A method for rapid propagation of iris avens through tissue culture and forming balls in a bottle

Abstract

The invention relates to a method for rapid propagation of iris tataricus by tissue culture and in-bottle ball formation, and the most suitable culture medium and suitable culture conditions for each stage of iris tataricus are analyzed and compared by exploring the growth of iris tataricus in culture mediums with different concentrations and under different culture conditions. The invention not only provides a reference for iris tataricus tissue culture experiments, but also provides a reference for subsequent mass propagation and popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation and relates to a method for rapid tissue culture and in-bottle ball formation of iris avens. Background Art

[0002] Iris Linnaeus belongs to the Iridaceae family and is also known as yellow bird gum flower, corn lily, and small iris. It is a small bulbous flower. There are more than 50 bulbs in the genus Iris Linnaeus, which are distributed in South Africa. It is a perennial flowering bulbous plant that can bloom again and easily grow bulbs. The flower shape of iris is peculiar and the colors of the flowers are also very rich. There are usually 6-12 flowers in an inflorescence. The inflorescence stands upright, and the round and plump small flowers bloom in clusters from bottom to top. There are dark round spots at the base of the petals, which are very obvious. When in full bloom, it is full of exotic style and can be used as cut flowers.

[0003] However, Iris granatum is not widely used in my country at present, and its application ratio in gardens is relatively low. Moreover, most of Iris granatum in my country are imported from abroad, with a small number of bulbs and a relatively low ramet propagation rate. Therefore, it is hoped that the problem of shortage of Iris granatum bulb resources can be solved through tissue rapid propagation technology, and at the same time, it is also hoped that the best conditions for the growth and reproduction of Iris granatum can be selected through tissue culture experiments. Summary of the invention

[0004] In view of this, the present invention provides a method for rapid propagation of Iris gracilis by tissue culture and in-bottle ball formation. By observing and recording the growth of Iris gracilis in culture media of different concentrations and under different culture conditions, the most suitable culture media and suitable culture conditions for each stage of Iris gracilis are analyzed and compared.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for rapid propagation of iris granatum by tissue culture and ball formation in a bottle, comprising induction, proliferation and rooting of iris granatum; the specific steps are as follows:

[0007] (1) Induction of Iris tataricus:

[0008] The adventitious buds on the treated iris bulbs are cut off, the green leaf parts are removed, and the impurities in the fleshy bulbs are cleaned and inoculated into the induction medium respectively, wherein the induction medium is based on MS and 6-BA and NAA are added;

[0009] (2) Proliferation of Iris tataricus:

[0010] The induced corms were cut and inoculated into proliferation medium to count the proliferation of adventitious buds.

[0011] (3) Rooting of Iris tataricus:

[0012] Adventitious buds of Iris tataricus with emerald green leaves, long stems and the same height were selected and transferred to rooting medium for rooting culture. The rooting rate and growth status were observed, and finally the most suitable rooting medium for Iris tataricus was screened out.

[0013] Furthermore, the steps of processing the iris bulbs are as follows:

[0014] 1) Select the "Superstar" iris variety with good growth, peel the selected iris bulbs on a clean bench, and keep the milky white fleshy bulbs; then drip detergent into it to stir and rinse, then wash it with sterile water, soak it in mercuric chloride, shake it, and finally wash it with sterile water until the explants are completely disinfected;

[0015] 2) In a clean bench, use sterilized tweezers to pick up the explant and place it on a pad of dry sterile filter paper. Use a scalpel to cut off the outer fleshy part of the explant, leaving only the growth point. Unscrew the cap of the culture bottle, tilt the culture medium in your hand, so that the mouth of the culture bottle is burned and sterilized over the flame, and place the tweezers over the flame to burn. Use tweezers to pick up the explant and insert it into the surface of the culture medium, exposing the top of the growth point, and then evenly rotate the bottle mouth over the flame to burn and sterilize it.

[0016] 3) Cover the culture bottles with caps, mark them, and then send the culture bottles containing the explants into light culture and dark culture environments, respectively, at a constant temperature of 25°C for record and observation.

[0017] Furthermore, the concentration of 6-BA in the induction medium is 1.0-4.0 mg / L, the concentration of NAA is 0.01-0.1 mg / L, sucrose and agar are also added to the induction medium, and the pH of the induction medium is 5-6.

[0018] Furthermore, the proliferation medium is based on MS as the basic medium, and 6-BA and NAA are added; wherein the concentration of 6-BA is 4.0 mg / L, and the concentration of NAA is 0.1 mg / L.

[0019] Furthermore, the rooting medium is 1 / 2MS medium added with NAA, and the concentration of NAA in the rooting medium is 0.1-0.7 mg / L.

