Fund Project: Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings
Roses are a common flower variety in people's daily lives. With the development of flower cultivation techniques, micrografting of rose tissue-cultured seedlings has become a subject of study for many agricultural experts. Today, the editor of Huinong Network would like to tell you about a fund project: Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings.
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 the combination of tissue culture and grafting techniques, and is a grafting technique in which grafting is performed onto rootstocks cultivated in a greenhouse or rootstocks grown in test tubes. At present, micrografting technology has been widely used in the research and production of fruit trees, flowering trees and shrubs, and others.
Many excellent rose varieties have low seed-setting rates, seeds that do not germinate easily, or cuttings that are difficult to root. For these varieties, grafting is an efficient means of propagation.
2 Scope
These regulations specify the facilities related to rose micrografting, the selection and disinfection of substrate materials, the selection and disinfection of rootstocks and scions, the micrografting technical methods, and post-grafting management, among other contents. These regulations apply to the production of rose micrografting.
3 Normative References
The following documents are indispensable for the application of this document. Only the dated versions apply to this document. For any undated referenced document, the latest version (including all amendments) applies to this document. NY/T 2306-2013 Technical Regulations for Rapid Propagation of Flower Seedlings by Tissue Culture.
4 Cultivation of Rootstocks and Scions
4.1 Selection of Rootstocks
Rootstocks generally use varieties with good growth vigor and strong resistance to pests and diseases. Thornless rose (Rosa multiflora var. inermis) can be selected as the rootstock for rose micrografting. Rootstocks can be propagated by cuttings and tissue culture. The scions for rose micrografting are generally propagated by tissue culture.
4.2 Cutting Propagation of Rootstocks
In rose micrografting, scions cultured in culture flasks can be grafted onto rootstocks in a greenhouse or onto rootstocks in petri dishes. Select branches of the current year that are plump, elastic, and have not yet sprouted flower buds. Cut the upper part into a flat opening, and use a knife to cut the lower part into a horse-ear shape (about 45-60 degrees) at 5-8 mm below the node. In spring, summer, and autumn, keep the top two small leaves and remove the rest; in winter, when cutting in a greenhouse, all leaves should be removed. Use vermiculite or river sand as the substrate, and use plug trays or flower pots; generally, 50-cell plug trays are used. The general cutting depth is 3-5 cm. When using flower pots or non-perforated plug trays, keep the plant spacing at 5-6 cm, then water thoroughly. Place the cut roses in a cool place and pay attention to moisture retention. If the temperature is not high, a plastic film can be used to build an arched tunnel. At a temperature of 20-28 degrees C, roots will form in 30 d. After the roses survive, there is no need to transplant them in a hurry. Wait until the roots age and turn brown before transplanting, which gives a higher survival rate.
4.3 Tissue Culture of Rootstocks and Scions
4.3.1 Explant Treatment
Select young rose stems free of pests and diseases and rinse them under running water for 3 h. Remove the leaves and cut them into stem segments 1.5 cm long, each segment bearing one bud. First disinfect with 70% alcohol for 15 s, then rinse 3 times with sterile water, then disinfect with NaClO at an effective concentration of 2% for 10 min, and rinse 3 times with sterile water. Place the disinfected stem segments with the buds facing upward
in the initiation medium MS + 6-BA 3.0 mg/L + NAA 0.15 mg/L, with a culture temperature of 24 degrees C plus or minus 2 degrees C, a fluorescent light source, a light intensity of 3000-4500 lx, and a photoperiod of 12 h/d.
4.3.2 Subculture
Cut off the young shoots that sprouted in the initiation culture and inoculate them onto the subculture proliferation medium MS + 6-BA 2.0 mg/L + GA3 1.0 mg/L, with a culture temperature of 24 degrees C plus or minus 2 degrees C, a fluorescent light source, a light intensity of 3000-4500 lx, and a photoperiod of 16 h/d. When the young rose stems used as scions grow to 2-3 cm, grafting can be carried out.
4.3.3 Rooting Culture
When the young stems of the thornless rose used as rootstock grow to 2-3 cm, cut the clustered seedlings into individual plants and transfer them to the rooting medium 1/2 MS + IBA 1.0 mg/L + NAA 0.5 mg/L. After the seedlings have rooted, micrografting can be carried out.
5 Grafting
5.1 Preparation Before Grafting
Before grafting, prepare scissors, a scalpel, tweezers, and Parafilm. Rootstocks in the greenhouse need to be watered thoroughly 1-2 d before grafting to ensure sufficient moisture. Before grafting, streptomycin at 500 times dilution + kasugamycin at 600 times dilution can be sprayed on the rootstocks in the greenhouse to prevent bacterial invasion.
