CN109258377B

CN109258377B - Method for creating germplasm of watercress

Info

Publication number: CN109258377B
Application number: CN201811354640.7A
Authority: CN
Inventors: 周艳; 周庆; 张梅; 胡瑾; 周洪英; 李依嫚; 冯佑鸿
Original assignee: Guizhou Botanical Garden (guizhou Landscape Science Institute Guizhou Plant Institute)
Current assignee: Guizhou Botanical Garden (guizhou Landscape Science Institute Guizhou Plant Institute)
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2022-05-17
Anticipated expiration: 2038-11-14
Also published as: CN109258377A

Abstract

The invention discloses a method for creating germplasm of watercress, which comprises the following steps: culturing the mother material of green broad-bean green; culturing the male parent material of the watercress 'red pigment'; cross pollination; ovary and seed development; aseptic seeding; subculturing; rooting culture; training seedlings and transplanting. The method combines the conditions of natural conditions and climate, and researches the scientific problems of parental cultivation, flowering phase regulation, hybrid pollination, ovary and seed development, tissue culture of the second generation of hybridization and the like of the watercress. The method can be used for cultivating excellent hybrid orchid progeny materials with the characteristics of the chlorophyll and the erythrogenin of the broad-leaved orchid, can enrich the variety types of the broad-leaved orchid, create new varieties and develop the breeding way of the broad-leaved orchid.

Description

Method for creating germplasm of watercress

Technical Field

The invention belongs to the technical field of orchid breeding, and particularly relates to a method for creating a germplasm of watercress.

Background

The leaves of the broad blue plant are elegant and elegant, and the flower type is modesty, so that the broad blue plant has great development value as an excellent gardening flower and is the first to draw the fingers in the national blue. Due to the difference of growth environments, the watercress is influenced by factors such as complex landform and landscape, various climate types, abundant underground mineral reserves and the like, the flower color is extremely rich and changeable, and the cultivation and creation of germplasm resources are not neglected. Currently, the techniques commonly used in the industry are such that: the first is cultivation technology, and the technical process is as follows: the cultivation agent is selected from seeds with good ventilation, and certain purple laterite is mixed during cultivation, so that sufficient illumination and ventilation are required (Yuan, 2005, rural practical technology). Secondly, tissue culture, the technology of which is summarized as follows: washing with running water for 1h, 30s with 75% alcohol, 20min with 0.1% mercuric chloride, and washing with sterile water for 5 times, wherein the optimal seed germination culture medium comprises: MS +1mg/L BAP + 10% coconut water; protocorm proliferation medium: improved kyoto2mg/L BAP + NAA 1 mg/L; and (3) differentiation and strong seedling culture medium: modified 1/2MS +1mg/LIBA +1g/L activated carbon + 5% coconut water. The seedling hardening and transplanting matrix has the best transplanting effect with aquatic weeds (Bu Chao Yang, 2010, plant physiology communication). At present, the watercress is popularized and planted in most provinces of the whole country, but is naturally distributed or some traditional varieties, the number of newly developed varieties is very small, corresponding germplasm creating technologies are lacked, and the increasingly pursuing requirements of the public cannot be met.

In summary, the problems of the prior art are as follows: the original variety of the bean cotyledon is more than the traditional variety, and the corresponding germplasm creating technology is lacked.

Disclosure of Invention

In view of the above, the invention provides a method for creating a germplasm of a watercress, which combines the advantages of resources thereof, artificially creates a germplasm resource of the watercress through a hybridization technology and a tissue culture technology, and contributes to the development of new varieties of orchids.

