CN115708481A - Tissue culture method of dwarf ginger flower - Google Patents
Tissue culture method of dwarf ginger flower Download PDFInfo
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Abstract
The invention discloses a tissue culture method of dwarf ginger flower, which comprises the steps of seed pretreatment, aseptic seedling and hypocotyl callus induction, callus proliferation and greening, cluster bud induction and proliferation, rooting and seedling strengthening and transplanting. The method comprises the steps of taking a fruit with yellow pericarp and without cracking, cutting the base and the tail of the fruit by a blade, sterilizing, cutting the sterilized fruit, and sowing seeds on a callus induction culture medium for culture until hypocotyls of aseptic seedlings form a white callus wound surface; cutting off the part, root and seed above hypocotyl of aseptic seedling, transferring white callus to callus proliferation and greening culture medium, and inducing and proliferating cluster bud on cluster bud induction and proliferation culture medium; cutting off buds along the bud base part, transferring the cut buds to a strong seedling rooting culture medium for culturing for 30-40 d, hardening seedlings under natural illumination for 12-15 d, taking out the seedlings, and transplanting to obtain a regenerated plant of the dwarf ginger flower. The method has low cost, lays a foundation for tissue culture rapid propagation and genetic engineering research of the dwarf ginger flower, and is favorable for large-scale production and application of dwarf ginger flower seedlings.
Description
Technical Field
The invention relates to a plant tissue culture method, in particular to a tissue culture method of dwarf ginger flower.
Background
The short ginger flower (Hedychium brevicaule D.Fang) belongs to the Zingiber (Hedychium) plant of Zingiberaceae (Zingiberaceae), produces Guangxi in China, has the plant height of 20-60 cm and the flowering phase of 1-3 months; the bract is reddish brown, the flower is pure white, the filament is as long as or slightly longer than the lipped, and the anther is 9mm long; has strong orchid fragrance and extremely short raw material (Huxiu, etc. 2010; wu De o, etc. 2016). The rhizome of the flower of dwarf ginger can be used for cough, asthma due to phlegm and bronchial asthma, and can also be potted and appreciated (Wu De O, et al 2016). Therefore, the dwarf ginger flower has high economic value and market development and utilization prospect in the aspects of medicine and appreciation.
The propagation method of the bush mainly depends on rhizome cutting and seed propagation (Huxiu, et al 2011). Because the tillering number of the dwarf ginger flower is small, the propagation speed of the cut rhizome is slow; the time from sowing to flowering of the seeds is two to three years, and therefore, the two propagation methods are inefficient, and cannot provide sufficient seedlings for the marketization application thereof, resulting in great difficulty in the enlargement of the cultivation scale. The tissue culture technology is adopted to obtain regeneration plants by inducing callus, and an effective way can be provided for large-scale propagation of bottle seedlings.
Tissue culture studies of plants of the genus zingiber have focused on flowers of white ginger (h.coronarium) (pandorinhua, et al 2005, 2011; xiao, et al 2016, hu, et al 2020), flowers of red ginger (h.coccineum) (reddish, et al 2014; entada, et al 1989), flowers of golden ginger (h.gardnerianum) (reddish, et al 2017), h.muluense (Hamidou, et al 2008) and h.bouguisonianum (Hamidou, et al 2009). Adopting a stem tip growth point of H.muluense as an explant to induce callus, pre-culturing the callus in a suspension culture mode, and then performing somatic embryo induction on the suspension culture; salicylic acid and silver nitrate are used for regulating and controlling the H.boussignianium callus proliferation and somatic embryo induction processes, and the method has a certain effect of improving the frequency of somatic embryogenesis (Hamidou, et al 2008, 2009). Using hypocotyl or adventitious bud of aseptic seedling germinated from mature seed of flowers of zingiberis albus as explant, and when inoculating on MS +1 mg/L6-BA +0.2mg/L NAA for culture, reproducing and producing complete plant by inducing cluster bud (friendship, etc. 2005, 2011); taking immature filaments of the white ginger flower as explants, inoculating the immature filaments on a culture medium of MS +4 mg/L2, 4-D +4mg/L NAA +1 mg/L6-BA, and inducing callus (ShouWang, et al 2016) through culture for 180 days; the pseudostem segment of the white ginger flower is taken as an explant, and is inoculated on a culture medium of MS + 3-5 mg/L6-BA +2mg/L TDZ +0.01mg/L NAA to induce protocorm-like (Hu, et al 2020). Taking inflorescence axis and bract of red ginger flower as explant, inoculating on MS +1 mg/L6-BA +2mg/L NAA culture medium, and obtaining regeneration plant (Cyclamen, et al 1989) by inducing callus to form adventitious bud; the filament and anther of the red ginger flower are taken as explants, inoculated on a culture medium of MS +4 mg/L2, 4-D +4mg/L NAA +1 mg/L6-BA +30g/L sucrose +7g/L agar, and cultured for 120 days to induce callus (showy red, etc. 2014). Taking the bud top end of 0.5-2.0 cm of the axillary bud of the rhizome of the golden ginger flower as an explant, and inoculating the explant on an MS +5.0 mg/L6-B +0.2mg/L NAA culture medium to induce adventitious buds (Tulip-Yan, etc. 2017).
