In vitro culture, any tissue of garlic needs suitable substrate, nutrition, temperature, humidity, light and other conditions to culture virus-free test-tube seedlings and test-tube bulbs. At the same time, appropriate and moderate hormones are needed to induce and promote callus, rooting, germination, growth and development, seedling formation and bulb expansion. The specific requirements are as follows:

① Nutritional requirements:

Inorganic nutrition: Nitrogen, phosphorus, potassium, sulfur, calcium, magnesium and other major elements and trace elements such as iron, manganese, copper, zinc, boron, cobalt and molybdenum are essential nutrients for the growth and development of garlic. The active components in the culture medium exist in the form of ions, and one type of ions can be provided by more than one salt.

Organic nutrition: including nitrogen source and carbon source. In order to make tissues grow well, it is often necessary to supplement one or more vitamins and amino acids in the culture medium, among which vitamin B 1 is essential, and vitamin B3, vitamin B6 and inositol can promote the growth of garlic cultured tissues.

② Substrate: Agar medium is usually used as the substrate for virus-free garlic tissue culture, and the concentration is generally 0.5% ~ 1.0%.

③ Hormones: In addition to nutrients, in order to promote the growth of tissues and organs, it is usually necessary to supply one or several plant growth regulators from outside sources, and the demand for these substances varies greatly with explant tissue, culture stage and purpose of increase or decrease.

Auxin: Auxin is used to induce cell division and root differentiation in tissue culture. Commonly used auxins are NAA (naphthylacetic acid), IAA (indoleacetic acid), IBA (indolebutyric acid), NOA (naphthoxyacetic acid), 2,4-D (dichlorophenoxyacetic acid) and so on. Among them, NAA and IBA are widely used for rooting, and they can cooperate with cytokinin to promote stem proliferation. 2,4-D is indispensable for callus induction of explants. Different explants have different requirements for 2,4-D concentration. 0.1.25mg/L 2,4-D MS medium is needed for the shoot tip and 0.5mg/L 2,4-D MS medium for the stem of test-tube seedlings.

Cytokinin: mainly promotes cell division and differentiation of adventitious buds of callus or organs, helps axillary buds to be liberated from the inhibition of apical dominance, and promotes stem proliferation. Commonly used cytokinins are BAP (benzylaminopurine) or 6-BA(6- benzyladenine), ZT (zeatin) and KT (kinetin), of which 6-BA is the most commonly used.

Gibberellin: It has the functions of promoting multiple buds at garlic shoot tips, stabilizing microtubules and antagonizing bulb formation. There are more than 20 kinds of gibberellin, which are mainly supplied by roots. Rooting can promote the expansion of leaf sheath cells and facilitate the formation of bulbs. In the process of garlic bulb formation, gibberellin content first decreased and then increased, and the decrease of gibberellin content was beneficial to bulb formation. It is a fact that long sunshine induces the decrease of gibberellin content in garlic plants, so the promotion of bulb formation by long sunshine is realized by affecting the endogenous gibberellin level.

Ethylene: ethylene stimulates the lateral expansion of leaf sheath cells by changing the shape of cell wall and the arrangement direction of cellulose microfibrils, rather than the longitudinal extension. In addition, ethylene can also inhibit leaf growth, induce leaf senescence, and change the relative ratio of leaf body and leaf sheath length, which is beneficial to bulb formation.

④ pH requirement: Before sterilization, the pH value of the culture medium is generally adjusted to pH 5 ~ 6. When pH is 6, the culture medium will harden. However, Xiong (1999) observed that garlic seedlings grew best and proliferated fastest at pH=6.4. Liu (1995) thinks that the optimum medium for bud differentiation, hairy roots and bud growth of Xuzhou white garlic and Taicang white garlic is pH = 7.5 (slightly alkaline).

⑤ Temperature and illumination requirements:

Temperature: In garlic plantlet culture, the temperature is generally 15 ~ 20℃, and lower temperature and larger temperature difference are beneficial to cultivate strong seedlings; In vitro garlic seedlings must go through a low temperature (< 20℃) vernalization stage, and then enter a higher temperature and longer and stronger light conditions to induce the formation and expansion of garlic bolts. The suitable temperature for garlic bulb formation and expansion in vitro is 20 ~ 25℃.

Illumination: the requirement of illumination is essentially the requirement of light period, light intensity and light quality.