In order to solve the technical problem, the invention discloses a method for creating germplasm of watercress, which comprises the following steps:

step 1, selecting and culturing a mother material:

selecting a female parent material to be cultivated in a mixed matrix, placing a special orchid cultivation pot serving as a container for cultivation in a greenhouse, and regularly watering, fertilizing and preventing and treating plant diseases and insect pests;

step 2, selecting and culturing male parent materials:

selecting male parent materials to cultivate in a mixed matrix, placing a container for cultivation as a special orchid culture pot in a greenhouse, and periodically watering, fertilizing and preventing and treating plant diseases and insect pests;

step 3, cultivation and flowering phase regulation of the parent material: the flowering phase regulation and cultivation adopts the measures of illumination regulation, nutrition regulation and humidity regulation, and comprehensive regulation and control;

step 4, cross pollination:

the best pollination time of the female flowers is 3-4 days after blooming, when pollinating, firstly removing pollen blocks of the female flowers of the female parent, and then lightly placing the collected pollen blocks of the male parent on the stigma of the female parent by using small tweezers; the column cap has mucus, so that the pollen block does not fall off; to prevent the pollinated flower from being pollinated by the insect again, the petals on the female parent flower can be removed; writing the label with a pencil, and hanging the label on the flower branch; recording is well done, parents and pollination date are written clearly;

step 5, ovary and seed development:

observing the change of the pollinated flowers every day after pollination and preventing damage, wherein the ovary begins to expand after the female parent pollinates for 10 days, and the hybridization is considered to be successful; comprehensively regulating and controlling illumination, moisture and nutrition of plants;

step 6, sterile seeding:

after pollinating seeds for 180 days, aseptically sowing the seeds into a culture medium for culturing; after sowing for 30-40 days, the seeds germinate into white protocorms; after 60 days, most protocorms grow up and turn green;

and 7, protocorm proliferation culture:

transferring the germinated protocorm to a proliferation culture medium for culture; after 30d, differentiating and sprouting;

step 8, rooting culture:

transferring the larger rootless seedlings into a rooting culture medium, culturing for 25d to grow 3-5 fleshy roots, wherein the average seedling height is 2 cm;

step 9, hardening off and transplanting:

transferring the culture bottle to room temperature for hardening treatment, extending the hardened seedlings into the bottle by using a small bamboo stick, and slightly moving out; during transplanting, roots and tender shoots are not damaged as much as possible, then the seedlings are placed in water for rinsing, and a basal medium is cleaned and dried; when the seedlings are transplanted in a pot, the flowerpot is soaked for 12 hours, taken out and dried in the sun to be clean and free of foreign bacteria, broken rubbles are put into the flowerpot as a bottom layer, seedling hardening matrix is put on the flowerpot, a small hole is dug on the flowerpot after the flowerpot is flattened by hands, then the roots of the seedlings are put into the hole, the seedlings are fixed by spreading the matrix, and water is sprayed on the seedlings after the seedlings are planted to enable the seedlings and the matrix to be more tightly fixed;

step 10, management after transplanting:

transplanting the seedlings and then sending the seedlings into a greenhouse; the ventilation is enhanced, and the manual spraying is combined; spraying 10% potassium dihydrogen phosphate water solution every 7 days; after 4-6 weeks of survival, adjusting the illumination intensity, shading for 30%, keeping the matrix wet, and spraying 800-fold 1000-fold chlorothalonil or carbendazim for 1 time every 7 days.

Optionally, the female parent material in the step 1 is flat and smooth in leaf surface, glossy, free of virus diseases and free of insect attack; the pseudobulb is full, the root system is stout, the growth vigor is strong and the force of the watercress is vitality; the mixed matrix is ceramsite, orchid special soil and humus soil with the mass ratio of 1:2: 1.

Optionally, the male parent material in the step 2 is glossy and dark green on leaf surface; the watercress "red pigment" with full pseudobulb, stout root system, strong growth vigor and no disease and pest infection; the mixed matrix is ceramsite, orchid special soil and humus soil with the volume ratio of 1:2: 1.

Optionally, the flowering phase regulation and cultivation in step 3 adopts measures of illumination regulation, nutrition regulation and humidity regulation, and the comprehensive regulation and control specifically comprises the following steps: receiving full light from 10 months to 3 months in the next year, shading 30% in 4 months and 50% in 5-9 months; after the pseudobulb is mature, entering a flower bud differentiation stage, applying a high-P fertilizer with the mass ratio of N to P to K being 1:3:2 to promote flower growth; at the same time, water is sprayed on the ground all the time, the ground is kept moist, the relative humidity of air is more than 60%, and a proper microclimate environment is created, so that the parents can meet each other in the florescence of 4-5 months.

