Application of cell engineering in plants

⑴ Application of micropropagation technology

Micro-propagation technology, that is, using plant organs, tissues, cells or protoplasts as explants, carries out plant regeneration under the condition of in vitro culture. The application of micropropagation technology can not only overcome the serious separation of offspring caused by sexual reproduction of highly heterozygous species, such as papaya in Australia; It can be used for rapid propagation of famous or endangered species, such as pineapple and strawberry. The main tree species regenerated by micropropagation technology are papaya, citrus, longan, litchi, apple, pear, grape, strawberry and banana. So as to realize commercial production.

Virus-free seedlings such as apples and strawberries can be obtained by shoot tip culture or micro-grafting. In addition, in the process of tissue culture, such as callus culture, cell suspension culture and protoplast culture. By changing the conditions such as pH value, temperature and ion concentration, the variation can be increased and excellent mutants can be screened, which opens up a new way for the breeding of new varieties.

Callus, suspension cells and protoplasts are good receptor materials for gene transformation, and plant regeneration in vitro is also an important link to realize plant genetic transformation.

In addition, micropropagation technology provides a new method for germplasm preservation. Under the condition of in vitro culture, many germplasm resources can be preserved for a long time through slow growth and low temperature treatment, and can be collected, exchanged, preserved and applied between different countries and regions, that is, to establish a "gene bank" to realize the global sharing of germplasm resources. For example, the Leuven Research Center of the Catholic University of Belgium has a large number of banana germplasm banks kept in test tubes.

⑵ Mass cell culture and production of useful secondary metabolites.

Mass culture of useful secondary metabolites is another important application field of plant cell engineering. Through cell engineering technology, it stimulates the synthesis and accumulation of some important secondary metabolites in plants, and then separates and purifies them, such as some precious drugs, spices and pigments. And realize the industrialized production of plant products.

As early as 1964, China began to cultivate ginseng cells. After 1980, Chinese researchers have carried out a lot of cell culture and research on plants such as Arnebia euchroma, Notoginseng Radix, Taxus chinensis, Artemisia annua, Rhodiola sachalinensis, Saussurea medusa and so on, and used bioreactor to carry out small-scale and pilot-scale cell culture on medicinal plants. Among them, the pilot scale of Arnebia euchroma in Xinjiang reached 100L, and a small amount of shikonin was produced, which was used to develop cosmetics and antibacterial, antiviral and antitumor drugs. Large-scale cell culture of Taxus China has also achieved initial results, and valuable anticancer drug paclitaxel has been obtained from cell culture, but the yield needs to be improved.

⑶ Application of haploid technology

Haploid breeding and related research have been widely used in agricultural and horticultural plants. Haploid plants were obtained by Blakeslee et al. (1922) and Kostoff( 194 1 year) respectively, which was beneficial to mutation detection and screening of resistant cell lines and greatly shortened the breeding time. In addition, haploid plays an important role in gene mapping and gene transfer research.

Naturally occurring haploids are rare and limited to several plants. Anther culture is an important way to form haploid. Since the first successful anther culture in 1964, remarkable progress has been made in anther culture technology, especially in rice, wheat, corn and other crops. At present, the successful fruit tree varieties are mainly annona (Nair et al., 1983), papaya (Litz and Conover, 1978), four citrus varieties (Chen, 1985) and longan (Tang and Wei, 1984). 1983), apples (Zhang et al., 1990), pears (Jordan, 1975), grapes (Rajasekaran and, 1979), etc. Xue Guangrong et al. (1980) successfully induced haploid plants by culturing haploid pollen of oriental strawberry (tetraploid).

Anther culture is mainly affected by genotype, anther development stage, pretreatment and culture conditions. The main problem is that the frequency of haploid induction is low, and it is difficult to distinguish the diploid formed by haploid spontaneous doubling from the diploid formed by somatic tissue. For example, Fowler et al. (197 1 year), Nishi et al. (1974) and Rosati et al. (1975) used the anthers of octoploid strawberry as materials to induce callus and differentiate plants, and found that the regenerated plants were still octoploid and developed from asexual organs.

In addition to anther culture, haploid cells such as egg cells, helper cells and antipodal cells of plants can differentiate into haploid embryos or callus through in vitro culture. Many attempts have been made to culture ovules and ovaries, but in most cases, the growth stops at the callus stage.

