CN111096118B

CN111096118B - Seed germination method for Balanophora japonica

Info

Publication number: CN111096118B
Application number: CN201911371333.4A
Authority: CN
Inventors: 郝冰; 赵秀; 杨生超
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2021-05-28
Anticipated expiration: 2039-12-26
Also published as: CN111096118A

Abstract

A method for seed germination of Balaophora plants, comprising: pre-culturing seeds before germination: washing mature Balaophora plant seeds with sterile water, and pre-culturing for 24-48 hours in a dark condition at the room temperature of 25-35 ℃; collecting the roots of the host plants: the host plant root is root without parasitic Balanophora japonica and has diameter less than 1 cm; extracting a host root extract, grinding the host plant root into powder by using liquid nitrogen, adding 50% v/v methanol solution for extraction for 2 times respectively, volatilizing the filtrate to obtain an extract, wherein the specific gravity of the extract is 1.3-1.7; seed germination: preparing the extract into a root extract solution with the concentration of 5ppm to 20ppm by using a 50% v/v methanol solution, culturing the pre-cultured seeds, and culturing the seeds in the dark at the temperature of between 25 and 35 ℃ until the seeds germinate. The method can ensure that the germination of seeds of the Balanophora japonica can reach 83.83 percent, and lays a good technical foundation for the artificial planting of the Balanophora japonica.

Description

Seed germination method for Balanophora japonica

Technical Field

The invention belongs to the technical field of agricultural seed production, and particularly relates to a germination method of Balanophora japonica plant seeds.

Background

The Balanophora japonica family is a total parasitic plant and depends on a host plant for survival, and the Balanophora japonica family has 22 species in China, wherein the Balanophora japonica genus (Balanophora) has 21 species, including Balanophora cryptophysa jordo ex S.Y.Chang et Tam, Balanophora japonica L.J.Balanophora L.R.Br.), Balanophora japonica L.Balanophora japonica L.J.Ex.Ex.Bl.C, Balanophora japonica L.Ex.Balanophora L.K.K., Balanophora japonica L.K., Balanophora japonica L.K.K., Balanophora japonica L.K.K.K., Balanophora japonica L.K., Balanophora.K.K., Balanophora japonica L.K.K.K., Balanophora.K.K.K., Balanophora.K., Balanophora.K.K.K., Balanophora C, Balanophora Balanophora.K.K.K.K.K.K.K., Balanophora.K.K.K.K.K., Balanophora.K.K., Balanophora.K.K.K.K.K.K.K., Balanophora, Balanophora.K.K.K.K.K.K.K.K.K.K.K.K.K.K., Balanophora (Balanophora.K.K.K.K.K.K.K.K.K.K.K., Balanophora, Balanophora involucrata is called Rhodococcus (Balanophora involucrata hook. f.), Balanophora japonica Makino, Balanophora glabrata Hayata, Guangzhou 40656Khostrona is called Aeginea either (Balanophora tobiracola Makino), Aeginea brachypoma (Balanophora abbrevia), Aeginea brachypoma (Balanophora abbrevaria), Aeginea indica (Balanophora obbreva), Aeginea indica (Balanophora funga), and Aeginea peltata (Rhopaleaceae, Balanophora peltata) belonging to genus Balanophora.

The whole plant of Balanophora japonica has medicinal value and has the effects of sobering up, protecting liver, resisting inflammation, easing pain, resisting aging, resisting oxidation, resisting tumor activity and the like (Tuonangjun and the like, research on chemical components and pharmacological action of Balanophora japonica, Chinese national folk medicine, 26: 7: 73-77 at the end of 4 months in 2017). The Balanophora japonica plants are mainly distributed in deep mountain jungles of Guangdong, Fujian, Jiangxi, Guizhou, Yunnan and the like in China, have the advantages of low yield, strong growth seasonality, short growth period and rare resources, and become necessary with the improvement, development and utilization of people on the understanding of medicinal values of the Balanophora japonica plants. At present, no artificial planting method for seeds of Balaophora javanica is available, and no related report for promoting germination of Balaophora javanica seeds is available.