Optionally, the step 5 of comprehensively regulating and controlling the illumination, moisture and nutrition of the plant specifically comprises the following steps: keeping 60% -70% of illumination intensity, spraying 1-2 times of water in 1 week and spraying 10% of potassium dihydrogen phosphate aqueous solution in volume ratio in 2 weeks according to weather conditions.

Optionally, the culture medium in step 6 is MS +6-BA 0.5mg/L + NAA 0.05mg/L + coconut milk 100mL/L, the volume percentage of agar in the culture medium is 1.0%, the volume percentage of sucrose in the culture medium is 3.0%, and the pH value is 5.6; the culture conditions were: culturing at 24 + -1 deg.C in dark for 1 week, and culturing under illumination of 2000lx for 12 h/d.

Optionally, the proliferation culture formula in step 7 is: 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice, wherein the volume percentage of agar in the culture medium is 1.0%, the volume percentage of sucrose is 3.0%, the pH value is 5.6, the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d.

Optionally, the formula of the rooting medium in step 8 is as follows: 1/2MS + IBA2.0+1.0g/L AC, the volume percentage of agar in the culture medium is 1.0%, the volume percentage of sucrose is 3.0%, and the pH value is 5.6; the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d.

Optionally, the seedling exercising time in the step 9 is to move the culture bottle to room temperature for 3 days, then open the bottle cap, and place the culture bottle on the shelf for 4-12 hours; hardening off the seedling substrate: the volume ratio of the water moss to the wood dust is 1: 2.

Optionally, the greenhouse in the step 10 is shaded by 50%, the temperature is 20-25 ℃, and the humidity is 50-70%.

Compared with the prior art, the invention can obtain the following technical effects:

1) the invention reasonably formulates a breeding target and selects the parental material with high ornamental value and strong stress resistance. By comprehensive regulation and control measures such as illumination regulation, nutrition regulation, humidity regulation and the like, a proper microclimate environment is created, so that the florescence of parents meet each other.

2) The cross pollination technology adopted by the invention has the advantages of simple method, simple and convenient operation steps, easy popularization and application and pollination success rate of more than 64 percent.

3) In the matrix proportion, the sterile seeding proportion is MS +6-BA 0.5mg/L + NAA 0.05mg/L +100mL/L coconut milk, and the seed germination rate reaches 72%. The protocorm proliferation culture formula is 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice, and the differentiation rate reaches 85%. The rooting culture formula is 1/2MS + IBA2.0+1.0g/L AC, and the rooting rate reaches 82%.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

Example 1

A method for creating the germplasm of the watercress comprises the following steps:

step 1, selecting and culturing a mother material:

selecting flat leaf surface, luster, no virus disease and no invasion of pests; 100 pseudo bulbs are full, the root system is thick, the growth vigor is strong, and the viable watercress 'green leaves' are cultivated in a mixed matrix of ceramsite, special soil for orchid and humus soil in a mass ratio of 1:2:1, a container for cultivation is a special cultivation pot for orchid, the special cultivation pot is placed in a greenhouse, and watering, fertilizing and pest control are performed regularly.

Step 2, selecting and culturing male parent materials:

selecting glossy and dark green leaves; 100 broad-leaved orchid red pigment plants with full pseudobulb, thick root system, strong growth vigor and no disease and pest infection are cultivated in a mixed matrix of ceramsite, orchid special soil and humus soil in a volume ratio of 1:2:1, a container for cultivation is a orchid special cultivation pot, the orchid special cultivation pot is placed in a greenhouse, and watering, fertilizing and pest control are performed regularly.