The present inventors have conducted studies on tissue culture of bush ginger, and found that pseudostem segments of bush ginger are used as explants, and inoculated with a protocorm-like medium (Hu, et al 2020): when MS +5 mg/L6-BA +2mg/L TDZ +0.01mg/L NAA are cultured, the pseudostem segment does not sprout; callus induction medium (cymbidium, et al 1989) used in literature was inoculated with the rachis of dwarf ginger as explant: when MS +1 mg/L6-BA +2mg/L NAA is cultured, no callus appears on the rachis; adventitious buds of aseptic seedlings of flowers of dwarf ginger are used as explants, and inoculated into a cluster bud propagation medium used in the literature (friendship bear, et al 2005, 2011): when MS +1 mg/L6-BA +0.2mg/L NAA is cultured, the phenomenon of slow bud growth occurs, and the formula of the culture medium listed in the literature is not suitable for tissue culture of the dwarf ginger flower. In addition, the literature only reports the process that callus of flowers of white ginger, flowers of red ginger, H.muluense and H.bougigannium regenerates plants through a somatic embryo induction way (Xiaowang, et al 2016; shogayan, et al 2014 Hamidou, et al 2008, 2009), and at present, the tissue culture technology that flowers of dwarf ginger directly regenerate plants through callus is still lacking. Therefore, it is necessary to invent a tissue culture method of the dwarf ginger flower to solve the above problems.
Disclosure of Invention
The invention aims to provide a tissue culture method for inducing callus and plant regeneration by hypocotyl of a bush sterile seedling. The invention can provide an effective tissue culture system for the large-scale in vitro propagation of the seedlings of the dwarf ginger flowers.
The invention comprises the following steps:
(1) Pretreatment of seeds: taking the fruits of dwarf ginger flowers with yellow pericarps and without cracking, putting the fruits into an ultra-clean workbench, cutting off the pedicles and the tails of the fruits by using a blade, dipping the surfaces of the fruits by using sterile cotton balls with alcohol solution to wipe the surfaces of the fruits, then sterilizing the fruits by using mercury bichloride solution, putting the fruits into sterile water for cleaning, taking out the fruits, putting the fruits into sterile dishes, and drying the water on the surfaces of the fruits to obtain the sterilized dwarf ginger flowers.
(2) Sterile seedling and hypocotyl callus wound surface induction: cutting the sterilized dwarf ginger flower fruits obtained in the step (1) along the fruit edges by using a blade on a super-clean workbench, sowing bright red seeds on a callus induction culture medium, and culturing for 115-135 d until hypocotyls of germinated aseptic seedlings are differentiated to form wound surfaces of callus; the sterile seedling and hypocotyl callus induction culture medium comprises: MS + 6-benzylaminopurine 6.0-9.0 mg/L + thidiazuron 6.0-8.0 mg/L + naphthylacetic acid 0.03-0.05 mg/L + coconut juice 25.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(3) Callus proliferation and greening: on an ultra-clean workbench, white callus formed by differentiating hypocotyl of the aseptic seedling obtained in the step (2), cutting off parts, roots and seeds above the hypocotyl of the aseptic seedling by using a blade, reserving the white callus, transferring the white callus to a callus proliferation and greening culture medium, and culturing for 60-90 days to obviously increase the volume of the callus and turn green; the callus proliferation and greening culture medium comprises: MS + 6-benzylamino adenine 5.0-6.0 mg/L + thidiazuron 0.5-0.6 mg/L + naphthylacetic acid 0.03-0.05 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(4) And (3) inducing and culturing cluster buds: transferring the green-turned callus obtained in the step (3) to a primary cluster bud induction culture medium on a superclean bench to induce cluster buds, and culturing for 35-45 days to obtain cluster bud masses; the cluster bud induction culture medium comprises: MS + 6-benzylamino adenine 5.0-6.0 mg/L + thidiazuron 0.5-0.6 mg/L + naphthylacetic acid 0.01-0.03 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(5) And (3) proliferating and culturing the cluster buds: cutting the cluster bud masses induced in the step (4) into small masses on a superclean workbench by using a blade, wherein each small mass contains 4-6 cluster buds, cutting off leaves and stem tips of the cluster buds on each small mass, reserving the base parts of the masses, inoculating a cluster bud multiplication culture medium, and culturing for 30-40 days to obtain the multiplied cluster buds; the cluster bud multiplication medium comprises: MS + 6-benzylamino adenine 4.0-5.0 mg/L + thidiazuron 0.4-0.5 mg/L + naphthylacetic acid 0.01-0.03 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(6) Rooting and seedling strengthening culture: selecting a robust plant with the plant height higher than 4cm and with 2-3 leaves from the seedling of the bush ginger obtained in the step (5), cutting off buds along the bud base part by a blade on a super clean workbench, inoculating the buds to a rooting and strong seedling culture medium, and culturing for 30-40 days to obtain a rooting seedling; the rooting and seedling strengthening culture medium comprises: MS, 1.0-1.2 mg/L of naphthylacetic acid, 20.0-30.0% of banana juice, 30-35 g/L of cane sugar, 12.5-13 g/L of carrageenan powder and pH 6.0-6.2.
(7) Domestication, transplantation and planting: selecting the dwarf ginger flower seedlings subjected to rooting culture in the step (6), selecting robust plants with the plant height of 6-8 cm, 3-5 leaves and 3-5 roots, placing the whole bottle in a greenhouse under natural illumination to harden the seedlings for 12-15 days, taking out the seedlings, cleaning the seedlings, and transplanting the seedlings in a root culture medium to obtain the regenerated dwarf ginger flower plants.
In order to further achieve the object of the present invention, preferably, in the step (1), the wiping with the cotton ball dipping alcohol solution comprises the following specific steps: firstly preparing 75% alcohol solution, and then dipping 75% alcohol solution by using a sterile cotton ball to wipe the surface of the fruit.
