Rapid propagation cultivation method of paphiopedilum maudiae

Abstract

The invention discloses a rapid propagation cultivation method of paphiopedilum magic emperor. The rapid propagation cultivation method comprises the steps of inducing leaf cluster buds, proliferating the leaf cluster buds, recovering the leaf cluster buds into normal adventitious buds, performing rooting and strong seedling culture on the adventitious buds and transplanting tissue culture seedlings. Compared with existing adventitious bud proliferation, the method adopting a leaf cluster bud proliferation mode is more efficient and stable, and rapid propagation cultivation of paphiopedilum maudiae is achieved; compared with adventitious bud proliferation, leaf clump bud proliferation is simpler and more convenient to operate, growing points of each adventitious bud need to be longitudinally cut in each time of adventitious bud proliferation culture, the leaf clump buds are longitudinally cut once before leaf clump bud proliferation inoculation, and each adventitious bud does not need to be longitudinally cut after each leaf clump bud is recovered into a normal adventitious bud; besides, before the tissue culture seedlings are transplanted, the tissue culture seedlings are subjected to seedling hardening, so that the leaf tip necrosis rate of the tissue culture seedlings can be reduced, and the transplanting effect of the tissue culture seedlings is improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation and cultivation method of Paphiopedilum mori. Background Art

[0002] Paphiopedilum, also known as slipper orchid, fairy slipper orchid, etc., is a general term for plants of the genus Paphiopedilum in the subfamily Cypripediodeae of the Orchidaceae family. Paphiopedilum plants have lip-shaped petals and a variety of colors, which are very strange and stunning. They are high-end products among flowers. Among them, Paphiopedilum Maudiae is a hybrid of Paphiopedilum Lawrenceanum and Paphiopedilum callosum.

[0003] For the cultivation of Paphiopedilum spp., the existing asexual propagation by tissue culture is an effective method to obtain Paphiopedilum spp. seedlings with stable and consistent traits; further, tissue culture of Paphiopedilum is carried out by adopting methods such as adventitious bud proliferation. However, the efficiency of the above-mentioned adventitious bud proliferation is low, which affects the propagation efficiency of Paphiopedilum spp. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the shortcomings of the prior art, the present invention provides a rapid propagation and cultivation method of Paphiopedilum demonii, which can solve the above technical problems.

[0006] (II) Technical solution

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for rapid propagation and cultivation of Paphiopedilum mori, comprising the following steps:

[0008] S1. Induction of leaf cluster buds: longitudinally cut the growth points of the adventitious buds of Paphiopedilum mori, inoculate them into leaf cluster bud induction medium for cultivation, and make the adventitious buds differentiate into leaf cluster buds;

[0009] The leaf cluster bud induction medium contains 5-20 g sucrose, 0.25-5 mg plant growth regulator, 50-100 mL coconut juice, 3-4.5 g agar per liter, and the rest is 1 / 4-1 / 2 MS medium;

[0010] S2. Proliferation of leaf buds: longitudinally cutting the growth point of the leaf buds, inoculating into leaf bud proliferation medium for cultivation, so that the leaf buds proliferate;

[0011] S3, the leaf cluster buds are restored to normal adventitious buds: the leaf cluster buds are inoculated into a leaf cluster bud restoration medium for cultivation, so that the leaf cluster buds are restored to adventitious buds;

[0012] The leaf cluster bud recovery medium contains 15-20 g sucrose, 50-80 mL coconut juice, 3-3.5 g peptone, 3-4.5 g agar per liter, and the rest is 1 / 2 MS medium;

[0013] S4, rooting and seedling cultivation of adventitious buds: inoculating the adventitious buds onto a rooting and seedling cultivation medium for cultivation to obtain tissue culture seedlings;

[0014] The rooting and seedling-strengthening culture medium is: 50-150 mL of coconut juice, 10-20 g of sucrose, 0.5 mg of NAA, 0.3 g of activated carbon, 5 g of agar, and the rest is 1 / 2 MS culture medium per liter;

[0015] S5. Transplantation of tissue culture seedlings: first harden the tissue culture seedlings, and then move the tissue culture seedlings into the cultivation medium for cultivation to obtain Paphiopedilum demonii seedlings.

[0016] Preferably, the plant growth regulator is TDZ or BA.

