CN113142059A - Method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting - Google Patents

Method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting Download PDF

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CN113142059A
CN113142059A CN202110570224.6A CN202110570224A CN113142059A CN 113142059 A CN113142059 A CN 113142059A CN 202110570224 A CN202110570224 A CN 202110570224A CN 113142059 A CN113142059 A CN 113142059A
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buds
rooting
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cluster
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刘芳
余注光
田湘
俞建妹
廖克波
陈碧珍
赵春梅
刘玉军
沈遐
梁小春
廖敏彤
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Guangxi Zhuang Autonomous Region Nanning Liangfengjiang National Forest Park
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
    • A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
    • A01G24/25—Dry fruit hulls or husks, e.g. chaff or coir
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention discloses a method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting, which comprises 4 steps of pretreatment, surface disinfection and sterilization of explants, primary bud culture, synchronous bud proliferation and rooting culture and seedling transplantation, wherein subculture buds are cut into cluster buds, every 3-5 buds are taken as a cluster, the cluster buds are inoculated into a bud proliferation and rooting culture medium, the culture lasts for 35-40 d, and the proliferation coefficient is 5-12; transplanting the multiple bud strong cluster buds, and continuously culturing multiple short and few cluster buds of the small buds in the bud multiplication and rooting synchronous culture medium. The method for synchronously culturing the bud multiplication and the rooting realizes the synchronous completion of the growth stages of multiplication, seedling strengthening, rooting culture and the like by using a culture medium, not only saves the steps and the cost of the rooting culture, but also shortens the seedling culture period, is a high-efficiency and low-cost production technology of the galangal tissue culture seedling of floral leaves, and is worthy of application and popularization in production.

Description

Method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting
Technical Field
The invention belongs to the technical field of plant asexual rapid propagation, and particularly relates to a method for synchronously culturing tissue culture buds, proliferated and rooted of galangal floral leaves.
Background
Flower and leaf of Alpinia galangaAlpinia zerumbet cv. Variegata), also known as Galangal, is a variant of flowers of Alpinia zerumbet belonging to the family Zingiberaceae. The galangal leaves are perennial herbaceous plants, the length of the leaves is up to 1m, the color of the leaves is dark green, and the leaves have golden stripes, and the galangal leaves are commonly used as foliage and flower-viewing plants for indoor pot culture and landscaping. The flower fragrance is beautiful in leaf color, beautiful in flower appearance, pure in flower fragrance and very high in ornamental value, and is highly favored by operators.
At present, the method for propagating the galangal seedlings by using the tillering and plant growing is mainly adopted, and the propagation method is limited by climatic factors, is slow in growth and is difficult to meet market demands. The seed reproduction method can also be adopted, but the separation caused in the sexual reproduction process of the seed reproduction causes more than 90 percent of the seedling leaves to turn green and lose the yellow-green tabling stripes. The tissue culture technology for propagating the buds by the buds not only can maintain the genetic stability of excellent characters of plants, but also can rapidly propagate seedlings in a short time, so the tissue culture technology is an effective way for promoting the rapid development of the galangal seedlings.
