With the continuous efforts of scientists all over the world, plant tissue culture technology has developed rapidly in recent decades. Using tissue culture, not only a large number of excellent clones can be produced, but also various generations needed by human beings can be obtained.
Haploid, triploid, polyploid and aneuploid can also be obtained thanks to substances. Cell fusion can break the boundaries of species and overcome the incompatibility of distant hybridization, which plays a great role in the cultivation of new plant varieties and germplasm improvement. Tissue culture of plant cells is an ideal material for analysis and research at the cellular level. From plant rapid propagation and anther culture to organelle culture, protoplast fusion and DNA recombination technology, plant tissue culture technology has been widely used in various fields of plant science, agriculture, forestry, industry, medicine and other industries, and has become the most vital subject in contemporary biological sciences.
1 Basic concepts, principles and experimental steps of plant tissue culture
1. 1 concept
Plant tissue culture is a technique to separate plant organs (root tips, stem tips, etc.). ), tissue (cambium, anther tissue, etc. ), cells (somatic cells, germ cells, etc. ), embryos (mature or immature embryos), protoplasts, etc. Cultured in artificially prepared culture medium under aseptic conditions, and given appropriate culture conditions to induce them to produce callus or latent buds or grow into complete plants.
1.2 principle
The basis of plant tissue culture is the totipotency of plant cells and the regeneration of plants. 1902, G. Haberlandt, a famous German botanist, put forward a viewpoint based on cytology theory that "organs and tissues of higher plants can divide continuously until a single cell, that is, plant somatic cells, has the potential to divide, reproduce and develop into a complete plant under appropriate conditions". 1943, White accidentally discovered the formation of buds in tobacco callus, which confirmed G. Haberlandt's argument.
Different plants need different growth conditions and use different media. Commonly used basic media are MT, MS, SH, N6, White, etc. In tissue culture, whether callus and embryoid can be formed is the key to cultivate new plants. Adding a certain concentration of exogenous hormones to the basic medium can induce callus, embryoid, adventitious buds, roots and other organs, and finally obtain regenerated plants or secondary substances.
Materials used for plant tissue culture are called explants, and their main forms are organs, embryos, single cells, protoplasts and so on. According to different explants, the types of culture media, culture conditions, and the types and proportions of exogenous hormones are also different. In plant tissue culture, there are many factors that affect the cultivation ability. The key to the success or failure of callus induction lies in the culture conditions, and plant hormones are the key factors to induce the differentiation of callus and green seedlings.
IAA, NAA and 2,4-D are the most commonly used auxins for callus induction, and the required concentration is 0.01~10 mg/L. The most commonly used cytokinins are KT and ABA, and the used concentration is 0.1~10 mg/L. The main functions of KT are ABA plays an important role in the occurrence and development of plant somatic embryos. Although the physiological functions of various plant hormones are relatively specific, the physiological effects of plants are the comprehensive expression of the interaction between different kinds of hormones.
1.3 test program
1.3. 1 The selection and preparation of culture medium is "blood" in plant tissue culture, and the composition and supply of blood are directly related to the growth and differentiation of culture, so it is very important to understand the composition, characteristics and preparation of culture medium.
1.3.2 sterilization is one of the important tasks in tissue culture, and physical or chemical sterilization methods are usually used. Culture medium is sterilized by moist heat such as normal pressure or high pressure cooking, instruments are sterilized by combustion, glassware and heat-resistant utensils are sterilized by dry heat, heat-labile substances are sterilized by filtration, surfaces of plant materials are sterilized by disinfectants, surfaces of objects are sterilized by chemical spraying, and spaces such as inoculation rooms are sterilized by ultraviolet rays or fumigation.
1.3.3 Inoculation: Cut the sterilized isolated organs such as roots, stems and leaves into sections or small pieces and put them into the culture medium. The whole inoculation process should be carried out under aseptic conditions.
L.3.4 Culture The culture is placed in a culture room with certain illumination and temperature conditions, so that it can grow, divide and differentiate to form callus or further differentiate into regenerated plants.
