CN109042333B

CN109042333B - Tissue culture and rapid propagation method for Angel red soft seed pomegranate

Info

Publication number: CN109042333B
Application number: CN201811093513.6A
Authority: CN
Inventors: 苑兆和; 火艳; 徐会丽
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Liuyuan Biological Technology Co ltd
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2020-05-22
Anticipated expiration: 2038-09-19
Also published as: CN109042333A

Abstract

The invention discloses a tissue culture and rapid propagation method of an angel red soft seed pomegranate, belonging to the field of plant tissue culture and comprising the following processes: collecting tender stem segments, sterilizing with 75% alcohol for 30 s, sterilizing with 0.1% mercuric chloride for 10 min, and inoculating on adventitious bud induction culture medium; after adventitious buds grow out, cutting off two ends of the leaves, cutting off veins, and inoculating the veins to a leaf callus induction culture medium; inoculating adventitious buds generated by stem induction and leaf callus differentiation to a proliferation culture medium; transplanting the single seedling to a rooting culture medium after the single seedling grows to 5-6 cm; when the root of the single seedling grows to 3-4cm, hardening the seedling. The invention has the advantages that: can induce the test-tube plantlet of the Angel red soft-seed pomegranate to quickly proliferate to generate a large amount of adventitious buds, has the inductivity as high as 92.22 percent, and provides an effective way for industrial seedling culture.

Description

Tissue culture and rapid propagation method for Angel red soft seed pomegranate

Technical Field

The invention relates to the field of plant tissue culture, in particular to a tissue culture and rapid propagation method of an angel red soft seed pomegranate.

Background

Pomegranate (Punica granatumL.) of the genus punica of the family Lythraceae (Lythraceae), (L.), (PunicaL.) fruit trees have high nutritional value and health care function, in recent years, pomegranate is more and more popular in the consumer market, and the pomegranate industry develops rapidly at home and abroad. The soft-seed pomegranate has soft seed coat, degraded kernel and large seed, and has high value in fresh food consumption. At present, the proportion of soft seed varieties in pomegranate cultivation in China is relatively small, the market price is 2-4 times that of common varieties, and the market prospect is wide. The Angel red soft seed pomegranate is an excellent soft seed pomegranate variety introduced from the United states, has medium fruit size, red peel, sour and sweet taste, high juice yield, short and small plant type, high quality and high yield.

Because the variety of the Angel red soft-seed pomegranate is precious and has less resources, the conventional breeding method can not meet the current production and market demands, and the advantages of high plant tissue culture propagation speed, maintenance of the excellent properties of the original variety, annual industrial production and the like can not be realized. At present, no tissue culture rapid propagation research report of the Angel red soft seed pomegranate exists.

Disclosure of Invention

In order to overcome the technical problems, the invention provides a tissue culture and rapid propagation method of the Angel red soft seed pomegranate, which is simple and strong in operability, provides a high-efficiency and feasible way for rapidly and stably propagating the Angel red soft seed pomegranate, and lays a theoretical foundation for industrially producing excellent seedlings.

In order to achieve the purpose, the invention adopts the following technical scheme:

a tissue culture and rapid propagation method of Angel red soft seed pomegranate comprises the following steps:

1) collecting tender stem as explant, sterilizing with 75% alcohol for 30 s, sterilizing with 0.1% mercuric chloride for 10 min, and cleaning with sterile water;

2) the stem segments after disinfection are inoculated on an adventitious bud induction culture medium with the formula of WPM + 2g/L PVP + 0.5mg/L NAA + 1.0 mg/L6-BA.

3) After adventitious buds grow out, selecting the leaves generated in the induction process, cutting off the two ends of the leaves, cutting off veins, and inoculating the cut leaves to a leaf callus induction and differentiation induction culture medium with the formula of WPM + 0.5mg/L NAA + 1.0 mg/L6-BA.

4) Adventitious buds induced from stem segments and adventitious buds induced from leaf callus differentiation were inoculated onto enrichment medium of WPM +0.1 mg/L NAA + 1.0 mg/L6-BA.

5) Cutting single seedling with strong growth and 5-6cm length for rooting culture in 1/2MS, 0.7mg/L IBA and 0.2 g/L PVP.

6) Taking out the tissue culture seedling with root length of 3-4cm from the culture chamber, transferring the culture bottle into a greenhouse, performing intensive light bottle-closing seedling hardening for about 5-20d, preferably 50% -70% of shading degree, opening the bottle mouth, and placing for 3-7 d; transplanting when the root length of the tissue culture seedling reaches 5-6cm and the plant grows well. The transplanting matrix is prepared by mixing turf and vermiculite according to the ratio of 2:1, fully mixing and sterilizing at high temperature. The temperature of the transplanting environment is 25-28 ℃, the humidity is 80% -85%, and the illumination time is 12-14 h per day.

