliferation media (the basic medium is 1 / 2MS). After 30 days, the proliferation of adventitious buds was counted, and the proliferation coefficient was calculated. At least 30 bud pieces were inoculated for each treatment, and repeated 3 times. As shown in Table 4 below.

[0085] Table 4 Effects of different concentrations of cytokinin and auxin combinations on bud proliferation

[0086]

[0087] When the adventitious buds (3 buds per cluster) induced from the leaves of Saxifraga bronchialis tissue - cultured seedlings were inoculated into 6 bud proliferation media, the adventitious buds could all proliferate. After 30 days, more adventitious buds proliferated around the bud pieces (as Figure 5 shown). It can be seen from Table 4 that good proliferation effects were achieved on all 6 media, and there were differences in the bud proliferation coefficients, which were between 2.50 and 4.03. Among them, the combinations of 6 - BA and NAA and TDZ and NAA had relatively good overall bud proliferation effects. The medium with the combination of 6 - BA 1 mg·L -1 + NAA 0.2 mg·L -1 had the best bud proliferation effect, and the bud proliferation coefficient reached 4.03. While on the bud proliferation media with the combination of KT and NAA, the bud proliferation coefficients were slightly lower, between 2.19 and 2.95.

[0088] Example 3

[0089] This example is about the effect of different concentrations of IBA on rooting.

[0090] The proliferated adventitious buds were cut into cluster buds and then transferred into three kinds of media containing indolebutyric acid (IBA) (0, 0.2, and 0.5 mg·L -1 ) in 1 / 2MS. After 30 days, the rooting situation of the cluster buds was observed and the rooting rate was counted. At least 30 cluster buds were inoculated for each treatment, and the experiment was repeated 3 times. The results are shown in Table 5 below

[0091] Table 5 Effect of different concentrations of IBA on the rooting of cluster buds

[0092]

[0093] As can be seen from Table 5, after the well - growing cluster buds were inoculated onto the three rooting media, the seedling survival rate and the rooting rate were both relatively high, and the difference between them was not obvious, with the highest reaching 100%. However, there were differences in the rooting time and the number of roots of the cluster buds on different media. When the IBA concentration was 0.5 mg·L -1 , the cluster buds rooted quickly. Generally, roots could be seen growing out 20 days after inoculation, and the number of root branches could reach 10.81. When the IBA concentration was 0 or 0.2 mg·L -1 , the cluster buds rooted slightly slower. Generally, roots could be seen growing out 30 days after inoculation, and the average number of root branches was between 4.53 and 6.56. By comparison, it shows that 1 / 2MS + IBA 0.5 mg·L -1 is the best rooting medium for cluster buds( Figure 6 and 7 shown)

[0094] Example 4

[0095] This example is about the effect of different substrates on the survival rate of transplanted seedlings.

[0096] As can be seen from Table 6 below, when the rooted tissue - cultured seedlings (conventional tissue - cultured seedlings) were transplanted into two substrates, the transplanting survival rate of the tissue - cultured seedlings could reach over 95.56%, and they grew well, with over 94% of the plants growing new leaves, indicating that both substrates of peat soil + perlite + vermiculite (volume ratio 2:1:1) and perlite + vermiculite (volume ratio 1:2) were suitable for the transplantation of Saxifraga bronchialis L. tissue - cultured seedlings. The substrate of perlite + vermiculite (volume ratio 1:2) had a higher seedling survival rate when transplanted( Figure 8 shown). As can be seen from Table 7 below, after culturing by the present invention, when the cluster buds passed through 500 mg·L -1After the treatment with IBA, they were transplanted onto two substrates. The rooting rate of the two substrates reached over 95.56%, the number of roots was over 3.32, and the transplanting survival rate could reach 97.78%. New leaves grew on the plants, indicating that it was feasible for the cluster buds to root in vitro after being treated with auxin on two substrates: peat soil + perlite + vermiculite (volume ratio 2:1:1) and perlite + vermiculite (volume ratio 1:2).