At present, the related reports in the tissue culture aspect of galangal flowers and leaves include that Mebejia and the like respectively use two culture media of MS + BA1.0mg/L + IAA0.2mg/L and MS + NAA0.2mg/L to carry out enrichment culture and rooting culture research, and transplantation is carried outThe survival rate reaches 90 percent; the mature prunus mume carries out cluster bud multiplication by using MS +6-BA2.0mg/L + KT0.5mg/L + IBA0.5mg/L, and carries out rooting induction by using 1/2MS + IBA0.25mg/L, wherein the maximum multiplication coefficient is 7.03, and the rooting rate of seedlings is 62.13%; in 2016, research on tissue culture industrial seedling raising technology of galangal flowers and leaves is carried out, and MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L + carrageenan 5.5g/L are screened out as an optimal proliferation culture medium; the most suitable rooting culture medium for strong seedlings is 3/4MS, NAA0.5mg/L + + sucrose 30g/L and carrageenan 5.5 g/L. Relevant patents granted by the country are: firstly, a method for improving the tissue culture propagation speed of the alpinia zerumbet is characterized in that the alpinia zerumbet plants are picked up 7-8 days in a sunny day continuously, the plants are hung upside down in a ventilated and shady place in a room and dried for 2-3 days, tubers of the plants are taken as explants, and after overlong leaves and root systems on the tubers are removed, washing soil under tap water, cutting tender shoots and old stems used as explant materials, cutting the explants into the size of 1-2 cm multiplied by 1-2 cm, soaking the explants in 75% alcohol for 30s, washing the explants once with sterile water, soaking the explants with 0.1% mercury bichloride for 20-25 min, washing the explants 5-6 times with sterile water, cutting off the outer bracts and peripheral redundant tissues of the sterilized explants, leaving stem tips or side buds containing growing points, keeping the size of the explants to be 1-1.5 cm multiplied by 1.5cm, and inoculating a primary induction bud culture medium (MS +6-BA 4.0-5.0 mg/L + KT 5.0mg/L + NaH).2PO4170mg/L + white sugar 3% + agar 0.6%), inducing adventitious buds, cutting into blocks of 1-2 cm when lateral buds of the explant sprout and the buds are 0.5-2 cm long, continuing to connect with an initial generation induction culture medium, performing generation-interrupted propagation for 3-4 times until 2-4 adventitious buds grow out of the base part of the blocks of the culture body, entering proliferation culture when the adventitious buds grow and proliferate stably, performing proliferation cutting on the blocks growing adventitious buds according to the growth direction, reserving 2-3 adventitious buds for each block, cutting off overlong leaves, inoculating 15 blocks in each bottle, inoculating into the proliferation culture medium which is MS + MgSO (magnesium sulfate)4•7H2O1180mg/L+ NaH2PO4170mg/L+Ca(NO3)2•4H2O440 mg/L +6-BA 2-3 mg/L + KT 3-5 mg/L + white sugar 3% + agar 0.6%. Cutting off solid buds which grow normally, have speckles on leaf surfaces and are 2-3 cm high from the bud clumps of the adventitious buds, inoculating the solid buds to a rooting culture medium, wherein the solid buds cannot reach the rooting standardThe culture mass is subjected to proliferation cutting for proliferation culture, and the rooting culture medium is 1/2MS + NAA0.5mg/L + white sugar 3% + agar 0.6%. ② a method for establishing a callus regeneration system of galangal leaf, which comprises taking seeds as explants, respectively sterilizing and sterilizing with 75% alcohol, 0.1% mercuric chloride and 5% sodium hypochlorite solution for 1min, 10min and 3min to obtain plants, cutting off roots when the plant buds are 1-2 cm high, inoculating the plants to a callus culture medium to induce callus with an induction rate of 85.33-88.46%, inoculating the callus to an adventitious bud differentiation culture medium to induce adventitious buds, wherein the callus generates adventitious buds after 30 days, the proliferation rate is 8-10.7 times, and the callus is inoculated to a rooting culture medium to perform rooting culture when the adventitious buds are 3-4 cm high, and the adventitious buds root after 30 days, and the rooting rate is 95.16-98.85%. The components of the germination medium MO are as follows: k2SO4 800mg/L，MgSO4•7H2O 350 mg/L，KH2PO4180 mg/L，NH4NO3600mg/L，MnSO4•4H2O 22.5 mg/L，Ca(NO3)2•4H2O 556 mg/L，FeSO4•7H2O27.8 mg/L，Na2EDTA37.3 mg/L，ZnSO4•7H2O 8.6 mg/L，H3BO3 6.2 mg/L，CuSO4•5H2O 0.25 mg/L，Na2MOO4•2H20.25mg/L of O, 100 mg/L of inositol, 2.0mg/L of glycine, 11.0 mg/L of vitamin B, 60.5 mg/L of vitamin B and 0.5mg/L of nicotinic acid. The callus induction culture medium is M0+ 1-2 mg/L of chlortoluron + 0.5-1 mg/L of 2, 4-dichlorophenoxyacetic acid, the adventitious bud differentiation culture medium M2 is M0+ 1-2 mg/L of chlortoluron + 0.05-0.1 mg/L of 2, 4-dichlorophenoxyacetic acid, and the rooting culture medium is 1/2M0+ 0.5-1 mg/L of ABT1 rooting powder. The reports are both secondary culture and rooting culture, and no research report of synchronous bud proliferation and rooting exists.