Disclosure of Invention

In order to solve the technical problems that the resources of Balanophora japonica are rare and seeds of Balanophora japonica cultured artificially are not germinated, the invention provides a Balanophora japonica seed germination method, which comprises the following steps:

(1) pre-culturing seeds before germination: washing mature Balaophora seeds with sterile water, putting the seeds into a container filled with wetting filter paper, and pre-culturing the seeds for 24 to 48 hours in a dark condition at the room temperature of between 25 and 35 ℃;

(2) collecting the root of the host plant

Digging host plant roots corresponding to Balaophora japonica plants, wherein the host plant roots are roots which are free of parasitic Balaophora japonica plants and have the diameter of less than 1cm, putting the dug host plant roots into a deep color container, and spraying sterile water to keep the relative humidity of air in the deep color container at 60-80% for later use;

(3) extracting root extract of host plant

Cleaning collected host plant roots, airing surface water, weighing 50 g of the roots, placing the roots into a mortar, adding liquid nitrogen, grinding the roots into powder, taking the powder out, placing the powder into the beaker, adding 200ml of 50% v/v methanol solution, extracting a host plant root extract, performing suction filtration by using filter paper to obtain a filtrate I and filter residue, adding 50ml to 100ml of 50% v/v methanol solution into the filter residue, repeatedly extracting the mixture once, performing suction filtration by using the filter paper to obtain a filtrate II, combining the filtrate I and the filtrate II to obtain a filtrate, volatilizing the filtrate to be in an extract state, wherein the specific gravity of the extract is 1.3 to 1.7;

(4) seed germination

Putting filter paper in a culture dish, wetting the filter paper by distilled water, putting the pre-cultured seeds in the step (1) on the wetted filter paper, taking the extract obtained in the step (3), preparing the extract into a root extract solution with the concentration of 5-20 ppm by using a 50% v/v methanol solution, dropwise adding the root extract solution on the pre-cultured seeds until the seed coat surface of the pre-cultured seeds is wetted, covering a culture dish cover, sealing by using a sealing film, and culturing in dark at 25-35 ℃ until the seeds germinate.

Further, the dark container in the step (2) is a brown glass bottle.

Further, the concentration of the root extract solution in the step (4) is 5ppm to 10 ppm.

Further, the mature Balanophora japonica plant seeds in the step (1) are mature Balanophora japonica plant seeds, and the host plant roots in the step (2) are ficus microcarpa plant roots.

Compared with the prior art, the invention has the beneficial effects that:

the invention fills the blank in the Balanophora plant production industry, the germination rate of Balanophora plant seeds reaches 83.83%, and a good technical foundation is laid for the artificial planting of Balanophora plants. The method has no adverse effect on the environment, and is favorable for environmental protection and sustainable utilization of the Balanophoraceae plants after the Balanophora japonica plants are successfully planted.

Drawings

FIG. 1: example 1 Effect diagram of seed germination rate of Balanophora japonica plants.

FIG. 2: example 2 seed germination rate effect diagram of Balanophora Turcroftiana plant.

FIG. 3: and contrasting with a seed germination rate effect diagram of the balanophoraceae plants.

In FIGS. 1-3, the abscissa indicates the concentration of the host plant root extract solution, and the ordinate indicates the germination rate of Balanophora japonica Balsamiferae plant seeds

Detailed Description

The present invention is further illustrated by the following examples, each of which is a conventional method without specific description.

Example 1

(1) Seed collection and seed storage

Through long-term observation of wild Balaophora L, the mature sign of Balaophora L seeds is that flowers and appendages on inflorescences can be peeled off by hands, and the mature seeds of Balaophora L are collected and put in a self-sealing bag together with the inflorescences to be brought back for later use. Wrapping the seeds with filter paper during long-term storage, and keeping the humidity at 65% -75%. The Balanophora involucrata plant material in the test is Balanophora involucrata hook.f.

