A cultivation method of Alsophila spinulosa

Abstract

The present invention relates to the field of plant tissue culture technology, and in particular to a method for cultivating Alsophila spinulosa. The method comprises the following steps: inoculating a disinfected spore suspension into a culture medium for cultivation; transferring germinated spore prothallus to an induction GGB culture medium for cultivation; cutting the GGB into small pieces and transferring them to a proliferation culture medium for cultivation; inducing sporophytes and rooting using an incomplete tissue culture method to obtain rooted seedlings; transferring the rooted seedlings to an outdoor greenhouse for cultivation to obtain tissue culture seedlings, and transplanting the tissue culture seedlings onto a mixed substrate to obtain Alsophila spinulosa seedlings. The method provided by the present invention has the characteristics of a high spore germination induction rate, a large GGB proliferation multiple, a high sporophyte induction rate, a high rooting rate, a well-developed root system, sturdy rooted seedlings, a high transplant survival rate, robust seedling growth, and low seedling cultivation costs, thereby providing an efficient and stable tissue culture and rapid propagation technology system for Alsophila spinulosa using the GGB pathway.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a cultivation method of Alsophila spinulosa. Background Art

[0002] The white tree also known as Sphaeropteris brunoniana belongs to the genus Sphaeropteris in the family Cyatheaceae. It is a tree-like fern with a tall, straight stem and deeply three-pinnate leaves. Its petioles are straw-colored and often covered with white powder. Each lobe contains 7-9 pairs of sori, located between the leaf margin and the main vein, without a sori cap. A Class II protected plant in my country, it is primarily found in Hainan, Yunnan, and Tibet, in China, at the margins of evergreen broad-leaved forests and in valleys at altitudes of 500-1150 meters. Its status as a relict species, its unique leaf shape, and its graceful appearance make it of great value for ornamental, scientific research, and conservation.

[0003] As a fern, Alsophila spinulosa's reproductive system differs significantly from other terrestrial plants in that it has independent gametophyte and sporophyte generations. The gametophyte generation, also known as the haploid generation, develops from spores into early gametophytes, which then grow into mature prothallus with antheridia and archegonium, where sperm and egg cells undergo fertilization. From the fertilized egg, the sporophyte generation (diploid generation) begins, where the young sporophyte gradually develops through mitosis into a mature sporophyte with sporangia. These two generations alternate, forming a cycle. Fern gametophytes are tiny, and sperm are single-celled, unprotected, swimming individuals that must swim into the archegonium in natural water. Therefore, their reproductive process is highly susceptible to environmental influences. It takes approximately one year for spores to germinate and for the next generation of sporophytes to mature, significantly impacting their distribution and population renewal. At present, the tissue culture of Alsophila albus is still at the stage of spore germination and gametophyte development. Therefore, in order to protect and utilize this precious protected species, a complete tissue culture regeneration system should be established to achieve rapid propagation of Alsophila albus seedlings. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for cultivating Alsophila spinulosa, which solves the problem of cultivating Alsophila spinulosa seedlings.

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

[0006] The present invention provides a method for cultivating Alsophila spinulosa, comprising the following steps:

[0007] 1) sterilizing spores of Alsophila spinulosa and inoculating them on a germination medium for germination culture to obtain spore prothallus;

[0008] The germination medium comprises: 1 / 10 MS medium + 11 g / L carrageenan, pH value is 5.8;

[0009] 2) inoculating the sporophyte obtained in step 1) into a green spheroid culture medium to culture green spheroids, thereby obtaining green spheroids;

[0010] The green spheroid culture medium includes: 1 / 2MS culture medium + 0.2mg / L naphthaleneacetic acid, pH value is 5.8;

[0011] 3) inoculating the green spheroids obtained in step 2) into a proliferation culture medium for proliferation culture to obtain proliferated green spheroids;

[0012] The proliferation culture medium comprises: 1 / 2MS culture medium + 0.1 mg / L naphthaleneacetic acid + 0.1 mg / L indolebutyric acid, with a pH value of 5.8;

[0013] 4) treating the proliferated green spheroids obtained in step 3) using an incomplete tissue method and then inoculating the spheroids into a matrix for rooting culture to obtain Alsophila spinulosa seedlings;

[0014] The matrix includes peat and perlite, and the volume ratio of the peat to the perlite is 1:1.

