Fund project: technical specification for micro-grafting of tissue culture seedlings of Chinese rose

Rose is a common flower variety in people's daily life. with the development of flower culture technology, micro-grafting of rose tissue culture seedlings has become the research object of many agricultural experts. Today, the editor of Huinong Network is going to talk to you about a fund project: technical regulations for tissue culture seedlings micro-grafting of rose.

1 introduction

As one of the basic means of biotechnology, micro-grafting plays an important role in the progress of social science and technology. Micrografting is a combination of tissue culture and grafting, which is a grafting technique on rootstocks cultured in greenhouse or in test tubes. Now micro-grafting technology has been widely used in the research and production of fruit trees, flowers and trees.

Many excellent rose varieties have low seed setting rate, seeds are not easy to germinate or cuttings are difficult to take root. For these varieties, grafting is an efficient means of propagation.

2 range

This regulation stipulates the facilities related to rose micro-grafting, the selection and disinfection of substrate materials, the selection and disinfection of rootstocks and scions, the technical methods of micro-grafting, the management after grafting and so on. This procedure is applicable to the production of rose micro-grafting.

3 normative citation documents

The following documents are essential for the application of this document. The dated version applies to this document. For all undated reference documents, the latest version (including all amendment orders) applies to this document. Technical regulations for tissue culture and rapid propagation of flower seedlings in NY/T 2306mur2013.

4 rootstock and scion cultivation

4.1 selection of rootstocks

Rootstocks generally choose varieties with good growth and strong resistance to diseases and insect pests. The thornless rose can be selected as the rootstock of rose micro-grafting. Rootstocks can be propagated by cutting and tissue culture. The scions of rose micrografting are generally propagated by tissue culture.

4.2 cutting of rootstocks

The micrografting of rose can graft the scion cultured in the culture bottle to the rootstock in the greenhouse or the rootstock in the petri dish. Select the branches with full growth, elasticity and ungerminated flower buds in the current year. The upper part is cut into a flat mouth, and the lower part is cut into a Mal shape (about 45-60 °) at the 5~8mm under the node. The uppermost two leaflets are left in spring, summer and autumn, and the rest leaves are removed. All leaves should be removed when cutting in the greenhouse in winter. The matrix uses vermiculite or river sand, use cave pots or flowerpots, generally choose 50-hole hole pots. Generally, the cutting depth is 3~5cm, keep the plant spacing in 5~6cm with flowerpots or pots without holes, and then pour water thoroughly. Put the cut rose in a cool place, pay attention to moisturizing, if the temperature is not high, you can use plastic film to set up the arch shed. When the temperature is 20-28 ℃, it will take root in 30 days. After the rose has survived, there is no need to rush to transplant. The survival rate is higher when the roots are aged and turned brown.

4.3 tissue culture of rootstock and scion

4.3.1 explant treatment

The young stems of rose without diseases and insect pests were selected and washed under tap water for 3 hours. Except for leaves, cut the stem segment of 1.5cm with one bud per segment, first disinfect it with 70% alcohol for 15 seconds, then rinse it with sterile water for 3 times, then disinfect 10min with 2% effective concentration of NaClO and rinse with sterile water for 3 times. Put the sterilized stem bud upward.

The culture temperature was 24 ℃ ±2 ℃, the light intensity was 3000~4500lx and the light time was 12h/d.

4.3.2 subculture

The buds that initiated the culture and germination were cut off and inoculated on the subculture medium MS+6-BA2.0mg/L+ GA31.0mg/L. The culture temperature was 24 ℃ ±2 ℃, fluorescent light source, light intensity 3000~4500lx and light time 16h/d. When the tender stem of rose used as scion grows to 2~3cm, it can be grafted.

4.3.3 rooting culture

When the tender stem of the thornless rose as a rootstock grows to 2~3cm, the clustered seedlings are cut into plants and transferred to the rooting medium 1/2MS+IBA 1.0mg/L+NAA 0.5mg/L, and micro-grafting can be carried out after the seedlings take root.