Photoperiod: the photoperiod of in vitro seedling culture is 12 hours. The length of photoperiod is very important for bulb formation, and longer sunshine conditions are beneficial to bulb expansion. However, the requirements of bulb formation on photoperiod response vary with the ecotype of garlic varieties. For example, garlic varieties that are slow to respond to low temperature generally do not form bulbs under 12 hours of sunshine, but they can form bulbs after 16 hours of sunshine, but their development is slow; However, garlic varieties sensitive to low temperature developed well under 12 hours sunshine.

Light intensity: In a certain light period, bulbs are easier to form under strong light than under weak light. When the light is strong, the synthesized carbohydrate will increase, and its accumulation is the material basis of bulb expansion. And induced bulb formation, even in weak light, as long as the light time exceeds the critical period, bulb formation can begin.

Light quality: Using incandescent lamp to supplement light for garlic can obviously promote the formation and expansion of test tube bulbs. The light balance φ value of photosensitive pigment has a great relationship with light quality, which is usually measured by the light quantum ratio of R∶FR (red light: far red light). With the decrease of R∶FR value, the bulb expansion speed is accelerated, and light quality plays an important role in bulb formation, which may be related to its endogenous ethylene production.

(2) preparation of virus-free garlic tissue culture medium

① Prepare concentrated stock solution: concentrate 20 times of macro elements, 200 times of trace elements, 200 times of iron salts and 200 times of organic matters except sucrose. When preparing these four stock solutions, each component should be dissolved separately and then mixed with each other.

The stock solutions of various hormones should be prepared separately. If it is insoluble in water, it should be dissolved with a small amount of appropriate solvent first, and then diluted with distilled water. According to the required hormone concentration level, the stock solution concentration can be 0.00 1 ~ 0.05438+0 micromole/liter. Auxin is generally dissolved in 95% alcohol or 0.0 1 micromole/liter sodium hydroxide, the latter is more effective. Cytokinins are generally dissolved in 0.5 ~ 1 micromole/l hydrochloric acid or dilute sodium hydroxide solution. Gibberellin is easily soluble in cold water, but GA3 is unstable and easily decomposed after being dissolved in water. It is best to use 95% alcohol as mother liquor.

All stock solutions should be stored in suitable plastic or glass bottles and kept in the refrigerator. The iron salt storage solution must be stored in a brown glass bottle. Shake the bottle gently before using these stock solutions. If sediment, suspended matter or microbial contamination is found, it must be removed immediately. When preparing stock solution and culture medium, distilled water or deionized water and high-purity chemical reagents should be used.

(2) A step of preparing a culture medium.

A. Weigh the specified amount of agar and sucrose, add water to 2/3 of the final volume, and heat to dissolve. Since sucrose is soluble, it can also be added after agar is dissolved.

B, adding a certain amount of various stock solutions respectively. If vitamins and hormones need to be added after autoclaving for special reasons, the solution of these substances can be disinfected through a microporous filter (with a pore size of 0.22 ~ 0.45 micron) after adjusting the pH value.

C. adding distilled water to the culture medium to the final volume.

D. After thorough mixing, use 0. 1 micromole/liter. Use sodium hydroxide and 0. 1 micromole/liter hydrochloric acid to adjust the pH of the culture medium.

E. Sub-package the culture medium (even liquid) into the selected culture container (about 1/3 container volume) while it is hot.

F seal the bottle mouth with a cotton plug wrapped in gauze or other suitable plug or cover such as aluminum foil and kraft paper.

G. sterilize with 12 1℃, 1. 15× 105 Pascal 15 ~ 20 minutes.

H. Let the culture medium be cooled at room temperature and then stored at low temperature. All culture containers must be marked so that they can be clearly identified after autoclaving and storage.

(3) Techniques for micropropagation of virus-free garlic

① The technical system of virus-free garlic tissue culture can be test-tube seedlings or test-tube bulbs. Bud regeneration and proliferation can be directly induced from explants to maintain the stability of species, or callus can be induced first and then proliferated and differentiated. Therefore, garlic tissue culture can be divided into four ways: one is to induce explants to regenerate test-tube seedlings; Secondly, explants are directly induced to form test-tube seedlings; The third is to induce callus to regenerate plants and form small bulbs in test tubes; Fourthly, the test-tube seedlings formed by direct germination form small bulbs in the test tube.