5.2 Grafting
The grafting method adopts the cleft grafting method. When the rootstock is a tissue-cultured seedling: under aseptic conditions, select a rootstock that has already rooted in the rooting medium, remove the terminal bud of the tissue-cultured seedling, leave about 2 cm of stem segment, remove the lower lateral buds with tweezers, and make a vertical cut 0.2-0.5 cm deep from the top of the rootstock. Select a scion about 1 cm long with a thickness similar to the rootstock, and cut its base into a wedge shape (with a length similar to the depth of the rootstock cut). Insert the trimmed scion into the rootstock so that it fits as closely as possible with the rootstock, then secure it with Parafilm. Place it in the tissue culture room to continue culturing. After the scion survives, hardening can be carried out and it can be transplanted.
When the rootstock is a cultivated seedling in the greenhouse: select robust tissue-cultured scion seedlings and place them in a hardening room for hardening. After the tissue-cultured seedlings have adapted to the external environment, grafting can be carried out. Grafting should be carried out in a shady environment. Select the current year's young stem as the rootstock, remove the terminal bud, remove the lateral buds below the top, and make a vertical cut 0.2-0.5 cm deep from the top of the rootstock. Select a scion about 1 cm long with a thickness similar to the rootstock, and cut its base into a wedge shape (with a length similar to the depth of the rootstock cut). Insert the trimmed scion into the rootstock so that it fits as closely as possible with the rootstock, then secure it with Parafilm.
Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings
6 Post-Grafting Management
Grafted seedlings cultured in petri dishes can simply be placed in the culture room to continue culturing. Grafted seedlings in the greenhouse need to maintain humidity within 4 d after grafting is completed; bagging or building a small tunnel can be used to retain moisture. At the same time, be careful to use a shade net for shading. After 5 d, use the criterion that the seedlings do not wilt, and gradually open the bags or small tunnel until they enter a normal environment for management.
For seedlings grafted in petri dishes, after the scion survives and grows sufficiently, hardening can be carried out. Place the petri dish in the hardening room, first loosen the lid, and pay attention to observing the seedlings. If signs of wilting appear, the lid needs to be tightened again. Use the criterion that the seedlings do not wilt, and finally open the lid completely. At this point, the seedlings can be transplanted into pots.
The above is what the editor of Huinong Network has compiled regarding
Roses are a common flower variety in people's daily lives. With the development of flower cultivation techniques, micrografting of rose tissue-cultured seedlings has become a subject of study for many agricultural experts. Today, the editor of Huinong Network would like to tell you about a fund project: Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings.
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 the combination of tissue culture and grafting techniques, and is a grafting technique in which grafting is performed onto rootstocks cultivated in a greenhouse or rootstocks grown in test tubes. At present, micrografting technology has been widely used in the research and production of fruit trees, flowering trees and shrubs, and others.
Many excellent rose varieties have low seed-setting rates, seeds that do not germinate easily, or cuttings that are difficult to root. For these varieties, grafting is an efficient means of propagation.
2 Scope
These regulations specify the facilities related to rose micrografting, the selection and disinfection of substrate materials, the selection and disinfection of rootstocks and scions, the micrografting technical methods, and post-grafting management, among other contents. These regulations apply to the production of rose micrografting.
3 Normative References
The following documents are indispensable for the application of this document. Only the dated versions apply to this document. For any undated referenced document, the latest version (including all amendments) applies to this document. NY/T 2306-2013 Technical Regulations for Rapid Propagation of Flower Seedlings by Tissue Culture.
4 Cultivation of Rootstocks and Scions
4.1 Selection of Rootstocks
Rootstocks generally use varieties with good growth vigor and strong resistance to pests and diseases. Thornless rose (Rosa multiflora var. inermis) can be selected as the rootstock for rose micrografting. Rootstocks can be propagated by cuttings and tissue culture. The scions for rose micrografting are generally propagated by tissue culture.