Preferably, in the step (1), the specific steps of sterilizing with mercuric chloride solution and then placing into sterile water for cleaning are as follows: sterilizing with 0.2% mercuric chloride solution for 22-25 min; then the mixture is put into sterile water to be cleaned for 5 to 6 times, and each time lasts for 3 to 4min.
Preferably, in the step (2), the wound surface of the hypocotyl callus is compact white callus.
Preferably, in step (3), the callus is volumetrically increased and turned green into a compact, green-textured tissue mass.
Preferably, in steps (2), (3), (4), (5) and (6), the medium is sterilized at 125 ℃ for 35 to 40min.
Preferably, in the steps (2), (3), (4), (5) and (6), the culture temperature is 25 to 27 ℃, the illumination intensity is 2500 to 2800lx, and the illumination time is 14 to 15h/d.
Preferably, in steps (2), (3), (4), (5) and (6), the pH of the medium is adjusted with an appropriate amount of 1mol/L sodium hydroxide solution.
The innovation point of the invention is that the seed germination and hypocotyl callus induction are carried out synchronously, the same culture medium is used, during the culture process, the hypocotyl of the aseptic seedling germinated from the seed contacts with the culture medium to form a callus wound surface, further the callus is obtained, and the callus is gradually transferred to a callus proliferation and greening culture medium and a cluster bud induction and proliferation culture medium to induce and proliferate the cluster buds, so as to obtain the regeneration plant of the bush ginger flower.
Compared with the prior art, the invention has the outstanding advantages and beneficial effects that:
(1) High efficiency of explant sterility: according to the method, the yellow and uncracked dwarf ginger flower fruit is taken, so that the phenomenon that the fruit is easy to pollute after being sterilized after being cracked is effectively avoided; after the fruit base and the fruit tail are cut off by a blade, the surface of the fruit is wiped by dipping 75% alcohol solution with an aseptic cotton ball, and then the fruit is sterilized for 22-25 min by 0.2% mercuric chloride solution; and then the seeds are put into sterile water for cleaning for 5 to 6 times, each time lasts for 3 to 4min, the seeds are taken out and put into sterile dishes to dry the moisture on the surfaces of the fruits, and sterile seeds are obtained.
(2) Induction of high-quality callus and plant regeneration thereof: the invention broadcasts the germ-free seed of the bush in the germ-free seedling and hypocotyl callus induction culture medium (MS + 6-benzylamino adenine 6.0-9.0 mg/L + thidiazuron 6.0-8.0 mg/L + naphthylacetic acid 0.03-0.05 mg/L + coconut juice 25.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4), which can make the hypocotyl of the germ-free seedling differentiate to form callus wound surface, and then obtain high quality callus, then transfer to callus proliferation and green transfer culture medium, cluster bud induction and proliferation culture medium to induce and proliferate cluster buds, root culture and transplant to obtain a large number of regeneration plants.
(3) The operation is simple, the cost investment is low: the method takes the seeds of the bush as explants, and the hypocotyl induction callus culture medium, the callus proliferation and greening culture medium, the cluster bud induction and proliferation culture medium, the rooting and seedling strengthening culture medium and the transplanting step of the germinated aseptic seedlings of the bush are all materials required by conventional tissue culture, so the required equipment is simple and the cost is low.
(4) The method for hypocotyl callus induction and plant regeneration of the bush aseptic seedling lays a foundation for in vitro rapid propagation and genetic transformation research of the bush; is beneficial to the large-scale popularization and application of the dwarf ginger flower seedlings.
Drawings
FIG. 1 is a photograph showing the differentiation of hypocotyl of aseptic seedling of Ardisia japonica into callus.
FIG. 2 is a photograph of callus after cutting off the portion above the hypocotyl, the root and the seed of a sterile seedling of Zingiber officinale.
FIG. 3 is a photograph of hypocotyl-induced callus proliferation and greening of a sterile seedling of Ardisia japonica.
FIG. 4 is a photograph of the primary culture of cluster buds of flowers of Ardisia japonica.
FIG. 5 is a photograph showing the multiplication culture of multiple shoots of flowers of Ardisia japonica.
FIG. 6 is a photograph of rooting culture of tissue culture seedlings of flowers of Ardisia japonica.
FIG. 7 is a photograph showing the transplantation of tissue culture seedlings of flowers of Ardisia japonica.
FIG. 8 is a photograph of the non-sprouting of pseudostem segment-induced protocorm-like bodies of flowers of Japanse bush (comparative example 1).
FIG. 9 is a photograph showing no germination of the rachis-induced callus of Zingiber officinale (comparative example 2).
FIG. 10 is a photograph showing the slow growth of adventitious bud-induced multiple shoots of a sterile seedling of Shigella (comparative example 3).
Detailed Description
For better understanding of the present invention, the present invention will be further described with reference to the following drawings and examples, which should not be construed as limiting the scope of the present invention.
MS is an international universal culture medium, and comprises the following components in a preparation method:
MS macroelement mother liquor is 100 times: 1000mL of culture medium is prepared, and 10mL of mother liquor is taken. The preparation method of the MS macroelement mother liquor comprises the following steps: 165g of ammonium nitrate, 190g of potassium nitrate, 44g of calcium chloride dihydrate, 37g of magnesium sulfate heptahydrate and 17g of monopotassium phosphate are respectively weighed, are respectively dissolved in sterilized water and then are mixed, and the volume is 1000mL.