[0097] Table 6 Transplanting of tissue-cultured seedlings with conventional rooting

[0098]

[0099] Table 7 Rooting of cluster buds in vitro

[0100]

[0101] The present invention uses the leaves of Saxifraga bronchialis tissue-cultured seedlings as explants and establishes an efficient regeneration system for Saxifraga bronchialis leaves. The media suitable for inducing callus and adventitious buds are 1 / 2MS + 6-BA + NAA, 1 / 2MS + KT + NAA, and 1 / 2MS + TDZ + NAA; the medium suitable for shoot proliferation is 1 / 2MS + 6-BA + NAA; the medium suitable for rooting of cluster shoots is 1 / 2MS + IBA; the substrates suitable for transplanting tissue-cultured seedlings are peat soil + perlite + vermiculite (volume ratio 2:1:1) and perlite + vermiculite (volume ratio 1:2). The tissue-cultured seedlings treated with IBA can root in vitro, and the tissue-cultured seedlings grow well after transplantation, providing new ideas for the seedling breeding, genetic breeding of Saxifraga bronchialis and the development and utilization of Saxifraga plants.

[0102] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, several improvements and refinements made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A method for efficient regeneration of Saxifraga macrostegia, characterized in that, The steps are as follows: (1) Material treatment: Select the leaves of Saxifraga bronchialis tissue culture seedlings with a size of 0.5 cm × 0.5 cm as explants; (2) Induction of callus and adventitious buds: Inoculate the explants onto the callus induction medium for culture to induce adventitious buds. Among them, the callus induction medium consists of 1 / 2MS medium and the first growth regulator group; The first growth regulator group is 1 to 4 mg·L -1 of KT and 0.2 mg·L -1 of NAA; or the first growth regulator group is 0.05 to 0.4 mg·L -1 of TDZ and 0.2 mg·L -1 of NAA; (3) Proliferation of adventitious buds: Inoculate the adventitious buds obtained in step (2) into the proliferation medium to proliferate the adventitious buds. Among them, the proliferation medium consists of 1 / 2MS medium and the second growth regulator group; The second growth regulator group is 1-2 mg·L -1 of KT and 0.2 mg·L -1 of NAA; or the second growth regulator group is 0.1-0.2 mg·L -1 of TDZ and 0.2 mg·L -1 of NAA; (4) Rooting culture in vitro: The proliferated adventitious buds obtained in step (3) are divided into cluster buds and then transferred to a rooting medium for culture to obtain rooted cluster buds. Among them, the rooting medium consists of 1 / 2 MS medium and IBA, and the concentration of IBA is 0 - 0.5 mg·L -1 ; (5) Hardening-off and transplanting: Harden off the rooted shoot seedlings and then transplant them into the substrate to cultivate and form new plants.

2. The method for efficient regeneration of Saxifraga macrostegia as described in claim 1, characterized in that, In step (2), the culture temperature is 25 ± 1 °C. The explants are cultured in the dark for 15 d and then transferred to light for continuous culture until adventitious buds are induced. Among them, when cultured under light conditions, the light intensity is 27 - 36 μmol·m -2 ·s -1 , and the light duration is 12 h·d -1 .

3. The method for efficient regeneration of Saxifraga macrostegia Hance as claimed in claim 1, characterized in that, In step (3), the proliferation culture of adventitious buds is carried out under light conditions, with the light intensity being 27 - 36 μmol·m -2 ·s -1 , and the light duration being 12 h·d -1 .

4. The method for efficient regeneration of Saxifraga macrostegia Hance as described in claim 1, characterized in that, In step (4), in-bottle rooting culture is carried out in the dark.

5. The method for efficient regeneration of Saxifraga macrostegia Hance as claimed in claim 1, characterized in that, In step (5), the substrate is a mixture formed by peat soil, perlite and vermiculite. Among them, the volume ratio of peat soil, perlite and vermiculite is 2:1:

1.

6. The method for efficient regeneration of Saxifraga macrostegia Hance as claimed in claim 1, characterized in that, In step (5), the substrate is a mixture formed by perlite + vermiculite. Among them, the volume ratio of perlite and vermiculite is 1:

2.

7. The method for efficient regeneration of Saxifraga macrostegia Hance as described in claim 1, characterized in that, In step (5), the culture conditions are: the culture temperature is 25 ± 1 °C, the light intensity is 27 - 36 μmol·m -2 ·s -1 , and the light duration is 12 h·d -1 .

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