阅读说明：本技术 野生竹荪组织分离制备母种方法 (Method for preparing mother seeds by tissue separation of wild dictyophora indusiata ) 是由 刘鹏 郑治洪 曹永直 彭成江 邵先强 文玉朝 于 2021-08-20 设计创作，主要内容包括：本发明公开了野生竹荪组织分离制备母种方法,涉及农业科技技术领域。本发明的野生竹荪组织分离制备母种方法,所述方法是以自然野外生长的竹荪菌蕾或开伞竹荪为原材料,经过组织分离得到菌柄包被,最后培养并纯化后得到母种。本发明公开了野生竹荪组织分离制备母种方法,能够有效的利用采集的竹荪野生资源,解决菌蕾成熟或已开伞的竹荪难以有效分离的难题,为竹荪的优质种源收集和良种选育奠定了技术基础。(The invention discloses a method for preparing mother seeds by tissue separation of wild dictyophora indusiata, and relates to the technical field of agricultural science and technology. The method for preparing the mother seeds by tissue separation of wild dictyophora indusiata comprises the steps of taking natural wild dictyophora indusiata buds or dictyophora indusiata as raw materials, obtaining stipe coatings through tissue separation, and finally obtaining the mother seeds after culture and purification. The invention discloses a method for preparing mother seeds by tissue separation of wild dictyophora indusiata, which can effectively utilize collected wild dictyophora indusiata resources, solve the problem that the dictyophora indusiata with mature fungus buds or opened mushrooms is difficult to effectively separate, and lay a technical foundation for high-quality provenance collection and fine seed breeding of the dictyophora indusiata.)

1. A method for preparing mother seeds by tissue separation of wild dictyophora indusiata is characterized in that dictyophora indusiata buds growing in the wild or open-umbrella dictyophora indusiata are used as raw materials, stipe coating is obtained by tissue separation, and finally, the mother seeds are obtained by culture and purification.

2. The method for preparing mother seeds by tissue separation of wild dictyophora indusiata according to claim 1, wherein for the dictyophora indusiata buds which are not opened, the specific operation of obtaining stipe coating by tissue separation is as follows: soaking the surface of the sterilized tissue in 75% alcohol on a sterile super clean bench for disinfection, then absorbing the surface moisture by using sterile absorbent paper, longitudinally cutting off buds by using a burning scalpel, clamping off stipe by using sterile tweezers to expose stipe coating, and transferring the tissue with the size of 5mm square of the stipe coating clamped into a mother culture medium for culture.

3. The method for preparing mother seeds through tissue isolation of wild dictyophora indusiata according to claim 1, wherein for dictyophora indusiata, the specific operation of obtaining stipe coating through tissue isolation is as follows: cutting off stipe at the position of the stipe, cleaning the fruit body upside down with 75% alcohol, washing with sterile water, wiping with sterile paper, separating the stipe with sterile tweezers, removing the stipe, clamping tissue with the stipe coating size of 5mm square with the sterile tweezers, and transferring into a mother culture medium for culture.

4. The method for preparing mother seeds through tissue isolation of wild dictyophora indusiata according to any one of claims 1-3, wherein the method specifically comprises the following steps:

s1: collecting natural wild-grown Dictyophora Indusiata buds and Dictyophora Indusiata to obtain separated raw materials;

s2: obtaining a sterile separation material stipe coating through sterile treatment, and transferring the stipe coating into a mother culture medium for culture;

s3: culturing until hypha germinates and transfers to a wood chip culture medium to obtain a wood chip culture medium mother seed;

s4: purifying and rejuvenating hypha tips to obtain purified mother seeds, transferring the purified mother seeds to stock seeds and cultivated seeds, and finally cultivating the mother seeds by imitating wild planting to obtain sporophores;

s5: performing systematic breeding by separating fruiting body tissue, performing systematic cultivation for 3-5 years, and performing artificial domestication to obtain stable Dictyophora Indusiata stock seed for reproduction and production.

5. The method for preparing mother seeds through tissue isolation of wild dictyophora indusiata according to claim 4, wherein the mother seed culture medium comprises the following raw materials per liter: 40g of green petiole sawdust, 10g of arrow bamboo leaves, 200g of potatoes, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of monopotassium phosphate and 0.5g of magnesium sulfate.

