CN106942052B - A kind of industrial seedling rearing and artificial cultivation method of dryopteris fragrans - Google Patents
A kind of industrial seedling rearing and artificial cultivation method of dryopteris fragrans Download PDFInfo
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- CN106942052B CN106942052B CN201710121011.9A CN201710121011A CN106942052B CN 106942052 B CN106942052 B CN 106942052B CN 201710121011 A CN201710121011 A CN 201710121011A CN 106942052 B CN106942052 B CN 106942052B
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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
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of high yield artificial cultivation methods of dryopteris fragrans sporinite, belong to dryopteris fragrans technical field of cultivation.Method provided by the present invention is that the mature spore of dryopteris fragrans is prepared into sterile spore suspension, it is cultivated after sterile spore suspension is inoculated in fluid nutrient medium the synchronous sprouting of first dark processing, continue to cultivate in solid medium to long be inoculated in the gametophyte stage, obtains the good dryopteris fragrans gametophyte of upgrowth situation；Dryopteris fragrans gametophyte is removed to be moved into cultivate after cleaning and is cultivated in the soil matrix for having floating fern gametophyte, mature dryopteris fragrans sporinite is obtained.The present invention overcomes the Period Process by tissue cultures and soil incubation inducing spore body is longer, and wild dryopteris fragrans are easy dead problem in Induction Process, suitable for the factorial production of dryopteris fragrans sporinite, and to realize that the large-scale production of dryopteris fragrans sporinite provides strong technical support.
Description
Technical field
The present invention relates to a kind of industrial seedling rearing of dryopteris fragrans and artificial cultivation methods, belong to dryopteris fragrans cultivation technique
Field.
Background technique
Dryopteris fragrans (Dryopteris fragrans (L.) Schott) are a kind of wild perennial pteridophytes, are squama
Hair Pteridiaceae Dryopteris, fresh blade are rubbed broken with fragrance, also referred to as spiceleaf shield-fern.Under most hygrophilous and hot environments
The pteridophyte of growth is completely different, and the growing environment of dryopteris fragrans is extremely special, be born in the talcum slope formed after volcanic eruption,
In lava gap and volcano magma seam.Worldwide, dryopteris fragrans are distributed in China, Japan, Korea, Mongolia, Russia
And North America, the severe cold areas that height above sea level is about 1000~1700m.In China, it is five big to be distributed mainly on Heilongjiang Province for dryopteris fragrans
Even pond area.
Dryopteris fragrans can be low temperature resistant, remains to survive under the conditions of -20 DEG C, but dryopteris fragrans slow growth, usually has
Longer growth period needs to form mature sporinite by cultivating one's ability for many years.In recent years, dryopteris fragrans are few to psoriasis, blueness
The treating for skin disease such as year acne, psoriasis have good curative effect and widely are paid close attention to, with good development prospect.But
It is since ecological environment is by destruction and excavation of the mankind to its medical value, wild dryopteris fragrans resource is by serious shadow
It rings.It is being lured by the longer and wild dryopteris fragrans of the Period Process of common tissue cultures and soil incubation inducing spore body
It is easy death during leading, therefore, how artificial cultivation technique high-yield culturing dryopteris fragrans to be utilized to become skill in the prior art
Art problem, does not also there is relevant report in the prior art.
Summary of the invention
In order to realize the artificial soil culturing of dryopteris fragrans, a large amount of mature dryopteris fragrans sporinite plant, this hair are obtained
It is bright to provide the industrial seedling rearing and artificial cultivation method of a kind of dryopteris fragrans, the technical solution adopted is as follows:
The purpose of the present invention is to provide a kind of industrial seedling rearing of dryopteris fragrans and artificial cultivation method, this method is group
Culture method, comprising the following steps:
Step 1: the mature spore of dryopteris fragrans is prepared into sterile spore suspension, sterile spore suspension is inoculated with
First dark processing is cultivated after synchronizing sprouting in culture medium, is inoculated in solid medium to length to gametophyte stage
Continue to cultivate, obtains the good dryopteris fragrans gametophyte of upgrowth situation；
Step 2: the dryopteris fragrans gametophyte that step 1 obtains, which is removed, to be moved into cultivate after cleaning floating fern gametophyte
It is cultivated in soil matrix, obtains mature dryopteris fragrans sporinite.
Fluid nutrient medium described in step 1 of the present invention is the Knop ' s fluid nutrient medium of improvement, is contained in the culture medium
0.25g NaH2PO4, 0.25g KNO3, 0.25g MgSO4, 1.0g Ca (NO3)2With distilled water 1000mL, pH 5.7-5.8.