Disclosure of Invention
The invention aims to overcome the defects of the conventional culture of galangal floral leaf tissue culture seedlings in the prior art, and discloses a method for synchronously culturing the tissue culture buds of galangal floral leaves by proliferation and rooting, which improves the propagation efficiency, reduces the production cost and can realize the synchronous culture of the tissue culture buds of galangal flowers and leaves by proliferation and rooting.
The invention is realized by adopting the following technical scheme:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in 3 days continuously in the weather, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 3-5 cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on an ultra-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 3-4 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking the buds in the solution for 16 min, cleaning the buds with sterile water for 3-4 times, stripping off outer leaf sheaths of axillary bud rhizomes, and inoculating the buds in a primary culture medium for primary culture of the buds, wherein the buds are 1.5-3 cm high;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 3-5 buds form a cluster, each cluster bud contains 1-2 colorful and robust buds with the height of 3-4 cm, and then transferring the cluster buds into a subculture multiplication rooting culture medium for culturing for 35-40 d, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS- (100-300) mg/L NH4NO3(3-5) mg/L6-BA + (1-2) mg/L NAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 15-28 ℃ of temperature, 70-85% of humidity and 70-80% of shading degree, and fertilizing and protecting at the seedling stage, wherein the seedlings can be taken out of the nursery or put into the big cup to culture the big seedlings when the height of the seedlings is more than or equal to 15 cm.
Preferably, the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/L CH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8. The CH is hydrolyzed casein. On the basis of the MS culture medium, a certain amount of 6-BA, NAA, CH, agar powder and sucrose are added, so that the rooting quantity can be increased, nutrient components necessary for root growth are provided, and the robust growth of the root system is promoted.
Preferably, the primary bud culture, the secondary bud multiplication and rooting synchronous culture are all carried out in a culture room with 80% of wall bodies being double-layer glass windows.
Preferably, the illumination time of the culture chamber is 11-14 h/d, the temperature is 22-30 ℃, and the illumination intensity is 4000-8000 LX.
Preferably, in the synchronous culture of the subculture multiplication and rooting of the buds, after the cluster buds are transferred to a subculture multiplication and rooting culture medium, the cluster buds are firstly placed in natural light with the illumination intensity of 1500-2500 LX and cultured for 15 days at the temperature of 25 ℃, and then the culture is continued under the conditions with the temperature of 22-28 ℃ and the illumination intensity of 4000-8000 LX.
Preferably, the matrix in step (4) comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use. The matrix prepared from the coconut chaff, the chaff and the perlite has the characteristics of good air permeability, good water holding capacity, difficult hardening and the like, can provide trace elements and nutrients necessary for the growth of seedlings, and can meet the growth of the galangal seedlings of the flower leaves without adding organic fertilizers.
Compared with the prior art, the technical scheme has the following beneficial effects:
1. the invention realizes the subculture bud multiplication and rooting one-step method rapid propagation of the galangal floral leaf, adopts cluster buds for subculture multiplication and rooting culture, only uses one culture medium, and simultaneously completes the processes of bud propagation, seedling strengthening and rooting culture, shortens the seedling culture period, effectively reduces the production cost, and the tissue culture seedling grows robustly and has excellent characters and stable heredity, thus being an advanced technology for high-quality and high-efficiency production of the galangal floral leaf tissue culture seedling.