6. The method for preparing mother seeds through wild dictyophora indusiata tissue isolation, according to claim 5, characterized in that raw materials of a mother seed culture medium further comprise nutrition slow-release particles, the nutrition slow-release particles take nutrition slow-release capsules as inner cores, composite film layers are wrapped outside the inner cores, and camphorwood extracting solution is filled between the inner cores and the composite film layers.

7. The method for preparing mother seeds through tissue separation of wild dictyophora indusiata according to claim 6, wherein the nutrient sustained-release capsule takes nutrient solution as a capsule core and a sodium alginate/collagen composite material as a capsule shell.

8. The method for preparing mother seeds through tissue isolation of wild dictyophora indusiata according to claim 4, wherein the wood chip culture medium comprises the following raw materials in parts by weight: 78 parts of green petiole sawdust, 20 parts of wheat bran, 1 part of monopotassium phosphate, 0.5 part of magnesium sulfate and 0.5 part of gypsum.

9. The method for preparing mother seeds through tissue isolation of wild dictyophora indusiata according to claim 4, wherein in the step of S3, hyphae are transferred to a wood chip culture medium when germinating to 1-2 cm.

Technical Field

The invention relates to the technical field of agricultural science and technology, in particular to a method for preparing mother seeds by tissue separation of wild dictyophora indusiata.

Background

Bamboo fungus (Dictyophora indusiata (Vent. ex Pers) Fisch) also known as bamboo fungus and bamboo ginseng, and there are 4 common and edible varieties: dictyophora indusiata, Dictyophora brevifolia, Dictyophora echinovolvata and Dictyophora rubrovolvata are cryptophysalodes parasitizing at the root of dried bamboo, are in a shape similar to a net-shaped dry white snake skin, have dark green caps, a snow-white cylindrical stipe and a pink egg-shaped bacteroid, and have a fine and white net-shaped skirt at the top end of the stipe spread downwards from the lid, which are called as Xue skirt fairy, mountain precious flower, fungus flower and fungus queen in the fungus. The bamboo fungus is rich in nutrition, aromatic in flavor and delicious in taste, and is listed as one of 'grass eight delicacies' since ancient times.

The fungus buds of young basidiomycete dictyophora phalloidea are spherical, have three layers of coatings, and are thin, smooth, grey white or light brown red; glue is applied to the middle layer; the inner envelope is tough and fleshy. The coating is cracked when the mushroom is mature, the pileus is ejected out by the stipe, the stipe is hollow and is 15-20 cm high, white, and the appearance of the stipe consists of spongy small holes; the coating is sent to the lower part of the stalk to form a bacterium tray; the pileus grows on the top end of the handle and is in a bell shape, the surface of the pileus is uneven and is in a grid shape, and basidiospores are densely distributed in the concave part; a white netted bacterium curtain is covered under the cover, and the length of the white netted bacterium curtain is more than 8 centimeters; the spores are smooth, transparent and oval, and the diameter of the spores is 3-3.5 multiplied by 1.5-2 microns.

At present, the bamboo fungus strain is obtained by mainly collecting immature eggs and performing tissue separation by using an agar culture medium, but the success rate is not very high, and adverse phenomena such as expansion and the like can also occur in the culture process.

Disclosure of Invention

Aiming at the problems, the invention aims to disclose a method for preparing mother seeds by tissue separation of wild dictyophora indusiata, which can effectively utilize collected wild dictyophora indusiata resources, solve the problem that buds are mature or the dictyophora indusiata opened is difficult to effectively separate, and lay a technical foundation for high-quality seed source collection and fine seed breeding of the dictyophora indusiata.

Specifically, the method for preparing the mother seeds by tissue separation of wild dictyophora indusiata comprises the steps of taking natural wild dictyophora indusiata buds or dictyophora indusiata as raw materials, obtaining stipe coatings by tissue separation, and finally culturing and purifying to obtain the mother seeds.