Solid medium described in step 1 of the present invention is 1/2MS+ sucrose+Solid agar culture, is contained in the culture medium
1/2MS, 3.0% sucrose (quality), 0.7%~0.8% agar (quality), pH 5.7-5.8.
The condition of culture described in step 1 and step 2 of the present invention is 22 DEG C ± 5 DEG C, illumination 12h-16h, dark 8h-
12h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
As the further preferred scheme of the present invention, the condition of culture described in step 1 and step 2 is 25 DEG C, illumination
16h, dark 8h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
Soil matrix described in step 2 of the present invention is made of being configured as turfy soil and vermiculite according to volume ratio for 3:1.
The number of floating fern gametophyte described in step 2 of the present invention is 1 plant~5 plants.Preferred embodiment is 1 plant~3 plants.
Preferably, it cleans described in step 2 and is cleaned using 22 DEG C ± 5 DEG C of clear water.
It is a further object of the present invention to provide the industrial seedling rearing of another dryopteris fragrans and artificial cultivation method, the party
Method is earth culture method, comprising the following steps:
Step 1: the mature spore of dryopteris fragrans is prepared into sterile spore suspension, sterile spore suspension is inoculated with
In the container for being loaded with soil matrix, whole container moisturizing is covered with plastic film after watering into soil matrix, in 22 DEG C
± 5 DEG C are cultivated, and the good dryopteris fragrans gametophyte of upgrowth situation is obtained；
Step 2: the dryopteris fragrans gametophyte that step 1 obtains, which is moved into cultivation, to be had in the soil matrix of floating fern gametophyte
It is cultivated, obtains mature dryopteris fragrans sporinite.
It is by turf as soil matrix described in soil matrix described in the preferred solution of the invention step 1 and step 2
Soil and vermiculite are according to made of volume ratio 3:1 configuration.
The number of floating fern gametophyte described in step 2 of the present invention is 1 plant~5 plants.Preferred embodiment is 1 plant~3 plants.
The condition of culture described in step 1 and step 2 of the present invention is 22 DEG C ± 5 DEG C, illumination 12h-16h, dark 8h-
12h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
As the further preferred scheme of the present invention, the condition of culture described in step 1 and step 2 is 25 DEG C, illumination
16h, dark 8h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
The present invention utilizes ecological principle, passes through main plant object and annual aquatic thick stalk floating fern (Ceratopteris
Thalictroides) common sowing connection life, floating fern root system and dryopteris fragrans root system syntrophism.In floating fern root system fixing soil
Moisture so that soil remained moist, moisture is fully absorbed and used.In addition, soil moisture is also beneficial to sperm and ovum
The combination of cell forms sporinite, can be improved the survival rate of dryopteris fragrans sporinite.Aerial part increases plastic film covering,
Invalid transpiration in soil is changed into the effective use of plant, improves the growth rate of dryopteris fragrans, one kind is formed and quickly obtains
The plant strain growth mode of largely mature dryopteris fragrans sporinite is obtained, to achieve the purpose that realize dryopteris fragrans industrial seedling rearing.
The invention has the advantages that:
1, the induction and training mode of the invention by dryopteris fragrans sporinite is studied, and establishes androconium hair for the first time
The high yield artificial cultivation method of fern, in the case where keeping normal edaphic condition, implantation can be fixed sufficiently and be utilized the present invention
The annual aquatic thick stalk floating fern of moisture, by by dryopteris fragrans and annual aquatic thick stalk floating fern (Ceratopteris
Thalictroides) common sowing connection life, makes floating fern root system and dryopteris fragrans root system syntrophism.Wherein floating fern can increase
Ventilatory capacity, sufficiently fixed moisture, so that soil remained moist, moisture is adequately absorbed and utilized, and it is rising to reduce soil inavailable
Effect, can speed up the formation of dryopteris fragrans sporinite, and improve the inductivity of dryopteris fragrans sporinite, meanwhile, on the ground
Part increases plastic film covering, and the invalid transpiration in soil is changed into the effective use of plant, further increases androconium hair
The growth rate of fern ultimately forms a kind of plant strain growth mode for being quickly obtained a large amount of mature dryopteris fragrans sporinites, to reach
The purpose of dryopteris fragrans sporinite the factorial production is realized, to realize that the large-scale production of dryopteris fragrans sporinite provides effectively
Technical support, while laying a good foundation for the sustainable uses of dryopteris fragrans plant resources.