In a further technical scheme, the WPM minimal medium in the steps 2), 3) and 4) comprises 6.5g/L of agar and 30 g/L of sucrose, and comprises the following components: NH (NH)₄NO₃400mg/L、Ca(NO₃)2·4H₂O 556mg/L、CaCl₂·2H₂O96mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、K₂SO₄990mg/L、H₃BO₃6.2mg/L、MnSO₄·H₂O22.4mg/L、ZnSO₄·7H₂O 8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.25mg/L、FeSO₄·7H₂O₂7.8mg/L、Na₂37.3mg/L of EDTA, 100mg/L of inositol, 2mg/L of glycine, 0.5mg/L of nicotinic acid, 11.0mg/L of vitamin B and 60.5mg/L of vitamin B.

In a further technical scheme, the MS minimal medium in the step 5) comprises 6.5g/L of agar and 30 g/L of sucrose, and the following components: NH (NH)₄NO₃1650mg/L、KNO₃1900mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、CaCl₂·2H₂O 440 mg/L、KI 0.83mg/L、H₃BO₃6.2mg/L、MnSO₄·4H2O 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.025mg/L、CoCl₂·6H₂O0.025mg/L、FeSO₄·7H₂O 27.8mg/L、Na₂37.25mg/L EDTA, 100mg/L inositol, 2mg/L glycine, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, 10.1 mg/L vitamin B.

In a further technical scheme, the pH value of the culture medium in the steps 2), 3), 4) and 5) is adjusted to 5.8.

Has the advantages that:

1. the invention adopts a disinfection mode of alcohol and mercury bichloride, can effectively remove residual germs in explants, and has an average pollution rate of only 37.78% and a browning rate of 22.22%.

2. In the stage of inducing adventitious buds and callus, an MPW culture medium is screened, the induction effect is the best, and reaches 93.33%, and the browning rate is only 6.67%; the browning phenomenon is severe in the induction process of MS and B5 culture media, the induction rates are respectively 11.11% and 8.89%, and the browning rates are respectively 88.89% and 91.11%.

2. The invention screens out the agar concentration and the pH value of the culture medium with the optimum agar concentration and pH value of 6.5g/L and pH5.8, the average browning rate is 28.89 percent, and the average inductivity is 66.67 percent.

2. The PVP is added in the adventitious bud induction stage and the rooting culture stage, so that the browning is effectively prevented. The average browning rate is reduced from 91.11% to 34.44% in the adventitious bud induction process, and the induced adventitious bud has good growth, extended leaves and dark green color. In the process of rooting, the browning of the roots is effectively inhibited, the roots are thick and long, and the growth force is vigorous.

4. In the adventitious bud induction stage, the highest number of adventitious buds are induced by the stem segments when 1.0mg/L of 6-BA and 0.5mg/L of NAA are added, and the average induction rate reaches 91.11 percent.

5. In the stage of leaf callus induction and differentiation, when NAA is 0.5mg/L and 6-BA is 1.0mg/L, the number of the leaves for inducing the callus is the largest, the induction rate and the adventitious bud differentiation rate are respectively 92.22% and 87.78%, and the adventitious buds differentiated from the callus on the culture medium have good growth vigor and the leaves are stretched.

6. In the proliferation culture stage, when 1.0mg/L of 6-BA and 0.1mg/L of NAA are added, the adventitious buds grow well, the base part of the stem has callus, and the proliferation coefficient can reach 3.65 at most.

7. In the rooting culture stage, when the IBA is added to the rooting culture medium at the concentration of 0.7mg/L, the rooting effect is best, the highest rooting effect can reach 61.11 percent, the roots are thick and long, and the growth force is vigorous.

Drawings

FIG. 1 growth of Angel Red Soft seed pomegranate for contamination prevention study

FIG. 2 adventitious bud growth on media treated with different combinations of pH and agar concentration

FIG. 3 leaf callus induction and adventitious bud growth on differentiation Medium

FIG. 4 growth of adventitious bud by multiplication culture

FIG. 5 rooting culture and transplanting and seedling hardening.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Examples

The WPM minimal medium in the steps 2), 3) and 4) comprises 6.5g/L of agar and 30 g/L of sucrose, and comprises the following components: NH (NH)₄NO₃400mg/L、Ca(NO₃)2·4H₂O 556mg/L、CaCl₂·2H₂O 96mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、K₂SO₄990mg/L、H₃BO₃6.2mg/L、MnSO₄·H₂O 22.4mg/L、ZnSO₄·7H₂O 8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.25mg/L、FeSO₄·7H₂O₂7.8mg/L、Na₂37.3mg/L of EDTA, 100mg/L of inositol, 2mg/L of glycine, 0.5mg/L of nicotinic acid, 11.0mg/L of vitamin B and 60.5mg/L of vitamin B.

