After the transgenic plants of tobacco and potato were first obtained in 1983, McGrananhan et al. [1] published in 1988.
The first transgenic fruit tree-walnut was obtained. 1990, they obtained the insect-resistant gene walnut. Zairen
Three years after scientists obtained transgenic field crops through particle bombardment, particle bombardment was successfully applied to papaya [2]
. In 1994, Kalinski counted the patents of transgenic plants, including skillful planting and apples. Domestic researchers
Attach great importance to transgenic research. Huang Xuesen and others [3] published the genetic transformation of grapes; Cheng Jiasheng et al. [4]
The technology of apple gene transfer was introduced. Subsequently, transgenic apple seedlings [5j; Fu Runmin and others.
Genetic engineering and its application in fruit trees are systematically introduced [6, 7]
; State Key of Tropical Crop Biotechnology, Chinese Academy of Tropical Agricultural Sciences
The work of transgenic papaya has been carried out in the laboratory [8 ~ 1 1]
; Citrus Research Institute of Chinese Academy of Agricultural Sciences and South China Agricultural University have developed antibacterial skin.
Studies on Citrus Gene Transfer [12,13]; Shenyang Agricultural University has obtained transgenic plants of the main apple variety' New Jonah Gold '[ 14 ~ 16].
Fujian Agricultural University has developed longan [17] and passion fruit [18].
Genetic transformation and transgenic research of subtropical fruit trees such as bat moth; Magnificent
China Agricultural University and others promoted the progress of fruit tree genetic engineering in methodology [19,20].
. Transgenic or genetic transformation is also carried out.
Multi-unit [2 1 ~ 24]
. The research on fruit tree transgene has become a hot spot. The author tries to summarize the literature of fruit tree transgenic so far, and also puts forward his own views on some problems. If there is anything wrong, please criticize and correct me. Plant gene transformation method 1. 1 Agrobacterium-mediated gene transformation method
Among all kinds of gene transformation methods that have been discussed at present, Agrobacterium gene transformation method is the most clearly studied one [25]
. This method uses the natural genetic engineering system in nature, namely the pathogenic process of Agrobacterium tumefaciens and Agrobacterium rhizogenes, to transform artificially processed genes into human plant cells. Root cancer farmers
Bacteria infect plants and cause crown gall. Coronary thin tumor has two states [26]
One is an unorganized tumor, which proliferates on hormone-free medium, but it has no apical advantage and is difficult to take root. The plasmid related to the induction of coronary thin tumor is Ti plasmid (Tum. : inducingplas-mid). Agrobacterium rhizogenes can produce many hairy roots at the infected site, which is related to Ri plasmid. Only plants that can be infected by Agrobacterium can use Agrobacterium as gene carrier. Agrobacterium mainly infects dicotyledonous plants, and monocotyledonous plants are generally insensitive to these two pathogens, but there have been many cases of infection recently, and gymnosperms can also be artificially inoculated to cause disease.
At present, there are three commonly used Agrobacterium transformation methods [
25〕
(1) Direct infection of plant wound: cut a wound from newly germinated seeds, immature embryos, cotyledons or stem segments with a knife, inoculate Agrobacterium tumefaciens cultivated overnight, and then culture tumor tissue on hormone-free medium. This method has a short experimental period, but there are often untransformed cells in tumor tissues, which are easy to form chimeras. (2) Leaf disc transformation method: take the leaf disc with the diameter of 3-smm from the tender and fresh leaves with a hole punch, soak it in the overnight cultivated Agrobacterium liquid for a few seconds, then transfer it to the culture medium for 2-3 days, and then transfer the leaf disc to the selective culture medium containing antibiotics to induce seedling differentiation, so that the transformed cells can directly regenerate plants. This method is simple and feasible, and can quickly obtain transgenic plants. (3) Protoplast culture method: When protoplast regenerates cell wall and divides, its behavior is similar to that of plant wound cells. At this time, Agrobacterium can infect protoplasts. Agrobacterium tumefaciens (1: 100) was added to the protoplast and cultured for 24~40h. Then, the protoplasts were sterilized by centrifugation and cultured on the selected medium to induce the callus to regenerate plants. Because of the genetic homogeneity of protoplasts, transformed cells are single independent cells, and the transformants obtained generally do not have chimeras, which has great application value. Direct transformation of 1.2DNA
Due to the limitation of the host range of Agrobacterium, it is difficult to transform cereals as the main part of crops by Agrobacterium. Therefore, in the 1980s, the technology of direct introduction of DNA was developed, that is, bare DNA was directly transferred into plants after special treatment.