[0015] Preferably, the disinfection method in step 1) comprises: soaking in a 75% by volume ethanol solution for 20 seconds, washing three times with sterile water, then soaking in a 4% to 5% by mass NaClO solution for 4 minutes, and washing four times with sterile water.

[0016] Preferably, the conditions for germination culture in step 1) include: first culturing in the dark for 24 hours, and then culturing in the light for 10 to 20 days;

[0017] The conditions of the light culture include: light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0018] Preferably, the conditions for the GGB culture in step 2) include: a culture time of 60 days, a light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0019] Preferably, the conditions of the proliferation culture in step 3) include: a time of 30 days, a light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0020] Preferably, the diameter of the GGB in step 3) is 0.7 cm.

[0021] Preferably, the step 4) incomplete tissue method comprises: mincing the proliferated GGB into pieces with a diameter of 0.3 to 0.5 cm, and mixing with 1 / 2 MS liquid culture medium at a volume ratio of 1:20;

[0022] The diameter of the proliferating GGB is 0.3 to 0.5 cm.

[0023] Preferably, the rooting culture conditions in step 4) include: a temperature of 25-28°C, a time of 30-60 days, and a light intensity of 15-20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0024] Preferably, after the rooting culture, the rooted seedlings obtained are hardened and then inoculated into a mixed medium for culture to obtain Alsophila spinulosa seedlings.

[0025] Preferably, the mixed matrix comprises peat and perlite, and the volume ratio of the peat to perlite is 2:1;

[0026] The culture time is 20 days.

[0027] The beneficial effects of the present invention are:

[0028] The present invention is a rapid propagation technology using spores of Alsophila spinulosa as explants, and has the characteristics of high spore germination induction rate, large GGB proliferation multiples, high sporophyte induction rate, high rooting rate, well-developed root system, strong rooted seedlings, high transplant survival rate, and robust seedling growth. It provides a set of efficient and stable GGB tissue culture rapid propagation technology system for Alsophila spinulosa. The present invention is applicable to the factory production of Alsophila spinulosa seedlings, realizes the rapid propagation of Alsophila spinulosa seedlings, protects the wild germplasm resources of this tree species, alleviates the problems of endangered and scarce germplasm resources of this tree species, and lays a preliminary foundation for future breeding work and provides material guarantee. DETAILED DESCRIPTION

[0029] The present invention provides a method for cultivating Alsophila spinulosa, comprising the following steps:

[0030] 1) sterilizing spores of Alsophila spinulosa and inoculating them on a germination medium for germination culture to obtain spore prothallus;

[0031] The germination medium comprises: 1 / 10 MS medium + 11 g / L carrageenan, pH value is 5.8;

[0032] 2) inoculating the sporophyte obtained in step 1) into a green spheroid culture medium to culture green spheroids, thereby obtaining green spheroids;

[0033] The green spheroid culture medium includes: 1 / 2MS culture medium + 0.2mg / L naphthaleneacetic acid, pH value is 5.8;

[0034] 3) inoculating the green spheroids obtained in step 2) into a proliferation culture medium for proliferation culture to obtain proliferated green spheroids;

[0035] The proliferation culture medium comprises: 1 / 2MS culture medium + 0.1 mg / L naphthaleneacetic acid + 0.1 mg / L indolebutyric acid, with a pH value of 5.8;

[0036] 4) treating the proliferated green spheroids obtained in step 3) using an incomplete tissue method and then inoculating the spheroids into a matrix for rooting culture to obtain Alsophila spinulosa seedlings;

[0037] The matrix includes peat and perlite, and the volume ratio of the peat to the perlite is 1:1.