5 grafting

5.1 preparation before grafting

Scissors, scalpels, tweezers and Parafilm film should be prepared before grafting. The rootstocks in the greenhouse need to be watered 1-2 days before grafting to ensure sufficient water. Before grafting, the rootstocks in the greenhouse can be sprayed with 500 times of streptomycin + 600 times of Chunleimycin to prevent bacterial damage.

5.2 grafting

The grafting method was split grafting. When the rootstock is tissue culture seedling: under aseptic condition, the rootstock which has grown root in rooting medium is selected, the top bud of tissue culture seedling is removed, the stem segment of 2cm is left, the bottom lateral bud is removed with tweezers, and a cut with depth of 0.2~0.5cm is made from the top of rootstock. The scion which is about 1cm in length and similar in thickness to that of the rootstock is selected, and its base is cut into a wedge (the length is similar to the cut depth of the rootstock). Insert the cut scion into the rootstock, make it fit with the rootstock as much as possible, and then fix it with Parafilm film. Put it into the tissue culture room to continue the culture. after the scion survived, the seedlings can be trained and transplanted.

When the rootstock is the cultivated seedling in the greenhouse, the strong scion tissue culture seedlings are selected and put into the seedling training room, and the tissue culture seedlings can be grafted after they adapt to the external environment. Grafting should be carried out in a shady environment. The rootstock should select the tender stem of the same year, remove the terminal bud, remove the lateral buds below the top, and make a longitudinal cut with a depth of 0.2~0.5cm from the top of the rootstock. The scion which is about 1cm in length and similar in thickness to that of the rootstock is selected, and its base is cut into a wedge (the length is similar to the cut depth of the rootstock). Insert the cut scion into the rootstock, make it fit with the rootstock as much as possible, and then fix it with Parafilm film.

Technical specification for micrografting of tissue culture seedlings of Chinese rose

6 Management after grafting

The grafted seedlings cultured in the culture dish can be put into the culture room to continue to cultivate. The grafted seedlings in the greenhouse need to maintain humidity within 4 days after grafting, and can be bagged or built in a shed for moisturization. at the same time, pay attention to shading the sun with a shading net, and gradually open the bagging or shed 5 days later, according to the standard that the seedlings do not wilt. Until it is managed in a normal environment.

The seedlings grafted in the petri dish can be trained after the scion has survived and fully grown. Put the petri dish into the seedling training room, loosen the lid first, and observe the seedlings. If there are signs of wilting, you need to tighten the lid again, taking the wilting ratio of the seedlings as the standard, and finally completely open the lid, and then you can transplant the seedlings into the pot.

The above is the information compiled by the editor of Huinong Network

Rose is a common flower variety in people's daily life. with the development of flower culture technology, micro-grafting of rose tissue culture seedlings has become the research object of many agricultural experts. Today, the editor of Huinong Network is going to talk to you about a fund project: technical regulations for tissue culture seedlings micro-grafting of rose.

1 Introduction

As one of the basic means of biotechnology, micrografting plays an important role in the progress of social science and technology. Micrografting is a combination of tissue culture and grafting techniques. It is a grafting technique to rootstock grown in greenhouse or in vitro. Now micrografting technology has been widely used in fruit trees, flowers and other research and production.

Grafting is an efficient propagation method for many excellent rose varieties with low seed setting rate, difficult seed germination or difficult rooting.

2 scope

This regulation specifies the facilities related to rose micrografting, selection and disinfection of substrate materials, selection and disinfection of rootstock and scion, technical methods of micrografting, management after grafting, etc. This regulation is applicable to the production of rose micrografting.

3 Normative references

The following documents are essential for the application of this document. Dated versions apply to this document. For undated references, the latest version (including all amendments) applies to this document. NY/T 2306-2013 Technical regulations for tissue culture and rapid propagation of flower seedlings.

4. Rootstock and scion cultivation

4.1 Selection of rootstock

Rootstocks are generally selected to grow well and have strong resistance to pests and diseases. Rosa spinosa can be selected as rootstock for micrografting of Rosa chinensis. Rootstocks can be propagated by cutting and tissue culture. Micrografted scions of Rosa chinensis are generally propagated in tissue culture.