4.2 Cutting Propagation of Rootstocks
In rose micrografting, scions cultured in culture flasks can be grafted onto rootstocks in a greenhouse or onto rootstocks in petri dishes. Select branches of the current year that are plump, elastic, and have not yet sprouted flower buds. Cut the upper part into a flat opening, and use a knife to cut the lower part into a horse-ear shape (about 45-60 degrees) at 5-8 mm below the node. In spring, summer, and autumn, keep the top two small leaves and remove the rest; in winter, when cutting in a greenhouse, all leaves should be removed. Use vermiculite or river sand as the substrate, and use plug trays or flower pots; generally, 50-cell plug trays are used. The general cutting depth is 3-5 cm. When using flower pots or non-perforated plug trays, keep the plant spacing at 5-6 cm, then water thoroughly. Place the cut roses in a cool place and pay attention to moisture retention. If the temperature is not high, a plastic film can be used to build an arched tunnel. At a temperature of 20-28 degrees C, roots will form in 30 d. After the roses survive, there is no need to transplant them in a hurry. Wait until the roots age and turn brown before transplanting, which gives a higher survival rate.
4.3 Tissue Culture of Rootstocks and Scions
4.3.1 Explant Treatment
Select young rose stems free of pests and diseases and rinse them under running water for 3 h. Remove the leaves and cut them into stem segments 1.5 cm long, each segment bearing one bud. First disinfect with 70% alcohol for 15 s, then rinse 3 times with sterile water, then disinfect with NaClO at an effective concentration of 2% for 10 min, and rinse 3 times with sterile water. Place the disinfected stem segments with the buds facing upward
in the initiation medium MS + 6-BA 3.0 mg/L + NAA 0.15 mg/L, with a culture temperature of 24 degrees C plus or minus 2 degrees C, a fluorescent light source, a light intensity of 3000-4500 lx, and a photoperiod of 12 h/d.
4.3.2 Subculture
Cut off the young shoots that sprouted in the initiation culture and inoculate them onto the subculture proliferation medium MS + 6-BA 2.0 mg/L + GA3 1.0 mg/L, with a culture temperature of 24 degrees C plus or minus 2 degrees C, a fluorescent light source, a light intensity of 3000-4500 lx, and a photoperiod of 16 h/d. When the young rose stems used as scions grow to 2-3 cm, grafting can be carried out.
4.3.3 Rooting Culture
When the young stems of the thornless rose used as rootstock grow to 2-3 cm, cut the clustered seedlings into individual plants and transfer them to the rooting medium 1/2 MS + IBA 1.0 mg/L + NAA 0.5 mg/L. After the seedlings have rooted, micrografting can be carried out.
5 Grafting
5.1 Preparation Before Grafting
Before grafting, prepare scissors, a scalpel, tweezers, and Parafilm. Rootstocks in the greenhouse need to be watered thoroughly 1-2 d before grafting to ensure sufficient moisture. Before grafting, streptomycin at 500 times dilution + kasugamycin at 600 times dilution can be sprayed on the rootstocks in the greenhouse to prevent bacterial invasion.
5.2 Grafting
The grafting method adopts the cleft grafting method. When the rootstock is a tissue-cultured seedling: under aseptic conditions, select a rootstock that has already rooted in the rooting medium, remove the terminal bud of the tissue-cultured seedling, leave about 2 cm of stem segment, remove the lower lateral buds with tweezers, and make a vertical cut 0.2-0.5 cm deep from the top of the rootstock. Select a scion about 1 cm long with a thickness similar to the rootstock, and cut its base into a wedge shape (with a length similar to the depth of the rootstock cut). Insert the trimmed scion into the rootstock so that it fits as closely as possible with the rootstock, then secure it with Parafilm. Place it in the tissue culture room to continue culturing. After the scion survives, hardening can be carried out and it can be transplanted.
When the rootstock is a cultivated seedling in the greenhouse: select robust tissue-cultured scion seedlings and place them in a hardening room for hardening. After the tissue-cultured seedlings have adapted to the external environment, grafting can be carried out. Grafting should be carried out in a shady environment. Select the current year's young stem as the rootstock, remove the terminal bud, remove the lateral buds below the top, and make a vertical cut 0.2-0.5 cm deep from the top of the rootstock. Select a scion about 1 cm long with a thickness similar to the rootstock, and cut its base into a wedge shape (with a length similar to the depth of the rootstock cut). Insert the trimmed scion into the rootstock so that it fits as closely as possible with the rootstock, then secure it with Parafilm.
Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings
6 Post-Grafting Management
Grafted seedlings cultured in petri dishes can simply be placed in the culture room to continue culturing. Grafted seedlings in the greenhouse need to maintain humidity within 4 d after grafting is completed; bagging or building a small tunnel can be used to retain moisture. At the same time, be careful to use a shade net for shading. After 5 d, use the criterion that the seedlings do not wilt, and gradually open the bags or small tunnel until they enter a normal environment for management.
