Chrysanthemum Flower Organ Culture Technology

Chrysanthemum is one of the four largest cut flowers in the world. The high regenerative capacity of floral organ cells makes it a good material for studying cell morphogenesis and also for the study of floral sex. In vitro flower bud culture helps to understand the role of the whole plant and endogenous and exogenous hormones in the sex determination of flower buds, so as to artificially control sex differentiation and be used for fruit and seed development research. Anther culture in flower organ culture can be used for haploid breeding and ovary culture can be used for fruit development studies. Therefore, flower organ culture is of great value both for theoretical research and production applications.

Principle

Flower organ culture refers to the aseptic culture of the whole flower organ and its components such as receptacle, petal, filament, stalk, ovary, stem and anther. The flower of a plant is one of the reproductive organs, and usually the flower consists of two parts, sterile and fertile. The calyx and petals are the sterile parts and the fertile parts are the stamens and pistils. Each part of the flower can be used as an in vitro culture material to obtain a regenerated plant. For chrysanthemum tissue culture, different explants, different basic medium and different hormones can be added according to different culture purposes.

Figure 1. Different stages of callus induction, somatic embryos and plant regeneration and direct organogenesis in Chrysanthemum morifolium. (Fardin, N.; et al. 2018)

Procedures

A. General Procedure of Flower Organ Culture

1. Medium and culture conditions: There are many kinds of medium suitable for chrysanthemum tissue culture, such as White, B₅, N₆, Morel, MS, etc. Currently, MS medium is mostly used. The suitable temperature range for chrysanthemum cultivation is 22-26 °C. The light is 12-16 h per day. Various hormones were added for cultivation. The light intensity is generally 30-55 μmol/(m²·s).
2. Material collection and disinfection: When collecting materials for flower organ culture, plants that grow vigorously and are free from diseases and insect pests should be selected. For the culture of flower organs such as petals, inflorescence rachis, filaments, anthers, pollen, ovaries, and styles, the whole buds (flowers) should be sterilized.
3. Inoculation culture: When culturing with whole flower buds, just insert the flower stalk into the solid medium. If a certain part of the flower organ is used, it is removed separately, cut into small pieces of 0.3-0.5 cm, inoculated into the culture medium, and put into the culture chamber for cultivation.
4. Callus induction and differentiation: Chrysanthemum tissue culture mainly includes adventitious bud pathway and callus regeneration pathway. The differentiation medium can be the same as the callus induction medium, but it may also be a medium with altered hormone concentrations.
5. Rooting and transplanting of tube seedlings: Rooting and transplanting of tube seedlings of chrysanthemum are generally easy.

B. Chrysanthemum Petal Culture Technology

1. Explant collection, disinfection and inoculation: 3-4 d before the flowering of the chrysanthemum, cut off the young flower buds that have not yet opened. Rinse with clean water first, then soak in 70% alcohol for 10-20 s, then disinfect with 2% sodium hypochlorite for 10-15 min according to the tenderness of the material, and then rinse with sterile water 4-6 times. Blot dry on sterile filter paper. Peel off the bracts of the young flower buds that have not yet opened, take out the young petals and cut them into 5 mm2 small pieces with a scalpel. After inoculation, they were cultured under conditions of a temperature of about 25 °C, a light intensity of 40 μmol/(m2·s), and 12 h of light per day.
2. Induction of callus: MS + (2-3) mg/L 6-BA + 1 mg/L NAA is used as the basic medium for callus induction, and 3-5 pieces are inoculated in each bottle. When the petals were cultured for about 10 d, they began to produce callus.
3. Differentiation culture: There are three ways of seedling differentiation in chrysanthemum petal culture as follows.
• Direct seedling formation. Adding high levels of cytokinin and high level of auxin in the medium can induce seedlings at one time.
• Seedling formation through embryoid. In some varieties, when the callus grows to about 30 d, a large number of green round grains can appear on the surface of MS medium with 2 mg/L BA + 1 mg/L NAA, which means that the embryoids are differentiated and formed, which are called somatic embryos (asexual embryos). After growing to 40-50 d, a large number of embryoid can be seen to produce regenerated plants.
• Differentiate into seedlings through callus. Some callus need to be transferred to the differentiation medium. The callus was cut into 5 mm size and inoculated into the culture medium, and after 20-30 d of culture, it could be induced to differentiate to obtain petal seedlings. In the differentiation medium, reduce the concentration of auxin or increase the concentration of cytokinin, transfer the formed callus to the differentiation medium of MS + 3mg/L BA + 0.01mg/L NAA, and then differentiate into adventitious buds.
4. Rooting: Rooting of chrysanthemum test-tube seedlings is generally relatively easy. There are two methods for rooting.
• Rooting by tube seedling culture. Cut out 2-3 cm rootless tender stems with 4-5 leaves, inoculate them on MS + 0.3 mg/L NAA rooting medium, cultivate for about a week to start rooting, so as to obtain complete plants.
• Rooting of tube seedlings from direct cuttings. In order to simplify the cultivation procedure, shorten the seedling time and reduce the production cost, the rootless seedlings of about 3 cm are cut directly and inserted into the perlite or vermiculite soaked with the rooting-promoting solution (auuxin or rooting powder). Direct cutting rooting requires loose and ventilated medium, shade after cutting, and the rooting rate reaches 95% to 100% after 10 d.
5. Transplanting and management methods: Humidity is very important to the survival rate of seedlings. The relative air humidity must be kept above 90% in the first few days. Within 10 d of transplanting, the sun should be properly shaded, avoid direct sunlight, and pay attention to a small amount of ventilation. The temperature should be kept at 25-28 °C.

C. Chrysanthemum Receptacle Culture Technology

1. Selection and disinfection of materials: Choose full flower buds with the typical characteristics of the variety. First remove the unopened flower buds from the mother plant, rinse them with clean water and distilled water, disinfect the surface with 70% alcohol for 30 s, then disinfect them in a saturated bleach solution for about 20 min, rinse them with sterile water 3 to 4 times after taking them out .
2. Inoculation culture and plant regeneration: Remove the sepals, petals, pistils and stamens on the flower buds with tweezers, cut off the receptacles and inoculate them on MS + 2mg/L 6-BA + 0.2mg/L NAA medium. Then they were cultured in a culture chamber at a temperature of about 26°C, a light intensity of 30 μmol/(m²·s), and a light duration of 10 h. A small amount of callus can be formed after about two weeks, and green bud points can be differentiated and grew into seedlings in about one month. The rootless seedlings were cut from the base of the callus and transplanted into the rooting medium, and they could grow into complete plants after 3 weeks.