MS microelement mother liquor is 100 times: 1000mL of culture medium is prepared, and 10mL of mother liquor is taken. The preparation method of the MS microelement mother solution comprises the following steps: respectively weighing 2.23g of manganese sulfate tetrahydrate, 0.86g of zinc sulfate heptahydrate, 0.62g of boric acid, 0.083g of potassium iodide, 0.25g of sodium molybdate dihydrate, 0.025g of copper sulfate pentahydrate and 0.025g of cobalt chloride hexahydrate, respectively adding sterilized water to dissolve, then mixing, and fixing the volume to 1000mL.
MS ferric salt mother liquor 100 times: 1000mL of culture medium is prepared, and 10mL of mother liquor is taken. The preparation method of the MS ferric salt mother liquor comprises the following steps: 3.73g of disodium ethylene diamine tetraacetate and 2.78g of ferrous sulfate heptahydrate are respectively weighed, and are respectively dissolved in sterilized water and then mixed, and the volume is fixed to 1000mL.
MS organic mother liquor 100 times: 1000mL of culture medium is prepared, and 10mL of mother liquor is taken. The preparation method of the MS organic mother liquor comprises the following steps: respectively weighing 0.05g of nicotinic acid, 0.05g of pyridoxine hydrochloride, 0.01g of thiamine hydrochloride, 10g of inositol and 0.2g of glycine, respectively adding sterilized water to dissolve, then mixing, and fixing the volume to 1000mL.
Example 1
A tissue culture method of dwarf ginger flowers comprises the following steps:
(1) Pretreatment of seeds: taking the fruits of dwarf ginger flowers with yellow pericarp and without cracking, placing the fruits in a superclean workbench, cutting off the pedicel and the tail of the fruits by a blade, dipping the surfaces of the fruits by using a sterile cotton ball to dip 75% alcohol solution for wiping, and then sterilizing for 25min by using 0.2% mercuric chloride solution; and cleaning in sterile water for 5 times, each time for 4min, taking out, air drying in sterile dish to remove water on the surface of the fruit, and collecting sterilized Ardisia japonica fruit.
(2) Sterile seedling and hypocotyl callus wound surface induction: cutting the sterilized fruit of the dwarf ginger flower obtained in the step (1) along the fruit ridge by using a blade, sowing bright red seeds on a callus induction culture medium, and culturing for 115 days until the hypocotyl of the germinated aseptic seedling is differentiated to form a wound surface of callus, which is compact and white callus (figure 1). The sterile seedling and hypocotyl callus induction culture medium comprises the following components: MS + 6-benzylaminopurine 9.0mg/L + thidiazuron 8.0mg/L + naphthylacetic acid 0.05mg/L + coconut juice 30.0% + sucrose 35g/L + carrageenan powder 13.5g/L, pH6.4.
(3) Callus proliferation and greening: and (3) white callus formed by hypocotyl differentiation of the aseptic seedling obtained in the step (2), cutting off parts, roots and seeds above the hypocotyl of the aseptic seedling by using a blade, reserving the white callus (figure 2), transferring the white callus to a callus proliferation and greening culture medium, and culturing for 60 days to obviously increase the volume of the callus and turn green (figure 3). The callus proliferation and greening culture medium comprises the following components: MS + 6-benzylaminopurine 6.0mg/L + thidiazuron 0.6mg/L + naphthylacetic acid 0.05mg/L + banana juice 30.0% + sucrose 35g/L + carrageenan powder 13.0g/L, pH6.2.
(4) And (3) inducing and culturing cluster buds: transferring the green-turning callus tissue obtained in the step (3) to an initial generation cluster bud induction culture medium to induce cluster buds, and culturing for 35d to obtain cluster bud masses (figure 4). The cluster bud induction culture medium comprises the following components: MS + 6-benzylamino adenine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.03mg/L + banana juice 20.0% + sucrose 40g/L + carrageenan powder 13.0g/L, pH6.2.
(5) And (3) carrying out multiplication culture on cluster buds: cutting the cluster bud clumps induced in the step (4) into small clumps by using a blade, wherein each small clump contains 5 cluster buds, cutting off leaves and stem tips of the cluster buds on each small clump, reserving base parts of the clumps, inoculating a cluster bud multiplication culture medium, and culturing for 40 days to obtain the multiplied cluster buds (figure 5). The cluster bud multiplication medium comprises the following components: MS + 6-benzylaminopurine 4.0mg/L + thidiazuron 0.4mg/L + naphthylacetic acid 0.01mg/L + banana juice 20.0% + sucrose 35g/L + carrageenan powder 13.0g/L, pH6.2.
(6) Rooting and seedling strengthening culture: selecting a robust plant with the plant height higher than 4cm and 3 growing leaves from the seedling of the bush ginger flower obtained in the step (5), cutting off buds along the bud base part by using a blade, inoculating the buds to a rooting and strong seedling culture medium, and culturing for 40 days to obtain a rooting seedling (figure 6). The rooting and seedling strengthening culture medium comprises the following components: MS, naphthylacetic acid 1.0mg/L, banana juice 20.0% + sucrose 30g/L + carrageenan powder 12.5g/L, pH6.0.
(7) Domesticating, transplanting and planting: selecting strong plants with the plant height of 6cm, 4 leaves and more than 3 roots of the seedlings of the dwarf ginger flowers after rooting culture in the step (6), putting the whole bottle in a greenhouse for seedling hardening under natural illumination for 15d, taking out the seedlings, cleaning the root culture medium, and transplanting to obtain the regeneration plants of the dwarf ginger flowers (figure 7).
In the above steps (2), (3), (4), (5) and (6), the sterilization condition of the medium was 125 ℃ for 40min.