[0017] Preferably, the leaf cluster bud proliferation culture medium contains 20 g sucrose, 0.75 mg TDZ per liter, and the rest is 1 / 2 MS culture medium.

[0018] Preferably, in step S3, the leaf cluster buds are inoculated into a leaf cluster bud recovery medium and cultured multiple times.

[0019] Preferably, in step S5, tissue culture seedlings with different leaf widths are respectively moved into different cultivation substrates for cultivation.

[0020] Preferably, the tissue culture seedlings with leaf width greater than 1.39 cm are transplanted into the first cultivation matrix; wherein the first cultivation matrix is ​​a mixed matrix of planting stone and peat soil in a volume ratio of 1:1, or the first cultivation matrix is ​​a mixed matrix of pine bark and peat soil in a volume ratio of 1:1.

[0021] Preferably, the tissue culture seedlings with a leaf width less than or equal to 1.39 cm are transplanted into the second cultivation medium; wherein the second cultivation medium is a mixed medium of planting stone and coconut bran in a volume ratio of 1:1.

[0022] (III) Beneficial effects

[0023] Compared with the prior art, the present invention provides a rapid propagation and cultivation method of Paphiopedilum sphaerocephala, which has the following beneficial effects: (1) The leaf cluster bud proliferation method adopted by the present invention is more efficient and stable than the adventitious bud proliferation method. According to experiments, the efficiency of leaf cluster bud proliferation is 5.05 times that of adventitious bud proliferation, thereby realizing the rapid propagation and cultivation of Paphiopedilum sphaerocephala; (2) The leaf cluster bud proliferation method of the present invention is simpler to operate than the adventitious bud proliferation method. Each time the adventitious bud proliferation is cultivated, each adventitious bud needs to be longitudinally cut at the growth point, while the leaf cluster bud proliferation method only needs to be longitudinally cut before inoculation, and each adventitious bud does not need to be longitudinally cut after it is restored to a normal adventitious bud; (3) In addition, before transplanting the tissue culture seedlings, the present invention first hardens the tissue culture seedlings, which can reduce the leaf tip necrosis rate of the tissue culture seedlings and improve the transplanting effect of the tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a flowchart of the steps of a rapid propagation and cultivation method of Paphiopedilum mori. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention 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.

[0026] The invention provides a rapid propagation and cultivation method of Paphiopedilum spatholobi. The Paphiopedilum spatholobi in the following embodiment is a seedling developed from a seed culture of Paphiopedilum spatholobi 170 days after pollination in a greenhouse of the South China Botanical Garden of the Chinese Academy of Sciences, and then an asexual propagation line (P.SCBG ZK68) is obtained through conventional adventitious bud proliferation for 3 years.

[0027] In addition, the composition of MS medium is shown in Table 1 below:

[0028] Table 1 MS medium components

[0029]

[0030] The 1 / 4 and 1 / 2 MS culture media referred to in the present invention refer to culture media prepared by reducing the amounts of macroelements in the MS culture media to 1 / 4 and 1 / 2 of the original amounts respectively (while the concentrations of other components remain unchanged).

[0031] Embodiment 1 of the rapid propagation and cultivation method of Paphiopedilum mori of the present invention:

[0032] S1. Induction of leaf cluster buds: longitudinally cut the growth point of the adventitious buds of Paphiopedilum mori, inoculate them into a leaf cluster bud induction medium for culture at a temperature of 25° C., so that the adventitious buds differentiate into leaf cluster buds. The lateral buds of Paphiopedilum mori are cut as explants, sterilized, and inoculated into an adventitious bud induction medium to obtain adventitious buds, which are further used for induction of leaf cluster buds. Specifically, the adventitious bud induction medium of the prior art can be used.

[0033] Many leaf primordia grow on the stem tip of the adventitious bud, which differentiate into leaves, producing too many leaves, which appear as morphologically abnormal adventitious buds. This type of adventitious bud has no apical dominance, and this abnormal adventitious bud is called a leaf cluster bud.

[0034] The leaf cluster bud induction medium contains 5 g sucrose, 0.25 mg TDZ (thidiazuron), 50 mL coconut juice, 3 g agar, and the rest is 1 / 4 MS medium per liter.

[0035] S2. Proliferation of leaf buds: Cut off the leaf buds from step S1, cut the growth points of the leaf buds longitudinally, and inoculate them into leaf bud proliferation culture medium for cultivation at a temperature of 25° C. to allow the leaf buds to proliferate.

