CN109526308B

CN109526308B - Embedding method for orchid artificial seeds

Info

Publication number: CN109526308B
Application number: CN201811541487.9A
Authority: CN
Inventors: 刘福平
Original assignee: SUBTROPICAL CROPS INSTITUTE OF FUJIAN PROVINCE
Current assignee: SUBTROPICAL CROPS INSTITUTE OF FUJIAN PROVINCE
Priority date: 2018-12-17
Filing date: 2018-12-17
Publication date: 2021-08-03
Anticipated expiration: 2038-12-17
Also published as: CN109526308A

Abstract

The invention discloses an embedding method of an orchid artificial seed, which comprises the following steps: 1) preparing endosperm hydrosol, wherein the endosperm hydrosol comprises 1-10% of gelling agent, 0.5-5% of thickening agent and nutrients and/or hormones required by seed germination according to mass volume ratio; 2) taking out the seeds from the fruits, putting the seeds into the endosperm hydrosol, standing the seeds and carrying out aseptic culture until the seeds absorb water and swell; 3) adding oxygen releasing agent 0.01-1%, stirring to make the seeds uniformly suspended in the endosperm liquor, immediately sucking the endosperm liquor containing the seeds by a suction pipe, dripping into the hardening solvent of the endosperm liquor, standing until the particles of the endosperm liquor are hardened into artificial seeds. The invention has simple process and low cost.

Description

Embedding method for orchid artificial seeds

Technical Field

The invention belongs to a plant seed seedling raising technology in the field of agriculture and forestry, and particularly relates to a method for embedding orchid seeds to produce artificial seeds.

Background

The artificial seed is a granule formed by embedding a tissue organ (a propagule such as a somatic embryo, a bud, a callus, a protocorm and the like) which is generated in the in vitro culture of a plant and can be developed into a complete plant in an artificial outer membrane containing nutrient substances and having a protection function. Most of orchidaceae plants have higher ornamental and medicinal values and are hot plants made of artificial seeds. The fruit of the orchids contains thousands of seeds, which are fine or powdery, the so-called embryo is actually a mass of tens of undifferentiated cells and has no endosperm tissue. At present, many reports are reported on cymbidium, butterfly orchid, dendrobium officinale, bletilla striata and the like in aseptic seeding (non-symbiotic germination) of orchids seeds, and two ways for the seeds to continue to develop after absorbing water and expanding into small spheres are provided, namely, the small spheres develop into small seedlings through original (round) bulbs; secondly, after the small ball body extends into a root stem, the small ball body develops into a small seedling (such as Wangbeyong and the like, and a plurality of ways of orchid seed aseptic germination and embryo development processes, Yunnan plant research 2006,28(4): 399-402). There are also some patents on the artificial seed inclusion of orchids being protocorms germinated from seeds, such as application No.: 201610547741.0, 201210382702.1, 201110284493.2, and the like. The artificial seeds in granulation are convenient to store and transport, and can realize mechanized sowing, but the general artificial seeds of orchids only embed one protocorm, and once the protocorm dies, the whole artificial seeds do not germinate and are wasted. The embedded protocorm is not in a dormant period but rapidly buds and grows roots, so that the storage and transportation time of the artificial seeds is short at normal temperature.

The seeds of the orchids are very fine, one artificial seed is embedded with one natural seed (propagule) according to the traditional method, the operation is difficult, and not every natural seed has vitality, and the germination rate is larger due to the variety difference, so that the normal germination of every natural seed can be ensured only by embedding multiple natural seeds in one artificial seed. The existing embedding method of artificial seeds is most commonly used by a hydrogel sodium alginate coating technology, alginate capsules have poor air permeability, inhibit the respiration of somatic embryos, and reduce the life of the somatic embryos (Sun Jing III, Main edition of Gui Yanlin plant cell engineering experiment technology, Beijing: scientific publishing company, 1990:233.), and tiny fragile orchid seeds are embedded in sodium alginate endosperm particles, so that the respiratory metabolism is obviously influenced by insufficient oxygen supply.

Disclosure of Invention

The invention mainly aims to provide a method for embedding an artificial seed of an orchid plant.

