A tissue culture rapid propagation method of small guavacotone

Abstract

The application discloses a tissue culture and rapid propagation method of small grass Pittosporum, and first discovers that small grass Pittosporum leaves can successfully induce callus and directly induce regenerated buds, and establishes a tissue culture and rapid propagation system of small grass Pittosporum callus and regenerated buds, obviously improves the propagation speed, improves the propagation coefficient and shortens the propagation period, can provide a large number of excellent seedlings with the same traits as the parent in a short period, and has the advantages of keeping the excellent traits of the parent, being simple and easy to operate, and causing little damage to the parent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant propagation technology, in particular, the present application relates to a tissue culture and rapid propagation method of Scaevola hainanensis Hance. BACKGROUND

[0002] Mangrove forest has important ecological significance and great economic value, but most of the mangrove plants are sensitive to low temperature. The temperature drop, irregular cold or frost has an important influence on the growth and distribution of them, and people have been trying to expand the planting range of mangrove plants, and the introduction or cross-region afforestation needs a large number of seedlings for practice. At present, the vegetation restoration of mangrove wetland in China mostly uses seedlings, which is not conducive to maintaining the excellent traits of mother trees, and often appears the problems of seedling trait separation, quality difference and the like, resulting in low afforestation success rate and slow forestation. Therefore, tissue culture propagation is an effective technical means to rapidly expand the number of seedlings.

[0003] Scaevola hainanensis Hance grows in the salt field by the sea or grows with mangrove forest, and the community size is generally 5m*6m in small patchy shrub distribution. Compared with Scaevola taccada, Scaevola hainanensis Hance has a more compact landscape effect and stronger development and application value. The molecular clustering result shows that the genetic diversity level of Scaevola hainanensis Hance is extremely low, and as a rare plant, it also faces the risk of extinction at the molecular level, so it is urgent to expand the number of seedlings. SUMMARY

[0004] Therefore, the present application provides a tissue culture and rapid propagation method of Scaevola hainanensis Hance, which is simple and easy to operate, has less damage to the mother body, short propagation cycle, high propagation efficiency and coefficient, and provides technical support for large-scale expansion of Scaevola hainanensis Hance for better mangrove wetland ecological restoration.

[0005] The present application adopts the following technical scheme:

[0006] In a first aspect,

[0007] The present application provides a tissue culture and rapid propagation method of Scaevola hainanensis Hance, which comprises the following steps:

[0008] S1: taking the tender leaves on the 1-year-old branches of Scaevola hainanensis Hance, washing, disinfecting and absorbing water, then cutting off the leaf tip and leaf base, and cutting the remaining part into leaf explants with an area of 0.12-0.15cm 2 , that is, the aseptic leaf explants;

[0009] S2: under sterile conditions, the sterile leaf explants are inoculated on callus induction medium for 30-40 days to obtain callus; the callus induction medium is MS medium added with 0.1 mg / L 6-BA, 0.5-1.2 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05;

[0010] S3: the callus with similar growth is cut into pieces with an area of 0.5-0.7 cm 2 S3: the callus with similar growth is cut into pieces with an area of 0.5-0.7 cm

[0011] S4: the explant with shoots is inoculated on adventitious shoot proliferation growth medium for 30-35 days to obtain elongated adventitious shoots; the adventitious shoot proliferation growth medium is MS medium added with 0.5 mg / L 6-BA or 1 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05;

[0012] S5: the adventitious shoots are inoculated on the rooting induction medium described above for 30-35 days to obtain small grass pittosporum tissue culture seedlings; the rooting induction medium is MS medium added with 0.5-1.5 mg / L IAA or 0.5-2.0 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05;

[0013] S6: the small grass pittosporum tissue culture seedlings with 3-4 regenerated roots are taken out, washed with tap water to remove residual culture medium, and transplanted into a seedling raising pot containing a seedling raising medium; the seedling raising medium is peat soil: perlite with a volume ratio of 3:1;

[0014] S7: after the small grass pittosporum tissue culture seedlings are transplanted, sufficient rooting water is poured, then a transparent film is covered and cultured in a room for 7 days; then it is moved to the outside to continue the film covering and moisture retaining culture for 20 days, during which the film is opened once in the morning and once in the evening to reduce the humidity (half an hour each time, and the leaf surface is sprayed with clean water after each opening); finally the film is opened and the seedling raising pot is filled with water to ensure that the water level is higher than the base of the seedling raising pot.

[0015] Preferably, in step S1, the specific steps of cleaning, disinfecting and drying are as follows: washed with ultrapure water for 1 min, immersed in 75% ethanol for disinfection on an ultraclean workbench, then sterilized with 10% NaClO solution for 5-10 min (more preferably 5 min), 2 drops of Tween-20 are added during sterilization, and then washed with sterile water for 4 times, and the leaf surface is dried on sterile cutting paper.

[0016] Preferably, the callus induction medium is MS medium added with 0.1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05.

[0017] Preferably, the rooting induction medium is MS medium added with 0.5 mg / L IAA or 1.5 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05.