[0036] Preferably, in step S2, leaf cluster buds with different bud lengths are inoculated into different leaf cluster bud proliferation culture media for culture, so as to better improve the proliferation coefficient of leaf cluster buds with different bud lengths: leaf cluster buds (small buds) with a bud length of 0.8-2.4 cm are inoculated into the first leaf cluster bud proliferation culture medium, and the first leaf cluster bud proliferation culture medium contains 15 g sucrose and 10 mg BA (benzylaminopurine) per liter, and the rest is 1 / 2MS culture medium; and leaf cluster buds (large buds) with a bud length of 2.5-5 cm are inoculated into the second leaf cluster bud proliferation culture medium, and the second leaf cluster bud proliferation culture medium contains 20 g sucrose and 0.75 mg TDZ per liter, and the rest is 1 / 2MS culture medium.

[0037] S3. Leaf cluster buds are restored to normal adventitious buds: the leaf cluster buds proliferated in step S2 are cut, and the leaf cluster buds are further inoculated into leaf cluster bud recovery medium for two cultures at a culture temperature of 25° C., so that the leaf cluster buds are restored to adventitious buds.

[0038] The leaf bud recovery medium is: 15 g sucrose, 50 mL coconut juice, 3 g peptone, 3.5 g agar per liter, and the rest is 1 / 2 MS medium. In this embodiment 1, the leaf buds are induced by the leaf bud induction medium containing TDZ. In the first culture of the leaf buds in step S3, the leaf buds are not transformed into normal adventitious buds, and normal adventitious buds appear after the second culture.

[0039] S4. Rooting and seedling cultivation of adventitious buds: the adventitious buds are separated from the clumped adventitious buds in step S3 and inoculated on a rooting and seedling cultivation medium for cultivation to obtain tissue culture seedlings at a cultivation temperature of 25°C.

[0040] Among them, the rooting and seedling strengthening culture medium is: each liter contains 100mL of coconut juice, 10g of sucrose, 0.5mg of NAA (naphthaleneacetic acid), 0.3g of activated carbon, 5g of agar, and the rest is 1 / 2MS culture medium.

[0041] S5. Transplantation of tissue culture seedlings: first harden the tissue culture seedlings; then move the tissue culture seedlings into a cultivation medium for cultivation to obtain Paphiopedilum demonii seedlings.

[0042] The seedling hardening method is: transfer the tissue culture seedlings from the tissue culture room to the greenhouse, place each tissue culture bottle on a shelf, and leave it without opening the bottle cap for 2 weeks to allow the tissue culture seedlings in the bottle to adapt to the temperature and light of the greenhouse in advance.

[0043] Preferably, in step S5, tissue culture seedlings with different leaf widths (the widest leaf width of each tissue culture seedling) are respectively transplanted into different cultivation substrates for cultivation, so as to correspondingly improve the growth rate and survival rate of different tissue culture seedlings after transplanting: tissue culture seedlings with leaf width greater than 1.39 cm are transplanted into the first cultivation substrate, which is a mixed substrate of planting stone and peat soil in a volume ratio of 1:1; tissue culture seedlings with leaf width less than or equal to 1.39 cm are transplanted into the second cultivation substrate; wherein the second cultivation substrate is a mixed substrate of planting stone and coconut bran in a volume ratio of 1:1.

[0044] Embodiment 2 of the rapid propagation and cultivation method of Paphiopedilum mori of the present invention:

[0045] S1. Induction of leaf cluster buds: The growth points of the adventitious buds of Paphiopedilum mori were cut longitudinally and inoculated into leaf cluster bud induction medium for cultivation at a temperature of 25°C to allow the adventitious buds to differentiate into leaf cluster buds.

[0046] The leaf cluster bud induction medium contains 15 g sucrose, 5 mg BA, 100 mL coconut juice, 4 g agar per liter, and the rest is 1 / 2 MS medium.

[0047] S2. Leaf bud proliferation: Cut the leaf buds from step S1, cut the growth points of the leaf buds longitudinally, and inoculate them into leaf bud proliferation medium for cultivation at a temperature of 25° C. to proliferate the leaf buds. The leaf bud proliferation medium is the same as that in the first embodiment, and will not be described in detail here.

