Asparagus (Asparagus officinalis) is a perennial herbaceous vegetable with dioecious sex. Asparagus seeds are expensive, so stem segments are often used for rapid propagation. Stem segment culture has the advantages of simple culture technology, fast propagation speed, and high propagation coefficient. A large number of plants can be propagated from one explant within a year. It can accelerate the propagation of improved varieties and precious plants. The seedlings propagated by stem segment culture have less variation, high propagation coefficient, and good seedling quality. It is an important means to solve the rapid propagation of asexual plants that cannot be propagated by seeds.

Principle

Stem segment culture refers to the in vitro culture of stem segments without buds and with axillary (lateral) buds or petioles (young stem cuttings including tubers, bulbs, and bulbs), including young stems and lignified stem segments. In general, the culture of stem segments without buds must involve the participation of plant hormones, while the culture of stem segments with buds can be done without the participation of plant hormones, or with the addition of a small amount of plant hormones. However, in order to promote the elongation of axillary buds, the concentration of cytokinin must be higher than the concentration of auxin. The main purpose of stem segment culture is to carry out rapid in vitro propagation of plants, followed by the study of the division potential and omnipotence of stem cells, as well as the induction of cell mutations and the acquisition of mutants.

Depending on the growth type and growth environment of the stem, some can grow in the air, while others can grow underground (rhizomes, tubers, corms, bulbs). In nature, the stem plays a supporting and conducting role in the plant body, and it is often used as an organ for asexual propagation. Using stems as culture materials, a variety of test tube plants have been obtained, and stem segment culture technology has been increasingly widely used. In plant tissue culture, due to the large plasticity of the cells of tender stem segments (i.e., branches that have sprouted or newly drawn out that have not yet fully lignified in the current year), in vitro culture is easy to succeed, so it has become a commonly used material in tissue culture, especially tender stems that are young in the developmental stage.

Procedures

A. General procedures for stem segment culture

1. Selection and disinfection of explants

In terms of explant selection, for woody plants, stem segment culture is generally easier to succeed with young annual stem segments than perennial stem segments. Take young and tender stem segments that are healthy, free of pests and diseases, and growing for culture. The survival rate of the base of the stem is lower than that of the top segment, and the survival rate of the lateral buds is lower than that of the top buds, so the top explants should be used first, but each new shoot has only one top bud, and axillary buds should also be used. The axillary buds on the upper part of the stem also have a good culture effect. Plant buds have dormant and growing periods, and explants should not be taken during the dormant period, otherwise the survival rate will be very low. For the stem segment culture of bulbous flowers with modified stems (corms, bulbs), their propagation can be divided into balls or scales for the in vitro culture, to achieve the purpose of a large number of proliferation. Bulbs are usually cultivated underground, and the contamination rate is relatively high.

2. Inoculation

Under sterile conditions, use a sterile knife to cut the stem segment into 0.5-1.0 cm internode segments. If it is a bulb, cut the base with small scales and then cut the base, so that each base has an axillary bud. Then transfer it to the culture medium.

3. Culture medium

Select different culture media according to the culture material and explant type. After culture, callus tissue will grow on the cut of the stem segment, especially the base cut, and show a slight increase. The buds begin to grow, and sometimes clustered buds appear, thus obtaining sterile seedlings.

Generally, the surface-sterilized stem segments are cut into several centimeters long with nodes under sterile conditions and inoculated on a solid culture medium. After culture, the stem segment with buds can directly germinate from the axillary buds into seedlings, or form callus tissue through induced dedifferentiation, and then form adventitious buds through redifferentiation. Cut the regenerated seedlings and transfer them to the rooting culture medium for culture, and then a complete plant can be obtained.

The time required for disinfection of stem segments at different parts is different. Disinfectants such as sodium hypochlorite solution, mercuric chloride, and saturated bleaching powder can be used. Stem segments without axillary buds generally form white protrusions at the edges of the cuts at both ends, forming callus tissue, and adventitious buds may form from the protrusions later. The formation of stem segment callus tissue and the production of adventitious buds are related to the ratio of auxin and cytokinin in the culture medium. In a culture medium containing a high concentration of auxin, the frequency of callus tissue directly formed by stem segments is higher, while in a culture medium with a high concentration of cytokinin, the frequency of adventitious buds formed by callus tissue is higher.