4.2 Rootstock cuttings

Rose micrografting can graft scions cultured in flasks onto rootstocks in greenhouses or in petri dishes. Select branches that are full, elastic and ungerminated in flower bud. The upper part is cut into a flat mouth, the lower part is cut into a horse ear shape (about 45~60°) at 5~8mm below the node with a knife, the top two leaflets are kept in spring, summer and autumn, and the rest leaves are removed. All leaves should be removed in winter when cutting in the greenhouse. Substrate use vermiculite or river sand, use hole pots or flower pots, generally choose 50 hole pots. Generally, the cutting depth is 3~5cm, and the plant spacing can be maintained at 5~6cm with flowerpots or holeless pots, and then watered thoroughly. Put the rose cuttings in a cool place, pay attention to moisture, if the temperature is not high, plastic film can be used to erect arches. Rooting will occur in 30 days at 20~28℃. After the roses survive, do not rush to transplant. The survival rate is higher when the roots are aged and turned brown.

4.3 Tissue Culture of Rootstock and Scion

4.3.1 Explant treatment

selecting young stems of roses without diseases and insect pests, and washing the young stems under tap water for 3 hours. Remove leaves, cut into 1.5 cm long stem segments with a bud in each segment, disinfect with 70% alcohol for 15s, then rinse with sterile water for 3 times, then disinfect with effective concentration of 2% NaClO for 10min, rinse with sterile water for 3 times. Put the sterilized stem bud up

Put it in MS+6-BA 3.0mg/L+ NAA 0.15mg/L culture medium, culture temperature 24℃±2℃, fluorescent lamp light source, illumination intensity 3000~ 4500lx, illumination time 12h/d.

4.3.2 Subculture

The buds were cut off and inoculated on MS+6-BA 2.0mg/L+ GA3 1.0mg/L medium at 24℃±2℃ under fluorescent light with illumination intensity of 3000~ 4500lx and illumination time of 16h/d. Grafting can be carried out when the tender stem of the rose used as scion grows to 2 - 3 cm.

4.3.3 Rooting culture

When the tender stem of Rosa spinulosa as rootstock grows to 2~3cm, the clustered seedlings are cut into individual plants and transferred to rooting medium 1/2MS +IBA 1.0mg/L+NAA 0.5mg/L. After rooting, micrografting can be carried out.

5 Grafting

5.1 Preparation before grafting

Scissors, scalpels, tweezers and Parafilm membrane should be prepared before grafting. The rootstock in the greenhouse needs to be watered 1~ 2 days before grafting to ensure sufficient water. Before grafting, streptomycin 500 times solution + kasugamycin 600 times solution can be sprayed on rootstocks in greenhouse to prevent bacterial damage.

5.2 grafting

The grafting method was cleavage grafting. when the rootstock is a tissue culture seedling, selecting the rootstock which has grown roots in a rooting culture medium under an aseptic condition, removing the terminal bud of the tissue culture seedling, leaving a stem segment of about 2cm, removing the lateral bud at the bottom by tweezers, and longitudinally cutting an incision with the depth of 0.2 - 0.5cm from the top of the rootstock. Select scions about 1 cm long and similar in thickness to the rootstock, and cut their bases into wedges (length similar to the rootstock incision depth). The cut scion is inserted into the rootstock to make it fit with the rootstock as much as possible, and then fixed with Parafilm membrane. Put into the tissue culture room to continue culture, scion survival after seedling training and transplanting.

When the rootstock is cultivated seedlings in a greenhouse, robust scion tissue culture seedlings are selected and put into a seedling training room to train seedlings, and grafting can be carried out after the tissue culture seedlings adapt to the external environment. Grafting should be carried out in a shady environment. Rootstock should be selected from the tender stems of the current year, the terminal buds should be removed, the lateral buds below the top should be removed, and a cut with a depth of 0.2~0.5cm should be cut longitudinally from the top of the rootstock. Select scions about 1 cm long and similar in thickness to the rootstock, and cut their bases into wedges (length similar to the rootstock incision depth). The cut scion is inserted into the rootstock to make it fit with the rootstock as much as possible, and then fixed with Parafilm membrane.