(4) embryo culture

Embryo culture in vitro is the earliest tissue culture technology directly applied to plant improvement. Embryo culture can overcome the decline of embryos after hybridization, ensure the success of intraspecific or interspecific hybridization, or be used for the culture of plants with difficulty in asexual reproduction. Embryo culture can also overcome seed dormancy and abortion. Magdalita et al. (1996) and Drew et al. (1997) respectively performed interspecific hybridization on papaya to obtain suitable embryos, and then cultured the embryos to promote the success of hybridization. Jordan (1992) obtained callus, but not regenerated plants.

Australia International Agricultural Technology Research Center successfully cultivated hybrid embryos of papaya and its wild species, and obtained hybrid offspring. The excellent characters of wild species such as resistance and high sugar content were inherited. Litchi is one of the fruit trees that are difficult to be cultured in vitro. Kantharajah et al. (1992) cultured immature embryos of litchi with a length of 3 mm ... Other tree species regenerated by immature embryo culture include avocado, annona and papaya. Yao Qiang (1990) cultured immature embryos of peach, nectarine and peach for 60 days to obtain regenerated plants. J.Button et al. (1975) obtained complete plants from embryogenic callus of sweet orange in vitro.

5. Protoplast culture and somatic hybridization.

Protoplast is a single cell with its cell wall removed, and it is the smallest unit that can regenerate complete plants in vitro. Each protoplast contains all the genetic information of the individual, and under suitable culture conditions, it has the totipotency to regenerate individuals similar to its parents. The main purpose of protoplast culture is to overcome the obstacle of distant hybridization through protoplast fusion, realize somatic hybridization and produce hybrid offspring. In the process of protoplast culture, there are often a lot of variations, from which excellent mutants can be screened. Protoplast can absorb foreign organelles, viruses, DNA and other macromolecular genetic materials, and is an ideal tool for genetic transformation. In addition, a large number of protoplasts obtained at the same time have genetic homogeneity, which can establish a good experimental system for biological disciplines such as cell biology, developmental biology, cell physiology and cytogenetics.

Lizz( 1986) isolated the protoplast of papaya, and Krikorian et al. (1988) isolated the protoplast of banana, but neither of them obtained the cells that continued to divide. Nyman et al. (1987, 1988) first reported the protoplast culture and plant regeneration of strawberry varieties Sengana and Canaga. In 1992, they obtained regenerated plants from protoplasts of young leaves and petioles of strawberry plantlets in vitro. Infante et al. separated protoplasts from leaves and petioles of test-tube seedlings of forest strawberry alpine nutrition system to obtain regenerated plants. Callus and suspension cells are important materials for preparing protoplasts, but only a few tree species have successfully isolated protoplasts from callus or suspension cells of deciduous fruit trees, and the most successful tree species is kiwifruit. Cai Qigui et al. (1988) isolated protoplasts from calli of Actinidia chinensis and obtained regenerated plants. Kovalenko et al. (1990) and Ochatt et al. (1988) used suspension cell lines to separate protoplasts from colt cherry and European grape, respectively, to obtain regenerated plants.

Lin et al. (1997) isolated protoplasts from embryogenic callus to obtain regenerated plants. Yi Ganjun et al. (1997) also isolated the protoplast of citrus (Hongjiang Citrus) from embryogenic callus, and obtained regenerated plants. However, the isolation of protoplasts from mesophyll was not successful. Ma et al. (1998) isolated and cultured the protoplasts of Prunus armeniaca. Under suitable conditions, the protoplast of Prunus armeniaca was deformed for 4-5 days, and the first division began in 5-6 days. Small cell clusters of 15-20 cells can be formed in about 20 days, and miniature callus can be formed after 60 days. After subculture, callus can induce adventitious buds and roots to form complete plants. Ding Aiping et al. (1994) studied the protoplast culture and plant regeneration of apple. Protoplasts were isolated from suspension cell lines established from embryogenic callus to obtain regenerated plants.

After removing the cell wall, plant cells can fuse with each other like fertilization, and genetic material recombination between incompatible parents in conventional hybridization can be realized, thus opening up a new field of somatic hybridization. Somatic hybridization has been widely used in plant breeding, and has made remarkable progress in cytoplasmic male sterility and disease resistance. At the same time, somatic hybrid plants with economic value were also obtained on woody fruit trees.

At present, there are two most effective fusion systems: PEG- high pH/Ca2+ method and electric shock fusion method.

The first case of somatic hybridization was achieved by protoplast fusion of tomato and potato. Protoplast fusion technology has been widely used in citrus interspecific hybridization. Ohgawary fused the protoplasts of sweet orange and flying dragon to obtain somatic hybrid plants.

American scholar Grosser fused the protoplast of sweet orange suspension culture cells with the protoplast of Eupatorium adenophorum callus, and obtained autotetraploid somatic hybrid plants. S.distcha has excellent characteristics such as disease resistance, cold tolerance and salt tolerance, and is suitable as the rootstock of citrus.