2, the present invention overcomes longer by the Period Process of tissue cultures and soil incubation inducing spore body and wild
Raw dryopteris fragrans are easy dead problem in Induction Process, and method of the invention substantially reduces the growth week of dryopteris fragrans
Phase, and the sporogenesis sporinite of method culture dryopteris fragrans maturation of the invention is utilized, cultivation cycle only needs 90 days.Usually exist
Formation dryopteris fragrans maturation spore is extremely difficult to sporinite is formed under natural conditions, and spore germination rate is about 0%~7%, this
The method of invention also substantially increases the inductivity of dryopteris fragrans sporinite, and tissue culture method dryopteris fragrans sporinite inductivity is up to
66.67%, earth culture method dryopteris fragrans sporinite inductivity is up to 86.67%.
3, the present invention will make between dryopteris fragrans and Parkeriaceae plant, and the two produces association effect, and the present invention provides for the first time
One biological control measure promotes the maturation of dryopteris fragrans sporinite, and the large-scale production for realization dryopteris fragrans sporinite provides
Strong technical support.Production cost is low simultaneously, cultivation management difficulty is low and cost, it is easy to accomplish dryopteris fragrans sporinite
The factorial production.
Detailed description of the invention
Fig. 1 is the dryopteris fragrans seedling induced by dryopteris fragrans gametophyte.
Fig. 2 is the dryopteris fragrans seedling of tissue culture dryopteris fragrans gametophyte induction.
Fig. 3 is the dryopteris fragrans seedling of earth culture dryopteris fragrans gametophyte induction.
Fig. 4 is mass propgation dryopteris fragrans sporinite.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the present invention should not be limited by the examples.
MATERIALS METHODS in following embodiment is as follows:
1 test material
Pteridophyte dryopteris fragrans pick up between the rock seam of Heilongjiang Province Wudalianchi slag.
2 condition of culture
Minimal medium is the Knop ' s formula (NaH of improvement2PO4 0.25g、KNO3 0.25g、MgSO4 0.25g、Ca
(NO3) 21.0g, distilled water 1000mL).Gametophyte growth medium is 1/2MS+ sucrose+agar solid medium, wherein
Comprising MS (containing a great number of elements, microelement and organic principle), then add 3.0% sucrose and the agar of 0.7-0.8%.
Culture medium is 5.7-5.8 with sodium hydroxide or hydrochloric acid adjustment pH value before high pressure sterilization；
22 DEG C ± 5 DEG C of cultivation temperature (preferably 25 DEG C), illumination 12h-16h (preferably 16h), dark 8h-12h is (preferably
For 8h), light intensity is about 150 μm of olm-2·s-1-180μmol·m-2·s-1, remain moist in incubation.
Embodiment 1:
Present embodiments provide the industrial seedling rearing and artificial cultivation method (tissue culture method) of a kind of dryopteris fragrans, specific proved recipe
Method is as follows: 1, the growth of gametophyte
It takes the spore of mature dryopteris fragrans to be placed in 1.5mL centrifuge tube, instills sterile water, sufficiently oscillation makes into suspension
Liquid, 4 000 r/min are centrifuged 1min, precipitate spore all, discard supernatant liquid.The 0.1% of about 1.0mL is instilled in centrifuge tube again
HgCl2Solution, sterilize 1min, and aseptic water washing 4~5 times, sterile spore suspension is obtained with above-mentioned centrifugal method.
Solid medium uses 1/2MS, sucrose concentration 3.0%, agar concentration 0.7%-0.8%.Culture medium packing
It is spare after high pressure sterilization (121 DEG C, 20min) in triangular flask.
Fluid nutrient medium culture is will to improve Knop ' s fluid nutrient medium direct packaging in triangular flask, high pressure sterilization (121
DEG C, 20min) after it is spare.
Soil matrix is that the turfy soil of high pressure sterilization (121 DEG C, 20min) and vermiculite have been prepared with the ratio of volume ratio 3:1
It forms, is divided in spare in polypots.