[0038] The present invention sterilizes the spores of Alsophila spinulosa and inoculates them on a germination medium for germination culture to obtain spore prothallus; the germination medium includes: 1 / 10MS medium + 11g / L carrageenan, and the pH value is 5.8. In the present invention, the disinfection method preferably includes: soaking in a 75% ethanol solution by volume for 20s, washing with sterile water 3 times, then adding a 4% to 5% NaClO solution by mass to soak for 4min, and washing with sterile water 4 times. In the present invention, the conditions for the germination culture preferably include: dark culture for 24h, and then light culture for 10 to 20d; the conditions for light culture include: light intensity of 15 to 20μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0039] The present invention inoculates the obtained protothallus into a green spheroid culture medium for green spheroid culture to obtain green spheroids (abbreviated as GGB); the green spheroid culture medium comprises: 1 / 2 MS medium + 0.2 mg / L naphthaleneacetic acid, pH value is 5.8. In the present invention, the conditions for the green spheroid culture preferably include: a time of 60 days, a light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0040] The green spheroids obtained are inoculated into a proliferation culture medium for proliferation culture to obtain proliferating green spheroids; the proliferation culture medium comprises: 1 / 2 MS medium + 0.1 mg / L naphthaleneacetic acid + 0.1 mg / L indolebutyric acid, with a pH of 5.8. In the present invention, the proliferation culture conditions preferably include: a duration of 30 days, a light intensity of 15 to 20 μmol / (m2·s), and a photoperiod of 12 hours / day. In the present invention, the diameter of the green spheroids is preferably 0.7 cm.

[0041] The present invention uses an incomplete tissue method to treat the obtained proliferating green spheroids, and then inoculates them into a substrate for rooting culture to obtain Alsophila spinulosa seedlings; the substrate includes peat and perlite, and the volume ratio of the peat to perlite is 1:1. In the present invention, the incomplete tissue method preferably includes: chopping the proliferating green spheroids to a diameter of 0.3-0.5 cm, and mixing them with 1 / 2 MS liquid culture medium at a volume ratio of 1:20; the diameter of the proliferating green spheroids is preferably 0.3-0.5 cm. In the present invention, the conditions for the rooting culture preferably include: a temperature of 25-28°C and a time of 30-60 days. In the present invention, after the rooting culture, the obtained rooted seedlings are preferably hardened and inoculated into the mixed substrate for culture to obtain Alsophila spinulosa seedlings. The present invention does not specifically limit the conditions for hardening, and conventional hardening methods can be used. In the present invention, the mixed matrix preferably includes peat and perlite, and the volume ratio of the peat to perlite is 2:1; the culturing time is preferably 20 days.

[0042] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A rapid propagation method for Alsophila spinulosa using the GGB tissue culture pathway comprises the following steps:

[0045] 1. Collect mature but uncracked sporangium leaves of Alsophila spinulosa on a sunny day, dry them naturally, sieve them, and place them in a 4℃ refrigerator for later use.

[0046] 2. Place the spores in step 1 in sterile water and shake for 3 hours, then wash them with sterile water three times, soak them in 75% alcohol for 20 seconds, wash them with sterile water three times, soak them in 5% NaClO solution for 4 minutes, wash them with sterile water four times, and finally add sterile water to make 0.25 g mL -1 Spore suspension.

[0047] 3. The spore suspension treated in step 2 was inoculated into the germination induction medium, cultured in a dark environment at 25°C for 24 hours, and then transferred to light culture. 2 ·s), photoperiod of 12h / d, temperature of 25℃, 10 days of germination, germination rate of 75%, wherein the germination medium is 1 / 10MS, carrageenan concentration is 11g / L, pH is 5.8.

[0048] 4. The germinated and grown spore prothallus after the culture in step 3 was transferred to the induction GGB medium and cultured under a light intensity of 20 μmol / (m 2·s), a photoperiod of 12h / d, and a temperature of 25°C for 60 days to obtain GGB, with an induction rate of over 90%. The GGB induction medium was 1 / 2MS medium supplemented with NAA 0.2 mg·L -1 , PH5.8.