1.2. 1 polyethylene glycol: PEG)PEG) PEG is the mediator of protoplast fusion, which can affect the permeability of plasma membrane. When plasmid DNA*** is cultivated, the protoplast actively absorbs foreign DNA molecules, and foreign genes can be integrated into the plant genome for expression and transmission. After PEG treatment, a large number of protoplasts contacted with DNA molecules, and then under the action of high Ca "+and high pH solution, plasma membrane charges were redistributed, and some temporarily variable pores appeared on the surface, and DNA molecules entered the protoplasts. When the protoplast was washed with culture solution to change the conditions of PEG, high CaZ+ and high pH, the protoplast returned to its original shape, and the DNA molecules that entered the protoplast could enter fine cells.
In the nucleus, integrated into chromosomes [25]
. This method is relatively simple and easy to operate, without expensive instruments and equipment, but it needs a protoplast preparation, culture and regeneration system.
1.2.2 electroporation: the protoplasts and DNA molecules are mixed in a buffer, and placed in a small container for short-time high-pulse electrical treatment, so that the pores on the cell membrane can be restored and new osmotic pores (3-4nm) are generated, so that the DNA molecules in direct contact with the protoplast membrane can enter the cells. The size and number of reversible pores in plasma membrane depend on electric field strength and puncture time. At present, this method is mostly limited to marking genes, aiming at establishing efficient transformation.
System giant [27]
.
1.2.3 gene bombardment, also known as partile blanching or high-speed particle microprotrusion, is a method of mechanical gene transfer. By adding Ca'+ and spermidine, DNA was adsorbed on the surface of tungsten and gold particles to make DNA microspheres, which entered human plant cells under the instantaneous force of gene gun. The biggest advantage of this method is its wide application. However, due to the influence of bombardment speed, target cell distance, diameter and speed of micro-projectile, shooting diffusion radius, quantity and structure of exogenous DNA, the transformation frequency is low, and there are some factors such as high price, difficult to exclude chimeras and difficult to select transformants, which limit its application.
1.2.4 Other transformation methods besides the above-mentioned common methods, such as microinjection, liposome fusion, ultrasonic shock, micro-laser electrophoresis, etc. It has been applied to plant gene transformation. Three factors affecting gene transformation of fruit trees
2.2. 1 Agrobacterium tumefaciens infectivity and vector influence
The infectivity of Agrobacterium is directly related to the transformation rate and is also one of the main factors for the success or failure of transformation. The infectivity of Agrobacterium depends on its own characteristics and the sensitivity of recipient plants to it. As far as Agrobacterium is concerned, its infectivity is related to the species of Agrobacterium and the structure of vector plasmid. Agrobacterium can be divided into three types, namely nopine type, Oct type and agrobacine type. Nop strain is characterized by its chromosome background of C58, fast growth, no pilling and simple operation. Commonly used engineering strains are C58, pGV3850, A208SE, etc. Oct strain is characterized by its chromosome background Achs, slow growth and strain culture.
Nodulation in the culture process is not conducive to the operation in the transformation experiment, and it is not easy to wash off after * * * culture. Commonly used engineering strains are LBA4404 and LBA 104. Suc strain is characterized by its chromosome background of A28 1 or A 136, which has the characteristics of Nop strain. Commonly used engineering bacteria are EHA 10 1 and EHA 105, which have excellent characteristics such as fast growth and no pilling, and both of them are highly contagious. We made a comparative study on the infectivity of the strain to various fruit trees. The results showed that EHA 105 was an ideal fruit tree transformation carrier system. The strain has the characteristics of fast growth, no pilling, easy transformation, easy elution after culture, sensitive to Cef, strong infectivity and wide host range. Using this strain, we have successfully obtained many transgenic plants such as apples, cherries and strawberries. 2.2.2 Activation of vir Gene in Agrobacterium tumefaciens
The activation of vir gene is the domain valve of Agrobacterium transformation. Vir region contains 7 operons virA, b, c, d, e, f, g and h * * * and 24 genes, all of which play a regulatory role and are related to the processing and transfer of T-DNA. The activation of vir gene plays an important role in Agrobacterium transformation, so any factor that can enhance the activation of vir gene can improve its infectivity. Acetylsyringone (As) is a common inducer of vir gene. AS for the use of inducers, the following three methods are commonly used: ① As is added to the liquid culture of Agrobacterium, and the adding time is generally 4~6 hours before the preparation of the infected solution of engineering bacteria. (2) adding as to the culture medium and explants of Agrobacterium; (3) adding AS to the liquid culture medium of Agrobacterium and the culture medium of Agrobacterium. This method seems to be the most reliable, but it is used too much as an inducer. However, many experiments show that transformation can be achieved without inducer. In addition, in the transformation of apple leaves, it was found that the addition of betaine (lmmol/L) or proline (lumol/L) simultaneously with as had a synergistic effect on the activation of vir gene. It was found that pH value had obvious influence on the activation of vir region. Theoretically, when the pH value of AS-containing medium is 5.0~5.6, the gene induction in vir region reaches the highest level, and the pH value changes by 0.3, which has obvious influence on the transformation rate of many plants. Octopus and agrobacine strains need lower pH value than carmine strains. Usually, the pH value of Agrobacterium is 7.2. The virus genes of this virulent strain are inactive. The pH value in plant tissue culture medium is usually 5.8, which is beneficial to the activation of vir gene. In the process of inducing the activation of vir gene, attention should be paid to adjusting the pH value of the medium. In addition, the activation of vir gene is also affected by other factors, such as temperature, activation of virD and virG must be lower than 28℃; Yeast extract should also be added to the culture medium; Sugar or high concentration of inositol in culture medium can promote the expression of vir gene.