[6] Transformation

The rapid development of molecular biology has led to a new revolution in plant science. After years of exploration, people have a deep understanding of biology and genetics at the molecular level. Combined with tissue culture technology, molecular biology technology has been applied to the modification and change of plant genome.

Because of the identity of gene coding, useful genes in any organism (such as viruses, fungi and insects) can be transferred to plants. The introduction of genes (such as insect-resistant or disease-resistant genes) leads to the emergence of new genotypes or the improvement of genotypes, so that insect-resistant or disease-resistant genotypes can be selected.

At present, the target genes that have been isolated or applied mainly include those that resist pests and diseases, those that resist abiotic stress, those that improve crop yield and quality, and those that change other plant traits.

There are many methods to introduce foreign genes into plant cells, such as Agrobacterium plasmid-mediated method (including Ri plasmid of Ti plasmid), plant virus vector-mediated method, DNA direct introduction method (including PEG-mediated, liposome-mediated and other chemical-induced DNA direct transformation methods, electrical stimulation method, ultrasonic wave, microinjection, laser microbeam, particle bombardment and other physical-induced DNA direct transformation methods) and germplasm system-mediated gene transformation methods (including pollen tube introduction method, germ cell soaking method and so on). ). At present, the most commonly used and effective methods are Agrobacterium tumefaciens-mediated method and particle bombardment. Since the first successful Agrobacterium-mediated transformation of 1983 in tobacco and potato, about 120 plants have been transformed by this method. Agrobacterium-mediated method is very effective for dicotyledonous plants, but it is also used for monocotyledonous plants. Gene bombardment can be used as both callus and recipient suspension cells, which is very effective for monocotyledons.

2. The application of cell engineering in animals.

(1) Rapid propagation of excellent and endangered varieties and new varieties.

Abdominal pregnancy improves the utilization rate of breeding stock. In 1930s, embryo transfer of sheep and goats was successful. 1982 American scholars obtained the world's first test-tube cow. Through in vitro fertilization, nuclear transfer technology, embryo segmentation, embryo fusion and other technologies to achieve the purpose of rapid reproduction, but also to create high-yield dairy cows, lean pigs and other new varieties. Breeding rare animals such as giant panda and Siberian tiger through embryo engineering and cloning technology.

⑵ Using animal cell culture to produce active products and drugs.

Major vaccines, antibodies, etc. 1975, the University of Cambridge obtained monoclonal antibodies for the first time by using animal cell fusion technology. 300L and 1000L culture tanks have been used to produce monoclonal antibodies and gray myelitis vaccine respectively. In 1990s, a kind of "living cell therapy" with living cells as therapeutic agent appeared in the world, which mainly proliferated, expanded or injected patients' autologous cells in vitro. This method has potential therapeutic effects on cancer, leukemia, diabetes, burns, AIDS and so on.

(3) tissue engineering for medical organ repair or transplantation

Using cell engineering technology, a small number of normal cells of human residual organs can be propagated in vitro, so as to obtain organs with the same function and no rejection required by patient organ transplantation. For example, some bones, cartilage, blood vessels and skin are being cultivated in the laboratory, and the liver, pancreas, heart, breast, fingers and ears are being grown and shaped in the laboratory.

(4) Bioreactors for transgenic animals.

Compared with traditional animal cell culture, transgenic animal pharmaceutical technology has high benefits, and transgenic animals are natural gene pharmaceutical factories. 1992, Shanghai Institute of Medical Genetics bred the first transgenic test-tube cow carrying human protein gene in China. In 2000, a transgenic goat carrying human α antitrypsin gene was bred in China, and specific drugs for treating chronic emphysema, congenital pulmonary fibrosis cyst and other diseases can be extracted from transgenic goat milk.

3. The application of cell engineering in energy and environmental protection.

In order to obtain a strain that can decompose cellulose hydrolysate and produce ethanol efficiently, Candida with strong cellobiose utilization ability was fused with Saccharomyces cerevisiae with high ethanol production. The obtained fusant not only uses cellobiose as the only carbon source, but also has higher ethanol production capacity than its parents.

Four strains of Streptomyces viridis TTA and Streptomyces xikang 75viz were fused, and their ability to degrade corn stalk cellulose was improved by 155%~264% compared with their parents.

Through electrofusion, Saccharomyces cerevisiae and Candida Guillermo were fused, and a strain which can produce ethanol from xylose and cellobiose was screened out, which is of great significance to the utilization of cellulose renewable resources and the reduction of environmental pollution.