[0049] 5. Cut the GGB obtained in step 4 into small balls with a diameter of approximately 0.7 cm and inoculate them into proliferation medium. After culturing for 30 days, proliferated GGB were obtained. Repeat the above operation for 5 generations to obtain a large number of GGB with a proliferation coefficient of 6.1. The GGB proliferation medium consists of 1 / 2 MS medium supplemented with 0.1 mg / L NAA and 0.1 mg / L IBA, and a pH of 5.8.

[0050] 6. Chop the GGB obtained in step 5 into pieces with a diameter of 0.3 cm and mix them with 1 / 2 MS liquid medium at a volume ratio of 1:20. Sow 2 mL of the mixture containing the protothallus fragments per hole evenly on a planting tray filled with a high-pressure steam sterilized peat: perlite (volume ratio 1:1) matrix. After sowing, cover the tray with a transparent lid and spray regularly to keep it moist. 2 s), a photoperiod of 12 h / d, and a temperature of 25°C for 60 days to induce sporophytic seedlings and rooting, thereby obtaining rooted seedlings with a sporophyte conversion rate of 87%.

[0051] 7. The rooted seedlings obtained in step 6 were subjected to open-cover hardening treatment. After hardening treatment, the tissue culture seedlings were transplanted to a mixed matrix consisting of peat soil: perlite (volume ratio 2: 1), watered thoroughly to allow the roots to settle, and then covered with a film and a shade net. After 20 days of transplanting, the shade was reduced and managed according to the conventional seedling management method to obtain the white spinulosa seedlings with a survival rate of 90%.

[0052] Example 2

[0053] A rapid propagation method for Alsophila spinulosa using the GGB tissue culture pathway comprises the following steps:

[0054] 1. Collect mature but uncracked sporangium leaves of Alsophila spinulosa on a sunny day, dry them naturally, sieve them, and place them in a 4℃ refrigerator for later use.

[0055] 2. Place the spores in step 1 in sterile water and shake for 3 hours, then wash them twice with sterile water, soak them in 75% alcohol for 20 seconds, wash them three times with sterile water, soak them in 5% NaClO solution for 4 minutes, wash them four times with sterile water, and finally add sterile water to make 0.25 g mL -1 Spore suspension.

[0056] 3. The spore suspension treated in step 2 was inoculated into the germination induction medium, cultured in a dark environment at 25°C for 24 hours, and then transferred to light culture. 2 ·s), photoperiod of 12h / d, temperature of 25℃, germination for 12 days, germination rate of 69%, wherein the germination medium is 1 / 10MS, carrageenan concentration is 11g / L, pH is 5.8.

[0057] 4. The germinated and grown spore prothallus after the culture in step 3 was transferred to the GGB medium for inducing green spheroids and the spores were cultured under a light intensity of 20 μmol / (m 2 ·s), a photoperiod of 12h / d, and a temperature of 25°C for 60 days to obtain GGB with an induction rate of 92%. The GGB induction medium was 1 / 2MS medium supplemented with NAA 0.2mg·L-1, pH 5.8.

[0058] 5. Cut the GGB obtained in step 4 into small balls with a diameter of approximately 0.7 cm and inoculate them into proliferation medium. After culturing for 30 days, proliferated GGB were obtained. Repeat the above operation for 5 generations to obtain a large number of GGB with a proliferation coefficient of 7.8. The GGB proliferation medium consists of 1 / 2 MS medium supplemented with 0.1 mg / L NAA and 0.1 mg / L IBA, and a pH of 5.8.