2. The invention firstly puts NH in each liter of MS culture medium on the basis of MS culture medium4NO3The amount of the active ingredients is reduced by 100-300 mg, and then a certain amount of 6-BA, NAA, IBA, agar powder and sucrose are added. Not only can increase the rooting quantity and promote the root growth, but also can reduce NH in the secondary rooting culture aiming at the characteristic of strong sprouting capacity of the galangal leaves4NO3The amount of the N is used for solving the problem that the rooting rate of buds is low due to the fact that the N content in a culture medium is high in the bud growth process.
3. In the pretreatment step, the rhizome axillary buds are cut into 3-5 cm sections, the rhizome axillary buds are rhizome sections with axillary buds, and if the rhizome axillary buds are too long, the difficulty in surface disinfection and sterilization is easily caused; if the axillary buds of the rootstock are too short, the obtained sterile living bodies are easy to be few, and the subsequent culture is not facilitated.
4. The invention synchronously performs bud multiplication and rooting, and has simple process, short production period and strong operability. The natural light-emitting light is used as a light source, so that the power cost of seedling production is effectively reduced, and the technical popularization and application prospect is wide.
Drawings
FIG. 1 shows a tissue culture seedling of Alpinia floral leaf obtained by culturing in Experimental example 2 in the presence of 2 shoots per cluster.
FIG. 2 shows a tissue culture seedling of Alpinia floral leaf obtained by culturing in Experimental example 2 in the presence of 3 shoots per cluster.
FIG. 3 shows a Alpinia floral leaf tissue culture seedling obtained by culturing in the case where each cluster bud contained 4 shoots as described in Experimental example 2.
FIG. 4 shows a Alpinia floral leaf tissue culture seedling obtained by culturing in the case where each cluster bud contained 5 shoots as described in Experimental example 2.
FIG. 5 shows a Alpinia floral leaf tissue culture seedling obtained by culturing in Experimental example 2 in the presence of 6 shoots per cluster.
FIG. 6 shows a Alpinia floral leaf tissue culture seedling obtained by culturing in Experimental example 2 in the presence of 7 shoots per cluster.
FIG. 7 shows a Alpinia floral leaf tissue culture seedling obtained by culturing in Experimental example 2 in the presence of 8 shoots per cluster.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. The specific experimental conditions and methods not indicated in the following examples are generally conventional means well known to those skilled in the art.
Example 1:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in continuous sunny days for 3 days, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 4cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on a super-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 3 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 4 times, peeling off outer leaf sheath of axillary bud rhizome, inoculating in primary culture medium for primary culture of bud with bud height of 2 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 5 buds are a cluster, each cluster bud contains 2 buds with the height of 4cm, obvious and strong leaves, then transferring the cluster buds into a subculture multiplication rooting culture medium, culturing for 35-40 d, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short and few roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-100 mg/L NH4NO3+5 mg/L6-BA +2 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the primary culture of the bud andthe bud subculture multiplication and rooting synchronous culture are carried out in a culture room with 80% of wall bodies being double-layer glass windows; the illumination time of the culture room is 12h/d, after the cluster buds are transferred to a subculture proliferation rooting culture medium, the cluster buds are placed under natural light with the illumination intensity of 1500-2500 LX and cultured for 15d at the temperature of 25 ℃, and then the culture is continued under the conditions with the temperature of 25 ℃ and the illumination intensity of 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 22 ℃ of temperature, 80% of humidity and 75% of shading degree, and culturing in shade, and after the fertilization and the management in the seedling stage, when the height of the nursery stock is more than or equal to 15 cm, taking out of the nursery or culturing the nursery stock in the big cup; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Example 2:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in 3 days continuously in the weather, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 3cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on a super-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 4 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 3 times, peeling off outer leaf sheath of axillary bud rhizome, inoculating in primary culture medium for primary culture of bud with bud height of 2 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 4 buds are a cluster, each cluster bud contains 1 bud which is 3.5cm high, has obvious colorful and strong leaves, then transferring the cluster buds into a subculture multiplication rooting culture medium, culturing for 35-40 d, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short and few roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-300 mg/L NH4NO3+5 mg/L6-BA +1 