The MS minimal medium in the step 5) comprises 6.5g/L of agar and 30 g/L of cane sugar, and the following components: NH (NH)₄NO₃1650mg/L、KNO₃1900mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、CaCl₂·2H₂O440 mg/L、KI 0.83mg/L、H₃BO₃6.2mg/L、MnSO₄·4H2O 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.025mg/L、CoCl₂·6H₂O 0.025mg/L、FeSO₄·7H₂O27.8mg/L、Na₂37.25mg/L EDTA, 100mg/L inositol, 2mg/L glycine, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, 10.1 mg/L vitamin B.

The pH value of the culture medium in the steps 2), 3), 4) and 5) is adjusted to 5.8.

Test comparison and analysis

Experimental materials: selecting young stem of American-introduced 'Angel Red' soft seed pomegranate as test material, which is collected from resource garden of pomegranate in Bama base of Nanjing forestry university, marking the collected young stem, placing the marked young stem in an ice box, bringing the young stem back to a laboratory, and placing the young stem in a refrigerator at 4 ℃ for standby, wherein the preservation time of the collected young stem is not more than one week.

The main reagents are as follows: 6-BA (6-Benzylaminopurine ), NAA (NapHthalene-acetic acid), IBA (3-indole butyric acid), PVP (PolyVinylpyrrolidone ), sucrose, agar, WPM Medium (Woody plant Medium), MS (Murashige and Skoog Medium), B5 (B5 Medium), which were produced by Shanghai Biotechnology, Inc., wherein PVP was directly added to the Medium and sterilized at high temperature.

The main instruments used are: SW-CJ-1F type superclean bench, MLS.3020 type autoclave, oven, electronic balance, pH meter, JZ-II inoculating instrument sterilizer, refrigerator, alcohol burner, Waters pure water system, pipette of Eppendorf Co, Germany, etc.

A data statistical formula:

contamination rate = number of contaminated explants/total number of inoculated explants × 100%;

mortality = number of dead explants/total number of inoculated explants × 100%;

browning rate = number of browned explants/total number of inoculated explants × 100%;

adventitious bud induction = number of adventitious buds germinated/number of adventitious buds uncontaminated after inoculation × 100%;

callus induction = number of explants forming callus/total number of explants inoculated × 100%;

adventitious bud differentiation rate = number of calli forming adventitious buds/total number of calli × 100%;

proliferation factor = number of adventitious buds transferred/number of adventitious buds transferred × 100%;

rooting rate = number of rooted plants/total number of inoculated plants × 100%.

1. Explant sterilization time screening

In order to screen out the optimal disinfection time treatment combination of the young and tender stem segments of the pomegranate, two disinfectants of alcohol and mercury bichloride are selected to study the disinfection treatment of the soft-seed pomegranate, wherein the disinfection time of 75% alcohol is selected from 3 levels: 10 s, 20 s, 30 s. The 0.1% mercuric chloride treatment time was selected at 3 levels: 6 min, 8 min and 10 min. The experimental design is detailed in table 1. The stem sections are washed and placed on a super clean bench, further disinfected by ethanol and mercury bichloride, and washed by sterile water after disinfection. And cutting off the part of the disinfected stem with buds, which is contacted with the disinfectant, on the filter paper, inserting the cut stem with buds into a culture medium, observing the pollution condition of the inoculated explants after 2 w, observing the pollution characteristics of the explants, further judging the cause of pollution, and counting the pollution rate.

TABLE 1 Disinfection protocol

Treatment combination	Alcohol Disinfection time(s)	Mercuric chloride disinfection time (min)
			1	10	8
2	10	10
			3	10	12
4	20	8
			5	20	10
6	20	12
			7	30	8
8	30	10
			9	30	12

After the stem sections are cultured for 10 days, the pollution number, the browning number and the induction condition of the budded stem sections are investigated, the difference of the pollution rate, the browning rate and the explant induction condition of different treatment combinations is obvious, and the results are shown in table 2:

TABLE 2 comparison of the effectiveness of different disinfection treatments

Note: a. the numerical value indicates the number of repetitions.

As can be seen from table 2, the different treatment combinations differ significantly with respect to the contamination of explants under different treatment conditions: treatment 1 was the highest group, Aa; treatment 4 was higher than the other groups, independently the next highest group and independently the Bb group; treatment 9 had an average contamination rate of 27.78% lower than treatment 8, but the average browning rate due to treatment 8 was very significantly lower than treatment 9 and the average induction rate of treatment 8 was very significantly higher than treatment 9. Therefore, considering the comprehensive consideration of the contamination rate, browning rate and induction condition of the explants, the treatment 8 is considered as the optimal disinfection operation of the germinated stem segments of the soft-seed pomegranate.

Therefore, the part selects the best disinfection operation of the pomegranate stem segment with buds, namely the pomegranate stem segment with buds disinfected by 75% of alcohol for 30 s and then disinfected by 0.1% of mercury bichloride for 10 min, wherein the explant pollution rate is 37.78%, the browning rate is 22.22% and the explant induction rate is 60%.