In the steps (2), (3), (4), (5) and (6), the culture temperature is 26 ℃, the illumination intensity is 2800lx, and the illumination time is 15h/d.
In the above steps (2), (3), (4), (5) and (6), the pH of the medium was adjusted with an appropriate amount of 1mol/L sodium hydroxide solution.
Survival rate after transplantation of tissue culture seedlings of example 1 [ note: survival rate = (number of surviving seedlings after 10d transplanting/total number of seedlings at transplanting) × 100%) ] can reach more than 90%; example 1 batches of tissue culture seedlings were obtained from the processes of pretreatment of seeds, induction of callus from hypocotyl of aseptic seedlings, callus proliferation and greening, induction and proliferation of cluster buds, rooting and strengthening, and transplantation.
Example 2
A tissue culture method of dwarf ginger flowers comprises the following steps:
(1) Pretreatment of seeds: taking the fruits of dwarf ginger flowers with yellow pericarp and without cracking, placing the fruits in a superclean workbench, cutting off the pedicel and the tail of the fruits by a blade, dipping the surfaces of the fruits by using a sterile cotton ball to dip 75% alcohol solution for wiping, and then sterilizing for 22min by using 0.2% mercuric chloride solution; and cleaning in sterile water for 3min for 6 times, taking out, air drying in sterile dish to remove water on the surface of the fruit, and collecting sterilized Ardisia japonica fruit.
(2) Sterile seedling and hypocotyl callus wound surface induction: cutting the sterilized fruit of the dwarf ginger flower obtained in the step (1) along the fruit ridge by using a blade, sowing bright red seeds on a callus induction culture medium, and culturing for 135d until hypocotyls of the germinated aseptic seedlings are differentiated to form a wound surface of the callus. The sterile seedling and hypocotyl callus induction culture medium comprises the following components: MS + 6-benzylaminopurine 6.0mg/L + thidiazuron 6.0mg/L + naphthylacetic acid 0.03mg/L + coconut juice 25.0% + sucrose 35g/L + carrageenan powder 13.0g/L, pH6.2.
(3) Callus proliferation and greening: and (3) differentiating the hypocotyl of the aseptic seedling obtained in the step (2) to form a white callus, cutting off parts, roots and seeds above the hypocotyl of the aseptic seedling by using a blade, reserving the white callus, transferring the white callus to a callus proliferation and greening culture medium, and culturing for 90d to obviously increase the volume of the callus and turn green. The callus proliferation and greening culture medium comprises the following components: MS + 6-benzylamino adenine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.03mg/L + banana juice 20.0% + sucrose 35g/L + carrageenan powder 13.0g/L, pH6.2.
(4) And (3) inducing and culturing cluster buds: transferring the green callus obtained in the step (3) to an initial cluster bud induction culture medium to induce cluster buds, and culturing for 45 days to obtain cluster bud masses. The cluster bud induction culture medium comprises the following components: MS + 6-benzylamino adenine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.01mg/L + banana juice 20.0% + sucrose 40g/L + carrageenan powder 13.5g/L, pH6.2.
(5) And (3) carrying out multiplication culture on cluster buds: cutting the cluster bud clumps induced in the step (4) into small clumps by using a blade, wherein each small clump contains 5 cluster buds, cutting off leaves and stem tips of the cluster buds on each small clump, reserving base parts of the clumps, inoculating a cluster bud multiplication culture medium, and culturing for 35d to obtain the multiplied cluster buds. The cluster bud multiplication medium comprises the following components: MS + 6-benzylamino adenine 4.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.02mg/L + banana juice 20.0% + sucrose 35g/L + carrageenan powder 13.5g/L, pH6.2.
(6) Rooting and seedling strengthening culture: selecting a robust plant with the plant height higher than 4cm and with more than 2 growing leaves from the seedling of the bush ginger flower obtained in the step (5), cutting off buds along the bud base part by a blade, inoculating the buds to a rooting and seedling strengthening culture medium, and culturing for 35d to obtain a rooting seedling. The rooting and seedling strengthening culture medium comprises the following components: MS, naphthylacetic acid 1.0mg/L, banana juice 25.0% + sucrose 30g/L and carrageenan powder 13g/L, pH6.2.
(7) Domestication, transplantation and planting: selecting robust plants with the plant height of 6cm, 3 leaves and more than 3 roots of the dwarf ginger flower seedlings subjected to rooting culture in the step (6), putting the healthy plants in a whole bottle under natural illumination of a greenhouse for hardening the seedlings for 12d, taking out the seedlings, cleaning the seedlings, and transplanting the seedlings in a root culture medium to obtain the regeneration plants of the dwarf ginger flowers.
In the above steps (2), (3), (4), (5) and (6), the sterilization condition of the medium was 125 ℃ for 40min.
In the steps (2), (3), (4), (5) and (6), the culture temperature is 27 ℃, the illumination intensity is 2500lx, and the illumination time is 14h/d.
In the above steps (2), (3), (4), (5) and (6), the pH of the medium was adjusted with an appropriate amount of 1mol/L sodium hydroxide solution.
The survival rate of the tissue culture seedlings in the embodiment 2 after transplantation can reach more than 85 percent; example 2A large number of tissue culture seedlings of Ardisia japonica were obtained from the processes of pretreatment of seeds, callus induction of hypocotyl of aseptic seedlings, callus proliferation and greening, cluster bud induction and proliferation, rooting and strengthening, and transplantation.