[0020] It can be seen from the above technical solution that, compared with the prior art, the method for rapid propagation and ball formation in a bottle of Iris tataricus provided by the present invention has the following excellent effects:

[0021] The present invention uses the bulbs of Ixia 'Giant' as the test material, observes and records the growth of Ixia in different concentrations of culture medium and different culture conditions, and analyzes and compares the most suitable culture medium and suitable culture conditions for each stage of Ixia. The test results show that the contamination rate of Ixia in the primary culture period reaches half; the culture medium of MS+4.0mg / L 6-BA+0.1mg / LNAA is conducive to achieving the purpose of proliferation, the number of proliferation plants is 135, and the proliferation multiple is 9; the adventitious buds screened out have good rooting on the 1 / 2MS+0.1mg / LNAA culture medium, and the rooting rate reaches 52.5%; and it is the preferred choice for the bulbing of Ixia under dark culture conditions.

[0022] The invention not only provides a reference for iris tissue culture experiments, but also provides a reference for subsequent mass propagation and promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0024] Figure 1 These are the growth conditions of the first generation axillary buds of Iris tatarica; A and B represent the fungal contamination during the axillary bud formation period of Iris tatarica; C and D represent the normal growth conditions of Iris tatarica without contamination.

[0025] Figure 2 Comparison of adventitious bud proliferation; a 1.0mg / L 6-BA+0.1mg / L NAA; b 2.0mg / L6-BA+0.1mg / LNAA; c 3.0mg / L 6-BA+0.1mg / LNAA; d 4.0mg / L 6-BA+0.1mg / LNAA; e 4.0mg / L 6-BA+0.05mg / LNAA; f4.0mg / L 6-BA+0.01mg / LNAA.

[0026] Figure 3 Comparison of adventitious bud rooting; a 1 / 2MS+0.1mg / L NAA; b 1 / 2MS+0.3mg / LNAA; c 1 / 2MS+0.5mg / LNAA; d 1 / 2MS+0.7mg / LNAA.

[0027] Figure 4 This is a diagram showing the effect of light on sphering in a bottle; A: dark culture; B: light culture. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention and the drawings of the specification to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The embodiment of the invention discloses a method for rapid tissue culture propagation and in-bottle ball formation of Iris tataricus.

[0030] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0031] The technical solution of the present invention will be further described below in conjunction with specific experiments.

[0032] 1. Experimental Materials and Methods

[0033] 1. Test materials

[0034] The iris bulbs used in the experiment of the present invention are provided by Xiangcheng Science and Technology Park. The seed bulbs reserved in 2019 are selected, and the iris bulbs without diseases and insect pests, healthy and plump, smooth and complete surface are selected as explants. Agar, sucrose, MS culture medium mother solution (macroelements, iron salts, organic matter, trace elements), naphthaleneacetic acid (NAA) and 6-BA are used as the test.

[0035] The equipment includes: clean workbench, culture room, scalpel, tweezers, enamel cup, culture bottle, alcohol lamp, etc.

[0036] 2. Test methods

[0037] Select the 'Superstar' iris of the valley variety that grows well, peel the selected iris of the valley bulb on the clean bench, keep the milky white fleshy bulb and place it in an enamel tank. Then add 3 drops of detergent, stir and rinse continuously for 5 minutes, and rinse under the tap for 20-30 minutes. Then wash it three times with sterile water, soak it in mercuric chloride for 10 minutes and shake it, and finally wash it three times with sterile water until the explant is disinfected. In the clean bench, use sterilized tweezers to clamp the explant and place it on a pad of dry sterile filter paper. Use a scalpel to cut off the outer fleshy part of the explant, leaving only the growth point. Then gently unscrew the bottle cap, tilt the culture medium in your hand, so that the mouth of the culture bottle can be burned and sterilized above the flame, and also place the tweezers above the flame to burn, and keep the burned tweezers in the air for more than ten seconds to cool it down to avoid burning or burning the explant to be inoculated. Then use tweezers to clamp the explant and insert it into the surface of the culture medium, exposing the top of the growth point. Then rotate the bottle mouth evenly over the flame of the alcohol lamp to burn and sterilize it. Finally, cover the culture bottle cap and write your culture material, culture medium name, inoculation date and name on the cap. Finally, send it to the light culture and dark culture environment, and record and observe in the culture room with a constant temperature of 25℃.

[0038] 3. Induction method of Iris tataricus

[0039] Use MS as the basic culture medium, add 1.0 mg / L 6-BA and 0.1 mg / L NAA, add 30 g / L sucrose, 5.2 g / L agar, adjust the pH value to about 5.8, and inoculate the treated Iris bulbs into the induction culture medium, one per bottle, for a total of 60 bottles.