FIG. 1 shows the growth of an angel red soft seed pomegranate for contamination prevention study, wherein FIG. 1-1 shows the contamination phenomenon induced by adventitious buds; FIG. 1-2 shows adventitious buds induced from stem segments after they have been sterilized; FIGS. 1-3 show the adventitious bud formation after 2 w inoculation of the stem segment; FIGS. 1-4 show the adventitious bud formation after 3 w inoculation of the stem segment; FIGS. 1 to 5 show the generation of adventitious buds after 4 w of stem inoculation; FIGS. 1-6 show the adventitious bud formation after stem segment inoculation of 5 w.

2. Screening of minimal Medium

The browning degree and the growth condition of explants in different culture media are greatly different, and MS and B are selected in order to screen out a more suitable culture medium for tissue culture of soft-seed punica granatum₅And 3 common culture media including WPM (woody plant Medium) are used for researching the influence of different culture media on the induction of adventitious buds in the inoculation process of the soft-seed pomegranate, the disinfected stem sections are respectively inoculated on the 3 different culture media, and the browning rate and the induction rate of the adventitious buds on the different culture media are counted after 2 w.

TABLE 3 Effect of different media on explant browning and induction rates

Culture medium	Average browning rate (%)	Average induction ratio (%)
			MS	88.89Aa	11.11Bb
B₅	91.11Aa	8.89Bb
			WPM	6.67Bb	93.33Aa

2 w later statistics of different culture media MS and B₅And influence of WPM on explant browning rate and induction condition. From the above table, it can be seen that the different culture media have very significant difference in induction effect on the soft seed pomegranate explant. Wherein the WPM culture medium has the best induction effect on the soft seed pomegranate stem segment, which reaches 93.33 percent, and the difference is remarkably higher than that of other groups. And MS and B₅The browning phenomenon is serious in the induction process of the culture medium, and the induction rates are 11.11% and 8.89% respectively.

For the browning rates of explants on different media of soft seed punica granatum, the 3 treatment combination differences were very significantly divided into 2 groups, wherein the browning rate of explants inoculated in WPM media was significantly lower than that of the other groups, which was the optimal group. In summary, the following steps: the most suitable induction culture medium for the soft-seed pomegranate is a WPM culture medium.

3. Medium pH and agar concentration screening

The influence of the different processing combinations of main research pH value and agar concentration to explant pollution and browning is all selected for the culture medium, and 30 explants are established to every processing, and the experiment is repeated 3 times all, and agar concentration selects 3 levels in the experiment: 6.0 g/L, 6.5g/L and 7.0 g/L. pH was selected at 3 levels: 5.4, 5.8 and 6.2. The experimental design is detailed in table 3.

TABLE 4 media pH and agar concentration design

Treatment of	Agar concentration (g/L)	pH value
			1	6	5.4
2	6	5.8
			3	6	6.2
4	6.5	5.4
			5	6.5	5.8
6	6.5	6.2
			7	7.0	5.4
8	7.0	5.8
			9	7.0	6.2

TABLE 5 Effect of different pH and agar concentration combinations on explant browning and inductivity

As can be seen from the above table and FIG. 2 (FIG. 2, 2-1 is the adventitious bud growth at the agar concentration of 7.0g/L and pH of 5.8; 2-2 is the adventitious bud growth at the agar concentration of 6.5g/L and pH of 5.8), the explant browning rate shows a rule of decreasing and then increasing with the increase of the agar concentration when the pH is constant; when the concentration of the agar is 6g/L, the browning degree of the explant is obviously reduced along with the increase of the pH value, and the induction rate is gradually increased; when the concentration of the agar is 6.5g/L, the browning degree of the explant shows a trend of firstly decreasing and then increasing along with the increase of the pH value, and the induction rate increases and then decreases; when the concentration of the agar is 7.0g/L, the browning degree of the explant is obviously increased along with the increase of the pH value, and the induction rate is gradually reduced.

In summary, considering the influence of the pH value and agar concentration of the culture medium on the browning and induction of the explant, it is considered that when the concentration of agar added to the culture medium is 6.5g/L and the pH value is 5.8, the influence of the combination of the pH value and agar concentration on the browning of the explant is minimal, and the agar concentration is set to be 6.5g/L and the pH value is set to be 5.8 in the culture medium at different stages of the study.

4. Screening of concentration of anti-browning agent

In the experiment, the browning phenomenon of the stem section in 3-4 d after inoculation is found to be serious, and in order to research the influence of the browning prevention agent PVP (polyvinylpyrrolidone) on the browning degree induced by adventitious buds, the addition concentration of the PVP is set to be 4 levels in the experiment: 0.2 g/L, 4g/L and 6g/L, wherein the experiments are carried out by adopting a single-factor random block experiment, and the browning rate is counted after 20 days of culture.