Because it is easy to mutate through callus pathway, it should be used with caution in the process of virus-free propagation in line with the principle of maintaining the excellent characteristics of varieties.

② General steps of virus-free garlic tissue sterile culture:

A. Put garlic cloves (or chopped garlic stems) into a glass bottle, disinfect the surface with 75% alcohol for 5 minutes (or 40 seconds) on an ultra-clean workbench, and then add a few drops of activator into a disinfectant (such as 0.2% mercuric chloride solution) to disinfect for 20 minutes or 12 minutes. Shake the glass bottle 2 ~ 3 times during disinfection.

B. After disinfection, pour out the disinfectant, add a proper amount of sterile distilled water, shake it evenly for several times, and pour out the water, and repeat this for 3-4 times.

C. take out the materials and put them into sterilized Petri dishes.

D. While sterilizing garlic materials, the equipment to be used is sterilized by soaking them in 95% alcohol, taking them out, burning them on the flame of alcohol lamp, and using them after cooling. All instruments often need to be disinfected after each use 1 time.

E. Use these sterilized instruments to cut off the suitable explant stem tips from the surface sterilized materials.

F. Open the lid or plug of the culture container and inoculate the explants on the culture medium. If a glass container is used, bake the bottle mouth for a few seconds on the flame of an alcohol lamp, and then quickly seal it with a bottle cap or a cork.

G. Sterilization of culture media and instruments shall be carried out by conventional methods, namely autoclaving.

③ Rapid propagation of virus-free garlic in vitro:

A. directly induce explants to form buds and roots. Adventitious buds and roots are directly induced from explants, and generally go through the process of primary culture, adventitious bud induction and rooting culture. All kinds of explants can regenerate complete plants on MS, B5 and LS media.

The existence of cytokinin is the premise of bud formation. Higher concentration of BA can promote bud formation, and adding 1 ~ 2 mg/L BA can induce bud formation. Hormone-free medium is beneficial to rooting, NAA is added to the medium to promote rooting, and BA is added to inhibit rooting.

Different sources of explants need different hormones. Only 0. 1 mg/L NNA MS medium is needed for flower bud regeneration. Low concentration of auxin (NAA) combined with high concentration of cytokinin (KT) is the best combination for differentiation from petal buds of garlic. The proliferation of buds requires a higher concentration of auxin than cytokinin. Adding 0. 1 mg/L zeatin (ZT) can promote the differentiation of adventitious buds from stem segments with tender leaves and form clustered buds. Adding a certain concentration of GA3 is beneficial to shoot tip induction. The existence of penicillin is beneficial to the formation of buds and reduces pollution.

The effect of storage temperature on ovules varies with explants. Garlic cloves can promote the formation of buds and roots after storage at 5℃. However, the low temperature of 5 ~ 7℃ for 60 days only promoted the germination rate and did not affect the number of buds. The germination ability of different parts is very different. Only the jointed stem segment culture can induce germination, and the other parts of the stem segment are invalid.

B. domestication and field growth of test-tube seedlings. Generally speaking, test-tube seedlings need to be domesticated and cultured with vermiculite, rock wool and perlite. As a substrate, it is watered with nutrient solution. Inoculation with sphaerococcus mosses during transplanting can promote the rooting of test-tube seedlings and improve the survival rate. The survival rate of transplanting seedlings with small bulbs was 92%, which was significantly higher than that of transplanting seedlings with small bulbs (53%).

④ Techniques for micropropagation of virus-free test-tube bulbs: Generally, the culture of test-tube bulbs goes through three stages: primary culture, bud proliferation and test-tube bulb formation. Examples of garlic bulb formation in vitro culture include explant initiation, callus induction and rooting of test-tube seedlings. Using explants such as stem tip, stem disk, stem disk with young leaves, heavy scales and flower buds, test-tube bulbs can be formed on MS, B5 or LS medium.

Low concentrations of hormones (BA, BA) promote bulb formation, and even hormone-free medium is superior. When shoot tips were used as explants, low concentrations of hormones promoted the formation of bulbs; When the stem disk was explant, 2 mg/L BA promoted bulb formation, and high concentration BA promoted bud formation. NAA is beneficial to the formation of bulblet roots, and 1.0 mg/L NAA can form 80% bulblet roots in the medium. Bulbs were formed on MS medium of 2 mg/L NAA+0.05 mg/L BA, without hormone swelling.