[0048] S3. The leaf cluster buds are restored to normal adventitious buds: the leaf cluster buds proliferated in the above step S2 are cut, and the leaf cluster buds are further inoculated into a leaf cluster bud recovery medium for a culture at a temperature of 25° C., so that the leaf cluster buds are restored to adventitious buds.

[0049] The leaf bud recovery medium contains 20 g sucrose, 80 mL coconut juice, 3.5 g peptone, 4.5 g agar per liter, and the rest is 1 / 2 MS medium. In the second embodiment, the leaf buds are induced by the leaf bud induction medium containing BA, and the leaf buds are transformed into normal adventitious buds in the first culture of step S3.

[0050] S4. Rooting and seedling cultivation of adventitious buds: the adventitious buds are separated from the clumped adventitious buds in step S3 and inoculated on a rooting and seedling cultivation medium for cultivation to obtain tissue culture seedlings at a cultivation temperature of 25°C.

[0051] Among them, the rooting and seedling strengthening culture medium is: each liter contains 120mL of coconut juice, 20g of sucrose, 0.5mg of NAA, 0.3g of activated carbon, 5g of agar, and the rest is 1 / 2MS culture medium.

[0052] S5. Transplantation of tissue culture seedlings: first harden the tissue culture seedlings; then move the tissue culture seedlings into a cultivation medium for cultivation to obtain Paphiopedilum demonii seedlings.

[0053] Preferably, in step S5, tissue culture seedlings with different leaf widths are respectively moved into different cultivation substrates for cultivation, so as to correspondingly improve the growth speed and survival rate of different tissue culture seedlings after transplantation: tissue culture seedlings with leaf width greater than 1.39 cm are moved into a first cultivation substrate, which is a mixed substrate of pine bark and peat soil in a volume ratio of 1:1; tissue culture seedlings with leaf width less than or equal to 1.39 cm are moved into a second cultivation substrate; wherein the second cultivation substrate is a mixed substrate of planting stone and coconut bran in a volume ratio of 1:1.

[0054] Compared with the prior art, the present invention provides a rapid propagation and cultivation method of Paphiopedilum mori, which has the following beneficial effects: (1) The leaf cluster bud proliferation method adopted by the present invention is more efficient and stable than the adventitious bud proliferation method. The proliferation coefficient of the adventitious bud proliferation method is affected by the length of the adventitious bud. Generally, the longer the adventitious bud is, the more nodes and dormant buds there are on its stem, and more adventitious buds may grow. The stem nodes of the leaf cluster bud tend to grow thicker and form a composite stem with a large number of leaf primordia and leaves growing on the stem. The thicker the composite stem of the leaf cluster bud is, the more leaf primordia and leaves it contains. The axillary buds at the base of the leaf primordia and leaves grow normal adventitious buds under the culture conditions without adding TDZ or BA. Therefore, the proliferation efficiency of the leaf cluster bud is mainly affected by the thickness of its composite stem. The effect is not related to its length. According to experiments, it is difficult to obtain 10.64 adventitious buds by culturing one adventitious bud for six months, while the leaf cluster bud proliferation culture can restore 53.72 normal adventitious buds after six months of cultivation. Therefore, the efficiency of leaf cluster bud proliferation is 5.05 (53.72 / 10.64) times that of adventitious bud proliferation, realizing the rapid propagation and cultivation of Paphiopedilum morifolium. (2) The leaf cluster bud proliferation of the present invention is simpler than the adventitious bud proliferation. Each time the adventitious bud proliferation is cultivated, each adventitious bud needs to be longitudinally cut at the growth point, while the leaf cluster bud proliferation needs to be longitudinally cut once before inoculation. After each leaf cluster bud is restored to a normal adventitious bud, it is not necessary to longitudinally cut each adventitious bud. (3) In addition, before transplanting the tissue culture seedlings, the present invention first The tissue culture seedlings are hardened. After hardening, the Paphiopedilum orchid has gradually adapted to the greenhouse environment. The stomata of the leaves are more autonomous in closing under high temperature and low humidity conditions than those of unhardened seedlings, thereby avoiding the metabolic imbalance of water and causing the leaf tips to wilt and necrosis. The leaf tip necrosis rate of the tissue culture seedlings can be reduced. According to experiments, the leaf tip necrosis rate of the unhardened seedlings was 83.72%, and the leaf tip necrosis rate of the hardened seedlings was 19.44%, thereby improving the transplanting effect of the tissue culture seedlings. (4) The present invention inoculates leaf cluster buds of different bud lengths into different leaf cluster bud proliferation culture media for cultivation, thereby better improving the proliferation coefficients of leaf cluster buds of different bud lengths. (5) The present invention finds that the most suitable cultivation substrates required for tissue culture seedlings of different leaf widths are different, so different The tissue culture seedlings with the same leaf width are respectively moved into different cultivation substrates for cultivation, so as to correspondingly improve the growth speed and survival rate of different tissue culture seedlings after transplantation: the growth of tissue culture seedlings with larger leaf width is promoted by the peat soil in the cultivation substrate, and the tissue culture seedlings are relatively easy to absorb inorganic salts in the peat soil, but the aeration of the peat soil is worse than that of the plant stone and pine bark, and the root system of the tissue culture seedlings with larger leaf width is relatively developed and can extend to the gap between the substrate and the plastic pot, thereby avoiding the disadvantage of poor aeration in the first cultivation substrate and making full use of its nutrient-rich advantage, so that the first cultivation substrate makes the tissue culture seedlings with larger leaf width have a higher survival rate and a faster growth rate; and coconut bran is added to the second cultivation substrate to improve the aeration of the substrate, so as to ensure the growth speed and survival rate of the tissue culture seedlings with smaller leaf width.