The technical scheme of the invention is as follows:

the embedding method of the orchid artificial seeds comprises the following steps:

1) preparing endosperm hydrosol for later use, wherein the endosperm hydrosol comprises 1-10% of gelling agent, 0.5-5% of thickening agent and nutrients and/or hormones required by seed germination according to mass volume ratio;

2) taking out the seeds from the fruits, putting the seeds into the sterile embryo emulsion sol, and standing and culturing until the seeds absorb water and swell;

3) adding oxygen releasing agent 0.01-1%, stirring to make the seeds uniformly suspended in the endosperm liquor, immediately sucking the endosperm liquor containing the seeds by a suction pipe, dripping into the hardening solvent of the endosperm liquor, standing until the particles of the endosperm liquor are hardened into artificial seeds.

Wherein, the nutrition and hormone required by seed germination are determined according to the requirements of each orchid plant.

Preferably, the sol comprises 3-5% of gelling agent, 1-2% of thickening agent and nutrients and/or hormones required for seed germination by mass ratio.

The fruit is mature or nearly mature. Preferably, the mature fruit is uncracked.

Preferably, the gelling agent is sodium alginate.

Preferably, the thickener is at least one of sodium carboxymethylcellulose, carrageenan (preferably tau carrageenan) and polyvinylpyrrolidone.

Preferably, the oxygen release agent is at least one of hydrogen peroxide, ammonium persulfate, urea peroxide and sodium percarbonate.

Preferably, the embryo emulsion hardener is a soluble calcium salt.

Preferably, the soluble calcium salt comprises at least one of calcium chloride, calcium sulfate and calcium lactate, the concentration is 1-5%, and the solidification time of endosperm liquid is 10-40 min.

Preferably, in step 2), 0.2 to 0.6 g of seeds per liter of sol is dosed.

Another object of the present invention is to provide an embedding composition for seeds of orchids, comprising, by mass to volume:

1-10% of gelling agent, 0.01-0.5% of oxygen release agent, 0.5-5% of thickening agent and nutrients and hormones required for seed germination.

Preferably, the gelling agent is sodium alginate; the oxygen release agent is at least one of hydrogen peroxide, ammonium persulfate, urea peroxide and sodium percarbonate; the thickening agent is at least one of sodium hydroxymethyl cellulose, carrageenan and polyvinylpyrrolidone.

The invention soaks and absorbs a certain amount of natural seeds, mixes and solidifies the natural seeds with good transparency with natural hydrogel, nutrient components, hormone, thickening agent and endosperm liquid of oxygen release agent to obtain artificial seeds containing a plurality of natural seeds, and the artificial seeds can germinate under proper conditions.

(1) The orchid natural seeds are very fine and light, float in the embryo milk and are difficult to be uniformly dispersed in the endosperm liquid, and when artificial seeds are prepared in batches, a small amount of natural seeds contained in each artificial seed are greatly different in density when the artificial seeds are absorbed from the endosperm liquid each time (for example, when the artificial seeds with the diameter of 0.3-0.6cm are prepared and about 0.01-0.08 ml of embryo milk is solidified each time) and are solidified into particles drop by drop. The invention pre-cultures seeds in endosperm liquid, the water absorption expansion specific gravity of the embryo is increased, which is beneficial to the suspension of the seeds in the endosperm liquid, the thickening agent is added in the endosperm liquid to increase the viscosity, the embryo emulsion is slightly stirred, the seeds are easily and uniformly dispersed in the endosperm liquid medium and can not quickly float up or sink, thus the density of the natural seeds contained in the artificial seeds prepared in the same batch is more consistent, the natural seeds in the artificial seed particles are more uniformly distributed, and the nutrition of the artificial endosperm can be more uniformly supplied to all the natural seeds. If the content of the thickening agent is less than 0.5%, the viscosity is small, the suspension effect is poor, and if the content of the thickening agent is more than 5%, the viscosity is too large, the seed dispersion is not facilitated, and the solidification effect of the sodium alginate is also influenced.

(2) Adding an oxygen releasing agent into the embryo emulsion to gradually release oxygen for the respiration of the embedded natural seeds.

(3) The oxygen release agent (when meeting water) is firstly converted into active oxygen and then slowly converted into oxygen, and the active oxygen has an antibacterial effect, is beneficial to the anticorrosion of artificial seeds and resists the infection of germs. If the content of the oxygen releasing agent is less than 0.01%, the released oxygen is insufficient, the effect on root growth is not obvious, and if the content of the oxygen releasing agent is more than 0.5%, the oxygen releasing agent may have a toxic effect on seeds.