B. Asparagus stem segment culture technology

There are two different ways to use stem segments for tissue culture of Asparagus: one is to directly differentiate into seedlings from terminal buds or axillary buds, but the propagation coefficient is relatively low; the other is to form seedlings after callus proliferation, which has a higher propagation coefficient. The key points of the rapid propagation of Asparagus with stem segments with terminal buds or axillary buds are as follows.

1. Explant collection and disinfection

Cut the tender stems with disease-free and insect-free leaves that have not yet unfolded from the field, rinse them in water for 40-50 min, and then disinfect the surface. First disinfect with 70% alcohol for 3 min, transfer to a saturated bleaching powder solution and soak for about 10 min, and shake continuously, or disinfect with 0.1% HgCl2 for 8 min, or disinfect with 2% sodium hypochlorite for 15 min. Rinse with sterile water 4-5 times, cut into stem segments with 1-2 buds about 1 cm long or cut into discs with 1-2 buds, and inoculate them on the medium of MS + 0.1 mg/L NAA + 0.1 mg/L KT (or 0.1-0.3 mg/L BA) to induce the apical buds or axillary buds to regenerate new shoots. The outer scales should be removed for the culture of stem segments with apical buds. Axillary buds sprout 3 days after inoculation, axillary buds elongate after 1 week, and stems grow up to 5 cm after 4 weeks, and bud clusters are formed at the bud nodes of axillary buds. Later, the new shoots are cut into small segments according to a lateral bud as a unit, and transferred to MS medium containing 0.1 mg/L NAA and 1mg/L KT to induce the axillary buds to regenerate new shoots, and continue to subculture to expand the propagation of asexual lines.

2. Bud proliferation

The above-mentioned bud clusters are cut and inoculated, and bud clusters can be formed at each bud eye. This process is repeated, and subculture can be carried out to reproduce a large number of tender stems in a short period of time. The conditions for bud proliferation are 26-27°C, 14-16 h of light per day, and the indoor humidity is controlled at about 60%. This procedure is repeated to establish asexual lines of propagating material, and repeated succession culture can expand the propagating asexual lines.

3. Rooting

When the clustered stem seedlings grow to 4-5 cm in height, they are transferred to MS + 0.1 mg/L NAA medium for rooting. After 1 week, callus tissue forms at the base of the tender stems, and roots are formed after 8-10 weeks of culture. The difficulty of rooting of asparagus test tube seedlings is mainly affected by genotype and explant source. In addition, when the three auxins NAA, IBA and IAA are used together, the rooting effect is better than when used alone. Adding 50 mg/L P333 or 0.5 μmol/L pyridinol to the rooting medium has a significant promoting effect on rooting.

4. Transplantation

Transplant the rooted complete plants into a mixed matrix of peat and vermiculite (1:1) or garden soil: sand: peat (2:1:1), and can be transplanted into a seedbed or nutrient pot. The temperature of the plant transplanting should be maintained at 25-27°C, the humidity should be higher than 90%, and attention should be paid to ventilation. It will survive after growing for 2-3 weeks, and it can be transplanted to the open field after growing in the greenhouse for about 2 months.

In the process of rapid propagation of seedlings using stem segments of asparagus as explants, in addition to the above methods, you can also use the hormone-free MS basic medium with multiple axillary buds removed from the terminal buds to directly carry out cuttings to obtain rootless test tube seedlings, and then use conventional cutting technology to produce seedlings. Or use stem segments with terminal buds and multiple axillary buds, inoculate them into a culture medium containing cytokinins (such as 6-BA or KT) for rapid propagation of seedlings, and then use conventional cutting technology to carry out rapid propagation of asparagus seedlings.

Note:

Browning is a common problem for woody plants, especially for some woody plants with high phenolic content. The following measures can be taken to prevent browning and the accumulation of harmful substances.

• Adding an appropriate amount of activated carbon (0.5%-1%) to the culture medium can absorb some harmful substances and reduce the adverse effects of phenolic substances.
• Add anti-discoloration agents to the culture medium, such as 5% H2O2, 0.2-0.4mg/L vitamin C, 0.7% polyvinylpyrrolidone (PVP), etc.
• Reducing the light intensity in the culture room can reduce the oxidation rate of phenolic substances.

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