[0018] Preferably, in step S6, the seedling transplanting substrate is sterilized and disinfected in a high-pressure steam kettle at 80℃ for 1h before mixing.

[0019] Preferably, the indoor culture conditions of steps S1-S7 are as follows: temperature is set to 25±1℃, light intensity is 1500±10 lux, light cycle is 12h / 12h (light / dark), and air humidity is 50±5%.

[0020] The second aspect of the application is a tissue culture and rapid propagation method of small grass Pittosporum.

[0021] The application also provides a tissue culture and rapid propagation method of small grass Pittosporum, comprising the following steps:

[0022] 1) Take the young leaves on the 1-year-old branches of small grass Pittosporum, wash, sterilize, and dry, then cut off the leaf tips and leaf bases, and cut the remaining parts into leaf explants with an area of 0.12-0.15 cm 2 , that is, aseptic leaf explants;

[0023] 2) Inoculate the aseptic leaf explants into a leaf direct induction of regeneration bud medium and culture for 40-45 days to obtain regeneration buds; the leaf direct induction of regeneration bud medium is MS medium added with 0.5-2.0 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and pH is 5.8±0.05;

[0024] 3) Inoculate the regeneration bud leaf explants into a regeneration bud induction of adventitious bud medium and culture for 40-45 days to obtain more adventitious buds; the regeneration bud induction of adventitious bud medium is MS medium added with 0.5-1.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and pH is 5.8±0.05;

[0025] 4) Inoculate the regeneration buds of step 2) or the adventitious buds of step 3) into a rooting induction medium and culture for 30-35 days to obtain small grass Pittosporum tissue culture seedlings; the rooting induction medium is MS medium added with 0.5-1.5 mg / L IAA or 0.5-2.0 mg / L IBA, 30 g / L sucrose, and 7 g / L agar, and pH is 5.8±0.05;

[0026] 5) take out the small grass pittosporum tissue culture seedlings with 3-4 regenerated roots, wash the root residual culture medium with tap water, and transplant into the seedling pots containing the seedling transplanting substrate; the seedling transplanting substrate is peat soil: perlite with a volume ratio of 3:1;

[0027] 6) after transplanting the small grass pittosporum tissue culture seedlings, pour enough rooting water, then cover the transparent film and cultivate in the room for 7 days; then move to the outdoor to continue the film covering and moistening cultivation for 20 days, during which the film is opened once in the morning and evening each day to reduce the humidity (half an hour each time, and the leaf surface is sprayed with clean water after each time of opening); finally, the film is opened, and clean water is sprayed once in the morning and evening, and the water amount in the seedling pot is ensured to be higher than the base of the seedling pot.

[0028] Preferably, in step 1), the specific steps of washing, disinfecting and drying water are as follows: washing with ultrapure water for 1 min, immersing in 75% ethanol for disinfection on the ultraclean workbench, then sterilizing with 10% NaClO solution for 5-10 min (more preferably 5 min), adding 2 drops of Tween-20 during sterilization, and then washing with sterile water for 4 times, and drying the water on the surface of the leaf on the sterile cutting paper.

[0029] Preferably, the leaf direct induction of regenerated bud culture medium is MS medium added with 1.5 mg / L 6-BA, 30 g / L sucrose and 7 g / L agar, and the pH is 5.8±0.05.

[0030] Preferably, the rooting induction culture medium is MS medium added with 0.5 mg / L IAA or 1.5 mg / L IBA, 30 g / L sucrose and 7 g / L agar, and the pH is 5.8±0.05.

[0031] Preferably, in step 5), the seedling transplanting substrate is sterilized and killed in the high-pressure steam boiler at 80℃ for 1 h before mixing.

[0032] Preferably, the culture conditions in steps 1)-6) are as follows: the temperature is set to 25±1℃, the light intensity is 1500±10 lux, the light cycle is 12h / 12h (light / dark), and the air humidity is 50±5%.

[0033] The present application has the following beneficial effects:

[0034] (1) The present application first discovers that the small grass pittosporum leaf can successfully induce callus and directly induce regenerated buds, and establishes a tissue culture rapid propagation system of small grass pittosporum callus and regenerated buds, which significantly improves the propagation speed, enhances the propagation coefficient and shortens the propagation period, can provide a large number of excellent seedlings consistent with the parent traits in a short period, and also has the advantages of being able to maintain the excellent traits of the parent, being simple and easy to operate, and causing little damage to the parent, etc., solves the problems of low propagation rate, time period limitation and environmental limitation in the existing technology of cutting propagation and seed propagation, and fills the gap of small grass pittosporum in the field of tissue culture rapid propagation technology.

[0035] (2) The tissue culture and rapid propagation method of P. parvifolia provided by the application uses leaves as explants, which causes less damage to the parent plant than using other parts such as stems as explants, and the leaves are more tender, and more materials are available, thereby providing technical support for large-scale propagation of the endangered mangrove plant resource P. parvifolia. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Cutting diagram of P. parvifolia leaves.