The method has the beneficial effects that (1) the method directly embeds natural seeds, omits the step of sowing and culturing protocorms (which are used for embedding), and saves a large amount of culture medium cost; (2) when in sowing, each artificial seed has seed germination, so that the seed cost for seedling culture can be reduced; (3) the oxygen release agent releases active oxygen antibacterial agent, and is beneficial to resisting germ infection of artificial seeds in early storage and sowing. (4) The embryo embedded by the artificial seed is subjected to the process of continuously expanding and developing into protocorm or rhizome, and does not immediately leave leaves and grow roots, so the embryo embedded by the artificial seed has longer normal-temperature storage time than the embryo embedded by the conventional artificial seed. The artificial seed of the invention has simple preparation process, low cost and convenient use, and can be popularized and applied.

Drawings

Fig. 1 to 4 are diagrams of examples 1 of artificial dendrobium officinale seed germination entities, wherein:

FIG. 1 shows an artificial seed of Dendrobium officinale Kimura et Migo which has just germinated a plurality of green bud spots;

FIG. 2 shows the germination of leaves of the artificial seeds of Dendrobium officinale Kimura et Migo;

FIG. 3 shows that one artificial seed of Dendrobium officinale Kimura et Migo sprouts a plurality of plants;

FIG. 4 shows the germination of the artificial seeds of Dendrobium officinale into seedlings.

Fig. 5 to 8 are diagrams of the bletilla striata artificial seed germination entity of example 2, wherein:

FIG. 5 shows an artificial seed of bletilla striata;

FIG. 6 shows that each of the bletilla striata artificial seeds germinates;

FIG. 7 shows that one artificial seed of bletilla striata sprouts multiple shoots;

FIG. 8 shows the germination of bletilla striata artificial seeds into seedlings.

Detailed Description

The technical scheme adopted by the invention is as follows:

washing fruit of Orchidaceae plant with water, soaking in 70-75% ethanol for instant sterilization, soaking in 10% calcium hypochlorite solution for 15-30 min, and washing with sterile water for 2 times.

The gelling agent is sodium alginate, and the use concentration is 3-5%. The oxygen releasing agent is one of hydrogen peroxide, ammonium persulfate, urea peroxide and sodium percarbonate, and the use concentration is 0.03-0.3%. The thickening agent is sodium carboxymethylcellulose, and the use concentration is 1-2%.

Adding water into gelling agent, nutrient components required by seed germination, hormone and thickening agent, sterilizing under high pressure (121 ℃ for 20-30 minutes) to obtain sol, cooling, digging fruits under aseptic condition, taking out seeds, putting endosperm sol, putting 0.2-0.6 g of seeds per liter of sol, standing, performing aseptic culture until the seeds absorb water and swell, then adding oxygen release agent in aseptic environment, stirring to uniformly suspend the seeds in endosperm solution, immediately sucking endosperm solution containing the seeds by a suction pipe, dropwise adding 2% calcium chloride sterile solution, stirring in advance by the suction pipe to uniformly distribute the seeds in the endosperm solution, dropwise adding calcium chloride, standing for 10-40 minutes, and hardening the particles into artificial seeds.

Example one

Dendrobium officinale (Dendrobium officinale) fruit is washed by water, soaked in 75% alcohol on an ultra-clean bench for instantaneous sterilization, then soaked in 10% calcium hypochlorite solution for 20 minutes, and washed with sterile water for 2 times.

4 percent (W/V) of gelling agent sodium alginate, 1.0 percent (W/V) of thickener sodium carboxymethylcellulose, 0.5 mg/L of nutrient components and hormones of MS + NAA + 20 percent (V/V) of coconut juice + 2.0 percent (W/V) of cane sugar, mixing, sterilizing at high pressure, cooling to obtain sol, dissecting Dendrobium officinale fruit on a superclean bench, shaking off seeds to obtain sol, adding 0.3 g of seeds per liter of sol, standing for aseptic culture for 1 week to allow the seeds to swell and turn yellow-green, adding ammonium persulfate as an oxygen release agent with the concentration of 0.2 percent in a sterile environment, adjusting the pH value to 5.6, stirring to enable seeds to be uniformly suspended in endosperm liquid, immediately sucking sodium alginate sol containing the seeds by a suction pipe, dropwise adding a 2 percent calcium chloride solution sterilized in advance, standing for about 20 minutes, solidifying the particles, and washing with sterile water for 2 times to obtain the artificial seed particles.

The artificial seeds are aseptically cultured in a bottle, the seed coat is broken through at the bud point after 30 days at the conventional temperature and under the conventional illumination, each seed pellet germinates, each seed pellet can germinate 3 buds or more, and the seedlings can almost continue to grow leaves and root into tender seedlings. The conventional artificial seeds embedded with protocorms of the dendrobium officinale can sprout and break through seed coats in about 10 days, so that the artificial seeds can be stored for about 20 days at normal temperature more than the conventional artificial seeds.