[0040] 4. Methods for multiplying irises

[0041] Cut off the adventitious buds on the iris bulbs, remove the green leaves, clean the impurities in the fleshy bulbs, and inoculate them in MS as the basic culture medium, add 6-BA (1.0, 2.0, 3.0, 4.0 mg / L) and NAA (0.01, 0.05, 0.1), and add sucrose and agar. Cut the induced corms and inoculate them in the proliferation culture medium. Each bottle has 3 explants, and 15 bottles are inoculated respectively, and then sent to the culture room. After 30 days, the proliferation of adventitious buds was counted.

[0042] 5. Rooting method of iris

[0043] Select adventitious buds of Iris tataricus with green leaves, long stems, and the same length and height, transfer them to rooting medium for rooting culture, and inoculate the adventitious buds into 1 / 2MS medium with different concentrations of NAA (0.1, 0.3, 0.5, 0.7 mg / L). Inoculate 10 bottles of each variety, and 4 adventitious buds in each bottle. A total of 40 adventitious buds. Observe its rooting rate and growth status after 30 days, and finally select the most suitable rooting medium for Iris tataricus.

[0044] 2. Experimental Results and Analysis

[0045] (1) Effects of plant growth regulators and their dosage on the germination of primary axillary buds

[0046] Cut off the periphery of the sterilized explants, leaving only the middle growth point, and then inoculate them into the primary culture medium of MS+6-BA1.0mg / L+NAA0.1mg / L+sucrose 30g / L+agar 5.2g / L. Inoculate 60 bottles in total, with one axillary bud in each bottle. The results were observed after 14 days and found that ( Figure 1 ), mold appeared in the culture medium, and within a certain period of time, the number of fungal contaminations was nearly half; the iris bulbs without fungi began to grow slowly, and 3-4 small buds grew on one bulb.

[0047] It should be noted that, in the observation process after 14d, the first generation axillary buds found that mold appeared in the culture medium, and as time went on, the diffusion area of ​​the mold was wider. So always observe and find in time to take it out, so as not to infect other culture media. The contamination rate of the general first generation is very high, and the number of contaminations in the experiment of the present invention is nearly half. Therefore, the inoculation operation process in the tissue culture experiment should be more rigorous, the sterilization of the explant should also be carefully serious, and the selection of the explant should be excellent and easy to sterilize, so as to reduce the contamination rate of the explant.

[0048] (2) Effects of plant growth regulators and their dosage on adventitious bud proliferation

[0049] After the adventitious buds were induced in vitro by primary culture, they were inoculated onto culture media with different concentrations of 6-BA (1.0, 2.0, 3.0, 4.0 mg / L) and NAA (0.1, 0.05, 0.01 mg / L), and observed 30 days later.

[0050] Table 1 Effects of plant growth regulators and their dosage on adventitious bud proliferation (30-day observation)

[0051]

[0052] When NAA0.1mg / L is a single factor, 6-BA is (1.0, 2.0, 3.0, 4.0mg / L) multi-factor comparison: From Table 1 and combined Figure 2 It can be seen that the number of plants proliferated in the medium of 6-BA4.0mg / L+NAA0.1mg / L is 105, the multiplication factor is 7, the growth state is excellent, and the growth is tall and good; when 6-BA4.0mg / L is a single factor unchanged, the NAA gradient is (0.1, 0.05, 0.01mg / L) comparison: From the above table, it can be seen that the number of plants proliferated in the medium of 4.0mg / L 6-BA+0.01mg / L NAA is 135, and the multiplication factor is 9. It is better than 0.1mg / LNAA, the growth of plants is generally excellent and uniform, the root system is large and developed, and the germination rate is high.

[0053] It should be noted that in the process of plant tissue culture, culture medium is an important matrix for plant tissue culture. Therefore, different plant tissue regulators and concentrations will have a great impact on the growth of in vitro tissues. In the experiment of the present invention, with MS as culture medium, NAA hormone concentration unchanged, 6-BA as a variable, the proliferation of MS+4.0mg / L6-BA+0.1mg / LNAA is the best; with 6-BA hormone concentration unchanged, NAA as a variable, in the proliferation culture medium of MS+4.0mg / L 6-BA+0.01mg / LNAA, the number of proliferation strains and multiples are the highest in the experiment, and the growth is also the best, with a strong and developed root system. Therefore, selecting MS+4.0mg / L 6-BA+0.1mg / LNAA culture medium is more conducive to achieving the purpose of proliferation.