Further, for the bamboo fungus buds without opening the umbrella, the specific operation of obtaining the stipe coating by tissue separation is as follows: soaking the surface of the sterilized tissue in 75% alcohol on a sterile super clean bench for disinfection, then absorbing the surface moisture by using sterile absorbent paper, longitudinally cutting off buds by using a burning scalpel, clamping off stipe by using sterile tweezers to expose stipe coating, and transferring the tissue with the size of 5mm square of the stipe coating clamped into a mother culture medium for culture.

Further, for dictyophora indusiata, the specific operation of obtaining stipe coating by tissue separation is as follows: cutting off stipe at the position of the stipe, cleaning the fruit body upside down with 75% alcohol, washing with sterile water, wiping with sterile paper, separating the stipe with sterile tweezers, removing the stipe, clamping tissue with the stipe coating size of 5mm square with the sterile tweezers, and transferring into a mother culture medium for culture.

Further, the method specifically comprises the following steps:

s1: collecting natural wild-grown Dictyophora Indusiata buds and Dictyophora Indusiata to obtain separated raw materials;

s2: obtaining a sterile separation material stipe coating through sterile treatment, and transferring the stipe coating into a mother culture medium for culture;

s3: culturing until hypha germinates and transfers to a wood chip culture medium to obtain a wood chip culture medium mother seed;

s4: purifying and rejuvenating hypha tips to obtain purified mother seeds, transferring the purified mother seeds to stock seeds and cultivated seeds, and finally cultivating the mother seeds by imitating wild planting to obtain sporophores;

s5: performing systematic breeding by separating fruiting body tissue, performing systematic cultivation for 3-5 years, and performing artificial domestication to obtain stable Dictyophora Indusiata stock seed for reproduction and production.

Further, each liter of the mother culture medium comprises the following raw materials: 40g of green petiole sawdust, 10g of arrow bamboo leaves, 200g of potatoes, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of monopotassium phosphate and 0.5g of magnesium sulfate.

Further, the wood chip culture medium comprises the following raw materials in parts by weight: 78 parts of green petiole sawdust, 20 parts of wheat bran, 1 part of monopotassium phosphate, 0.5 part of magnesium sulfate and 0.5 part of gypsum.

Further, in the step S3, when the hyphae germinate to 1-2cm, the hyphae are transferred to a wood chip culture medium.

Furthermore, the raw materials of the mother culture medium also comprise nutrient slow-release particles, the nutrient slow-release particles take nutrient slow-release capsules as cores, composite film layers are wrapped outside the cores, and camphorwood extracting solution is filled between the cores and the composite film layers.

Furthermore, the nutrient sustained-release capsule takes nutrient solution as a capsule core and takes a sodium alginate/collagen composite material as a capsule shell.

Further, the preparation method of the nutritional sustained-release granules specifically comprises the following steps:

a1: dissolving sodium alginate in water to obtain a sodium alginate solution, adding a collagen solution into the sodium alginate solution, adjusting the pH to 6-7, adding ethanol, stirring and mixing uniformly, heating and refluxing for 30min, cooling to room temperature, adding a nutrient solution, stirring and mixing uniformly, spraying into a continuously stirred calcium chloride solution by adopting a high-pressure electrostatic spraying method, taking out microcapsules, and cleaning with deionized water to obtain a nutrient sustained-release capsule;

a2: weighing methyl cellulose, stirring and dissolving the methyl cellulose in deionized water to obtain a methyl cellulose solution, stirring and uniformly mixing the methyl cellulose solution, adding a chitosan solution, stirring and uniformly mixing the mixture, homogenizing the mixture for 10min at 10000r/min, dropwise adding camphor wood extract in the homogenizing process, adding nutrient slow-release microcapsules, performing ultrasonic dispersion, adding a cross-linking agent, stirring and reacting the mixture for 15min at the temperature of 25 ℃, filtering the mixture after the reaction is finished, washing a filter cake to be neutral by using the deionized water, and performing freeze drying to obtain the nutrient slow-release particles.