Example 3
A tissue culture method of dwarf ginger flower comprises the following steps:
(1) Pretreatment of seeds: taking the fruits of dwarf ginger flowers with yellow pericarp and without cracking, placing the fruits in a superclean workbench, cutting off the pedicel and the tail of the fruits by a blade, dipping the surfaces of the fruits by using a sterile cotton ball to dip 75% alcohol solution for wiping, and then sterilizing for 24min by using 0.2% mercuric chloride solution; and cleaning in sterile water for 5 times, each time for 4min, taking out, air drying in sterile dish to remove water on the surface of the fruit, and collecting sterilized Ardisia japonica fruit.
(2) Wound surface induction of sterile seedlings and hypocotyl calli: cutting the sterilized fruit of the dwarf ginger flower obtained in the step (1) along the fruit ridge by using a blade, sowing bright red seeds on a callus induction culture medium, and culturing for 125 days until hypocotyls of the germinated aseptic seedlings are differentiated to form a wound surface of the callus. The components of the sterile seedling and hypocotyl callus induction culture medium are as follows: MS + 6-benzylaminopurine 8.0mg/L + thidiazuron 7.0mg/L + naphthylacetic acid 0.04mg/L + coconut juice 25.0% + sucrose 40g/L + carrageenan powder 13.0g/L, pH6.2.
(3) Callus proliferation and greening: and (3) white callus formed by hypocotyl differentiation of the aseptic seedling obtained in the step (2), cutting off parts, roots and seeds above the hypocotyl of the aseptic seedling by using a blade, reserving the white callus, transferring the white callus to a callus proliferation and greening culture medium, and culturing for 80d to obviously increase the volume of the callus and turn green. The callus proliferation and greening culture medium comprises the following components: MS + 6-benzylamino adenine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.04mg/L + banana juice 30.0% + sucrose 40g/L + carrageenan powder 13.0g/L, pH6.4.
(4) And (3) inducing and culturing cluster buds: transferring the green callus obtained in the step (3) to an initial cluster bud induction culture medium to induce cluster buds, and culturing for 40 days to obtain cluster bud masses. The cluster bud induction culture medium comprises the following components: MS + 6-benzylaminopurine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.01mg/L + banana juice 30.0% + sucrose 40g/L + carrageenan powder 13.5g/L, pH6.4.
(5) And (3) carrying out multiplication culture on cluster buds: cutting the cluster bud clumps induced in the step (4) into small clumps by using a blade, wherein each small clump contains 4 cluster buds, cutting off leaves and stem tips of the cluster buds on each small clump, reserving base parts of the clumps, inoculating a cluster bud multiplication culture medium, and culturing for 30 days to obtain the multiplied cluster buds. The cluster bud multiplication medium comprises the following components: MS + 6-benzylamino adenine 5.0mg/L + thidiazuron 0.5mg/L + naphthylacetic acid 0.03mg/L + banana juice 30.0% + sucrose 40g/L + carrageenan powder 13.5g/L, pH6.4.
(6) Rooting and seedling strengthening culture: selecting a robust plant with the plant height higher than 4cm and with more than 2 growing leaves from the seedling of the bush ginger flower obtained in the step (5), cutting off buds along the bud base part by a blade, inoculating the buds to a rooting and seedling strengthening culture medium, and culturing for 30 days to obtain a rooting seedling. The rooting and seedling strengthening culture medium comprises the following components: MS, 1.2mg/L of naphthylacetic acid, 30.0% of banana juice, 35g/L of cane sugar and 13g/L of carrageenan powder, and the pH value is 6.2.
(7) Domestication, transplantation and planting: selecting strong plants with the plant height of 8cm, 3 leaves and more than 3 roots which are planted in the seedlings of the bush flowers after the rooting culture in the step (6), putting the whole bottle in a greenhouse under natural illumination for hardening the seedlings for 12d, taking out the seedlings, cleaning the seedlings, and transplanting the seedlings in a root culture medium to obtain the regeneration plants of the bush flowers.
In the above steps (2), (3), (4), (5) and (6), the sterilization conditions of the medium were 125 ℃ for 35min.
In the steps (2), (3), (4), (5) and (6), the culture temperature is 27 ℃, the illumination intensity is 2600lx, and the illumination time is 14h/d.
In the above steps (2), (3), (4), (5) and (6), the pH of the medium was adjusted with an appropriate amount of 1mol/L sodium hydroxide solution.
The survival rate of the tissue culture seedlings in the embodiment 3 after transplantation can reach over 86 percent; example 3A large number of tissue culture seedlings of Ardisia japonica were obtained from the processes of pretreatment of seeds, callus induction of hypocotyl of aseptic seedlings, callus proliferation and greening, cluster bud induction and proliferation, rooting and strengthening, and transplantation.
Comparative example 1
(1) Selection and disinfection of explants: selecting a pseudostem of a dwarf ginger plant with inflorescence, cutting off leaves, peeling off leaf sheaths layer by layer, cutting the pseudostem into pseudostem sections with the length of 4.0-6.0 cm along the pseudostem nodes, dipping 75% alcohol solution by using a sterile cotton ball to wipe the surface of the pseudostem sections, and sterilizing for 20min by using 0.1% mercuric chloride solution; then the mixture is put into sterile water to be cleaned for 5 to 6 times, and each time lasts for 3 to 4min. Taking out and putting into a sterile dish to air dry the water on the surface of the pseudostem section to obtain the sterilized pseudostem section of the dwarf ginger flower.