[0054] (3) Effects of adding different concentrations of NAA to the culture medium on rooting of adventitious buds

[0055] When the iris of the valley proliferates to a certain number, separate the seedlings in clusters, and inoculate the adventitious buds into 1 / 2MS culture medium with different concentrations of NAA (0.1, 0.3, 0.5, 0.7 mg / L) in the rooting medium. Inoculate 10 bottles for each formula, with 4 clusters of adventitious buds in each bottle, for a total of 40 plants. Then wait 30 days and observe their growth status.

[0056] Table (2) Effects of different concentrations of NAA on rooting of adventitious buds

[0057]

[0058] From Table 2 and combined Figure 3It can be seen that although the rooting rate of the adventitious buds of Iris tatarinowii is not very high, it can be seen from the comparison of four different hormones that when the concentration of NAA in the culture medium is 0.7 mg / L, the number of roots and the number of rooted plants are both 0, with no signs of rooting. The rooting status of the adventitious buds in the other three culture media is roughly the same, and the roots are relatively thick and off-white. The root length of the adventitious buds in the culture medium with an NAA concentration of 0.5 mg / L is ≤0.3 cm; the root length in the culture medium with an NAA concentration of 0.3 mg / L is ≤0.8 cm; the root length in the culture medium with an NAA concentration of 0.1 mg / L is ≤1 cm; and the number of rooted plants, the number of roots, and the rooting rate in the culture medium of 1 / 2MS+0.1 mg / LNAA are the highest.

[0059] It should also be noted that after 30 days of cultivation, it was found that the rooting rate decreased with the increase of NAA concentration. No rooting occurred in the medium with NAA concentration of 0.7 mg / L. The medium with NAA concentration of 0.1 mg / L had the highest rooting rate in this group of experiments, but it was only 52.5%. Therefore, it can be found from the experiment that rooting in tissue culture is difficult, and the rooting effect of Iris tataricus is not so ideal.

[0060] (4) Effects of dark and light conditions on the growth of Iris tataricus

[0061] The Iris truncatulae in different concentrations of culture medium were divided into two batches for comparative experiments. One batch was cultured under light illumination, and the other batch was cultured under dark conditions. Each batch had 30 bottles, with 1 plant in each bottle, for a total of 30 plants. The contamination rate and survival rate were calculated during the culture process, and their growth was observed during this period.

[0062] Table 3 Effects of dark and light conditions on the growth of Iris tataricus (30-day observation)

[0063]

[0064] From Table 3 and combined Figure 4 It can be seen that under constant temperature conditions, the survival rate and contamination rate of light culture and dark culture are not much different. The iris of the valley under light culture conditions grows faster, grows taller, and has green leaves; while the iris of the valley under dark culture conditions grows slowly, generally has no green leaves, has a large fleshy ball, a well-developed root system, and a milky white ball.

[0065] It should also be noted that in a constant temperature culture room, when the number of light culture and dark culture, culture medium concentration, and culture time are the same, the survival rate and contamination rate of the two are similar when the culture conditions under light culture and dark culture are compared. However, the balls of Iris truncatula in dark culture are larger and fuller, so the balls in dark culture are better than those in light culture.

[0066] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapid propagation of Iris tataricus and balling in a bottle, characterized in that: The following steps are involved: (1) Select healthy, plump, smooth and intact iris bulbs that are free of pests and diseases as explants; (2) Use a scalpel to remove the outer fleshy part of the explant, leaving only the growth point; (3) Induction of Iris tataricus: inoculate the treated Iris tataricus bulbs into the induction medium; The induction medium uses MS as the basic medium, adds 1.0 mg / L 6-BA and 0.1 mg / L NAA, 30 g / L sucrose, 5.2 g / L agar, and adjusts the pH value to 5.8; (4) Proliferation of Iris tataricus: Cut off the adventitious buds on the iris bulbs, remove the green leaves, clean the impurities in the fleshy bulbs, and inoculate them into the proliferation medium. Cut the induced corms and inoculate them into the proliferation medium. After 30 days, count the proliferation of the adventitious buds. The proliferation medium is based on MS as the basic medium, and is supplemented with 6-BA and NAA, and is also supplemented with sucrose and agar, wherein the concentration of 6-BA is 4.0 mg / L, and the concentration of NAA is 0.01 mg / L or 0.1 mg / L; (5) Rooting of Iris tataricus: Select adventitious buds of Iris tataricus with emerald green leaves, long stems, and the same length and height, transfer them into rooting medium for rooting culture, and inoculate the adventitious buds into 1 / 2MS medium supplemented with 0.1 mg / L NAA.

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