According to the invention, the nutrient slow-release particles are added in the mother culture medium, and the composite film layer is arranged on the outer surface of the nutrient slow-release particles, so that on one hand, the composite film layer protects the internal nutrient particles and is convenient to store, on the other hand, the composite film layer takes methylcellulose and chitosan as raw materials, and the chitosan is used as a natural antibacterial substance, so that the mother culture medium can be prevented from being infected by mixed bacteria to a certain extent, the infection rate of tissues is reduced, and the methylcellulose can also be used as a nutrient substance required by the growth of hypha of subsequently separated tissues. And in the process of preparing the mother culture medium, the external composite film layer absorbs water and swells to become loose, and the internal camphorwood extracting solution is released, so that the antibacterial performance of the mother culture medium is further improved, and the probability of the culture medium being infected by mixed bacteria can be further reduced to a certain degree. In addition, the nutrient slow-release particles are added in the mother culture medium, so that long-acting and continuous supply of nutrients in the culture process is ensured to a certain extent, the concentration of nutrient substances in the culture medium can be maintained at a proper concentration all the time, and the aims of promoting the rapid growth of hyphae and shortening the growth period are fulfilled.

Further, the camphorwood extracting solution is obtained by decocting camphorwood chips with water and then purifying, and specifically comprises the following steps: adding deionized water into camphor wood chips, decocting for 2 hours, filtering to obtain a primary filtrate, adding deionized water into filter residues, decocting for 1 hour twice, filtering to obtain a secondary filtrate, combining the filtrates, concentrating under reduced pressure to 2/3 volumes, adding a chitosan solution, stirring uniformly, performing electrophoresis treatment, and filtering to obtain a camphor wood extract.

The camphor wood extract contains compounds such as alkaloids, flavonoids, proteins and organic acids, and the content of the alkaloids in the camphor wood extract can be reduced by combining the added chitosan with electrophoresis treatment, so that the compounds such as flavonoids, proteins and organic acids beneficial to hypha growth are kept as much as possible.

The invention has the beneficial effects that:

the invention discloses a method for preparing mother seeds by tissue separation of wild dictyophora indusiata, which is characterized in that through tissue separation research on different parts of dictyophora indusiata buds with different maturity, the inventor finds that the separation effect of stipe coatings of the dictyophora indusiata is very good, the success rate is higher than that of stipe separation, and the phenomena of poor expansion and the like are avoided. The discovery that the stipe coating is used as a separated strain material solves the problem that the bamboo fungus with mature buds or opened umbrella is difficult to organize and separate to obtain the strain, and has important significance for collecting and preserving the resources of the bamboo fungus strain.

Drawings

Fig. 1 is a schematic diagram of the position and morphological structure of stipe coating in a dictyophora fungus bud according to an embodiment of the present invention;

fig. 2 is a schematic anatomical diagram of a dictyophora fungus bud in the first embodiment of the invention;

wherein, the fungus stalk 1, the spore layer 2, the fungus skirt 3, the fungus cap 4, the fungus stalk coating 5 and the fungus tray 6.

Detailed Description

The present invention will be described in detail with reference to specific examples below:

example one

S1: collecting three kinds of mature (roof fall, slight roof fall and no roof fall) Dictyophora Indusiata buds and Dictyophora Indusiata fruiting bodies in sunny days, wherein the fruiting bodies are not washed by rain water and the bottom of the stipe is not polluted to obtain separated raw materials;

preparing a mother culture medium and a wood chip culture medium, which specifically comprise the following steps:

according to a formula of 40g of green bar wood chips, 10g of arrow bamboo leaves, 200g of potatoes, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of monopotassium phosphate, 0.5g of magnesium sulfate and the balance of water, per liter of mother strain agar culture medium is prepared by a common existing preparation method of a common edible fungus culture medium, wherein the green bar wood chips, the potatoes and the arrow bamboo leaves are boiled for 30min to obtain filtrate, the prepared culture medium is subpackaged into test tubes, sterilized at 121 ℃ for thirty minutes and then cooled to 70 ℃ to place an inclined plane for separating mother strains, and the pH is natural.

The method comprises the steps of preparing a dry material wood chip culture medium per 100kg according to 78kg of green bar wood chips, 20kg of wheat bran, 1g of monopotassium phosphate, 0.5g of magnesium sulfate and 0.5kg of gypsum, soaking the green bar wood chips one day in advance, taking out the green bar wood chips the next day, draining, mixing the green bar wood chips according to a formula, fermenting for 24 hours, bottling, sterilizing at 123 ℃ for 2 hours, cooling to room temperature after the sterilization is finished, and performing transfer of agar mother seeds with natural pH.