[0055] It should be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0056] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid propagation of Paphiopedilum mori, characterized in that: The following steps are involved: S1. Induction of leaf cluster buds: longitudinally cut the growth points of the adventitious buds of Paphiopedilum mori, inoculate them into leaf cluster bud induction medium for cultivation, and make the adventitious buds differentiate into leaf cluster buds; The leaf cluster bud induction medium contains 5-20 g sucrose, 0.25-5 mg plant growth regulator, 50-100 mL coconut juice, 3-4.5 g agar per liter, and the rest is 1 / 4-1 / 2 MS medium; S2. Proliferation of leaf buds: longitudinally cutting the growth point of the leaf buds, inoculating into leaf bud proliferation medium for cultivation, so that the leaf buds proliferate; S3, the leaf cluster buds are restored to normal adventitious buds: the leaf cluster buds are inoculated into a leaf cluster bud restoration medium for cultivation, so that the leaf cluster buds are restored to adventitious buds; The leaf cluster bud recovery medium contains 15-20 g sucrose, 50-80 mL coconut juice, 3-3.5 g peptone, 3-4.5 g agar per liter, and the rest is 1 / 2 MS medium; S4, rooting and seedling cultivation of adventitious buds: inoculating the adventitious buds onto a rooting and seedling cultivation medium for cultivation to obtain tissue culture seedlings; The rooting and seedling strengthening medium is: 50-150 mL of coconut juice, 10-20 g of sucrose, 0.5 mg of NAA, 0.3 g of activated carbon, 5 g of agar, and the rest is 1 / 2 MS medium per liter; S5. Transplantation of tissue culture seedlings: first harden the tissue culture seedlings, and then move the tissue culture seedlings into the cultivation medium for cultivation to obtain Paphiopedilum demonii seedlings.

2. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 1, characterized in that: The plant growth regulator is TDZ or BA.

3. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 2, characterized in that: The leaf cluster bud proliferation culture medium contains 20g of sucrose and 0.75mg of TDZ per liter, and the rest is 1 / 2MS culture medium.

4. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 1, characterized in that: In step S3, the leaf cluster buds are inoculated into a leaf cluster bud recovery medium and cultured multiple times.

5. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 1, characterized in that: In step S5, the tissue culture seedlings with different leaf widths are respectively moved into different cultivation substrates for cultivation.

6. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 5, characterized in that: The tissue culture seedlings with leaf width greater than 1.39 cm are moved into the first cultivation medium; wherein the first cultivation medium is a mixed medium of planting stone and peat soil in a volume ratio of 1:1, or the first cultivation medium is a mixed medium of pine bark and peat soil in a volume ratio of 1:

1.

7. The rapid propagation and cultivation method of Paphiopedilum mori according to claim 5, characterized in that: The tissue culture seedlings with leaf width less than or equal to 1.39 cm are moved into the second cultivation medium; wherein the second cultivation medium is a mixed medium of planting stone and coconut bran in a volume ratio of 1:1.

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