1.3.5 Domesticated and transplanted test-tube seedlings are seedlings that grow under special environmental conditions, which are quite different from those that grow naturally, and can only be transplanted after domestication and adaptation to the natural environment.
2 Application of plant tissue culture
2. 1 rapid plant propagation and virus-free seedling production
The technology of plant rapid propagation began in 1960s, and the French Morchella successfully propagated orchids in large quantities through shoot tip culture, which opened the prelude to the research and application of plant rapid propagation technology. At present, there are more than 65,438+0,000 families and more than 65,438+0,000 species, some of which have developed into commodities. 80% ~ 85% of orchids in the world are virus-free and propagate rapidly through tissue culture. The cultivated plant species gradually developed from ornamental plants to horticultural plants, field crops, economic plants and medicinal plants. In China, similar research began in 1970s. Non-toxic potato seeds and sugarcane seedlings have been planted on a large scale in production, and more than 30 kinds of plants have been produced on a large scale or tested in the middle. Using tissue culture for rapid plant propagation and virus-free seedling production can not only save and cherish endangered species, but also solve the problem of lack of wild plant resources.
2.2 Plant anther culture and haploid breeding
Plant anthers are cultured into haploid plants, and then homozygous diploids can be obtained quickly after chromosome doubling, which will greatly shorten the breeding cycle. So far, hundreds of haploid plants have been obtained by pollen and anther culture in the world. The yield of rice lines cultivated by Indian scientists in this way increased by 15% ~ 49% compared with the control. South Korea has cultivated five rice varieties with high quality, disease resistance and lodging resistance. Since 1970s, more than 40 haploid plants developed from pollen or anthers have been cultivated in China, among which more than 65,438+00 species are the first in China. More than 100 homozygous inbred lines were obtained in maize. Rubber obtained diploid and triploid plants. During the Ninth Five-Year Plan period alone, 44 new crop varieties with high yield, high quality, stress resistance and disease resistance were cultivated, with a planting area of over 6.6 million hm2.
2.3 Plant embryo culture
In hybrid breeding, hybrid embryos often miscarry, so hybrid plants can be cultivated by taking out early-growing embryos and applying tissue culture methods. There have been more than 100 reports on the cultivation of immature embryos into plants. Scientists at home and abroad have obtained a variety of recombinant, cultivated and hybrid varieties of distant hybridization by using plant embryo culture technology.
2.4 Suspension culture of plant callus or cells
Plant secondary metabolites used to prevent and treat diseases can be produced by suspension culture of plant callus or cells. In recent years, this field has developed very rapidly. More than 400 kinds of plants were studied, and more than 600 kinds of secondary metabolites were isolated from cultured cells, of which more than 60 kinds were greater than or equal to the original plants in content, and more than 20 kinds were greater than19,6 in dry weight of the original plants. For example, diosgenin produced from callus and suspension cells of sweet potato is used to synthesize steroid drugs. Recently, the cell culture of taxol-Taxus chinensis, an anticancer drug, can be cultured in a 75t fermentor, which has reached the level of commercial production. In addition, Arnebia euchroma, ginseng, coptis root, geranium and so on. It has reached the level of commercialization. Catharanthus roseus, foxglove and tobacco have been industrialized; More than 20 kinds of plants, such as toothpick grass and safflower, are transitioning to commercialization.
2.5 Cell Fusion and Protoplast Culture
Starting from 1960, British scholar Cocking successfully isolated protoplasts from the roots of tomato seedlings for the first time. By 1990, more than 100 plants had been regenerated from protoplasts. More than 30 varieties of protoplast regenerated plants have been obtained in China, including important food crops and cash crops, such as soybean, rice, corn, wheat, millet, sorghum and cotton. Protoplast culture of woody plants, medicinal plants, vegetables and fungi has also made rapid progress. Intraspecific and interspecific somatic hybrid plants have been obtained abroad. Plant protoplast culture can also be applied to the study of foreign gene transfer, clonal variation and mutant screening, so it has attracted more and more attention.