After the sterile spore suspension of acquisition is inoculated in the Knop ' s culture medium of improvement the synchronous sprouting of first dark processing
It is cultivated, is inoculated in the culture medium of+0.8% agar of 1/2MS+3.0% sucrose and is cultivated to the gametophyte stage
15d。
2, the induction of sporinite
The tissue culture dryopteris fragrans gametophyte of acquisition is carefully removed from bottle with tweezers, and by the culture medium of attachment with 22
DEG C ± 5 DEG C of clear water is cleaned, and is moved into the polypots (number A0-A6) for filling soil matrix and is cultivated 60d.Soil matrix is
Turfy soil and vermiculite are formulated with the ratio of 3:1.Experiment uses single factor test RANDOMIZED BLOCK DESIGN, if 6 processing: A0 polypots
Middle cultivation has 3 plants of tissue culture dryopteris fragrans gametophytes, no floating fern gametophyte.A1Planting in polypots has 3 plants of tissue culture dryopteris fragrans gametophytes
With 1 plant of floating fern gametophyte.A2Planting in polypots has 3 plants of tissue culture dryopteris fragrans gametophytes and 2 plants of floating fern gametophytes.A3In polypots
Cultivation has 3 plants of tissue culture dryopteris fragrans gametophytes and 3 plants of floating fern gametophytes.A4Planting in polypots has 3 plants of tissue culture dryopteris fragrans gametophytes
With 4 plants of floating fern gametophytes.A5Planting in polypots has 3 plants of tissue culture dryopteris fragrans gametophytes and 5 plants of floating fern gametophytes.Each processing is done
3 repetitions irrigate matrix with distilled water, and plastic film covers moisturizing, cultivate in 22 DEG C ± 5 DEG C.Count dryopteris fragrans spore
Body inductivity, as shown in table 1.The dryopteris fragrans seedling induced by dryopteris fragrans gametophyte is as shown in Figure 1, tissue culture dryopteris fragrans
The dryopteris fragrans seedling of gametophyte induction is as shown in Fig. 2, mass propgation dryopteris fragrans sporinite is as shown in Figure 4.
1 tissue-cultured seedling of table grows up to the comparison of sporinite
As it can be seen from table 1 the method for the present invention is using the floating fern gametophyte of different number with dryopteris fragrans tissue culture gametophyte
Association grows sporinite.It was found that formation significant effect of the 1-3 plants of floating fern gametophytes to dryopteris fragrans sporinite, inductivity is high
Up to 66.67%.When wherein 2 plants of floating fern gametophytes are with the gametophyte association of dryopteris fragrans tissue culture, sporinite growth population is most, does not have
The sporinite growth population of floating fern gametophyte association is minimum, and experimental group and control group Comparative result are obvious, shows floating fern to androconium
Certain facilitation is played in the growth of hair fern sporinite.The experimental group and control group phase of dryopteris fragrans and floating fern co-incubation
Than induction time shortens 30d, and induction number increases by 1 times, and growth rate improves 25.00%.
Embodiment 2
Present embodiments provide the industrial seedling rearing and artificial cultivation method (earth culture method) of a kind of dryopteris fragrans, specific proved recipe
Method is as follows: 1, the growth of gametophyte
It takes the spore of mature dryopteris fragrans to be placed in 1.5mL centrifuge tube, instills sterile water, sufficiently oscillation makes into suspension
Liquid, 4 000 r/min are centrifuged 1min, precipitate spore all, discard supernatant liquid.The 0.1% of about 1.0mL is instilled in centrifuge tube again
HgCl2Solution, sterilize 1min, and aseptic water washing 4-5 times obtains sterile spore suspension with above-mentioned centrifugal method.
Soil matrix is that the turfy soil of high pressure sterilization (121 DEG C, 20min) and vermiculite have been prepared with the ratio of volume ratio 3:1
It forms, is divided in spare in polypots.
The spore suspension of acquisition is inoculated in the polypots for fill soil matrix and is cultivated, is poured into soil matrix
For water to soil moisture, plastic film covers whole container moisturizing, in 22 DEG C of ± 5 DEG C of culture 45d.Mature dryopteris fragrans are obtained to match
Daughter.
2, the induction of sporinite
The dryopteris fragrans gametophyte of acquisition is moved into the flexible plastic alms bowl for filling transplanting medium, using the random district's groups of single factor test
Design equally also designs 6 processing: B0Planting in polypots has 3 plants of earth culture dryopteris fragrans gametophytes, no floating fern gametophyte.B1Plastics
Planting in alms bowl has 3 plants of earth culture dryopteris fragrans gametophytes and 1 plant of floating fern gametophyte.B2Planting in polypots has 3 plants of earth culture dryopteris fragrans to match
Daughter and 2 plants of floating fern gametophytes.B3Planting in polypots has 3 plants of earth culture dryopteris fragrans gametophytes and 3 plants of floating fern gametophytes.B4Plastics
Planting in alms bowl has 3 plants of earth culture dryopteris fragrans gametophytes and 4 plants of floating fern gametophytes.B5Planting in polypots has 3 plants of earth culture dryopteris fragrans to match
Daughter and 5 plants of floating fern gametophytes.3 repetitions are done in each processing, irrigate matrix with distilled water, plastic film covers moisturizing, in 22
DEG C of ± 5 DEG C cultures.Count dryopteris fragrans sporinite inductivity.The results are shown in Table 2.The perfume (or spice) induced by dryopteris fragrans gametophyte
Shield-fern seedling as shown in Figure 1, earth culture dryopteris fragrans gametophyte induction dryopteris fragrans seedling as shown in figure 3, mass propgation is fragrant
Shield-fern sporinite is as shown in Figure 4.