Step 3, cultivation and flowering phase regulation of the parent material:

the flowering phase regulation and cultivation adopts the measures of illumination regulation, nutrition regulation, humidity regulation and the like, and the comprehensive regulation and control is realized; the sun is exposed from 10 months to 3 months in the next year, the sun is shielded for 30% in 4 months, and the sun is shielded for 50% in 5-9 months. After the pseudobulb is mature, entering a flower bud differentiation stage, and applying a high-P fertilizer with the mass ratio of N to P to K being 1:3:2 to promote flower growth. At the same time, water is sprayed on the ground all the time, the ground is kept moist, the relative humidity of air is more than 60%, and a proper microclimate environment is created, so that the parents can meet each other in the florescence of 4-5 months.

Step 4, cross pollination:

the best pollination time of the female flowers is 3-4 days after the flowers bloom, when pollinating, pollen blocks of the female flowers (female parent) are removed (castration), and then the collected pollen blocks of the male parent are gently placed on the stigma of the female parent by using small tweezers; to prevent the pollinated flower from being pollinated by the insect again, the petals on the female parent flower can be removed; the label is written with a pencil and hung on the flower branch. Recording is well done, parents and parents are written clearly, pollination date and the like are written;

step 5, ovary and seed development:

after pollination, the change of the pollinated flowers is observed every day to prevent damage, and the ovary begins to expand after pollination by taking the green of the watercress as a female parent for about 10 days, so that the hybridization can be considered to be successful. Comprehensively regulating and controlling illumination, moisture and nutrition of plants, keeping illumination intensity of 60-70%, spraying 1-2 times of water in 1 week and spraying 10% of potassium dihydrogen phosphate aqueous solution in volume percentage once in 2 weeks according to weather conditions.

Step 6, sterile seeding:

after pollinating seeds for 180 days, aseptically sowing the seeds into a culture medium of MS +6-BA 0.5mg/L + NAA 0.05mg/L +100mL/L coconut milk, wherein the volume percentage of agar in the culture medium is 1.0 percent, the volume percentage of sucrose in the culture medium is 3.0 percent, and the pH value is 5.6. The culture temperature is 24 +/-1 ℃, after dark culture for 1 week, the culture is carried out under illumination of 2000lx for 12 h/d. After 30-40 days of sowing, the seeds can be seen to germinate into white protocorms, and the germination rate reaches 72%. After 60 days, most protocorms grow up and turn green;

and 7, protocorm proliferation culture:

transferring the germinated protocorm into a culture medium of 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice, wherein the volume percentage content of agar in the culture medium is 1.0%, the volume percentage content of sucrose in the culture medium is 3.0%, the pH value is 5.6, the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d. After 30 days, seedlings are differentiated and emerge, and the differentiation rate reaches 85 percent.

Step 8, rooting culture:

transferring the larger rootless seedlings into 1/2MS + IBA2.0+1.0g/L AC culture medium containing 1.0 percent of agar by volume, 3.0 percent of sucrose by volume and pH5.6, wherein the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d. After 25 days of culture, 3-5 fleshy roots can grow, the average seedling height is 2cm, and the rooting rate reaches 82%.

Step 9, hardening off and transplanting:

and (3) moving the culture bottle to room temperature, storing for 3 days, opening the bottle cap, placing on a rack for 4-12h, stretching the trained seedlings into the bottle by using a small bamboo stick, and slightly moving out. When transplanting, the seedlings are placed in water for rinsing, and the basal medium is cleaned and dried. When the seedlings are transplanted in a pot, the flowerpot is soaked for 12 hours, taken out and dried in the sun to be clean and free of foreign bacteria, broken rubbles are put into the flowerpot as a bottom layer, seedling hardening matrix of water moss and wood chips (volume ratio is 1:2) is put on the flowerpot, a small hole is dug on the seedling hardening matrix after the seedling hardening matrix is flattened by hands, then the roots of the small seedlings are put into the hole, the matrix is paved to fix the small seedlings, and water is sprayed on the small seedlings after the seedlings are planted to enable the small seedlings and the matrix to be more tightly fixed;

step 10, management after transplanting:

transplanting the seedlings, and putting the seedlings into a greenhouse for shading by 50 percent, wherein the temperature is 20-25 ℃, and the humidity is 50-70 percent. The ventilation is enhanced, and the manual spraying is combined. Spraying 10% potassium dihydrogen phosphate water solution every 7 days. After 4-6 weeks of survival, adjusting the illumination intensity, shading for 30%, keeping the matrix wet, and spraying 800-fold 1000-fold chlorothalonil or carbendazim for 1 time every 7 days.