A higher concentration of sucrose (90 ~ 1.20g/L) is beneficial to bulb formation. Adding (5g/L) activated carbon to the culture medium can promote bulb formation. Long light and far red light promote bulb formation and expansion.

There are differences in quantity and quality of bulbs formed by different ecotypes under the same in vitro conditions. In vitro, there are significant differences in photoperiod requirements for bulb formation of spring and autumn varieties. Low temperature pretreatment at 3 ~ 4℃ promotes bulb formation, and spring garlic varieties must be pretreated at low temperature to form bulbs.

The induction of bulb formation by environmental conditions cannot be replaced by changing the composition of culture medium. Tube bulbs were transplanted directly without domestication. The size of test-tube bulbs is positively correlated with field germination rate, plant height and yield. The size and planting density of test-tube bulbs affect the yield and quality of bulbs. The bulb (late-maturing variety) can break dormancy at 35 ~ 20 ~ 5℃.

Xiong et al. (1999) used virus-free test-tube seedlings of Xuzhou white garlic as explants. After continuous culture of initial medium, subculture medium and bulb induction medium, the bulb induction rate reached 90%, the weight of fresh bulb was over 300 mg, and the bulb diameter reached15 mm.

⑤ Improve micropropagation coefficient:

A. select suitable explants. Bud is the basis of rapid propagation of virus-free garlic, and inducing explants to directly produce virus-free multiple buds can greatly improve the basis of rapid propagation.

There are obvious differences in the number of shoot tips among garlic varieties. The number of buds at the top of dormant bulbs is obviously less than that of bulbs that break dormancy. Low temperature treatment can obviously increase the number of buds, and the size of peeled buds also affects the number of buds. Large seedlings with 2 ~ 3 leaf primordia germinate more than small seedlings with 1 leaf primordia, but the virus-free rate of large seedlings is lower than that of small seedlings. When bulbs were heat-treated at 37℃ for 20 ~ 30 days, the virus-free rate of stem tip was similar to that of stem tip. Therefore, a number of buds with high virus-free rate can be obtained by first heat-treating the bulbs and then peeling off the big shoot tips for culture.

Garlic bolt can produce many aerial bulbs, which shows that it has strong axillary bud germination potential; At the same time, as a reproductive organ and meristem, the reproductive stem tip has more axillary bud primordia and stronger virus-free potential. There are obvious differences in the number of buds at the tip of reproductive stem among different garlic varieties, and many buds tend to grow weakly, so it is necessary to cut and separate many buds for strong seedling culture.

B. improve the generation multiplication coefficient. Cutting off the buds formed in the initial medium in time is the key link to improve the proliferation coefficient of subculture. The variety, concentration and proportion of hormones in the culture medium, as well as the induction, differentiation and bulb formation of callus, will affect the proliferation coefficient of generations.

Callus induction: Callus can be induced in MS, B5, modified B5(BDS), LS and N6. Different explants have different requirements for hormones. Shoot tips and leaves have good callus induction rate on the medium containing 1 ~ 2 mg/L IAA or 0.05 ~1.0 mg/l 2,4-d, and 2,4-d and KT are necessary to induce pedicels to form callus. After adding 2,4-D, flower buds will form callus. Anthers were cultured in B5 medium containing 2 mg/L NAA, and the callus induction effect was better. The formation of meristematic nodules (MRTs) requires low concentration of NAA (0.5 mg/L) and does not require KT, and the formation of nodules is negatively correlated with normal rooting ability. Low temperature storage promoted the formation of garlic callus, but low temperature pretreatment could not promote the formation of garlic callus without 2,4-D. Pretreatment of anthers at 5℃1~ 2 days is beneficial to callus induction.

Callus proliferation: Callus growth generally does not need light, and the suitable temperature is 25℃. In fixed-orbit shaking table culture, 70 rpm is beneficial to callus growth, and 150 rpm promotes ball formation, and then seedlings can be regenerated. Shoot tip callus proliferated and grew fastest in MS medium with high concentration of BA and NAAR. 2 mg/L IAA promoted callus growth, while IBA and BA did not.

