Plant cell tissue culture

1. The purpose of the experiment?

Plant cell and tissue culture is highly technical and requires aseptic operation. Through this experiment, we can master the conventional tissue culture technology and deepen our understanding of aseptic operation. ?

2. Experimental principle?

Plant totipotency: Any cell in a plant has the ability to grow and differentiate into a complete plant, which is called plant totipotency. ?

Plant tissue culture is a technique to cultivate sterile plants in vitro by using plant totipotency. Plant tissue culture refers to callus culture according to its original meaning. But today, its scope is expanding day by day, including sterile culture of plants and their isolated organs, tissues, cells and protoplasts. Therefore, there are several different levels of culture techniques, namely, whole, organ, tissue, cell and protoplasm culture techniques. What they mean is:

1. Plant culture. It refers to sterile culture with complete plant morphological materials (such as seedlings and larger plants) as explants.

2.？ Embryo culture. Mature or immature embryos isolated from ovules were used as explants for sterile culture in vitro.

3.？ Organ culture. It refers to sterile culture in vitro with roots, stems, leaves, flowers, fruits and other organs as explants, such as root tips and segments, stem tips, stem nodes and segments, Ye Yuan bases, leaves, petioles, leaf sheaths and cotyledons, petals, stamens (anthers, filaments), ovules, ovaries, fruits and other sterile culture in vitro.

4. Tissue culture. Aseptic in vitro culture refers to tissues separated from various parts of plants (such as meristem, cambium, xylem, phloem, epidermis, cortex, endosperm tissue, parenchyma, pith, etc.). ) or induced callus as explants. This is tissue culture in a narrow sense.

5. Cell culture. Refers to the isolated sterile culture with single free cells (such as somatic cells, pollen cells and egg cells separated from tissues by uricase) as inoculum.

6. Protoplast culture. Sterile culture in vitro, in which protoplasts with cell walls removed were used as explants.

In this experiment, tobacco and peas were used as materials. Tobacco is an important industrial raw material and one of four typical experimental plants (tobacco, Petunia, carrot and Brassica) in plant tissue culture. Its research is the most extensive, always ahead of other plant materials. At present, among 66 tobacco varieties, 16 has regenerated plants. There are 12 species that regenerate pollen plants by anther culture, 20 species that regenerate plants by protoplast culture, and 3 species and 12 species that regenerate hybrids by protoplast fusion within and between tobacco species. Peas (Pisum? Sativum) is a leguminous plant and an important food, feed and vegetable crop. It is also an experimental plant that geneticists and plant physiologists are interested in and willing to adopt. Roots, stems, cotyledons, hypocotyls, epicotyls, flower buds and other explants of pea can form callus, among which shoot tips, young embryos, young leaves and other organs can successfully regenerate plants. Shoot tip culture can include shoot tip meristem (growth point) as small as ten microns and shoot tip culture as large as several tens of millimeters or more. In practice, virus-free plants are often cultured at growing points to preserve fine varieties and improve yield and quality.

3. Experimental materials?

Instruments and equipment?

1. alcohol lamp, 100ml triangular flask, 9 cm Petri dish;

2. Tweezers, scissors and dissecting needles;

3. Double-barrel dissecting microscope;

4. Light incubator or greenhouse;

Materials and reagents?

1. Material? Tobacco aseptic seedling, pea seedling.

2. Reagent

(1)MS medium, plant hormones: NAA, 6-BA, etc.

(2) saturated bleaching powder solution (sodium hypochlorite);

(3)70% alcohol, 100% alcohol and alcohol cotton balls;

(4) Sterile water?

4. Experimental steps?

1. Rapid cutting propagation of tobacco aseptic seedlings-subculture: (completely aseptic operation required)?

One bottle of tobacco aseptic seedlings in each group, please be careful to avoid pollution. ?

( 1)? On the experimental platform, light the alcohol lamp, wipe your hands with alcohol cotton balls for disinfection, and open the triangular bottle with sterile tobacco seedlings. The bottle mouth of triangle bottle needs to be burned and disinfected on the outer flame of alcohol lamp before and after use. ?

(2)? Squeeze out 1 seedling gently with sterile tweezers, and cut the sterile seedling into small pieces of 0.5- 1 cm with sterile scissors, each piece is required to contain at least one growing point, and directly cut into triangular bottles filled with MS culture medium. Each piece must be in direct contact with the culture medium, and the triangular bottle mouth must be disinfected and resealed. ?

(3)? Cultured in a light incubator at 65438 05 ~ 25℃ and 65438 06 hours for 4 weeks, a complete plant can be grown, which can be used for field transplantation. ?

2. Shoot tip culture of pea seedlings?

⑴? Take 65,438+00 pea seedlings germinated for 6 days, simply take 65,438+0 cm stem tips, soak them in 70% alcohol for 65,438+0 minutes, then soak them in saturated sodium hypochlorite solution for disinfection for 65,438+05 minutes, rinse them with sterile water for 5 times (after this step, it is required to be sterile), absorb water with sterilized filter paper, and put them into sterilized Petri dishes. ?

⑵? Under the binocular dissection mirror, the young leaves are peeled off with a sterilized dissection needle to expose the growth cone, and the growth cone with two or three leaf primordia is picked with the sterilized dissection needle. ?

⑶? Move the picked stem tip to ms+10.7 μ mol/l NAA+4.4 μ mol/l? Callus formation was induced on 6- benzyladenine (6-BA) medium, and the Petri dish was sealed with a sealing film. ?

[4] Callus was cultured in a constant temperature incubator at 26℃ for six weeks, which could be used for rooting culture after subculture.