The technical effects of the invention are illustrated below with reference to specific experimental data:

1. influence of pollination time on pollination seed setting rate:

setting 4, 5, 7 and 10 days for 4 pollination time periods after the female flowers bloom, removing pollen blocks of the female flowers (female parents) (castration), slightly placing collected pollen blocks of the male parents on stigma of the female parents by using small tweezers, recording, and writing parent and parent pollinations, pollination dates and the like. Statistical analysis was performed on the post-pollination results (table 1). The result shows that the pollination time has important influence on the maturing rate, and the maturing rate is the highest by pollinating 3 days after the female parent flowers, and reaches 64 percent.

TABLE 1 Effect of different time on pollination seed setting

2. Effect of Medium composition on sterile seeding

The seeds were uniformly sown in 4 media of MS, MS +6-BA 0.5mg/L + NAA 0.05mg/L +100mL/L coconut milk, 1/2MS, 1/2MS +6-BA 0.5mg/L + NAA 0.05mg/L +100mL/L coconut milk, and aseptically cultured to observe the germination of the seeds (Table 2). The result shows that the components of the culture medium have important influence on seed germination, the difference of seed germination difference is extremely obvious among different treatments, and the germination rate of the coconut milk treated by the culture combination MS +6-BA 0.5mg/L + NAA 0.05mg/L +100mL/L is the highest and reaches 72%.

TABLE 2 Effect of Medium composition on seed Germination Rate

3. Effect of medium composition on protocorm proliferation culture:

the germinated protocorm is respectively transferred into 4 culture media of MS +6-BA 0.5mg/L + NAA0.1mg/L +100mL/L banana juice, MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L coconut milk, 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice, 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L coconut milk (Table 3). The results show that the difference of the proliferation culture effect of different culture combinations on protocorms is obvious, the culture effect of 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice is the best, and the differentiation rate reaches 85%.

TABLE 3 Effect of Medium composition on protocorm proliferation culture

4. Influence of the Medium composition on root culture:

the results of transferring the larger rootless seedlings to 1/2MS + IBA1.0mg/L, 1/2MS + IBA2.0 mg/L, 1/2MS + IBA1.0mg/L +1.0g/L AC, 1/2MS + IBA2.0 mg/L +1.0g/L AC 4 culture media (Table 4) show that the difference of the culture effect of different culture combinations on the rootage is obvious, the culture effect of 1/2MS + IBA2.0+1.0g/L AC is the best, and the rootage rate reaches 82%.

TABLE 4 Effect of Medium composition on root culture

The foregoing description shows and describes several preferred embodiments of the invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, and is not to be construed as excluding other embodiments, and that the invention is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The method for creating the germplasm of the watercress is characterized by comprising the following steps of:

step 1, selecting and culturing a mother material:

selecting flat leaf surface, luster, no virus disease and no invasion of pests; the pseudo bulbs are full, the root systems are thick, the growth vigor is strong, and the viable broad blue green is used as a mother material to be cultivated in a mixed matrix of ceramsite, special orchid soil and humus soil in a mass ratio of 1:2:1, a container for cultivation is a special orchid cultivation pot, the special orchid cultivation pot is placed in a greenhouse, and watering, fertilizing and pest control are carried out at regular intervals;

step 2, selecting and culturing male parent materials:

selecting glossy and dark green leaves; the watercress 'red pigment' with full pseudobulb, thick root system, strong growth vigor and no disease, insect and pest infection is used as a male parent material to be cultivated in a mixed matrix of ceramsite, special orchid soil and humus soil with the volume ratio of 1:2:1, a container for cultivation is a special orchid cultivation pot, the special orchid cultivation pot is placed in a greenhouse, and watering, fertilizing and pest control are carried out at regular intervals;