TABLE 6 Effect of different concentrations of PVP on the browning rate of adventitious buds

PVP concentration (g/L)	Average browning rate (%)	Average induction ratio (%)
			0	91.11Aa	5.56Cd
2	34.44Bb	56.67Aa
			4	22.22Cc	25.56Bb
6	18.89Cc	22.22Bc

As can be seen from the above table, after culturing the adventitious bud on the medium added with the anti-browning agent PVP for 20 days, the browning phenomena on the medium without the addition of PVP and the medium added with 2g/L of PVP are obviously different. The browning condition on the culture medium without the anti-browning agent is very serious, and the browning rate is as high as 91.11%. When the anti-browning agent PVP is added, the browning degree is obviously reduced, and meanwhile, the inductivity has the tendency of increasing firstly and then reducing. Researches find that the later growth vigor of the bud seedlings is poor due to the fact that the anti-browning agent is used at an excessively high concentration or for an excessively long time.

By comparison, for the influence of different concentrations of PVP on the browning rate of adventitious buds, the differences of 4 treatment combinations were very significantly divided into 3 groups: the browning rate in the medium added with 0g/L PVP is remarkably higher than that in other groups and is independently an Aa group; the browning rate in the medium added with 2g/L PVP is the second highest group, and independently is Bb group; the browning rates of the explants in the culture medium with 4g/L of PVP and the culture medium with 6g/L of PVP are obviously lower than those of other groups, namely the lowest group, and the explants are listed as the group Cc. While the influence of different concentrations of PVP on the adventitious bud induction rate, the differences of the 4 treatment combinations were significantly divided into 4 groups. Wherein the highest induction rate in the culture medium added with 2g/L PVP is an independent Aa group, and the lowest induction rate in the culture medium added with 0g/L PVP is an independent Cd group.

In conclusion, the influence of PVP with different concentrations on the browning condition and the adventitious bud induction condition is comprehensively considered, and the effect is considered to be the best when the PVP concentration is 2 g/L.

5. Adventitious bud induction

In order to induce shoot adventitious buds, this experiment added NAA and 6-BA to WPM medium to study the effect of growth regulator concentration on adventitious bud induction, wherein the concentration treatment combination of the two hormones is shown in Table 7. And selecting the stem section of the sterile culture system constructed in the previous stage as the explant, adding 2.0 g/L PVP into the culture medium, and after culturing for 21 d, counting the induction condition of the adventitious bud. And calculating the adventitious bud induction rate.

TABLE 7 adventitious bud Induction protocol

Treatment combination	NAA concentration (mg/L)	6-BA concentration (mg/L)
			CK	0	0
1	0.5	0.5
			2	0.5	1.0
3	0.5	1.5
			4	1.0	0.5
5	1.0	1.0
			6	1.0	1.5
7	1.5	0.5
			8	1.5	1.0
9	1.5	1.5

The stem segments containing buds are inoculated on adventitious bud induction culture media WPM +6-BA + NAA, so that adventitious buds are induced on all treated culture media, but the difference between the number of the induced adventitious buds and the growth condition of the adventitious buds is obvious between different treatments, and the result is shown in a table 8:

TABLE 8 Effect of NAA and 6-BA on adventitious bud Induction

Treatment combination	NAA concentration (mg/L)	6-BA concentration (mg/L)	Average induction ratio (%)
				CK	0	0	22.22±1.73Fh
1	0.5	0.5	87.78±1.75Aab
				2	0.5	1.0	91.11±2.15Aa
3	0.5	1.5	51.11±3.40Df
				4	1.0	0.5	83.33±5.37ABbc
5	1.0	1.0	88.89±1.55Aab
				6	1.0	1.5	59.78±1.39Ce
7	1.5	0.5	77.78±1.48Bcd
				8	1.5	1.0	75.56±2.19Bd
9	1.5	1.5	38.89±1.00Eg

After 3 w of culture, the adventitious bud induction condition is counted, and the adventitious bud induction rate is generally increased and then decreased along with the increase of the concentration of NAA and 6-BA, and the induction rate of the treatment with the growth regulator is higher than that of a control group without any hormone. Wherein, when the treatment 2 is that 1.0mg/L of 6-BA and 0.5mg/L of NAA are added into the WPM culture medium, the number of the adventitious buds induced by the stem segment is the highest, and the average induction rate reaches 91.11 percent; when 1.5 mg/L of 6-BA and 1.5 mg/L of NAA are added into the culture medium, the induction rate is the lowest and is only 38.89%. It was found that the promoting effect was the best when 6-BA was added to the medium at 1.0mg/L, and that the generation of adventitious buds was inhibited when the concentration exceeded 1.5 mg/L. In conclusion, the optimal adventitious bud induction culture medium of the soft-seed pomegranate is WPM + 0.5mg/L NAA + 1.0 mg/L6-BA.

6. Leaf callus induction and differentiation

In the experiment, the pollution phenomenon and browning phenomenon of the explant are very serious when the leaves are collected from the field for callus induction. In order to better induce the callus of the leaves, the leaves of aseptic seedlings grown in the adventitious bud inducing process are selected and utilized, and because the leaves are newly germinated from disinfected stem segments, germs in the leaves can be greatly reduced, the pollution is easier to control than the leaves collected from the field, and the efficiency of inducing the callus by the leaves can be improved. Leaves which are about 2cm long and have light green color and no obvious yellowing phenomenon are selected for callus induction, and the sizes of the leaves have no obvious influence on test results.