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the bud primary culture, the bud secondary multiplication and the rooting synchronous culture are all carried out in a culture room with 80 percent of wall bodies as double-layer glass windows; the illumination time of the culture room is 13 h/d, after the cluster buds are transferred to a subculture proliferation rooting culture medium, the cluster buds are placed under natural light with the illumination intensity of 1500-2500 LX and cultured for 15d at the temperature of 25 ℃, and then the culture is continued under the conditions with the temperature of 30 ℃ and the illumination intensity of 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning up the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 25 ℃ of temperature, 70% of humidity and 75% of shading degree, and culturing in shade, and after the fertilization and the management in the seedling stage, when the height of the nursery stock is more than or equal to 15 cm, taking out of the nursery or culturing the nursery stock in the big cup; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Example 3:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in continuous sunny days for 3 days, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 5cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on an ultra-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30S, then cleaning the axillary bud rhizome with sterile water for 4 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 4 times, peeling off outer leaf sheath of axillary bud rhizome, inoculating in primary culture medium for primary culture of bud with bud height of 1.5 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 5 buds are a cluster, each cluster bud contains 2 buds which are 3cm high, obvious in leaf color and strong, then transferring the cluster buds into a subculture multiplication rooting culture medium, culturing for 35-40 d, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short and few roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-200 mg/L NH4NO3+5 mg/L6-BA +1.5 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the bud primary culture, the bud secondary multiplication and the rooting synchronous culture are all carried out in a culture room with 80 percent of wall bodies as double-layer glass windows; the illumination time of the culture room is 11 h/d, after the cluster buds are transferred to a subculture proliferation rooting culture medium, the cluster buds are placed under natural light with the illumination intensity of 1500-2500 LX and cultured for 15d at the temperature of 25 ℃, and then the culture is continued under the conditions with the temperature of 28 ℃ and the illumination intensity of 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning up the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 28 ℃ of temperature, 75% of humidity and 80% of shading degree, and after seedling fertilization and management, taking out of the nursery or culturing the big seedlings in the big cup when the height of the seedlings is more than or equal to 15 cm; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Example 4:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in continuous sunny days for 3 days, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 3.5cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on a super-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 3 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 3 times, peeling off outer leaf sheath of axillary bud rhizome, inoculating in primary culture medium for primary culture of bud with bud height of 1.5 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 3 buds are a cluster, each cluster bud contains 1 bud which is 3cm high, has obvious colorful and strong leaves, then transferring the cluster buds into a subculture multiplication rooting culture medium, culturing for 35-40 d, wherein the bud multiplication coefficient is 5-12, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short and few roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-100 mg/L NH4NO3+3 mg/L6-BA +1.5 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the bud primary culture, the bud secondary multiplication and the rooting synchronous culture are all carried out in a culture room with 80 percent of wall bodies as double-layer glass windows; the illumination time of the culture room is 14 h/d, the cluster buds are transferred to a subculture proliferation rooting culture medium and then are placed under natural light with the illumination intensity of 1500-2500 LX at the temperature of 25 DEG CCulturing for 15 days, and then continuously culturing under the conditions that the temperature is 26 ℃ and the illumination intensity is 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning up the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 15 ℃ of temperature, 85% of humidity and 80% of shading degree, and after seedling fertilization and management, taking out of the nursery or culturing the big seedlings in the big cup when the height of the seedlings is more than or equal to 15 cm; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Example 5:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in continuous sunny days for 3 days, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 4.5cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on a super-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 3 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 4 times, peeling off outer leaf sheath of axillary bud rhizome, inoculating in primary culture medium for