[0037] Figure 2 Growth status diagram of P. parvifolia adventitious buds induced by different concentrations of plant growth regulators after 35 days, wherein: a: induction of bud proliferation in the medium added with 0.5 mg / L 6-BA, KT and TDZ and the medium without hormones; b: induction of bud proliferation in the medium added with 0.5, 1.0, 1.5 and 2.0 mg / L 6-BA; c: induction of bud proliferation in the medium added with different concentrations of 6-BA and KT; d: induction of bud proliferation in the medium added with 0.5 mg / L 6-BA and 0.1 mg / L NAA, IAA and IBA and the medium without hormones. Scale = 1 cm.

[0038] Figure 3 Growth status diagram of regenerated buds of leaf explants induced by different concentrations of hormones after 45 days of differentiation and regeneration;

[0039] Figure 4 Growth status diagram of adventitious roots of P. parvifolia induced by different concentrations of auxins after 35 days of differentiation;

[0040] wherein: a: MS medium without adding hormones; b: 0.5 mg / L NAA; c: 0.5 mg / L IAA; d: 2.0 mg / L IBA. Scale = 1 cm. DETAILED DESCRIPTION

[0041] The embodiments of the application are described in detail below, and the embodiments described below are exemplary and are intended to explain the application, and cannot be understood as a limitation of the application.

[0042] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0043] The materials, reagents, devices and the like used in the following examples can be prepared by commercial means or according to the methods disclosed in the literature unless otherwise specified.

[0044] Example 1: Establishment of aseptic system of P. parvifolia

[0045] The tender leaves of 1-year-old branches of Pittosporum undulatum were washed with ultrapure water for 1 min, and then sterilized with 75% ethanol for 1 min on a clean bench. The leaves were then sterilized with 2%, 5%, and 10% NaClO (w:v) solutions for 5 min, 10 min, and 15 min, respectively, with 2 drops of Tween-20 added during sterilization. The leaves were then washed with sterile water for 4 times, and then dried on a sterile cutting paper. The tips and bases of the leaves were then cut off, and the remaining parts were cut into explants with an area of 0.12-0.15 cm 2 (see Figure 1 ). The explants were placed on MS+1.0 mg / L 6-BA+0.1 mg / L NAA medium with the back of the leaves facing down. The contamination rate and browning rate were recorded after 10 days:

[0046] Contamination rate (%) = number of contaminated explants / number of inoculated explants x 100%

[0047] Browning rate (%) = number of browning explants / number of inoculated explants x 100%

[0048] The results showed that the contamination rate of P. undulatum leaf explants decreased significantly with increasing concentration of NaClO solution and sterilization time (Table 1). After sterilization with 2%-10% NaClO for 5-15 min, it was found that sterilization with 10% NaClO for 10 min resulted in the lowest contamination rate, but there was no significant difference in sterilization time at this concentration. To minimize the damage of NaClO to the explants, sterilization with 10% NaClO for 5 min was the best (the contamination rate and browning rate of the explants were reduced to 13.33% and 0.00%, respectively).

[0049] Table 1 Effect of different concentrations of NaClO solution and sterilization time on the sterilization of P. undulatum leaf explants

[0050]

[0051] Note: The data are mean ± standard error, and the same letter in the same column indicates a significant difference at the P<0.05 level. 15 explants were used as one replicate, and the experiment was repeated 3 times.

[0052] Example 2 Effect of different hormone ratios on the induction of callus from P. undulatum leaves

[0053] The sterile leaf explants were inoculated on medium without hormone addition (CK), medium with 6-BA (0, 0.1, 0.3, 0.5, 1.0 mg / L) + NAA (0.5, 1.2 mg / L), and medium with 6-BA (0.1 mg / L) + IAA, IBA, 2,4-D (0.5 mg / L) (MS medium with 30 g / L sucrose and 7 g / L agar, pH 5.8±0.05). The callus induction rate, mortality rate, fresh weight of explants, and rooting rate were recorded after 35 days of inoculation:

[0054] Callus induction rate (%) = number of callus induced explants / number of inoculated explants x 100%

[0055] Mortality rate (%) = number of dead explants / number of inoculated explants x 100%

[0056] Fresh weight (mg / plant) = weight of explants after callus induction - weight of explants at inoculation

[0057] Rooting rate (%) = number of rooted explants / number of inoculated explants x 100%