[0059] 6. The GGB obtained in step 5 was chopped into pieces with a diameter of 0.3 cm and mixed with 1 / 2 MS liquid medium at a volume ratio of 1:20. 2 mL of the mixture containing the protothallus fragments was sown evenly on a planting tray filled with a high-pressure steam sterilized peat: perlite (volume ratio 1:1) matrix. After sowing, the tray was covered with a transparent lid and sprayed regularly to maintain moisture. The seeds were cultured for 60 days under a light intensity of 20 μmol / (m2·s), a photoperiod of 12 h / d, and a temperature of 25°C to induce sporophyte seedlings and rooting, thereby obtaining rooted seedlings with a sporophyte conversion rate of 82%.

[0060] 7. The rooted seedlings obtained in step 6 were subjected to open-cover hardening treatment. After hardening treatment, the tissue culture seedlings were transplanted to a mixed matrix consisting of peat soil: perlite (volume ratio 2: 1), watered thoroughly to allow the roots to take root, and then covered with a film and a shade net. After 20 days of transplanting, the shade was reduced and managed according to the conventional seedling management method to obtain the white spinulosa seedlings with a survival rate of 90%.

[0061] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for cultivating Alsophila spinulosa, characterized in that: The following steps are involved: 1) sterilizing spores of Alsophila spinulosa and inoculating them on a germination medium for germination culture to obtain spore prothallus; The germination medium is: 1 / 10 MS medium + 11 g / L carrageenan, pH 5.8; 2) inoculating the sporophyte obtained in step 1) into a green spheroid culture medium to culture green spheroids, thereby obtaining green spheroids; The GGB medium is: 1 / 2MS medium + 0.2 mg / L naphthaleneacetic acid, pH value is 5.8; 3) inoculating the green spheroids obtained in step 2) into a proliferation culture medium for proliferation culture to obtain proliferated green spheroids; The proliferation culture medium is: 1 / 2MS culture medium + 0.1mg / L naphthaleneacetic acid + 0.1mg / L indolebutyric acid, pH value is 5.8; 4) treating the proliferated green spheroids obtained in step 3) using an incomplete tissue method and then inoculating the spheroids into a matrix for rooting culture to obtain Alsophila spinulosa seedlings; The matrix is ​​peat and perlite, and the volume ratio of the peat and perlite is 1:1; The incomplete tissue method in step 4) is as follows: mincing the proliferating green spheroids into pieces with a diameter of 0.3 to 0.5 cm, and mixing them with 1 / 2 MS liquid culture medium at a volume ratio of 1:20; the diameter of the proliferating green spheroids is 0.3 to 0.5 cm.

2. The cultivation method according to claim 1, characterized in that The disinfection method in step 1) is as follows: soaking in a 75% by volume ethanol solution for 20 seconds, washing with sterile water 2-3 times, then soaking in a 4-5% by mass NaClO solution for 4 minutes, and washing with sterile water 4 times.

3. The cultivation method according to claim 1, wherein The conditions for the germination culture in step 1) are: first culturing in the dark for 24 hours, and then culturing in the light for 10 to 20 days; The conditions of the light culture include: light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

4. The cultivation method according to claim 1, characterized in that The conditions for culturing the green spheroids in step 2) are as follows: 60 days, a light intensity of 15 to 20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

5. The cultivation method according to claim 1, characterized in that The conditions of the proliferation culture in step 3) are as follows: the culture time is 30 days, the light intensity is 15-20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

6. The cultivation method according to claim 1, characterized in that The diameter of the green spheroid in step 3) is 0.7 cm.

7. The cultivation method according to claim 1, characterized in that The rooting culture conditions in step 4) are as follows: temperature of 25-28°C, time of 30-60 days, light intensity of 15-20 μmol / (m 2 ·s), and the photoperiod was 12h / d.

8. The cultivation method according to claim 1, characterized in that After the rooting culture, the obtained rooted seedlings are hardened and inoculated into a mixed matrix for culture to obtain Alsophila spinulosa seedlings.

9. The cultivation method according to claim 8, characterized in that The mixed matrix is ​​peat and perlite, and the volume ratio of the peat to perlite is 2:1; The culture time is 20 days.