primary culture of bud with bud height of 3 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture bud into cluster buds with 4 buds each containing 2 colorful and strong buds of 3.5cm, transferring into subculture multiplication rooting medium, culturing for 35-40 days, and increasing bud multiplication coefficient5-12, transplanting multiple shoots with strong roots, and continuously culturing the multiple shoots with more buds and short and few roots in a shoot proliferation and rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-100 mg/L NH4NO3+4 mg/L6-BA +2 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the bud primary culture, the bud secondary multiplication and the rooting synchronous culture are all carried out in a culture room with 80 percent of wall bodies as double-layer glass windows; the illumination time of the culture chamber is 13 h/d, the temperature is 22 ℃, and the illumination intensity is 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning up the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 18 ℃ of temperature, 80% of humidity and 70% of shading degree, and culturing in shade, and after the fertilization and the management in the seedling stage, when the height of the nursery stock is more than or equal to 15 cm, taking out of the nursery or culturing the nursery stock in the big cup; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Example 6:
a method for synchronously culturing tissue culture buds of galangal flowers with proliferation and rooting comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in continuous sunny days for 3 days, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 4cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on a super-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 4 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking in the solution for 16 min, cleaning with sterile water for 3 times, removing outer leaf sheath of axillary bud rhizome, inoculating to primary culture medium, and performing primary generationCulturing until the height of the bud is 3 cm; the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 5 buds are a cluster, each cluster bud contains 1 bud which is 4cm high, has obvious colorful and strong leaves, then transferring the cluster buds into a subculture multiplication rooting culture medium, culturing for 35-40 d, wherein the bud multiplication coefficient is 5-12, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short and few roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS-300 mg/L NH4NO3+5 mg/L6-BA +2 mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8; the bud primary culture, the bud secondary multiplication and the rooting synchronous culture are all carried out in a culture room with 80 percent of wall bodies as double-layer glass windows; the illumination time of the culture chamber is 12h/d, the temperature is 30 ℃, and the illumination intensity is 4000-8000 LX;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning up the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 20 ℃ of temperature, 75% of humidity and 70% of shading degree, and carrying out seedling fertilization and management, wherein when the height of the nursery stock is more than or equal to 15 cm, the nursery stock can be taken out of the nursery or put into the big cup for culturing the big seedling; the matrix comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
Comparative example 1:
a method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting only differs from the method in example 1 in that: in the bud subculture proliferation and rooting synchronous culture in the step (3), cutting single buds with the height of 1.5-3 cm from the cut single buds, taking 6 buds as a bottle, then transferring the cut single buds into a subculture proliferation and rooting culture medium, culturing for 35-40 d, transplanting multiple and strong-rooted multiple buds, and continuously culturing the multiple buds with multiple small buds and short and few roots in the bud proliferation and rooting synchronous culture medium.
Experimental example 1:
and (3) performing multiplication and rooting synchronous culture on the galangal floral leaf tissue culture buds according to the methods of the examples 1-6 and the comparative example 1, observing and counting the rooting and transplanting survival conditions of the galangal floral leaf tissue culture seedlings, wherein specific results are shown in table 1.
TABLE 1 rooting and transplanting survival situation of tissue culture seedling of Alpinia galanga
Figure 358425DEST_PATH_IMAGE001
As can be seen from the data in Table 1, the method for cutting the secondary buds into cluster buds and then carrying out secondary multiplication and rooting culture according to the scheme of the invention is obviously superior to the conventional method adopting single-bud culture in the aspects of rooting quantity, rooting rate and survival rate.
Experimental example 2:
and (2) performing multiplication and rooting synchronous culture on the galangal floral leaf tissue culture buds according to the method in the example 1, shearing the secondary buds into cluster buds in the bud secondary multiplication and rooting synchronous culture in the step (3), culturing under the condition that each cluster bud contains 2 buds, 3 buds, 4 buds, 5 buds, 6 buds, 7 buds and 8 buds respectively, and observing and counting the rooting and transplanting survival conditions of the galangal floral leaf tissue culture seedlings, wherein the specific results are shown in a table 2 and attached figures 1-7.