In this fraction, 3 levels (mg/L) of NAA were selected: 0.05, 0.1, 0.15. 6-BA 3 levels (mg/L) were selected: 1. 1.5 and 2. The experimental design is detailed in table 5. The explants were selected from the light green leaves produced on the stem sections of the sterile culture line constructed in the previous stage. And after culturing for 20 days, counting the callus induction condition, continuously culturing for 20 days, counting the adventitious bud differentiation condition, and calculating the callus induction rate and the adventitious bud differentiation rate.

TABLE 9 callus induction and differentiation protocol

Treatment combination	NAA concentration (mg/L)	6-BA concentration (mg/L)
			1	0.05	1
2	0.05	1.5
			3	0.05	2
4	0.1	1
			5	0.1	1.5
6	0.1	2
			7	0.15	1
8	0.15	1.5
			9	0.15	2

Selecting the leaves generated on the stem section of the sterile culture system constructed in the previous stage, cutting off two ends of the leaves on an ultraclean workbench, inoculating the cut leaves into a culture medium added with different growth regulating substance treatment combinations (as shown in figure 3-1), culturing about 1 w to generate loose callus, further growing the callus about three weeks (as shown in figures 3-2 and 3-3), and culturing 4 w to differentiate partial callus into adventitious buds (as shown in figures 3-5), wherein in figure 3, figure 3-1 is the leaves inoculated on a WPM culture medium; FIGS. 3-2 and 3-3 show callus formed after three weeks of culture; FIGS. 3-4 show callus formed in control groups; FIGS. 3-5 are the adventitious buds differentiated after four weeks of culture: FIGS. 3 to 6 show the growth of adventitious buds after two months of culture on a medium containing 1.5 mg/L of NAA and 1.5 mg/L of 6-BA.

The effect of the treatment combination of NAA and 6-BA with different concentrations on the induction and differentiation of the leaf callus is obvious, and the results are shown in the table 10:

TABLE 10 influence of NAA and 6-BA on leaf callus induction rate and adventitious bud differentiation rate

Treatment combination	NAA concentration (mg/L)	6-BA concentration (mg/L)	Callus induction rate (%)	Adventitious bud differentiation Rate (%)
					CK	0	0	28.89±0.90Gg	38.89±1.60DEd
1	0.5	0.5	75.56±1.25Bb	61.11±3.14Bb
					2	0.5	1.0	92.22±1.47Aa	87.78±1.12Aa
3	0.5	1.5	61.11±1.17Cc	51.11±3.79Cc
					4	1.0	0.5	59.78±2.51Cc	55.56±0.57BCbc
5	1.0	1.0	88.89±1.68Aa	83.33±3.40Aa
					6	1.0	1.5	51.11±2.89Dd	42.22±0.90Dd
7	1.5	0.5	35.56±1.06Ff	33.33±2.78Ee
					8	1.5	1.0	42.22±3.43Ee	38.89±0.90DEd
9	1.5	1.5	38.89±1.55EFef	22.22±1.98Ff

The control group found that the adventitious bud was very slowly differentiated after the leaf callus induction was completed (see FIGS. 3-4), the adventitious bud could be differentiated only after about 60 days, and the difference of callus induction conditions in the culture medium with different concentrations of growth regulating substances was significant. When 1.5 mg/L NAA and 0.5 mg/L6-BA are added into WPM medium, the number of the leaf inducing callus is minimum, and is only 35.56%. When 0.5mg/L NAA and 1.0 mg/L6-BA are added into the culture medium, the number of the leaf blades for inducing the callus is the largest, the induction rate and the adventitious bud differentiation rate are respectively 92.22% and 87.78%, and the adventitious bud differentiated from the callus on the culture medium has good growth vigor and the leaf blades are stretched. Therefore, the best culture medium for inducing the leaf callus and differentiating the callus is WPM + 0.5mg/L NAA + 1.0 mg/L6-BA in the experiment.

7. Proliferation culture

The effect of 6-BA and NAA on explant seedling propagation was studied. Subculturing the explant seedling which grows vigorously, and carrying out a proliferation culture test when the seedling grows to 5-6 cm. Cutting the tender stem into 1 tender shoot, inoculating into different proliferation culture medium, 6 bottles each time, 5 stem segments per bottle. After 30 days of culture, the proliferation effect of different media on explant shoots was observed. The height and number of adventitious buds were counted and the proliferation coefficient (proliferation coefficient = number of adventitious buds transferred/number of adventitious buds transferred × 100%) was calculated.