Technical regulations for micrografting of rose tissue culture seedlings

6 Post-grafting management

The grafted seedlings cultured in the culture dish are put into the culture chamber for continuous culture. Grafted seedlings in greenhouse need to maintain humidity within 4 days after grafting, can be bagged or set up small shed for moisture, at the same time pay attention to shade with sunshade net, after 5 days to seedlings do not wilt as a standard, gradually open the bag or small shed, until into the normal environment for management.

After the grafted seedlings survive and fully grow in the culture dish, the seedlings can be trained, the culture dish is placed in a seedling training room, the cover is loosened first, and attention is paid to observing the seedlings. If there is a sign of wilting, the cover needs to be tightened again, and the seedling wilting ratio is taken as the standard. Finally, the cover is completely opened, and then the seedlings can be transplanted into the pot.

The above is the technical regulations for micro-grafting of rose tissue culture seedlings compiled by Huinong. Have you learned them? With the continuous improvement and progress of breeding technology, the technology of flower practitioners should also be constantly updated and updated, so as to make the flower breeding industry better and better.

Transplantation and management techniques of tissue culture seedlings of Guanyinlian

Guanyinlian, also known as black leaf taro, is a high-grade, unique style indoor foliage plant belonging to Alocasia genus of Araceae. At present, Guanyinlian is very popular in the international foliage plant market. It is widely used in high-grade indoor garden design and waterscape design.

1 Biological characteristics and main varieties

1.1 Living habits Guanyin lotus likes high temperature, high humidity ecological environment, growth temperature of about 20~30℃, like half shade. Suitable for planting in loose, well-ventilated soil rich in humus, the growing season requires soil and air to have greater humidity. Winter room temperature should be kept at 15~18℃, temperature below 15℃, growth stops, enter dormancy. During dormancy, water should be controlled and fertilization should be stopped.

1.2 There are about 60~70 species of plants in this genus, which are native to tropical Asia and America. Most of the leaves are large and special. Stems short, stout, fleshy, with underground stems. Leaves simple, arrow shaped, halberd shaped, cordate or ovate, leathery, 25~40cm long, 10~20cm wide, apex acute; petiole long, light green, leaf margin with 5~7 large dentate notches, main vein trident, lateral veins reaching notch. The leaves are thick green and rich in metallic luster. The underground part has fleshy tubers, and it is easy to tiller to form clustered plants. The plant height is 30~50cm.

1.3 The main varieties of Guanyinlian are:

A.sanderiana, also known as Alocasia. Long arrow-shaped leaves, silver-green leaves shiny, with gray-white veins, prominent main lateral veins.

Iowii has large leaves, broad heart shape, pale brownish green leaves and grayish green veins.

A.longiloba has arrow-shaped leaves, 30~50cm long, slender petioles, dark green leaves and silver-gray veins.

A.mortefontanensis leaves are 50cm long, arrow-shaped, pale blue-green, with silver and green veins.

A.watsoniano, large, ruffled, leathery leaves, blue-green with silver and white veins, purple abaxially.

A.koahdsii, arrow-shaped, pale green, silver-white veined.

The leaves of A.nacrirrguzo are large and thin, peltate, petiole long and dark green.

A. macrorhizavar.variegata has dark green leaves with milky white and light green patches.

C.cupreo, also known as large mosaic taro, leaves heart-shaped, ribs and pinnate lateral veins are dark green, obviously concave, purple leaf back. In addition, there are potato (A.cucullata), fragrant Guanyin (A.jodora), gray Guanyin (A.plumdea).

2. Transplantation technology of tissue culture seedlings

2.1 The purpose of cleaning tissue culture seedlings is to clean the culture medium, remove old leaves, rotten leaves and yellow leaves, and improve the survival rate.

Take a container (square plastic bucket) with a capacity of 50~100L and hold 2/3 of the water. Take out the whole cup of tissue culture seedlings, gently hold the rhizome part with both hands and shake it back and forth in water until the medium is scattered and fallen off, separate it with both hands, take the lump as the unit, pay attention to keep the lump intact; or separate it in water first, take the lump as the unit, gently rub the medium of the rhizome part with hands, and be careful not to hurt the seedlings. Cut off old tissues, rotten leaves, yellow leaves, diseased leaves, etc., and put 1 cup of seedlings into a clean basket after washing, and place them neatly according to the stem.