[0058] The results showed that all the treatments except 0.5 mg / L NAA, 1.2 mg / L NAA and 0.5 mg / L IAA + 0.1 mg / L 6-BA could induce callus from explants with 100.00% of callus induction rate, and all the treatments could induce callus with more than 90.00% of callus induction rate (Table 2). The treatments without NAA caused explants to die, and the treatment without hormone had the highest mortality rate (83.34%). The addition of 0.1 mg / L 6-BA to the medium with 0.5 mg / L IAA, IBA, 2,4-D and NAA could decrease the mortality rate (decreased to 44.45%, 36.11%, 27.78% and 0.00%, respectively). The medium with NAA could induce explants to root, and the rooting rates were 61.11% and 72.22% for the medium with 0.5 mg / L and 1.2 mg / L NAA, respectively. Compared with the medium with 0.5 mg / L and 1.2 mg / L NAA alone, the addition of 0.1-1.0 mg / L 6-BA could significantly increase the fresh weight of callus, and the medium with 0.5 mg / L NAA + 0.1 mg / L 6-BA was the most suitable for the increase of fresh weight of explants (62.13 mg). However, the combination of different concentrations of NAA and 6-BA did not significantly promote the increase of fresh weight of explants. The addition of 0.5 mg / L NAA to the medium with 0.1 mg / L 6-BA could significantly promote the increase of fresh weight of explants, and the addition of 0.5 mg / L 2,4-D to the medium with 0.1 mg / L 6-BA had the weakest effect. The medium with 0.5 mg / L IAA + 0.1 mg / L 6-BA, 0.5 mg / L IBA + 0.1 mg / L 6-BA and 0.5 mg / L 2,4-D + 0.1 mg / L 6-BA caused explants to die, and the callus induced by the medium with 0.5 mg / L IAA + 0.1 mg / L 6-BA and 0.5 mg / L IBA + 0.1 mg / L 6-BA was white, the callus induced by the medium with 0.5 mg / L 2,4-D + 0.1 mg / L 6-BA was light yellow, and the callus induced by the medium with 0.5 mg / L NAA + 0.1 mg / L 6-BA was light green and compact. Therefore, the medium with 0.5 mg / L NAA + 0.1 mg / L 6-BA was the most suitable for the induction of callus for the induction of adventitious buds of Leptodermis parviflora.

[0059] Table 2 Effects of different concentrations of auxin combined with 6-BA on callus induction from leaf explants of Syzygium subheterophyllum

[0060]

[0061] Note: Data are mean ± standard error, and different letters in the same column represent significant differences at the P <0.05 level. Callus induction rate, mortality rate, and rooting rate were calculated based on 12 explants as one replicate, and repeated three times. Fresh weight was measured based on one explant as one replicate, and repeated eight times. “-” represents not measured.

[0062] Example 3 Effects of different hormone combinations on adventitious bud differentiation from callus of Syzygium subheterophyllum

[0063] Callus explants that had grown for 40 days with similar growth vigor were cut into small pieces with an area of 0.5-0.7 cm 2 and inoculated in media without added hormones or with 0.5 mg / L TDZ, KT, and 6-BA (MS medium with 30 g / L sucrose, 7 g / L agar, pH 5.8±0.05). After 35 days, the bud rate, bud number, and mortality rate were calculated (when counting the regenerated buds, large buds and small buds were distinguished, two or more leaves were considered as a large bud, and one leaf or no leaf was considered as a small bud, and the subsequent statistics were consistent):

[0064] Bud rate (%) = number of bud explants / number of inoculated explants × 100%

[0065] Bud number (number / plant) = total number of buds / number of inoculated explants

[0066] Mortality rate (%) = number of dead explants / number of inoculated explants × 100%

[0067] The results showed that: from Table 3, neither no added hormones nor TDZ could induce callus to differentiate into adventitious buds. Compared with the no-hormone treatment, KT and 6-BA could significantly promote adventitious bud differentiation, but the bud rate and average bud number were smaller. Among them, 6-BA had the best effect on inducing adventitious bud differentiation, with a bud rate of 42.00% and no mortality.

[0068] Table 3 Effects of 0.5 mg / L TDZ, KT, and 6-BA on adventitious bud differentiation from callus of Syzygium subheterophyllum

[0069] Hormone class Bud emergence rate (%) Bud emergence number (piece / plant) Mortality rate (%) CK (no hormone) 0.00±0.00c 0.00±0.00c 0.00±0.00b TDZ 0.00±0.00c 0.00±0.00c 0.00±0.00b KT 13.89±2.78b 0.11±0.03b 25.00±4.8a 6-BA 41.67±0.00a 0.78±0.00a 0.00±0.00b

[0070] Note: Data are mean ± standard error, and different letters in the same column represent significant differences at the P <0.05 level. 12 explants as one replicate, repeated three times.

[0071] Example 4 Effects of different hormone combinations on the proliferation and growth of adventitious buds of Syzygium buxifolium

[0072] The explants with buds were inoculated in the medium (MS medium added with 30 g / L sucrose, 7 g / L agar, pH 5.8±0.05) added with different concentrations of TDZ, KT, 6-BA and NAA, and the total proliferation coefficient and the proportion of large buds were counted after 35 days:

[0073] Proliferation coefficient = the number of proliferated buds / the number of buds before explant differentiation

[0074] Proportion of large buds (%) = the total number of large buds after proliferation / the total number of buds after proliferation × 100%