For seedlings grafted in petri dishes, after the scion survives and grows sufficiently, hardening can be carried out. Place the petri dish in the hardening room, first loosen the lid, and pay attention to observing the seedlings. If signs of wilting appear, the lid needs to be tightened again. Use the criterion that the seedlings do not wilt, and finally open the lid completely. At this point, the seedlings can be transplanted into pots.
The above is what the editor of Huinong Network has compiled regarding the Technical Regulations for Micrografting of Rose Tissue-Cultured Seedlings. Have you all learned it? As cultivation techniques continue to improve and advance, the skills of flower industry practitioners must also be continuously updated and learned, so that the flower cultivation industry can become better and better.
Transplanting and Management Techniques for Alocasia Tissue-Cultured Seedlings
Alocasia, also known as black-leaf taro, is a plant of the genus Alocasia in the family Araceae. It is a high-grade, uniquely styled indoor foliage plant. At present, Alocasia is very popular in the international foliage plant market, and it is widely used in high-grade indoor courtyard design and waterscape design.
1 Biological Characteristics and Main Varieties
1.1 Growth Habits Alocasia prefers a high-temperature, high-humidity ecological environment. The optimum growth temperature is around 20-30 degrees C, and it prefers semi-shade. It is suitable for planting in loose, well-drained and well-aerated soil rich in humus. During the growing season, the soil and air are required to have relatively high humidity. In winter, the room temperature should be maintained at 15-18 degrees C. Below 15 degrees C, growth stops and the plant enters dormancy. During dormancy, water should be controlled and fertilizer should be stopped.
1.2 Morphological Characteristics There are about 60-70 species in this genus, native to tropical Asia and the Americas. Most have huge, distinctive leaves. The stem is short, stout, fleshy, and has underground stems. The single leaves are sagittate, hastate, cordate, or ovate, somewhat leathery, 25-40 cm long and 10-20 cm wide, with sharp tips; the petiole is long and light green; the leaf margin has 5-7 large tooth-like notches; the main vein is trifurcate, and the lateral veins extend directly to the notches. The leaves are dark green and rich in metallic luster. The underground part has fleshy tubers and easily tillers to form clustered plants. The plant height is 30-50 cm.
1.3 Main Varieties The varieties of the genus Alocasia that have been rapidly propagated by tissue culture and are popular in China include:
Alocasia sanderiana, also known as the silver-leaf Alocasia. The leaves are long sagittate, the silvery-green leaf surface is glossy, with grayish-white veins, and the main and lateral veins are prominently raised.
Alocasia lowii has large leaves, broadly cordate, with a pale brownish-green leaf surface and grayish-green veins.
Alocasia longiloba has sagittate leaves, 30-50 cm long, with slender, upright petioles, a dark green leaf surface, and silvery-grayish-green veins.
Alocasia mortefontanensis has leaves 50 cm long, long sagittate, pale blue-green, with silver and green vein patterns.
Alocasia watsoniana has large, wrinkled, leathery leaves, blue-green, with silver and white vein patterns, and a purple leaf underside.
Alocasia korthalsii has sagittate leaves, pale green, with silvery-white vein patterns.
Alocasia macrorrhizos has large, thin, peltate leaves, long petioles, and is dark green.
Alocasia macrorrhizos var. variegata has dark green leaves with milky-white and light green patches.
Alocasia cuprea, also known as large mosaic-leaf taro, has cordate leaves, with the midrib and pinnate lateral veins dark green and markedly sunken, and a purple leaf underside. In addition, there are also Alocasia cucullata, fragrant Alocasia (Alocasia jodora), and gray Alocasia (Alocasia plumdea).
2 Technical Process for Transplanting Tissue-Cultured Seedlings
2.1 Cleaning Tissue-Cultured Seedlings The purpose is to wash off the medium, remove old leaves, rotten leaves, and yellow leaves, and improve the survival rate.
Take a 50-100 L container (square plastic bucket) and fill it 2/3 with water. Take out a whole cup of tissue-cultured seedlings and gently hold the root-stem part with both hands, shaking back and forth in the water until the medium disperses and falls off. Separate them with both hands using clumps as units, taking care to keep the clumps intact; or first separate them in water using clumps as units, and gently rub and wash the medium off the root-stem part by hand, taking care not to injure the seedlings. Cut off old tissues, rotten leaves, yellow leaves, diseased leaves, etc. After each cup of seedlings is cleaned, place them in a clean basket, aligning them by the stems.