The standard of attachment mass: plant height 4~8cm (from base to petiole tip), with 1 leaf, plant thickness more than 3mm, if the number of leaves more than 1, the thickness does not count, no yellow leaves, basal callus mass diameter ≤ 1.5 cm, roots, no excessive growth, dwarf Guanyinlian plant height 3.5~ 6 cm.

2.2 When mutant seedlings are encountered, they are picked out and replanted (and labeled as mutant seedlings). Variant seedlings refer to seedlings with significant differences in characteristics, such as color and leaf shape, from the variety.

Tissue culture seedling refinement technique of Dendrobium nobile

Dendrobium nobile is a perennial epiphytic plant belonging to dendrobium orchid, mainly distributed in sichuan, guizhou, yunnan, guangdong, guangxi, taiwan and other provinces. It is a traditional and precious medicinal plant, which has the effects of nourishing yin and clearing heat, promoting body fluid and benefiting stomach, moistening lung and relieving cough, improving eyesight and strengthening body. Modern pharmacological research shows that Dendrobium also has anti-tumor, anti-aging, enhancing immunity, dilating blood vessels and anti-platelet aggregation effects. Therefore, Dendrobium is widely used in clinical practice and traditional Chinese medicine compound, and has broad market prospects. It has been listed as a rare and endangered species under special protection by the state.

Dendrobium nobile natural reproduction stand extremely low, slow growth coupled with predatory digging, wild resources have been seriously depleted. In order to improve propagation coefficient and reduce resource consumption, Yunnan Branch of Institute of Medicinal Plants, Chinese Academy of Medical Sciences adopts tissue culture rapid propagation technology to propagate seedlings, among which "seedling refinement" is an important link in the whole process of tissue culture seedling breeding, which will directly affect the survival rate of tissue culture seedlings if it is not well grasped.

1. Preparation before transplanting and management after transplanting

When the test-tube seedlings grow to 3~ 5 cm and have 4~5 roots, they can be transplanted. Before transplanting, strong seedlings should be cultivated and the seedlings should be opened. When transplanting, select the seedbed substrate first, and build a shade net to prevent direct sunlight.

1.1 Cleaning and disinfection of tissue culture seedlings

Gently but thoroughly wash off the culture medium stained on the roots to avoid mildew after planting, and pay attention to the seedlings without or less trauma. After cleaning, disinfect and sterilize with 0.1% carbendazim or 0.3%~0.5% potassium permanganate for 5 minutes.

1.2 Transplanting of tissue culture seedlings

Carefully insert the tissue culture seedlings into the prepared matrix, smooth them with hands, spray water to bury them, and spray 0.1% carbendazim or chlorothalonil.

1.3 Post-transplant management

After seedling training, special attention should be paid to heat preservation, moisture retention, shading and other management work in the first 3~5 days, mainly spraying, spraying every day, watering once every 2~3 days for substrate drying, spraying some nutrients one month after seedling transplantation, and investigating survival rate in 2 months.

2. The effect of different media on survival of Dendrobium

Three different substrates of bagasse + ceramsite, charcoal and ceramsite were used for seedling refinement (charcoal could not use new charcoal). The survival rate was counted after two months. The results are shown in Table 1.

It can be seen from the table that the survival rate of bagasse + ceramsite is higher than charcoal.

3. Growth of Dendrobium at different seedling training time

The growth of the same batch of seedlings on the same seedling refining substrate was investigated, and 100 seedlings were selected at fixed points for observation. The results are shown in Table 2.

Dendrobium nobile is a perennial epiphytic plant belonging to dendrobium orchid, mainly distributed in sichuan, guizhou, yunnan, guangdong, guangxi, taiwan and other provinces. It is a traditional and precious medicinal plant, which has the effects of nourishing yin and clearing heat, promoting body fluid and benefiting stomach, moistening lung and relieving cough, improving eyesight and strengthening body. Modern pharmacological research shows that Dendrobium also has anti-tumor, anti-aging, enhancing immunity, dilating blood vessels and anti-platelet aggregation effects. Therefore, Dendrobium is widely used in clinical practice and traditional Chinese medicine compound, and has broad market prospects. It has been listed as a rare and endangered species under special protection by the state.