[0075] The results showed that, compared with CK and the medium added with 0.5 mg / L TDZ, KT and 6-BA, 0.5 mg / L 6-BA and 0.5 mg / L TDZ had the best and the worst effects on bud proliferation, respectively, and the maximum proportion of large buds appeared in 0.5 mg / L KT (Table 4). When 0.5-2.0 mg / L 6-BA was added alone, the greater the concentration, the more unfavorable to bud proliferation, and the proliferation coefficient and the proportion of large buds decreased with the increase of the concentration, and when the concentration was more than 1.5 mg / L, not only the effect of 6-BA on bud proliferation was lower than that of CK, but also the existing buds died (Table 4). When the total concentration was constant (1.0 mg / L), among 1.0 mg / L 6-BA, 0.6 mg / L 6-BA+0.4 mg / L KT, 0.5 mg / L 6-BA+0.5 mg / L KT and 1.0 mg / L KT, the maximum values of the proliferation coefficient and the proportion of large buds appeared in 1.0 mg / L KT, while the minimum values appeared in 1.0 mg / L 6-BA and 0.6 mg / L 6-BA+0.4 mg / L KT, respectively (Table 4), and the higher the KT concentration, the more favorable to bud growth. When 0.5 mg / L 6-BA was combined with 0.1 mg / L NAA, IAA and IBA, the addition of IAA was the most favorable to improve the proliferation coefficient, but the least favorable to improve the proportion of large buds, and compared with the addition of 6-BA alone, all the three kinds of auxins had inhibitory effects on bud proliferation, and the number of bud points produced by explants was less than that of 0.5 mg / L 6-BA. Among all the treatments, the addition of 0.5 mg / L 6-BA alone made the proliferation coefficient reach the maximum (3.57), but most of the buds were small, the addition of 0.5 mg / L KT made the proportion of large buds reach the maximum, and the addition of 1.0 mg / L KT could make the buds grow better under the condition of higher proliferation coefficient. Figure 2

[0076] Table 4 Effects of different concentrations of plant growth regulators on the proliferation of Syzygium buxifolium

[0077] ​

[0078] Note: Data are means ± standard errors. Means followed by different letters within a column are significantly different at the P < 0.05 level. Each treatment was replicated three times, and each replicate consisted of nine explants.

[0079] Example 5 Effects of different hormone combinations on the direct induction of adventitious shoots from leaves of Syzygium subhorizontally

[0080] After sterilization, leaf explants were inoculated on media (MS medium supplemented with 30 g / L sucrose, 7 g / L agar, and pH 5.8 ± 0.05) containing 0.5 mg / L TDZ, KT, and 6-BA, and 6-BA (0.5, 1.0, 1.5, 2.0 mg / L) + NAA (0, 0.05, 0.1, 0.5, 1.0 mg / L), and the shoot rate, shoot number, and large shoot number were counted after 45 days:

[0081] Shoot rate (%) = number of explants with shoots / number of inoculated explants x 100%

[0082] Shoot number (number per explant) = total shoot number / number of inoculated explants

[0083] Large shoot number (number per explant) = total large shoot number after differentiation / number of inoculated explants x 100%

[0084] The results showed that 6-BA had a significant effect on the differentiation of regenerated shoots of S. subhorizontally. At the same concentration (0.5 mg / L), TDZ could not induce shoot differentiation, while the shoot rate, shoot number, and large shoot number were significantly greater with 6-BA than with KT. Compared with 6-BA + NAA, the use of 6-BA alone significantly improved the effect of explant differentiation (Table 5). The addition of 0.05-1.0 mg / L NAA to 6-BA at four concentrations gradually reduced the shoot rate, shoot number, and large shoot number of explants. Among them, 0.5-1.0 mg / L NAA reduced the shoot rate to less than 50%, the shoot number to less than 0.3, and the large shoot number to less than 0. Among the combinations of 6-BA and NAA, only the addition of low concentrations of NAA (0.05-0.1 mg / L) to 0.5 mg / L 6-BA resulted in a significantly greater shoot rate and shoot number than the addition of the same concentration of 6-BA alone, but a significantly fewer large shoots. When the concentration of 6-BA increased, the shoot rate and shoot number showed an increasing trend first and then a decreasing trend, but the large shoot number showed a gradually decreasing trend, with the maximum shoot rate and shoot number (greater than all treatments in this experiment) appearing after treatment with 1.5 mg / L 6-BA. Explants died only when 0.5 mg / L KT, 0.5 mg / L 6-BA, and 1.0 mg / L 6-BA were added alone, but the mortality rate was low (the maximum mortality rate was less than 17.00%). From the results, it can be concluded that 6-BA is the most effective hormone for the direct induction of adventitious shoots from leaves of S. subhorizontally, and the optimal concentration of 6-BA is 1.5 mg / L. The addition of NAA to 6-BA at low concentrations (0.05-0.1 mg / L) can improve the shoot rate and shoot number, but the large shoot number is significantly reduced. The addition of NAA to 6-BA at high concentrations (0.5-1.0 mg / L) can significantly reduce the shoot rate, shoot number, and large shoot number. The addition of KT to 6-BA at the same concentration (0.5 mg / L) can significantly reduce the shoot rate and shoot number, but the large shoot number is not significantly reduced. Figure 3It was found that the addition of 6-BA alone was beneficial to the differentiation and regeneration of bud from the leaf explants of P. parvifolia, but not conducive to the generation of callus. Under the same concentration of 6-BA, the addition of NAA above 0.5 mg / L would make the explants yellow and tend to die. In summary, 0.5-2.0 mg / L 6-BA had a significant induction effect (budding rate and bud number were more than 70.00% and 1.50, respectively), and 1.5 mg / L 6-BA made the budding rate and bud number reach the maximum (88.89% and 3.61, respectively).