TABLE 11 proliferation culture protocol

Treatment combination	6-BA concentration (mg/L)	NAA concentration (mg/L)
			1	0.5	0.1
2	0.5	0.3
			3	0.5	0.5
4	1	0.1
			5	1	0.3
6	1	0.5
			7	2	0.1
8	2	0.3
			9	2	0.5

Selecting well-growing parts in adventitious buds generated by leaf callus induction and stem direct induction for enrichment culture, periodically investigating the proliferation condition of the adventitious buds, finding that the proliferation condition of the adventitious buds is good after about four weeks, recording and counting the growth condition and proliferation coefficient of the bud seedlings, and obtaining the results shown in table 12 and figure 4; in FIG. 4, FIG. 4-1 is the adventitious bud just inoculated; FIG. 4-2 shows the proliferation culture after 2 weeks of treatment; FIGS. 4-3 and 4-4 show the proliferation culture after 3 weeks of treatment; FIGS. 4-5 are graphs of proliferative growth after 4 weeks of treatment; FIGS. 4-6 show the proliferation growth after 4 weeks of treatment.

TABLE 12 Effect of growth regulating substances on the multiplication factor of adventitious buds

Treatment combination	Coefficient of proliferation	Growth conditions
			1	2.07±0.15Ccd	Short and thick stem
2	2.69±0.08ABCbcd	Short and thick stem
			3	3.28±0.30ABab	Slender stem, extended leaves and dark green color
4	3.65±0.36Aa	Slender stem, wide leaf and dark green color
			5	3.38±0.33ABab	Slender stem, extended leaves and dark green color
6	2.80±0.10ABCbc	Short and thick stem
			7	2.70±0.10ABCbcd	Blade curling
8	2.56±0.23BCcde	Leaf curl, light green
			9	2.03±0.15Ce	Dark yellow leaves

The result shows that the growth regulating substances NAA and 6-BA have the promotion effect on the multiplication culture of the adventitious bud, but when the concentration of the 6-BA exceeds 2.0 mg/L, the growth of the bud seedling is easy to have the condition that the leaf is yellow and curled. When 1.0mg/L of 6-BA and 0.1mg/L of NAA are added into the culture medium, the adventitious buds grow well, the base part of the stem has callus, the multiplication coefficient can reach 3.65 at most, and the promotion effect is obvious compared with other treatment. Treatment 5 followed by a growth factor of 3.38. The lowest proliferation factor of treatment 9 was only 2.03, which had short and thick stems, significant leaf curl, and evidence of leaf yellowing. Test results show that the optimal proliferation culture medium of the soft-seed pomegranate is WPM +0.1 mg/L NAA + 1.0 mg/L6-BA.

8. Rooting culture

In a preliminary experiment, when the soft seed pomegranate rooting culture medium is not added with the anti-browning agent PVP, the root is seriously browned after the rooting culture is continued for about one week. To obtain thick and long roots, the effect of IBA and PVP on the rooting of soft seed punica granatum was studied in this section. IBA was applied using 3 concentration (mg/L) gradients: 0.5, 0.7 and 0.9. PVP was selected at 3 concentration levels (g/L): 0. 0.2, 0.4 (see table 13). The rooting culture medium is 1/2MS culture medium, and the rooting condition is observed periodically. After about 4 w, counting the rooting condition of the bud seedlings and calculating the rooting rate.

TABLE 13 rooting culture protocol

Treatment combination	IBA concentration (mg/L)	PVP concentration (g/L)
			1	0.5	0
2	0.5	0.2
			3	0.5	0.4
4	0.7	0
			5	0.7	0.2
6	0.7	0.4
			7	0.9	0
8	0.9	0.2
			9	0.9	0.4

After 30 days of light culture, it was observed that some seedlings had grown fine roots of about 1 cm, and the culture was continued for about 15 days, and most of the roots had grown to 2cm, as shown in Table 14.

TABLE 14 rooting Rate for different treatment combinations

Treatment combination	IBA concentration (mg/L)	PVP concentration (g/L)	Rooting percentage (%)	Root conditions
					1	0.5	0	50.00±1.26Cde	Severe root browning
2	0.5	0.2	30.43±1.09ABCbcd	Thin and short root
					3	0.5	0.4	31.33±3.09ABab	Thin and short root
4	0.7	0	44.44±3.76Aa	Severe root browning
					5	0.7	0.2	61.11±2.02ABab	Root of large and long
6	0.7	0.4	55.56±2.51ABCbc	Root of large and long
					7	0.9	0	56.67±2.51ABCbc	Severe root browning
8	0.9	0.2	32.22±2.78BCcde	Thin and short root
					9	0.9	0.4	18.89±1.51Ce	Small amount of wound healing, thin and short root

From table 14, it was found that the medium to which the anti-browning agent PVP was added was effective in suppressing the browning of the roots. Wherein, when the IBA concentration is 0.7mg/L, the rooting effect is best, the highest IBA concentration can reach 61.11%, the roots are thick and long, and the growth force is vigorous.

Therefore, the test result considers that the optimal rooting medium is 1/2MS + 0.7mg/L IBA + 0.2 g/L PVP.