step 4, cross pollination:

the best pollination time of the female flowers is 3-4 days after blooming, when pollinating, firstly removing pollen blocks of the female flowers of the female parent, and then lightly placing the collected pollen blocks of the male parent on the stigma of the female parent by using small tweezers; the column cap has mucus, so that the pollen block does not fall off; removing petals on the female parent flower to prevent the pollinated flower from being pollinated by the insect; writing the label with a pencil, and hanging the label on the flower branch; recording is well done, parents and pollination date are written clearly;

step 5, ovary and seed development:

step 6, sterile seeding:

and 7, protocorm proliferation culture:

step 8, rooting culture:

transferring the larger rootless seedlings into a rooting culture medium, culturing for 25d to grow 3-5 fleshy roots, wherein the average seedling height is 2cm, and the rooting culture medium comprises the following formula: 1/2MS + IBA2.0+1.0g/L AC, the volume percentage of agar in the culture medium is 1.0%, the volume percentage of sucrose is 3.0%, and the pH value is 5.6; the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d;

step 9, hardening off and transplanting:

step 10, management after transplanting:

and (3) transplanting the seedlings and then sending the seedlings into a greenhouse: the ventilation is enhanced, and the manual spraying is combined; spraying 10% potassium dihydrogen phosphate water solution every 7 days; adjusting the illumination intensity, shading for 30 percent, keeping the matrix wet, and spraying 800-fold and 1000-fold chlorothalonil or carbendazim for 1 time every 7 days after the survival of the transplanted seedlings 4-6 weeks;

the flowering phase regulation and cultivation in the step 3 adopts the measures of illumination regulation, nutrition regulation and humidity regulation, and the comprehensive regulation and control specifically comprises the following steps: receiving full light from 10 months to 3 months in the next year, shading 30% in 4 months and 50% in 5-9 months; after the pseudobulb is mature, entering a flower bud differentiation stage, applying a high-P fertilizer with the mass ratio of N to P to K being 1:3:2 to promote flower growth; spraying water on the ground at the same time, keeping the ground moist and the relative humidity of the air above 60%, creating a suitable microclimate environment, and enabling the parents to meet in the flowering phase of 4-5 months;

the comprehensive regulation and control of the illumination, the moisture and the nutrition of the plants in the step 5 specifically comprises the following steps: keeping 60% -70% of illumination intensity, spraying 1-2 times of water in 1 week and spraying 10% of potassium dihydrogen phosphate aqueous solution in volume ratio in 2 weeks according to weather conditions;

the culture medium in the step 6 is MS +6-BA 0.5mg/L + NAA 0.05mg/L + coconut milk 100mL/L, the volume percentage content of agar in the culture medium is 1.0%, the volume percentage content of sucrose in the culture medium is 3.0%, and the pH value is 5.6; the culture conditions were: culturing at 24 + -1 deg.C in dark for 1 week, and culturing under illumination of 2000lx for 12 h/d;

the proliferation culture formula in the step 7 is as follows: 1/2MS +6-BA 1.0mg/L + NAA0.1mg/L +100mL/L banana juice, wherein the volume percentage of agar in the culture medium is 1.0%, the volume percentage of sucrose is 3.0%, the pH value is 5.6, the culture temperature is 24 +/-1 ℃, and the illumination time is 12 h/d.

2. The method for creating the germplasm of the watercress according to claim 1, wherein the seedling exercising time in the step 9 is that the bottle cap is opened after the culture bottle is moved to room temperature for storage for 3 days, and the culture bottle is placed on a shelf for 4-12 h; hardening off the seedling substrate: the volume ratio of the water moss to the wood dust is 1: 2.

3. The method for creating the germplasm of Phaseolus vulgaris according to claim 1, wherein the greenhouse in step 10 is shaded 50%, the temperature is 20-25 ℃, and the humidity is 50-70%.