S2: obtaining the aseptic separation material stipe coating through aseptic processing, which specifically comprises the following steps:

for the non-opened Dictyophora Indusiata buds, cleaning the surface of the eggs, soaking in 75% ethanol for 1min, placing on a clean bench, placing the required test tube slant and separation tool on the operation bench, sterilizing with ultraviolet lamp for 30min, and opening the bench for tissue separation. Each egg is prepared by taking 6 tissues of spores, pileus, upper, middle and lower parts of stipe, stipe coating and the like, taking tissue blocks with the size of soybeans and putting the tissue blocks into a slope, and the operation and separation methods of eggs with different maturity are the same. Marking, putting the tissue blocks into a constant-temperature incubator for dark culture at 23 ℃, regularly observing every day, and recording the pollution and germination conditions of the tissue blocks at different parts in a culture medium;

for the open-umbrella dictyophora phalloidea, the fungus tray of the open-umbrella dictyophora phalloidea is cleaned, water is not dropped on the stipe and the inside of the fungus egg when the fungus tray is cleaned, then 75% alcohol is used for wiping the fungus tray and then the fungus tray is placed on a super clean bench, meanwhile, the required test tube inclined plane, a separating tool and the like are placed on an operation bench, an ultraviolet lamp is started for sterilization for 30 minutes, and then the workbench is opened for tissue separation. Dipping spores on the inner layer of a pileus on a burning and cooling inoculating needle to perform streaking on a culture medium, then removing a bacterium support, and taking a stipe which is not contacted with air to coat and inoculate the bottom of the stipe on the culture medium.

S3: separating the mother seeds, culturing at 23 deg.C in dark until mycelia germinate 1-2cm, transferring to wood chip culture medium to obtain wood chip culture medium mother seeds as shown in tables 1 and 2;

TABLE 1 Germination of Dictyophora Indusiata (Vent. Ex pers) Fisch eggs isolation

As can be seen from Table 1, the separated tissue began to germinate and grow white hyphae after 2 to 7 days. In each tissue, the germination rate of the spores is zero, and the spores are completely polluted; the germination rates of the stipes are not greatly different at different parts, the germination rates of the stipes with different maturity are greatly different, the more mature stipes of the eggs are lower in separation germination rate, secondary metabolites are generated after a certain time, and part of the stipes are extremely expanded to 2-3 times of the original volume; the pili can grow white, but cannot grow further. The germination rate of the stipe coating is the highest, the germination time is the shortest, and the pollution rate is lower. Therefore, spores and pileus are not suitable for tissue separation, the germination rates of various parts of stipe are not greatly different, and certain white hypha can grow. The stipe coating has the highest germination rate and the lowest pollution rate, and is more suitable for being used as a tissue object for separating mother seeds.

TABLE 2 germination status of fruiting body isolation

Location of a body part	Rate of contamination	Germination rate	Time of germination d
				Under stipe of fungus	75％	0	0
Mushroom stalk coating	15％	80％	3
				Spore	100％	0	0

As can be seen from Table 2, the fruiting body of Dictyophora Indusiata (Vent. Ex pers) Fisch has a high contamination rate, and mainly, most tissues are in contact with air after opening, so that infectious microbes are serious, and even though bottom tissues are taken, certain contamination is still inevitable. In the three parts, spores and stipes do not germinate, the spores are separated and are completely polluted, and tissues begin to expand after 3 days from the stipe parts; the stipe coating is positioned at the bottom of the stipe, the pollution probability is only 10 percent, and the germination rate is 80 percent. The hyphae after the stipe germination are white, the germination time is 3 days, and the hyphae are dense, thus being a better separation material. Therefore, the stipe coating is still the best material for tissue separation of the bamboo fungus after the umbrella is opened.

S4: purifying and rejuvenating hypha tips to obtain purified mother seeds, transferring the purified mother seeds to stock seeds and cultivated seeds, and finally cultivating the mother seeds by imitating wild planting to obtain sporophores;

s5: performing systematic breeding by separating fruiting body tissue, performing systematic cultivation for 3-5 years, and performing artificial domestication to obtain stable Dictyophora Indusiata stock seed for reproduction and production.