(2) Pre-culturing seeds before germination: washing mature Balanophora japonica plant seeds with sterile water for 3 times, putting the washed seeds into a container filled with wetting filter paper, wherein the container is a culture dish, putting tinfoil paper in the culture dish, putting 3 layers of filter paper on the surface of the tinfoil paper, adding sterile water to just soak the filter paper, namely wetting the filter paper, and pre-culturing the seeds for 24 hours in a room with the room temperature of 25 ℃ under the dark condition;

(3) collecting the root of the host plant

According to field investigation, selecting and digging host plant roots corresponding to Balanophora japonica plants, wherein the host plant roots are roots which do not have parasitic Balanophora japonica plants and have the diameter of less than 1cm, placing the dug host plant roots into a brown glass bottle, spraying sterile water to keep the relative humidity of air in the brown glass bottle at 60-70%, and placing the brown glass bottle into an ice box to be brought back to a laboratory for later use. The test host plant is Ficus microcarpa (Ficus microcarpa L.f.), and the irritant germinant is root extract of Ficus microcarpa with diameter less than 1 cm. Namely, the host plant corresponding to the balanophora fasciata is ficus microcarpa.

(4) Extracting root extract of host plant

Cleaning collected host plant roots with sterile water, airing the surface water, weighing 50 g of the roots, putting the roots into a mortar, adding liquid nitrogen, grinding the roots into powder, taking the powder out, putting the powder into a beaker, adding 200ml of 50% v/v methanol solution, standing the powder for 5min, performing suction filtration with filter paper to obtain filtrate I and filter residue, adding 100ml of 50% v/v methanol solution into the filter residue, repeatedly standing the mixture for 5min, performing suction filtration with filter paper to obtain filtrate II, combining the filtrate I and the filtrate II to obtain filtrate, volatilizing the filtrate to obtain an extract, wherein the specific gravity of the extract is 1.7 (the specific gravity of the extract is the mass ratio of the host plant roots to the extract).

(5) Seed germination

Preparing 4 culture dishes, putting two layers of filter paper in each culture dish, wetting the filter paper by using distilled water, putting 30 pre-cultured seeds in the step (2) on the wetted filter paper, taking the extract obtained in the step (4), preparing the extract into root extract solutions with the concentrations of 5ppm, 10ppm, 15ppm and 20ppm by using 50% v/v methanol solution, dripping the root extract solutions with different concentrations on the pre-cultured seeds to wet the seed coat surfaces of the pre-cultured seeds, covering the culture dish cover, sealing the culture dish cover by using a sealing film, culturing for 36 hours in dark at 25 ℃, taking out the seeds after germination is completed, and repeating the test for 3 times.

The calculation of the germination rate is put into the formula:

the germination rates of seeds of Balanophora japonica Makino under the treatment of 5ppm, 10ppm, 15ppm and 20ppm of root extract solution of Ficus microcarpa Makino for 36h are shown in figure 1. FIG. 1 shows that: in the culture described in example 1, the root extract solution concentrations were 5ppm, 10ppm, 15ppm, and 20ppm, respectively, for 36h of germination, and the germination rates of Balanophora japonica seeds were 61.04%, 56.63%, 53.49%, and 42.49%, respectively. (the germination rate is an average of 3 replicates).

Example 2

Example 2 the procedure was the same as in example 1 except that the following procedure was different.

(2) Pre-culturing seeds before germination: pre-culturing for 48 hours in a dark condition in a room with the room temperature of 30 ℃;

(3) collecting the root of the host plant

The relative humidity of air in the brown glass bottle is kept at 75-80%.