Problems and countermeasures in plant transgenic research
Through transgenic technology, we can optimize the agronomic traits of plants, obtain good economic benefits and make remarkable achievements ... Nevertheless, there are still some potential problems in plant transgenic research.
The digestive function of insects that feed on the leaves of plants will be damaged after the inhibitory genes that affect protease activity are introduced into plants. Will the leaves, fruits and seeds of transgenic plants do similar harm to people and animals? What will happen to people and animals after other kinds of genes are introduced into plants? Although there is no direct evidence to prove whether genetically modified plants are harmful to humans and animals or how harmful they are, this suspicion is worrying. 3.2 Drug resistance of pests and diseases.
Drug resistance of pests and diseases is a big problem that puzzles agricultural production. Recent research shows that if Bacillus thuringiensis insecticide 10~20 times is sprayed every year, the mortality of pests will be reduced by 50% after five years, and its half-life dose will be increased by 10 times. A similar situation may occur if transgenic plants that can produce Bacillus thuringiensis toxin protein by themselves are planted on a large scale. In addition, due to the co-evolution of organisms, resistant plants can survive under unfavorable conditions, so pests and diseases will rapidly develop their adaptability to bad habitats, and transgenic plants will gradually lose their resistance to pests and diseases. 3.3 Environmental and ecological balance issues
After the target gene was introduced into the plant, the characteristics of the plant were improved. However, if these genes are captured by some wild plants, especially some weeds, some new types of weeds will be produced. For example, herbicides are no longer effective in clearing weeds with herbicide-resistant genes. If the stress-resistant genes are captured by weeds, their adaptability to the environment and survival competitiveness will be enhanced, thus intensifying their proliferation and diffusion. Some rare plant species will be extinct due to competitive substitution, the genetic diversity of the same plant will be reduced, and the insect community will also be affected.
Although transgenic research has brought the above problems, with the deepening of research, people will seek new countermeasures to solve these problems. We should strengthen the management of transgenic research through legislation and other means to minimize the negative impact of transgenic research. By means of time, space and gene isolation, the spread of plant genes can be basically controlled and the harm to the ecological environment can be reduced.
Prospect of transgenic technology
It is the main goal of transgenic research to introduce foreign genes into the genomic DNA of recipient cells at a fixed point and quantitatively, and obtain new individuals with stable and efficient expression and inheritance. With the acceleration of biotechnology industrialization, transgenic technology will play an increasingly important role.
(1) Breeding super insect-resistant new varieties by multi-gene transformation Many viruses, fungi, bacteria and pests not only caused huge economic losses to fruit tree production, but also caused environmental pollution due to the extensive use of pesticides. Many insect-resistant genes have been cloned, such as cp, bt, icp, cpti, antimicrobial peptides, chitinase, Saussurea involucrata lectin, PRP gene and so on. It laid a foundation for cultivating new varieties with disease resistance, but these genes have strong specificity and can only be effective for a certain kind of pests and diseases. In recent years, the important progress of plant genetic engineering is the construction of artificial chromosome vector, the establishment of multi-gene transformation system and the realization of multi-gene transformation. These new advances laid a foundation for introducing many different types of insect-resistant genes into fruit trees, and also opened up a new way for cultivating super new varieties with general insect resistance.
(2) Using genetic engineering to cultivate new rootstocks. The cultivation of new rootstocks of fruit trees is an important research topic. The stress resistance, dwarfing, high yield and rooting ability of fruit trees are all related to rootstocks. At present, there are many stress-resistant genes, rooting hormone genes, dwarfing genes and so on. Introducing these genes into existing rootstocks will effectively promote the development of fruit industry. Moreover, rootstock genetic engineering breeding has almost no transgenic safety problems and is easily accepted by the society.
(3) Using antisense gene manipulation to cultivate new storage-resistant varieties. According to the principle of antisense RNA, the antisense gene is artificially constructed, and the partial sequence complementary to the target mRNA is introduced into cells, and then paired with it to form a complex, thus blocking the information flow of DNA from RNA to protein and regulating the expression of the target gene. The application of antisense gene manipulation technology in plant genetic engineering has achieved gratifying results.
(4) Improve fruit quality. So far, many genes related to fruit quality have been cloned. If these genes are introduced into fruit trees, such as polysaccharide synthase gene, trehalose synthase gene, sweet protein gene, lysine and other essential amino acid synthase genes, high-quality new varieties will be cultivated.