(2) Primary induction: cutting two ends of pseudostem nodes of the sterilized pseudostem section of the dwarf ginger flower obtained in the step (1) by using a blade on a clean bench, reserving pseudostems with the pseudostem nodes of 0.4-0.6 cm, and inoculating the pseudostems to a protocorm-like induction culture medium for culture: MS +5 mg/L6-BA +2mg/L TDZ +0.01mg/L NAA, and the temperature and time for sterilizing the medium, the temperature and amount of light for culturing and the time for culturing of the comparative example were the same as those of example 1. After 135d cultivation, there is no pollution, but no sprout in pseudostem segment.
Comparative example 2
(1) Selection and disinfection of explants: selecting the rachis of the dwarf ginger flower as an explant, cutting the rachis into segments of 3.0-4.0 cm by using a blade, dipping a sterile cotton ball into 75% alcohol solution to wipe the surface of the rachis segments, and disinfecting for 20min by using 0.1% mercuric chloride solution; then the mixture is put into sterile water to be cleaned for 5 to 6 times, and each time lasts for 3 to 4min. Taking out and putting into a sterile dish to air dry the water on the surface of the rachis segment, and obtaining the sterilized rachis segment of the dwarf ginger flower.
(2) Primary induction: cutting off two tail ends of the sterilized rachis segment of the dwarf ginger flower obtained in the step (1) by using blades on a clean bench, reserving the rachis segment of 1.2-1.5 cm, and inoculating the rachis segment to a callus induction culture medium for culture: MS +1 mg/L6-BA +2mg/L NAA, the sterilization temperature and time of the medium of this comparative example, the culture temperature, the amount of light and the time were the same as those of example 1. After 135d culture, no pollution exists, but no callus appears on the rachis segment.
Comparative example 3
(1) Selection and disinfection of explants: taking the fruits of dwarf ginger which have yellowed pericarps and are not cracked, putting the fruits into a clean bench, cutting off the pedicles and the tails of the fruits by using a blade, dipping the surfaces of the fruits by using a sterile cotton ball to dip 75% alcohol solution, and then disinfecting the surfaces of the fruits by using 0.2% mercuric chloride solution for 25min; and cleaning in sterile water for 5 times, each time for 4min, taking out, air drying in sterile dish to remove water on the surface of the fruit, and collecting sterilized Ardisia japonica fruit.
(2) Obtaining and primarily inducing aseptic seedlings: and (2) cutting the sterilized fruits of the dwarf ginger flowers obtained in the step (1) along the fruit edges by using a blade, and sowing bright red seeds on an MS culture medium until the seeds germinate aseptic seedlings. Taking the adventitious bud of the aseptic seedling of the bush ginger flower as an explant, inoculating the explant on a cluster bud multiplication culture medium for culture: MS +1 mg/L6-BA +0.2mg/L NAA. The temperature and time for sterilization, the temperature for cultivation, the amount of light irradiation and the time for the culture medium of this comparative example were the same as those of example 1. After 135d culture, the method has no pollution, but the bud grows slowly.
FIG. 1 shows photographs of the hypocotyl differentiation of the seedlings of the Ardisia japonica into callus. The hypocotyls of the seedlings of the dwarf ginger flower seeds are seen to expand to form white callus. FIG. 2 shows a photograph of callus after cutting off the portion above the hypocotyl, the root and the seed of a seedling of a flower of Zingiber officinale. FIG. 3 is a photograph showing hypocotyl-induced callus proliferation and greening of dwarf ginger seed seedlings. An enlarged greenish, compact mass of tissue is visible. FIG. 4 shows photographs of the first generation culture of cluster buds of flowers of Shibataea chinensis. FIG. 5 shows photographs of the multiplication culture of the cluster buds of the flowers of Shibataea chinensis. FIG. 6 is a photograph showing the rooting culture of a tissue culture seedling of flowers of Zingiber officinale. FIG. 7 shows a photograph of a tissue culture seedling transplantation of flowers of Ardisia japonica. FIG. 8 is a photograph showing the absence of sprouting in the pseudostem-segment-induced protocorm-like body of Zingiber officinale Roscoe (comparative example 1). FIG. 9 is a photograph showing the absence of sprouting in the rachis-induced callus of Zingiber officinale flower (comparative example 2). FIG. 10 is a photograph showing the slow growth of adventitious bud-induced buds of a sterile seedling of Zingiber officinale Roscoe (comparative example 3).
As can be seen from the comparative examples 1, 2 and 3, the pseudostem and the rachis of the dwarf ginger plant with the inflorescence and the adventitious bud of the aseptic seedling are taken as the explants, and the explant has no germination and the adventitious bud grows slowly; in the embodiment, the same culture medium (MS + 6-benzylaminopurine 6.0-9.0 mg/L + thidiazuron 6.0-8.0 mg/L + naphthylacetic acid 0.03-0.05 mg/L + coconut juice 25.0-30.0% + sucrose 35-40 g/L + carrageenan 13.0-13.5 g/L + pH 6.2-6.4) is used in the sterile seed germination and hypocotyl callus induction steps, during the culture process, the hypocotyl of the sterile seedling of the seed germination is contacted with the culture medium to form a callus wound surface, so that callus is obtained, and the callus is gradually transferred to a callus proliferation and greening culture medium and a cluster bud induction and proliferation culture medium to induce and proliferate cluster buds, so that a regenerated plant of the bush flower is obtained, and a tissue culture system of the bush flower is successfully established. The invention lays an important foundation for tissue culture rapid propagation and gene engineering research of the dwarf ginger flower.