Example two

The Bletilla striata (Bletilla striata) fruits are washed by water, soaked in 75% alcohol in a super clean bench for instant sterilization, then soaked in 10% calcium hypochlorite solution for 20 minutes, and washed by sterile water for 2 times.

3 percent of gelling agent sodium alginate, 1.5 percent of thickening agent polyvinylpyrrolidone and 3.0 percent (W/V) of MS and cane sugar as nutritional ingredients, mixing the components, sterilizing at high pressure by a conventional method, cooling to obtain sol, splitting bletilla striata fruits on a clean bench, shaking off seeds to the sol, adding 0.4 gram of seeds in each liter of sol, standing for aseptic culture for 15 days, swelling the seeds to turn yellow green, adding hydrogen peroxide as an oxygen release agent in an aseptic environment, 0.1 percent of concentration and 0.1 percent of sodium percarbonate, adjusting the pH to 5.6, stirring to enable the seeds to be uniformly suspended in endosperm liquid, immediately sucking the sodium alginate sol containing the seeds by a suction pipe, dripping 2 percent calcium sulfate solution which is sterilized at high pressure drop by drop, standing for about 30 minutes, solidifying particles, and washing for 2 times by using sterile water to obtain the artificial seed particles.

The artificial seeds are aseptically cultured in a bottle, the seed coats are broken through by green bud points after 25 days at the conventional temperature and under the conventional illumination, each seed pellet germinates, each seed pellet can germinate 3 buds or more, and the seedlings can almost continue to grow leaves and root into tender seedlings. The conventional artificial seeds embedded with bletilla striata protocorms can sprout and break through seed coats in about 10 days, so that the artificial seeds can be stored for about 15 days at normal temperature more than the conventional artificial seeds.

Claims

1. An embedding method of an orchid artificial seed comprises the following steps:

1) preparing endosperm hydrosol, wherein the endosperm hydrosol comprises 3-10% of gelling agent, 0.5-1.5% of thickening agent and nutrients and/or hormones required by seed germination according to mass volume ratio;

3) adding 0.01-1% of oxygen release agent into the endosperm liquid, stirring to make the seeds uniformly suspended in the endosperm liquid, immediately sucking the endosperm liquid containing the seeds by a straw, dropwise adding into a hardening solvent of the endosperm liquid, and standing until the particles of the endosperm liquid are hardened into artificial seeds; wherein the oxygen release agent is at least one of hydrogen peroxide, ammonium persulfate, urea peroxide and sodium percarbonate.

2. The method for embedding artificial seeds of orchids according to claim 1, wherein the method comprises: the sol comprises 3-5% of gelling agent, 1-2% of thickening agent and nutrients and/or hormones required for seed germination according to mass volume ratio.

3. The method for embedding an artificial seed of an orchid family plant according to claim 1 or 2, wherein the method comprises the following steps: the gelling agent is sodium alginate.

4. The method for embedding an artificial seed of an orchid family plant according to claim 1 or 2, wherein the method comprises the following steps: the thickening agent is at least one of sodium hydroxymethyl cellulose, carrageenan and polyvinylpyrrolidone.

5. The method for embedding an artificial seed of an orchid family plant according to claim 1 or 2, wherein the method comprises the following steps: the embryo emulsion hardening solvent is soluble calcium salt.

6. The method for embedding artificial seeds of orchids according to claim 5, wherein the method comprises: the soluble calcium salt comprises at least one of calcium chloride, calcium sulfate and calcium lactate, the mass ratio concentration is 1-5%, and the curing time is 10-40 minutes.

7. The method for embedding artificial seeds of orchids according to claim 1, wherein the method comprises: in the step 2), 0.2-0.6 g of seeds is added into each liter of sol.

8. An embedding composition for artificial seeds of orchids, comprising, by mass to volume:

3-10% of gelling agent, 0.01-0.5% of oxygen release agent, 0.5-1.5% of thickening agent and nutrients and/or hormones required for seed germination, wherein the oxygen release agent is at least one of hydrogen peroxide, ammonium persulfate, urea peroxide and sodium percarbonate.

9. The embedding composition for artificial seeds of orchidaceae plants according to claim 8, wherein the gelling agent is sodium alginate; the thickening agent is at least one of sodium hydroxymethyl cellulose, carrageenan and polyvinylpyrrolidone.