TABLE 2 rooting and transplanting survival situation of tissue culture seedling of Alpinia galanga
Figure DEST_PATH_IMAGE002
As can be seen from the data in Table 2 and the attached figures 1 to 7, when the number of buds in each cluster is 3 to 6, the number of the radicles of the cultured galangal mosaic tissue culture seedling is more, the radicles are stronger, the rooting rate and the survival rate are also obviously superior to other conditions, and the subculture multiplication and rooting culture of cluster buds are not suitable for each cluster with too many or too few buds.
Example 3:
the tissue culture bud proliferation and rooting synchronous culture of the galangal flowers and leaves is carried out according to the method described in the example 1, and in the bud secondary proliferation and rooting synchronous culture, the formulas of secondary proliferation and rooting culture media with different proportions (specifically shown in table 3) are adopted, the rooting condition of the galangal flowers and leaves tissue culture seedlings is observed and counted, and the specific results are shown in table 3.
TABLE 3 formulation of the subculture proliferation rooting medium and rooting conditions
Figure 636085DEST_PATH_IMAGE003
As can be seen from the data in Table 3, the culture medium adopted by the method of the invention can effectively improve the proliferation coefficient and the average rooting rate compared with the conventional MS culture medium, the average rooting rate obtained by the formulas 1, 2 and 7 is more than 80%, and the proliferation coefficient obtained by the formula 1 is obviously higher than that obtained by other formula combinations.
Experimental example 4:
the tissue culture bud proliferation and rooting synchronous culture of the galangal mosaic is carried out according to the method in the example 1, in the bud subculture proliferation and rooting synchronous culture, the illumination time of the culture chamber is 14 h/d, the cluster buds are transferred to a subculture proliferation and rooting culture medium, the cluster buds are firstly placed under natural light with the illumination intensity of 1500-2500 LX and are cultured for 15d under the condition of the temperature of 25 ℃, then the cluster buds are cultured under different illumination and temperatures (see table 4 specifically), and the rooting condition of the galangal mosaic tissue culture seedling is observed and counted, and the specific result is shown in table 4.
TABLE 4 Effect of different illumination intensities and temperatures on rooting
Figure DEST_PATH_IMAGE004
As can be seen from Table 4, the light intensity is 4000-8000 LX, the temperature is 25-30 ℃, and when the subculture period is 35-40 days, the obtained rooting rate and survival rate are obviously superior to other light and temperature conditions, and better rooting rate and survival rate cannot be obtained by independently adjusting the light intensity, the temperature or one of the conditions in the subculture period.
The experimental materials in the experimental examples 1 to 4 are prepared by selecting bagged galangal leaves which are bright and bright in leaf color, glossy, free of diseases and insect pests and strong in growth and are sold in the Guangxi Nanning flowery bird market, planting the bagged galangal leaves in a seedling breeding center nursery of a Liangfengjiang tree garden, and then planting rootstock axillary buds of cluster plants for more than half a year as explants.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (6)

1. A method for synchronously culturing tissue culture buds of galangal flowers and proliferating and rooting is characterized by comprising the following steps: the method comprises the following steps:
(1) pretreatment: collecting robust galangal flowers and leaves in 3 days continuously in the weather, cleaning soil at the roots, putting the whole plant in tap water for 3d, changing water 1 time every day, then cutting off all leaves and root systems, cutting off the rhizome with axillary buds to obtain the rhizome of the axillary buds, wherein the rhizome of each axillary bud is 3-5 cm long, then soaking in 5 per thousand of washing powder solution, brushing for 5min by using a soft brush, and then cleaning for 3 times by using purified water;