(4) Extracting root extract of host plant

Weighing 50 g of root, putting into a mortar, adding liquid nitrogen, grinding into powder, taking out the powder, putting into a beaker, adding 200ml of 50% v/v methanol solution, carrying out ultrasonic extraction on the root extract at the temperature of 25 ℃ for 10min, and carrying out ultrasonic frequency: 28KHZ, filtering with filter paper to obtain filtrate I and residue, adding 50ml of 50% v/v methanol solution into the residue, and ultrasonic extracting at 25 deg.C for 10min with ultrasonic frequency: 28KHZ, performing suction filtration by using filter paper to obtain a filtrate II, combining the filtrate I and the filtrate II to obtain a filtrate, and volatilizing the filtrate to obtain an extract with the specific gravity of 1.3.

(5) Seed germination

30 pre-cultured seeds of step (2) were placed on the wetted filter paper of each petri dish. Culturing at 30 deg.C in dark for 24 hr, and taking out the seeds.

The germination rates of seeds of balanophora fasciata under the treatment of the concentrations of ficus microcarpa root extract solutions of 5ppm, 10ppm, 15ppm and 20ppm for 24h are shown in a figure 2, and the figure 2 shows that: in the culture described in example 2, the root extract solution concentrations were 5ppm, 10ppm, 15ppm and 20ppm respectively, and the germination rates of Balanophora japonica seeds were 83.83%, 81.11%, 79.91% and 75.96% respectively.

Comparison: extracting root extract of host plant from root of host ficus microcarpa with diameter larger than 1cm, treating with root extract solution with concentration of 5ppm, 10ppm, 15ppm and 20ppm respectively as control, and allowing seeds of Balanophora japonica to germinate for 24h as shown in FIG. 3. The rest of the procedure was the same as in example 2. FIG. 3 shows: the host plant root extract is extracted from the host plant root with the diameter of more than 1cm, and the germination rates of the balanophoraceae wild rice seeds are respectively 43.12%, 42.58%, 39.67% and 35.41% under the treatment of root extract solutions with the concentrations of 5ppm, 10ppm, 15ppm and 20 ppm.

Claims

1. A method for germinating Balanophora japonica seeds is characterized in that:

(1) pre-culturing seeds before germination: washing mature Balaophora seeds with sterile water, putting the seeds into a container filled with wetting filter paper, and pre-culturing the seeds for 24 to 48 hours in a dark condition at the room temperature of between 25 and 35 ℃; the mature Balanophora japonica plant seeds are mature Balanophora chinensis plant seeds;

(2) collecting the root of the host plant

Digging host plant roots corresponding to Balaophora japonica plants, wherein the host plant roots are roots which are free of parasitic Balaophora japonica plants and have the diameter of less than 1cm, putting the dug host plant roots into a deep color container, and spraying sterile water to keep the relative humidity of air in the deep color container at 60-80% for later use; the host plant root is a ficus microcarpa plant root;

(3) extracting root extract of host plant

Cleaning collected host plant roots, airing surface water, weighing 50 g of the roots, placing the roots into a mortar, adding liquid nitrogen, grinding the roots into powder, taking the powder out, placing the powder into the beaker, adding 200ml of 50% v/v methanol solution, extracting a host plant root extract, performing suction filtration by using filter paper to obtain a filtrate I and filter residue, adding 50 ml-100 ml of 50% v/v methanol solution into the filter residue, repeatedly extracting the mixture once, performing suction filtration by using filter paper to obtain a filtrate II, combining the filtrate I and the filtrate II to obtain a filtrate, volatilizing the filtrate to be in an extract state, wherein the mass ratio of the host plant roots to the extract is 1.3-1.7;

(4) seed germination

2. The method of germination of Balaophora indica seed as claimed in claim 1, wherein:

the dark color container in the step (2) is a brown glass bottle.

3. The method of germination of Balaophora indica seed as claimed in claim 1, wherein:

the concentration of the root extract solution in the step (4) is 5ppm to 10 ppm.