Attached clump standard: plant height 4-8 cm (from the base to the top of the petiole), with 1 leaf, plant thickness 3 mm or more; if the number of leaves is more than 1, then thickness is not counted; no yellow leaves; callus mass diameter at the base ≤1.5 cm; rooted; no etiolation; for dwarf Alocasia, plant height 3.5-6 cm.
2.2 Removing Variant Seedlings When variant seedlings are encountered, pick them out and plant them separately (and mark them with labels as variant seedlings). Variant seedlings refer to seedlings that differ significantly from the characteristics of the variety, such as in color and leaf shape.
Hardening Techniques for Dendrobium nobile Tissue-Cultured Seedlings
Dendrobium nobile is a perennial epiphytic herb of the genus Dendrobium in the family Orchidaceae. It is mainly distributed in Sichuan, Guizhou, Yunnan, Guangdong, Guangxi, Taiwan, China, and other provinces (regions). Its stems are used as medicine. It is a traditional precious medicinal plant and has effects such as nourishing yin and clearing heat, promoting fluid production and benefiting the stomach, moistening the lungs and relieving cough, and improving eyesight and strengthening the body. Modern pharmacological studies have shown that Dendrobium also has effects such as anti-tumor, anti-aging, enhancing body immunity, dilating blood vessels, and inhibiting platelet aggregation. Therefore, Dendrobium is widely used in clinical practice and in traditional Chinese medicine compound prescriptions, and has broad market prospects. It has now been listed by the state as a rare and endangered species under key protection.
The natural reproduction rate of Dendrobium nobile is extremely low, and with slow growth plus predatory harvesting by humans, wild resources have been severely depleted. In order to increase the propagation coefficient and reduce resource consumption, the Yunnan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, uses tissue culture rapid propagation technology to propagate seedlings. Among these, "hardening" is an important link in the whole process of tissue culture seedling raising, and if it is not handled well, it will directly affect the survival rate of tissue-cultured seedlings.
1 Preparation Before Transplanting and Post-Transplanting Management
When test-tube seedlings have grown to 3-5 cm and have 4-5 roots, they can be transplanted. Before transplanting, it is first necessary to cultivate strong seedlings and carry out open-flask hardening. When transplanting, first select the seedbed substrate well, and set up a shade net to prevent direct sunlight.
1.1 Cleaning and Disinfection of Tissue-Cultured Seedlings
Gently but thoroughly wash off the medium adhering to the roots to prevent mold after planting, and take care that the seedlings are not injured or are injured as little as possible. After cleaning, disinfect and sterilize with 0.1% carbendazim or 0.3%-0.5% potassium permanganate for 5 minutes.
1.2 Transplanting of Tissue-Cultured Seedlings
Carefully insert the tissue-cultured seedlings into the prepared substrate, smooth them by hand, spray water to settle them in, and then spray 0.1% carbendazim or chlorothalonil.
1.3 Management After Transplanting
After hardening, special attention should be paid during the first 3-5 days to management work such as heat preservation, moisture retention, and shading. Spraying is the main method, with spraying every day. If the substrate is dry, water thoroughly once every 2-3 days. One month after transplanting the young seedlings, spray some additional nutrient elements. Investigate the survival rate after 2 months.
2 Effects of Different Hardening Substrates on the Survival of Dendrobium
Three different substrates, sugarcane bagasse + ceramsite, charcoal, and ceramsite, were used for hardening. After two months, the survival rates were counted, and the results are shown in Table 1.
It can be seen from the table that the survival rate with sugarcane bagasse + ceramsite was relatively high, while charcoal was relatively poor.
3 Growth of Dendrobium Under Different Hardening Times
A survey was conducted on the growth of the same batch of seedlings on the same hardening substrate, with 100 plants randomly selected for observation. The results are shown in Table 2.
Dendrobium nobile is a perennial epiphytic herb of the genus Dendrobium in the family Orchidaceae. It is mainly distributed in Sichuan, Guizhou, Yunnan, Guangdong, Guangxi, Taiwan, China, and other provinces (regions). Its stems are used as medicine. It is a traditional precious medicinal plant and has effects such as nourishing yin and clearing heat, promoting fluid production and benefiting the stomach, moistening the lungs and relieving cough, and improving eyesight and strengthening the body. Modern pharmacological studies have shown that Dendrobium also has effects such as anti-tumor, anti-aging, enhancing body immunity, dilating blood vessels, and inhibiting platelet aggregation. Therefore, Dendrobium is widely used in clinical practice and in traditional Chinese medicine compound prescriptions, and has broad market prospects. It has now been listed by the state as a rare and endangered species under key protection.