(2) and (3) sterilizing the surface of the explant: putting the axillary bud rhizome obtained in the step (1) on an ultra-clean workbench, disinfecting the axillary bud rhizome with 75% alcohol solution for 30s, then cleaning the axillary bud rhizome with sterile water for 3-4 times, and then using HgCl with the mass fraction of 1.25 per mill2Soaking the buds in the solution for 16 min, cleaning the buds with sterile water for 3-4 times, stripping off outer leaf sheaths of axillary bud rhizomes, and inoculating the buds in a primary culture medium for primary culture of the buds, wherein the buds are 1.5-3 cm high;
(3) bud subculture proliferation and rooting synchronous culture: cutting the subculture buds into cluster buds, wherein each 3-5 buds form a cluster, each cluster bud contains 1-2 colorful and robust buds with the height of 3-4 cm, and then transferring the cluster buds into a subculture multiplication rooting culture medium for culturing for 35-40 d, transplanting the cluster buds with multiple strong roots, and continuously culturing the cluster buds with multiple small buds and short roots in a bud multiplication rooting synchronous culture medium; the formula of the subculture proliferation rooting culture medium is as follows: MS- (100-300) mg/L NH4NO3(3-5) mg/L6-BA + (1-2) mg/LNAA +0.5 mg/L IBA +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8;
(4) transplanting the seedlings: taking out the multiple bud strong cluster buds obtained in the step (3), cleaning the bud multiplication and rooting synchronous culture medium adhered to the root system, then transplanting the cluster buds into a nutrition cup filled with the matrix, culturing under the environment conditions of 15-28 ℃ of temperature, 70-85% of humidity and 70-80% of shading degree, and fertilizing and protecting at the seedling stage, wherein the seedlings can be taken out of the nursery or put into the big cup to culture the big seedlings when the height of the seedlings is more than or equal to 15 cm.
2. The method for synchronously culturing the tissue culture buds, the proliferation and the rooting of the galangal flowers according to claim 1, which is characterized in that: the formula of the primary culture medium is as follows: MS +5 mg/L6-BA + 2mg/L NAA +200 mg/LCH +4.5 g/L agar powder +30 g/L sucrose, pH = 5.8.
3. The method for synchronously culturing the tissue culture buds, the proliferation and the rooting of the galangal flowers according to claim 1, which is characterized in that: the primary bud culture, the secondary bud multiplication and rooting synchronous culture are all carried out in a culture room with 80% of wall bodies being double-layer glass windows.
4. The method for synchronously culturing the tissue culture buds, the proliferation and the rooting of the galangal flowers according to claim 4, which is characterized in that: the illumination time of the culture chamber is 11-14 h/d, the temperature is 22-30 ℃, and the illumination intensity is 4000-8000 LX.
5. The method for synchronously culturing the tissue culture buds, the proliferation and the rooting of the galangal flowers according to claim 5, which is characterized in that: in the bud subculture multiplication and rooting synchronous culture, after the cluster buds are transferred to a subculture multiplication rooting culture medium, the cluster buds are placed under natural light with the illumination intensity of 1500-2500 LX and cultured for 15 days at the temperature of 25 ℃, and then the cluster buds are continuously cultured under the conditions with the temperature of 22-28 ℃ and the illumination intensity of 4000-8000 LX.
6. The method for synchronously culturing the tissue culture buds, the proliferation and the rooting of the galangal flowers according to claim 1, which is characterized in that: the matrix in the step (4) comprises the following components in parts by volume: 8 parts of coconut chaff, 1 part of rice husk and 1 part of perlite, uniformly mixing all the components of the matrix, spraying 800 times of 98% hymexazol liquid, stirring, sterilizing and filling into a plug for later use.
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CN115708481A (en) * 2022-10-25 2023-02-24 广东省农业科学院环境园艺研究所 Tissue culture method of dwarf ginger flower
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CN114680046A (en) * 2022-04-22 2022-07-01 海南茗卉农林科技发展有限公司 Tissue culture rapid propagation method for keeping stable characters of color leaf chimera ornamental plants
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