2 earth culture seedling of table grows up to the comparison of sporinite
From table 2 it can be seen that the method for the present invention is using the floating fern gametophyte of different number with dryopteris fragrans earth culture gametophyte
Association grows sporinite.It was found that formation significant effect of the 1-3 plants of floating fern gametophytes to dryopteris fragrans sporinite, inductivity is high
Up to 86.67%.When wherein 3 plants of floating fern gametophytes are with the gametophyte association of dryopteris fragrans tissue culture, sporinite growth population is most, and 5 plants
The sporinite growth population of floating fern gametophyte association is minimum, shows that excessive floating fern will affect the formation of dryopteris fragrans sporinite,
It is preferred that 1-3 plants of floating ferns and dryopteris fragrans association.Experimental group and control group Comparative result are obvious, show floating fern to dryopteris fragrans spore
Certain facilitation is played in the growth of body.Dryopteris fragrans are compared with the experimental group of floating fern co-incubation and control group, when induction
Between shorten 30d, induction number increases by 1 times, and growth rate improves 25.00%.
The present invention overcomes the Period Process by tissue cultures and soil incubation inducing spore body is longer and wild
Dryopteris fragrans dead problem is easy in Induction Process, realize the factorial production of dryopteris fragrans sporinite, and to realize
The large-scale production of dryopteris fragrans sporinite provides strong technical support.
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this
The people of technology can do various changes and modification, therefore protection of the invention without departing from the spirit and scope of the present invention
Range should subject to the definition of the claims.
Claims (8)
1. the industrial seedling rearing and artificial cultivation method of a kind of dryopteris fragrans, which comprises the following steps:
Step 1: the mature spore of dryopteris fragrans is prepared into sterile spore suspension, sterile spore suspension is inoculated in liquid
First dark processing is cultivated after synchronizing sprouting in body culture medium, is inoculated in solid medium to length to gametophyte stage
Continue to cultivate, obtains the good dryopteris fragrans gametophyte of upgrowth situation；
Step 2: the dryopteris fragrans gametophyte that step 1 obtains is removed and is moved into the soil that cultivation has floating fern gametophyte after cleaning
It is cultivated in matrix, obtains mature dryopteris fragrans sporinite；
Wherein: fluid nutrient medium described in step 1 is the Knop ' s fluid nutrient medium of improvement, contains 0.25g in the culture medium
NaH2PO4, 0.25g KNO3, 0.25g MgSO4, 1.0g Ca (NO3)2With distilled water 1000mL, pH 5.7-5.8；Step 1 institute
Stating solid medium is 1/2MS+ sucrose+Solid agar culture, in the culture medium containing 1/2MS, 3.0% (quality) sucrose,
0.7%-0.8% (quality) agar, pH 5.7-5.8.
2. the method according to claim 1, wherein the condition of culture described in step 1 and step 2 is 22 DEG C
± 5 DEG C, illumination 12h-16h, dark 8h-12h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
3. the method according to claim 1, wherein soil matrix described in step 2 be by turfy soil and vermiculite by
It is made of 3:1 configuration according to volume ratio.
4. the method according to claim 1, wherein the number of floating fern gametophyte described in step 2 is 1 plant -5 plants.
5. the industrial seedling rearing and artificial cultivation method of a kind of dryopteris fragrans, which comprises the following steps:
Step 1: the mature spore of dryopteris fragrans is prepared into sterile spore suspension, sterile spore suspension is inoculated in Sheng
In container equipped with soil matrix, whole container moisturizing is covered with plastic film after watering into soil matrix, in 22 DEG C ± 5 DEG C
It is cultivated, obtains the good dryopteris fragrans gametophyte of upgrowth situation；
Step 2: the dryopteris fragrans gametophyte that step 1 obtains is moved into cultivation has in the soil matrix of floating fern gametophyte and carry out
Culture obtains mature dryopteris fragrans sporinite.
6. the method according to claim 1, wherein soil described in soil matrix and step 2 described in step 1
Matrix is made of being configured as turfy soil and vermiculite according to volume ratio 3:1.
7. the method according to claim 1, wherein the number of floating fern gametophyte described in step 2 is 1 plant~5
Strain.
8. the method according to claim 1, wherein the condition of culture described in step 1 and step 2 is 22 DEG C
± 5 DEG C, illumination 12h-16h, dark 8h-12h, 150 μm of olm of light intensity-2·s-1-180μmol·m-2·s-1。
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