Example two

Compared with the first embodiment, the difference is that the used mother culture medium further comprises nutrient slow-release particles, the nutrient slow-release particles take nutrient slow-release capsules as inner cores, composite film layers are wrapped outside the inner cores, camphor wood extracting solution is filled between the inner cores and the composite film layers, the nutrient slow-release capsules take nutrient solution as capsule cores, sodium alginate/collagen composite materials are used as capsule shells, and the nutrient solution is nutrient solution used in the existing bamboo fungus culture process.

The camphorwood extracting solution is obtained by decocting camphorwood chips with water and then purifying, and specifically comprises the following steps: taking camphor wood chips, adding deionized water with the volume 8 times that of the camphor wood chips, decocting for 2 hours, filtering to obtain primary filtrate, adding deionized water with the volume 5 times that of the filter residues into the filter residues, decocting for 1 hour for the second time, filtering to obtain secondary filtrate, combining the filtrates, concentrating under reduced pressure to 2/3 volume, adding chitosan solution with the mass 2 wt% of 0.1 time that of the filtrate, stirring and mixing uniformly for electrophoresis, and filtering to obtain camphor wood extract.

The preparation of the nutrient sustained-release granules comprises the following steps:

a1: dissolving sodium alginate in water to obtain a sodium alginate solution with the concentration of 60g/L, adding a saturated collagen solution into the sodium alginate solution, wherein the mass ratio of sodium alginate to collagen is 2:1, adjusting the pH value to 6-7, adding absolute ethyl alcohol with the volume of 0.01 time of that of the sodium alginate solution, stirring and uniformly mixing, heating and refluxing for 30min at 30 ℃, cooling to room temperature, adding nutrient solution with the same volume of the sodium alginate solution, stirring and uniformly mixing, spraying into a continuously stirred calcium chloride solution by adopting a high-pressure electrostatic spraying method, taking out microcapsules, and cleaning with deionized water to obtain the nutritional sustained-release capsules;

a2: weighing methyl cellulose, stirring and dissolving the methyl cellulose in deionized water to obtain a methyl cellulose solution with the concentration of 4g/L, uniformly stirring and mixing, adding 6g/L of chitosan solution with the same volume, uniformly stirring, homogenizing for 10min under the condition of 10000r/min, dropwise adding a camphorwood extracting solution with the volume of 1/2 chitosan solution in the homogenizing process, adding nutrient slow-release microcapsules according to the solid-to-liquid ratio of 5g/L, ultrasonically dispersing, adding a cross-linking agent, stirring and reacting at the temperature of 25 ℃ and the speed of 300r/min for 15min, filtering after the reaction is finished, washing a filter cake to be neutral by using deionized water, and freeze-drying to obtain the nutrient slow-release particles.

The stock culture medium of this example comprises per liter: 40g of green common clubmoss herb chips, 10g of arrow bamboo leaves, 200g of potatoes, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 10g of nutrient slow-release particles and the balance of water, and the preparation method adopts a conventional method for preparation.

Comparative example 1

Compared with the two phases of the embodiment, the difference of the comparative example is that the camphorwood extract is directly used for preparing the nutrition sustained-release particles without electrophoresis treatment.

Comparative example No. two

Compared with the two phases of the embodiment, the difference of the comparison example is that the nutrition slow-release capsule is directly added into the mother culture medium.

According to the method, tissue separation is carried out to obtain stipe coating tissues of dictyophora open, then, according to the method of the first embodiment to the second embodiment, the dictyophora open is cultured at the temperature of 23 ℃ in a dark place, and the germination conditions are shown in tables 3 and 4.

TABLE 3 Germination of Dictyophora Indusiata (Vent. Ex pers) Fisch eggs isolation

	Rate of contamination	Germination rate	Time of germination d
				Example one	10％	80％	3
Example two	1％	95％	2
				Comparative example 1	5％	87％	2
Comparative example No. two	10％	85％	3

As can be seen from the data in Table 3, in combination with the data in tables 1 and 2, the infection rate of the isolated tissue was decreased and the time for hyphae to germinate was shortened by using the mother culture medium prepared in this example; and the infection rate of the separated tissue can be effectively reduced and the germination rate can be improved by adding the camphor wood extracting solution.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.