[0085] Table 5 Effect of different concentrations of hormones on the induction of the differentiation and regeneration of bud from the leaf explants of P. parvifolia

[0086]

[0087]

[0088] Note: The data are mean ± standard error, and different letters in the same column represent significant differences at the P < 0.05 level. There were 12 explants in each repeat, and the experiment was repeated three times.

[0089] Example 6 Effect of different hormone ratios on the induction of adventitious bud from the leaf explants of P. parvifolia regeneration bud

[0090] The leaf explants of the regeneration bud were inoculated in the medium (MS medium added with 30 g / L sucrose and 7 g / L agar, pH 5.8 ± 0.05) added with different concentrations of 6-BA, and the budding rate, bud number, large bud number, and mortality rate were counted after 45 d.

[0091] Budding rate (%) = number of budding explants / number of inoculated explants × 100%

[0092] Bud number (number / plant) = total bud number / number of inoculated explants

[0093] Large bud number (number / plant) = total large bud number after differentiation / number of inoculated explants × 100%

[0094] Compared with CK, 6-BA could significantly induce the differentiation of adventitious bud from the leaf explants of P. parvifolia regeneration bud, and increase the budding rate, bud number, and large bud number of the leaf explants of the regeneration bud (Table 6). The maximum values of the budding rate and large bud number appeared in 0.5 mg / L 6-BA (60.42% and 0.25, respectively), and gradually decreased with the increase of 6-BA concentration, and the maximum value of the bud number appeared in 1.5 mg / L 6-BA (1.23). The maximum mortality rate appeared in the treatment with the addition of 0.5 mg / L 6-BA, but did not exceed 15%.

[0095] Table 6 Effect of different concentrations of 6-BA on the induction of adventitious bud from the leaf explants of P. parvifolia regeneration bud

[0096]

[0097] Note: Data are mean ± standard error. Different letters in the same column indicate significant differences at the P < 0.05 level. Each treatment was repeated 4 times, with 12 explants as one replicate.

[0098] Example 7 Effects of different hormone ratios on adventitious root induction by adventitious buds of Pittosporum tobira

[0099] Adventitious buds with 5-7 true leaves were inoculated into MS medium (MS medium supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.8 ± 0.05) without hormone addition, 1 / 2MS medium (all nutrient elements were reduced by half, (1 / 2MS medium supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.8 ± 0.05)), and medium supplemented with 0.5, 1.0, 1.5, and 2.0 mg / L NAA, IAA, and IBA (MS medium supplemented with 30 g / L sucrose, 7 g / L agar, pH 5.8 ± 0.05). After 35 days, the rooting rate and rooting coefficient were calculated:

[0100] Rooting rate (%) = number of rooted explants / number of inoculated explants × 100%

[0101] Rooting coefficient = total number of roots / number of rooted and regenerated shoots

[0102] Results showed that compared with MS medium, 1 / 2 MS significantly reduced the rooting rate and rooting coefficient of adventitious buds (Table 7). When NAA, IAA, and IBA were added to MS medium, NAA treatment significantly increased the rooting coefficient but decreased the rooting rate compared with hormone-free MS medium. 1.0 mg / L NAA significantly decreased and increased the rooting rate and rooting coefficient, respectively, to their minimum and maximum values. Compared with MS medium, the addition of IAA and IBA significantly increased the rooting coefficient, but did not significantly increase the rooting rate. 2.0 mg / L IAA even significantly decreased the rooting rate (by 19.35%). In the IAA treatment, 0.5 mg / L IAA reached the maximum rooting rate and rooting coefficient. In the IBA treatment, the maximum rooting rate occurred at 1.0 mg / L IBA, and the maximum rooting coefficient occurred at 1.5 mg / L IBA. Among all treatments, 0.5 mg / L IAA and 1.5 mg / L IBA reached the maximum rooting rate and rooting coefficient, respectively. Although NAA can significantly increase the rooting coefficient, the adventitious roots that grow are short and thick and easily fall off ( Figure 4 Compared with MS, the addition of IAA and IBA can significantly promote the normal growth of adventitious roots ( Figure 4 a, c and d).

[0103] Table 7 Effects of different auxin concentrations on adventitious root induction in Pittosporum tobira

[0104]

[0105] Note: Data are means ± standard errors. Means with different letters within a column are significantly different at the P < 0.05 level. Rooting rate: 12 regenerated plants for one replicate, and triplicated. Half of all nutrient elements in 1 / 2MS medium. The basal medium not specified is MS medium.