2.6 Screening of Plant Cell Mutants
The selection of plant cell mutants began with 1959, and G. Melchers obtained temperature mutants in the suspension cell culture of snapdragon. In 1970, P.S.Carlson, H.Binding and Y.M. Heimer isolated tobacco auxotrophic cell, Petunia streptomycin resistant cell line and tobacco threonine resistant cell line respectively. Up to now, more than 100 plant cell mutants or variants have been screened from plant cell cultures of 45 species belonging to no less than 15 families. These include disease-resistant cell mutants, such as maize mutants resistant to leaf spot and wheat mutants resistant to scab and root rot; Cell mutants resistant to amino acids and their analogues, such as HYP-resistant mutants in Brassica napus [263; Cell mutants resistant to adversity stress, such as salt-tolerant mutants of rice and salt-tolerant mutants of wheat; Herbicide-resistant cell mutants and auxotrophic cell mutants, such as corn herbicide-resistant mutants; Screening plant height mutants, such as rice dwarf mutants.
2.7 Plant somatic embryos and artificial seeds
1958, Reinert first discovered somatic embryos (embryoids) in tissue culture of carrots. According to incomplete statistics, there are more than 100 species of plants that can produce a large number of embryoids, belonging to 43 families and 92 genera. Some important crops, such as rice, wheat, corn and pearl valley, can also produce embryoids by in vitro culture. These embryoids are embedded by sodium alginate and so on. Add artificial seed coat to form artificial seed. The advantages of artificial seeds are: fast propagation and high seedling rate; Not affected by climate, it can be industrialized all year round. In the early 1980s, the United States, Japan, France and other countries successively carried out research on artificial seeds, and China also carried out this research during the Seventh Five-Year Plan period, which was included in the national "863" high-tech research and development plan 1987.
2.8 cryopreservation of plant tissue cell cultures and establishment of germplasm bank
The discovery and confirmation of plant cell totipotency has opened up a new way for the long-term preservation of plant germplasm resources. Using liquid nitrogen cryopreservation technology can maintain a high survival rate, regenerate new plants and maintain the original genetic characteristics. For example, the establishment of cryopreservation germplasm bank for shoot apex meristem culture can not only prevent genetic variation and degradation of germplasm, but also preserve virus-free original seeds for a long time.
2.9 Plant Tissue Culture and Application of Transgenic Technology
The construction and research of the first T-DNA insertion mutant library in China laid a good technical and material foundation for the research of rice functional genomics in China, and made positive contributions to ensuring that China has a number of gene resources with independent intellectual property rights. In cooperation with the Key Open Laboratory of Rice Biology, Ministry of Agriculture, China Rice Research Institute and Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, a large-scale and efficient Agrobacterium-mediated transgenic technology system was established. Exogenous genes such as maize transposon AC-DS were introduced into rice immature embryos and seed-induced calli, and1.2000 independent T-DNA inserted strains were obtained, and a rice mutant database was constructed.
3 outlook
The research and application of plant tissue culture is one of the great achievements of scientific and technological progress in the 20th century, which provides many good experimental materials and effective ways for studying plant growth and development, resistance physiology, hormones, organogenesis and embryogenesis. With the continuous improvement of plant tissue culture methods, its application scope has been broadened accordingly. Because tissue culture is carried out under artificial control, it is easy to grasp the reasons for flower bud differentiation and flowering; Through embryo culture, hybrid or inbred species can be obtained; By separating haploid cells, homozygous diploid strains can be cultivated; Improve the diversity of breeding and shorten the breeding time; Through mutant screening, improve plant quality, enhance stress resistance and stress resistance ability, and expand plant growth range; Cryopreservation of somatic cells and establishment of gene bank can achieve the purpose of preserving species; Obtain secondary products with high medicinal value and industrial production, speed up the time of drug production, and reduce the passivity of relying solely on natural plants. Plant tissue culture technology has penetrated into all fields of scientific research, production and life, and will be improved day by day.
Heilongjiang Agricultural Science 2006, (3)