Callus differentiation: Callus differentiation usually requires light. Callus from different sources have different differentiation ability. Leaf primordium callus is easier to differentiate than shoot tip callus. The explants from anthers are most conducive to bud formation, proliferation and rooting, and deformed roots are easy to differentiate into adventitious buds, and the best is 0.5 mg/L IAA+5 mg/L BA. Because the callus formed by MRTs has good organogenesis ability, meristematic nodules can be used as good explants. Callus from small roots and pedicels can regenerate buds and roots more easily than those from big roots and pedicels.

The differentiation conditions of buds and roots are different: high concentration of NH4+ is beneficial to bud differentiation. BA is indispensable for seedling differentiation. No 2,4-D is beneficial to regeneration. Wang Honglong reported that high concentration of cytokinin and low concentration of auxin are beneficial to bud differentiation and can take root on hormone-free MS medium. Low concentrations of BA and NAA are beneficial to plant regeneration.

The differentiation of callus from different sources requires different hormones. The flower bud callus can differentiate into buds and roots in the presence of 2,4-d, and the root nodule meristem formed by the root tip of test-tube seedlings can regenerate into complete plants in MS medium of 0.5 mg/L NAA or 0.5 ~1.0 mg/l NAA+1.0 mg/l kt. Callus differentiation requires different hormones and different media. Adventitious buds need high concentration of KT (2 ~ 4 mg/L) in B5 medium and low concentration of BA(0.25 mg/L) in LS medium. The differentiation effect of anther callus on B5 medium was better than that on LS medium. Low temperature treatment of pedicel callus is necessary for bud differentiation. Contemporary plants are differentiated from calli. Because roots mainly grow from calli, not from the base of seedlings, roots and buds eventually separate, and the survival rate of transplanting is only 10% ~ 20%. The effective solution is to induce bulbs directly from seedlings.

Variation of callus: Chromosome variation during callus formation may be related to the type and concentration of growth regulators. Dolezel et al. found that 5 ~ 50 μ mol/L NAA increased the number of cells with abnormal mitosis, but when the total hormone concentration reached 500 μ mol/L, the mitotic activity was obviously inhibited and the mitosis was abnormal, so the lower concentration of hormone could normalize the mitosis of cells.

With the extension of subculture time, the variation is intensified. After 4 months of subculture, the diploid decreased from the initial 52% to 16%, and the regeneration frequency decreased. After one year of subculture, the bud differentiation rate decreased from 60% to 10%. 1 ~ 3 Gy can promote callus growth without affecting seedling regeneration, and can be used for mutant screening.

To sum up, all the factors that make test-tube seedlings grow healthily, improve the induction rate of test-tube bulbs and promote their expansion will affect the improvement of micropropagation coefficient of virus-free garlic.

⑥ Precautions:

A. virus detection. Although the shoot tips were carefully cut off and various treatments were carried out to eliminate the virus, only some cultures could produce virus-free plants. Therefore, for each plant produced by the shoot tip, it is necessary to detect the specific virus before producing the virus-free original seed as the mother plant. Many viruses in cultured plants have a delayed recovery period. Therefore, in the first 18 months, plants must be tested many times. Only those who have really eliminated a virus can be popularized and used in production. Because plants that have been tested for virus may still be reinfected, it is necessary to repeat the test during the breeding process and at all stages. These virus-free plants must reproduce under conditions that can eliminate any possibility of infection.

B. optimize the strain. The cultured offspring are classified according to the growth potential, and the plants with consistent growth are selected for subculture every time, so that the individual grows fully and will not be inhibited.

C, timely inducing bulb formation. In general, the culture of test-tube bulbs goes through three stages: primary culture, bud proliferation and test-tube bulb formation. However, the stage of bud proliferation cannot go on indefinitely, and bulb formation must be induced in time. The formation of test-tube bulbs requires certain seedling growth, and bulb formation should be induced in summer and autumn when the temperature is high, so as to reduce the production cost and connect with the field growing season.

D. update in time to prevent vitrification. When the sterile culture method is established, it is easy to propagate continuously, and it is not compared with the original parent plants, which may accumulate the variation that occurred in the early stage of culture. At the same time, according to the research experience, if repeated propagation in test tubes, the leaves of some cultures often become waterlogged and almost translucent, and the growth and reproduction speed of this test tube seedling will decrease, and finally even die, which is the vitrification phenomenon, which is difficult to completely eliminate. Therefore, attention should be paid to upgrading in time to prevent vitrification.