[0106] Example 8: Elaboration and transplanting of regenerated seedlings

[0107] The regenerated plants with similar growth vigor and 3-4 regenerated roots were carefully taken out from the medium (without damaging the roots), washed with tap water to remove the residual medium on the roots, and then transplanted into black seedling bags filled with substrate. The substrate ratio was as follows (v:v): (1) river sand: peat soil: garden soil = 1:1:1; (2) garden soil: peat soil = 1:1; (3) peat soil: perlite = 3:1. The substrate was sterilized in a high-pressure steam boiler at 80°C for 1 h before mixing. After placing the seedling bags in the seedling pots, enough rooting water was poured, then covered with transparent film and cultured indoors for 7 d. Then moved to the outdoor to continue the film-moisture culture for 20 d, during which the film was opened twice a day to reduce humidity (half an hour each time, and the leaf surface was sprayed with clean water after each opening). Finally, the film was opened and the plants were sprayed with clean water in the morning and evening, and the water level in the seedling pots was ensured to be above the base of the seedling bags. The survival rate was counted after 30 d of transplanting:

[0108] Survival rate (%) = number of survived plants / number of transplanted plants x 100%

[0109] The results showed that the survival rates of the regenerated seedlings of S. microphylla cultured with river sand: peat soil: garden soil = 1:1:1 (T1), garden soil: peat soil = 1:1 (T2), and peat soil: perlite = 3:1 (T3) were all above 65% (Table 8), but compared with T1, T2 and T3 significantly improved the survival rate (increased by 20.00% and 24.01%, respectively), and T3 reached the maximum (86.11%).

[0110] Table 8: Effect of different substrates on the survival of regenerated plants of S. microphylla after transplanting

[0111] Matrix ratio (v:v) Survival rate (%) River sand: peat soil: garden soil = 1:1:1 69.44±2.78b Garden soil: peat soil = 1:1 83.33±4.81a Peat soil: perlite = 3:1 86.11±2.78a

[0112] Note: Data are means ± standard errors. Means with different letters within a column are significantly different at the P < 0.05 level. Each treatment was repeated 3 times, and each replicate had 12 regenerated plants.

[0113] Example 9

[0114] Based on the above experimental verification, a method for successfully inducing callus from leaf explants and then rapidly propagating S. microphylla through tissue culture was obtained, and the specific steps were as follows:

[0115] S1: take the tender leaves on the 1-year-old branches of small grass Pittosporum, wash with ultrapure water for 1 min, immerse in 75% ethanol for sterilization on an ultraclean workbench, then sterilize with 10% NaClO solution for 5 min, add 2 drops of Tween-20 during sterilization, and then wash with sterile water for 4 times, dry the surface moisture of the leaves on a sterile cutting paper, then cut off the leaf tips and leaf bases, and cut the remaining parts into leaf explants with an area of 0.12-0.15 cm 2 , that is, the sterile leaf explants;

[0116] S2: under sterile conditions, inoculate the sterile leaf explants on a callus induction medium for culture for 40 d to obtain callus; the callus induction medium is MS medium added with 0.1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0117] S3: take the callus with similar growth vigor, cut into small pieces with an area of 0.5-0.7 cm 2 , inoculate on an adventitious shoot induction medium for culture for 35 d to obtain adventitious shoots; the adventitious shoot induction medium is MS medium added with 0.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0118] S4: inoculate the explants with shoots on an adventitious shoot proliferation growth medium for culture for 35 d to obtain elongated adventitious shoots; the adventitious shoot proliferation growth medium is MS medium added with 0.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0119] S5: inoculate the adventitious shoots on the rooting induction medium as described above for culture for 35 d to obtain small grass Pittosporum tissue culture seedlings; the rooting induction medium is MS medium added with 0.5 mg / L IAA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0120] S6: take out the small grass Pittosporum tissue culture seedlings with 3-4 regenerated roots, wash the root residual medium with tap water, and transplant into a seedling raising pot containing a seedling transplanting substrate; the seedling transplanting substrate is peat soil: perlite with a volume ratio of 3:1;

[0121] S7: after transplanting the small grass Pittosporum tissue culture seedlings, pour enough rooting water, then cover with a transparent film and culture in a room for 7 d; then move to the outdoors to continue the film covering and moisture retaining culture for 20 d, during which the film is opened once in the morning and once in the evening each day for half an hour, and after each opening, the leaf surface is sprayed with clean water; finally, the film is opened, and the leaf surface is sprayed with clean water once in the morning and once in the evening, and the water amount in the seedling raising pot is ensured to be higher than the base of the seedling raising pot.

[0122] The culture conditions in steps S1-S7 are as follows: temperature is set to 25±1℃, light intensity is 1500±10 lux, light cycle is 12h / 12h (light / dark), and air humidity is 50±5%.

[0123] Example 10

[0124] Based on the above experimental verification, a tissue culture rapid propagation method for directly inducing regenerated shoots from leaf explants of P. humilis is obtained, comprising the following steps:

[0125] 1) Take the tender leaves on the 1-year-old branches of P. humilis, wash them with ultrapure water for 1 min, immerse them in 75% ethanol for sterilization on an ultraclean workbench, then sterilize them with a 10% NaClO solution for 5 min, add 2 drops of Tween-20 during sterilization, and then wash them with sterile water for 4 times, dry the surface moisture of the leaves on a sterile cutting paper, then cut off the leaf tips and leaf bases, and cut the remaining parts into leaf explants with an area of 0.12-0.15 cm 2 , which are sterile leaf explants;