9. Hardening and transplanting seedlings

The aim of the experiment is to improve the survival rate of the transplantation. Taking out the tissue culture seedling with root length of 3-4cm from the culture room, transferring the culture bottle into a greenhouse, performing intensive light closed bottle hardening for about 5-20d, preferably 50% -70% shading degree, opening the bottle mouth, and placing for 3-7 d. After hardening off, selecting tissue culture seedlings with root length of 5-6cm and good growth vigor for transplanting, selecting grass carbon and vermiculite as transplanting matrix to be mixed according to the proportion of 2:1, and sterilizing the matrix at high temperature after mixing to kill various germs contained in the matrix. Strictly managing the temperature, humidity and illumination of the culture environment after transplanting, wherein the temperature is controlled to be 25-28 ℃, the humidity is 80-85%, the illumination time is controlled to be 12-14 h per day, nutrient in the matrix is supplemented by nutrient solution every 2 w in the transplanting process, the transplanting survival rate of the tissue culture seedlings is high, as shown in figure 5, and in the figure 5-1, the browning condition of 4 seedlings is treated; FIGS. 5-2 and 5-3 show the initial root growth of treatment 5; FIG. 5-4 shows the root growth after treating 5 rooting cultures for 45 days; 5-5 closing the bottle and hardening the seedling for 15d, and then transplanting; FIGS. 5 to 6 show the growth of the roots of the transplanted seedlings.

Claims

1. A tissue culture and rapid propagation method of Angel red soft seed pomegranate is characterized by comprising the following steps:

2) inoculating the disinfected stem segments on an adventitious bud induction culture medium with the formula of WPM + 2g/L PVP + 0.5mg/L NAA + 1.0 mg/L6-BA;

3) after adventitious buds grow out, selecting leaves generated in the induction process, cutting off two ends of the leaves, cutting off veins, and inoculating the leaves onto a leaf callus induction and differentiation induction culture medium with a formula of WPM + 0.5mg/L NAA + 1.0 mg/L6-BA;

4) inoculating adventitious buds generated by stem induction and adventitious buds generated by leaf callus differentiation to a proliferation culture medium with the formula of WPM +0.1 mg/L NAA + 1.0 mg/L6-BA;

5) cutting single seedling which grows strongly and grows for 5-6cm to carry out rooting culture, wherein the rooting culture medium is 1/2MS, 0.7 mg/LIBA and 0.2 g/L PVP;

6) taking out the tissue culture seedling with root length of 3-4cm from the culture chamber, transferring the culture bottle into a greenhouse, performing intensive light bottle-closing seedling hardening for about 5-20d, preferably 50% -70% of shading degree, opening the bottle mouth, and placing for 3-7 d; transplanting when the root length of the tissue culture seedling reaches 5-6cm and the plant grows well.

2. The tissue culture and rapid propagation method of the Angel red soft seed pomegranate according to claim 1, wherein the WPM minimal medium in the steps 2), 3) and 4) comprises 6.5g/L agar and 30 g/L sucrose, and comprises the following components: NH (NH)₄NO₃400mg/L、Ca(NO₃)2·4H₂O 556mg/L、CaCl₂·2H₂O 96mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、K₂SO₄990mg/L、H₃BO₃6.2mg/L、MnSO₄·H₂O 22.4mg/L、ZnSO₄·7H₂O8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.25mg/L、FeSO₄·7H₂O₂7.8mg/L、Na₂-EDTA37.3mg/L, inositol 100mg/L, glycine 2mg/L, nicotinic acid 0.5mg/L, vitamin B11.0mg/L, vitamin B60.5mg/L.

3. The tissue culture and rapid propagation method of the Angel red soft seed pomegranate of claim 1, wherein the MS minimal medium in the step 5) comprises 6.5g/L agar and 30 g/L sucrose, and the following components: NH (NH)₄NO₃1650mg/L、KNO₃1900mg/L、MgSO₄·7H₂O 370mg/L、KH₂PO₄170mg/L、CaCl₂·2H₂O 440 mg/L、KI 0.83mg/L、H₃BO₃6.2mg/L、MnSO₄·4H2O 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、NaMoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.025mg/L、CoCl₂·6H₂O 0.025mg/L、FeSO₄·7H₂O 27.8mg/L、Na₂EDTA37.25mg/L, inositol 100mg/L, glycine 2mg/L, nicotinic acid 0.5mg/L, vitamin B60.5mg/L, vitamin B10.1mg/L.

4. The tissue culture and rapid propagation method of the Angel red soft seed pomegranate according to claim 1, wherein the pH value of the culture medium in the steps 2), 3), 4) and 5) is adjusted to 5.8.

5. The tissue culture and rapid propagation method of the Angel red soft seed pomegranate according to claim 1, wherein in the step 6), the transplanting matrix is prepared by mixing turf and vermiculite in a ratio of 2:1, fully mixing, and sterilizing at high temperature; the temperature of the transplanting environment is 25-28 ℃, the humidity is 80% -85%, and the illumination time is 12-14 h per day.