Claims (8)
1. A tissue culture method of dwarf ginger flowers is characterized by comprising the following steps:
(1) Pretreatment of seeds: taking the fruits of the dwarf ginger flowers which are not cracked but have yellow pericarp, putting the fruits into a clean bench, cutting the pedicels and the tails of the fruits by a blade, dipping sterile cotton balls in alcohol solution to wipe the surfaces of the fruits, sterilizing by mercuric chloride solution, putting the fruits into sterile water for cleaning, taking the fruits out of sterile dishes, and airing the water on the surfaces of the fruits to obtain the sterilized fruits of the dwarf ginger flowers.
(2) Wound surface induction of sterile seedlings and hypocotyl calli: cutting the sterilized dwarf ginger flower fruits obtained in the step (1) along the fruit edges by using a blade on a superclean workbench, sowing bright red seeds on a callus induction culture medium, and culturing for 115-135 days until hypocotyls of the germinated aseptic seedlings are differentiated to form wound surfaces of callus; the sterile seedling and hypocotyl callus induction culture medium comprises: MS + 6-benzylaminopurine 6.0-9.0 mg/L + thidiazuron 6.0-8.0 mg/L + naphthylacetic acid 0.03-0.05 mg/L + coconut juice 25.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(3) Callus proliferation and greening: on an ultra-clean workbench, cutting off parts, roots and seeds above the hypocotyl of the aseptic seedling obtained in the step (2) by using a blade to form white callus through hypocotyl differentiation, reserving the white callus, transferring the white callus to a callus proliferation and greening culture medium, and culturing for 60-90 days to obviously increase the volume of the callus and turn green; the callus proliferation and green turning culture medium comprises: MS + 6-benzylamino adenine 5.0-6.0 mg/L + thidiazuron 0.5-0.6 mg/L + naphthylacetic acid 0.03-0.05 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(4) And (3) inducing and culturing cluster buds: transferring the green-turned callus obtained in the step (3) to a primary cluster bud induction culture medium on a superclean bench to induce cluster buds, and culturing for 35-45 days to obtain cluster bud masses; the cluster bud induction culture medium comprises: MS + 6-benzylamino adenine 5.0-6.0 mg/L + thidiazuron 0.5-0.6 mg/L + naphthylacetic acid 0.01-0.03 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(5) And (3) carrying out multiplication culture on cluster buds: cutting the cluster bud masses induced in the step (4) into small masses on a superclean bench by using a blade, wherein each small mass contains 4-6 cluster buds, cutting off leaves and stem tips of the cluster buds on each small mass, reserving the base parts of the masses, inoculating a cluster bud multiplication culture medium, and culturing for 30-40 days to obtain the multiplied cluster buds; the cluster bud multiplication medium comprises: MS + 6-benzylamino adenine 4.0-5.0 mg/L + thidiazuron 0.4-0.5 mg/L + naphthylacetic acid 0.01-0.03 mg/L + banana juice 20.0-30.0% + sucrose 35-40 g/L + carrageenan powder 13.0-13.5 g/L, pH6.2-6.4.
(6) Rooting and seedling strengthening culture: selecting a strong plant with a height of more than 4cm and 2-3 leaves, cutting off buds along a bud base by using a blade on a clean bench, inoculating the buds to a rooting and strong seedling culture medium, and culturing for 30-40 days to obtain a rooting seedling from the seedling of the dwarf ginger flower obtained in the step (5); the rooting and seedling strengthening culture medium comprises: MS, 1.0-1.2 mg/L of naphthylacetic acid, 20.0-30.0% of banana juice, 30-35 g/L of cane sugar, 12.5-13 g/L of carrageenan powder and pH 6.0-6.2.
(7) Domestication, transplantation and planting: selecting strong plants with the plant height of 6-8 cm, 3-5 leaves and 3-5 roots of the seedlings of the dwarf ginger flowers after rooting culture in the step (6), putting the whole bottle in a greenhouse under natural illumination, hardening the seedlings for 12-15 days, taking out the seedlings, cleaning the root culture medium, and transplanting to obtain the regeneration plants of the dwarf ginger flowers.
2. The tissue culture method of dwarf ginger flower according to claim 1, wherein in the step (1), the specific steps of dipping sterile cotton balls in the alcohol solution and wiping the sterile cotton balls are as follows: firstly preparing 75% alcohol solution, and then dipping 75% alcohol solution by using a sterile cotton ball to wipe the surface of the fruit.
3. The tissue culture method of dwarf ginger flower according to claim 1, wherein in the step (1), the steps of sterilizing with mercuric chloride solution and then placing into sterile water for washing comprise: sterilizing with 0.2% mercuric chloride solution for 22-25 min; then the mixture is put into sterile water for cleaning for 5 to 6 times, and each time lasts for 3 to 4min.
4. The tissue culture method of dwarf ginger flower according to claim 1, wherein in the step (2), the wound surface of hypocotyl callus is compact white callus.
5. The method for tissue culture of Ardisia japonica according to claim 1, wherein in step (3), the callus is increased in volume and turned green into a compact green tissue mass.
6. The tissue culture method of Ardisia japonica according to claim 1, wherein the sterilization conditions of the culture medium in steps (2), (3), (4), (5) and (6) are 125 ℃ for 35-40 min.
7. The tissue culture method of Ardisia japonica according to claim 1, wherein the culture temperature in steps (2), (3), (4), (5) and (6) is 25-27 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 14-15 h/d.
8. The tissue culture method of Ardisia japonica according to claim 1, wherein the pH of the culture medium in steps (2), (3), (4), (5) and (6) is adjusted with an appropriate amount of 1mol/L NaOH solution.
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