[0126] 2) Inoculate the sterile leaf explants on a leaf direct induction of regenerated shoots medium for 45 days to obtain regenerated shoots; the leaf direct induction of regenerated shoots medium is MS medium added with 1.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0127] 3) Inoculate the regenerated shoot leaf explants on a regenerated shoot induction of adventitious shoots medium for 45 days to obtain more adventitious shoots; the regenerated shoot induction of adventitious shoots medium is MS medium added with 0.5-1.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0128] 4) Inoculate the regenerated shoots of step 2) or the adventitious shoots of step 3) on a rooting induction medium for 35 days to obtain P. humilis tissue culture seedlings; the rooting induction medium is MS medium added with 1.5 mg / L IBA, 30 g / L sucrose, and 7 g / L agar, and the pH is 5.8±0.05;

[0129] 5) Take out the P. humilis tissue culture seedlings with 3-4 regenerated roots, wash the root residual medium with tap water, and transplant them into a seedling raising pot containing a seedling raising medium; the seedling raising medium is peat soil: perlite with a volume ratio of 3:1;

[0130] 6) After transplanting the good small grass Pittosporum tissue culture seedlings, pour enough rooting water, then cover the transparent film and cultivate in the room for 7d; then move to the outside to continue the film to keep wet cultivation for 20d, during which the film is opened once in the morning and evening to reduce humidity (half an hour each time, and after each opening, the leaf surface is sprayed with clean water); finally, the film is opened, and the seedling pots are sprayed with clean water in the morning and evening, and the water in the seedling pots is ensured to be higher than the base of the seedling pots.

[0131] Steps 1)-6) The culture conditions in the room are: temperature is set to 25±1℃, light intensity is 1500±10 lux, light cycle is 12h / 12h (light / dark), air humidity is 50±5%.

[0132] Although the embodiments of the present application have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-mentioned embodiments within the scope of the present application.

Claims

1. A method for tissue culture propagation of Survillea humilis, characterized in that, Comprise the following steps: S1: take the tender leaves on the 1-year-old branches of small grass Pittosporum, clean, disinfect, and dry the water, then cut off the leaf tip and leaf base, and cut the remaining part into leaf explants with an area of 0.12-0.15 cm 2 , which are aseptic leaf explants; S2: under sterile conditions, sterile leaf explants are inoculated on callus induction medium for 30-40 days to obtain callus; the callus induction medium is MS medium added with 0.1 mg / L 6-BA, 0.5-1.2 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05; S3: the long potential similar growth callus was cut into 0.5-0.7 cm2 2 Small pieces were inoculated on adventitious bud induction medium for 30-35 days to obtain adventitious buds; the adventitious bud induction medium was MS medium added with 0.5 mg / L 6-BA, 30 g / L sucrose, 7 g / L agar, and pH was 5.8±0.05; S4: the explants with buds are inoculated on adventitious bud proliferation growth medium for 30-35 days to obtain elongated growth of adventitious buds; the adventitious bud proliferation growth medium is MS medium added with 0.5 mg / L 6-BA or 1 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05; S5: the adventitious buds are inoculated on the rooting induction medium to induce rooting for 30-35 days to obtain small grass pittosporum tissue culture seedlings; the rooting induction medium is MS medium added with 0.5-1.5 mg / L IAA or 0.5-2.0 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.05; S6: small grass pittosporum tissue culture seedlings with 3-4 regenerated roots are taken out, washed with tap water to remove residual culture medium, and transplanted into a seedling raising pot containing a seedling raising medium; the seedling raising medium is peat soil: perlite with a volume ratio of 3:1; S7: after the small grass pittosporum tissue culture seedlings are transplanted, sufficient rooting water is poured, then a transparent film is covered, and indoor culture is carried out for 7 days; then it is moved to the outdoor to continue the film covering and moisture retaining culture for 20 days, during which the film is opened once in the morning and once in the evening each day for half an hour, and after each opening, the leaf surface is sprayed with clean water; finally, the film is opened, and the seedling raising pot is sprayed with clean water in the morning and in the evening, and the water amount in the seedling raising pot is ensured to be higher than the base of the seedling raising pot.

2. The method for tissue culture propagation of Survillea humilis according to claim 1, characterized in that, In step S1, the specific steps of cleaning, disinfecting and drying are as follows: the leaves are washed with ultrapure water for 1 min, then immersed in 75% ethanol for disinfection on an ultraclean workbench, then sterilized with 10% NaClO solution for 5-10 min, 2 drops of Tween-20 are added during sterilization, and then washed with sterile water for 4 times, and the leaf surface is dried on sterile cutting paper.

3. The method for tissue culture propagation of Survillea humilis according to claim 1, characterized in that, The rooting induction medium is MS medium added with 0.5 mg / L IAA or 1.5 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.

05.

4. The method for tissue culture propagation of Survillea humilis according to claim 1, characterized in that, The rooting induction medium is MS medium added with 0.5 mg / L IAA or 1.5 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH is 5.8±0.

05.

5. The method for tissue culture propagation of Survillea humilis according to claim 1, characterized in that, The culture conditions in steps S1-S7 are as follows: the temperature is set to 25±1℃, the light intensity is 1500±10 lux, the light cycle is 12 h light / 12 h dark, and the air humidity is 50±5%.

Images