1. 1.2 anther culture
Haploid obtained by pollen (microspore) culture technology has been successful in many plants. When haploids are obtained, diploids and naturally doubled diploids often appear. The disadvantage of this method is that it needs to induce callus and then differentiate into regenerated plants through tissue culture, but not every microspore can reproduce asexually. In addition, after diploid, it is difficult to grow or bear fruit because of adaptability problems. The low efficiency of anther culture and breeding, the low induction rate of green seedlings and the difficulty of chromosome doubling are also reasons. Theoretically, anther culture of maize is an effective way to quickly obtain pure lines of maize. But so far, the pure lines and hybrids obtained from maize anther culture are rarely used for production in a large area [5]. At present, studies at home and abroad show that the callus induction rate can reach 2% and the green seedling rate can reach 2%.
1. 1.3 Distant hybrid single parent chromosome disappeared.
In distant hybridization, it may be due to the unsynchronized division cycle of parental cells, which leads to the loss of chromosomes of one parent, thus producing haploids.
1. 1.4 delayed pollination induction
Delayed pollination, even if the pollen tube reaches the embryo sac, the egg cell can not be fertilized because of the loss of fertilization ability, but it is easy to divide after being stimulated by the pollen tube, so as to parthenogenesis and form haploid plants. Delayed pollination after emasculation can greatly improve the induction rate of parthenogenesis [6]. The reason may be that pollination promotes the formation of endosperm, thus ensuring the smooth development of parthenogenetic young embryos.
1. 1.5 radiation induction
After X-ray treatment, flowers or male pollen are irradiated by X-rays, and then pollinated by emasculated female parents, thus affecting fertilization and inducing parthenogenesis to produce haploids.
1. 1.6 haploid promoter gene
1950, a high-frequency haploid induction material was discovered by King's Seed Company of North Lupu, USA. EdCoe( 1956) discovered the value of this material, and introduced two dominant marker genes (R-nj, P 1) to control grain traits and plant traits. After two backcrosses and two selfings, it was named Stock6. In the breeding practice, with Stock6 as the male parent and induced breeding materials as the female parent, there are about 3% haploid embryos [7]. Hagberg( 1980) found a partially dominant haploid promoter (hap) mutant in barley, which controls the abortion or survival of abnormal embryos and endosperm, and its homozygous offspring can produce 10% ~ 15% haploids. Liu Jilin (200 1) introduced the maize induction line Stock6 from the United States, and used it as the male parent to induce haploids in six maize populations such as WBM and Liaolu. Some diploid materials were obtained by identifying the seeds, plant markers and plant fertility of induced offspring.
2 haploid level breeding-haploid identification
Haploid identification methods mainly include morphological identification, anatomical identification, cytogenetic identification, radiation, genetic markers, molecular markers and so on. Morphological identification and genetic markers are mainly used to identify maize induced haploids.
2. 1 morphological and anatomical identification methods
It is an intuitive and convenient method to identify haplotypes by morphological and anatomical characteristics. The number of chromatin in maize haploid cells is only half that of diploid cytoplasm, so its cells and nuclei become smaller. The size and number of stomata and guard cells in leaf epidermis can also be distinguished from diploid by anatomical observation. Because cells become smaller, vegetative organs and reproductive organs change, and plants become shorter, most haploids can be selected by visual inspection at seedling stage. Haploid is often much smaller than its standard type in normal growth state; At the early stage of seedling growth, the length difference between taproot and tooth sheath is obvious; At seedling stage, the first leaf of maize haploid is shorter; Comparing the size of embryo and peltate can also be used to preliminarily identify haploid. The plant height of maize haploid is about 70% of that of homozygous diploid sister plant, and the leaf area is about 35% of that of the latter. In addition, haploid plants are characterized by early flowering and long duration; Flower deformity, abortion, less fruit; Haploid plants are short, with smaller leaves, flowers, anthers and stomata, denser inflorescences, more flowers and shorter filaments, while the corresponding diploids are the opposite [8].
2.2 Cytogenetic identification methods
The most accurate method is to observe the number of chromosomes in somatic cells or meiotic splinter cell compressed by apical meristem, which can distinguish haploid from aneuploid. Using photometer to analyze the DNA content in the nucleus, but it can't distinguish haploid from aneuploid haploid, and the detection speed is slow, which requires instruments and equipment, so it is not suitable for large-scale identification in the field.
2.3 ray irradiation method
The sensitivity of plants to radiation changes with the change of multiple, which is a general law of biology. The basic method is to irradiate (for example, X-ray) a part of the blade with ionizing rays, and first see the green fading, and then radiation necrosis occurs. The key is to explore the best dose and irradiation time of different crops. If mastered well, this method can be one of the simplest and most practical identification methods.
2.4 Genetic marker method
Using genetic markers to screen haploids can reduce the errors often encountered in screening according to morphological differences. Therefore, using genetic markers has become the most reliable and effective haploid identification method. In maize, the famous Navajo marker gene R-nj was successfully used. The haploid induction lines of Coe's Stock6 and Song's Gaoyou 1 introduced genetic marker genes for grain and plant color development. Agricultural University Gaoyou 1 has high induction rate and easy propagation, which overcomes the important defects of Stock6. In the current hybrid generation, the embryo surface of the female parent grain can be enlarged and the mark is clearer.
The seeds induced by haploid induction lines can be divided into three types: the aleurone layer of endosperm is purple or purplish red, and the embryo is also purple or purplish red; The endosperm aleurone layer is marked with purple or purplish red, and the germ part is colorless. Aleurone layer and endosperm germ are not colored and unmarked [2].
2.5 molecular labeling method
Tang Feiyu et al. (2004) used SSR markers to detect diploids from parthenogenesis of maize, and achieved obvious results [9].
Resynthesis of Three Polyploids
Chromosome doubling can occur naturally and its frequency can be improved by selection. In addition, the method of artificial induction doubling has been developed.
3. 1 natural doubling
Most haploid male flowers are highly sterile. However, in a few cases, haploid tassels will naturally double completely or partially, thus producing normal pollen. There is evidence that a few somatic cells of a haploid plant are generally hermaphroditic and there are few normal pollen grains, so the frequency of haploid natural doubling only accounts for 1% of all haploid plants. The natural doubling rate of many materials is less than 5%, and some materials are not naturally doubled. Simple haploid natural doubling is difficult to meet the requirements of breeding practice, and artificial doubling must be carried out.
3.2 artificial doubling
Usually, the natural recovery rate of tassel fertility does not exceed 20%, so it is particularly necessary to double the number of chromosomes by chemical treatment. Generally speaking, treating haploid seedlings with 0.06% ~ 0.5% colchicine solution can increase the doubling rate of haploid tassels to 20% ~ 50%. Kato(2002) doubled the haplotypes with nitrous oxide gas, and obtained four inbred lines and hybrids [10] respectively.
Natural doubling characteristics can be improved by reselection. Using two DH lines as parent materials, the natural doubling frequency of newly selected haploid tassels can be increased by the next round of line selection, and the fertility recovery rate can be increased from 9.4% to 33%, even to 43%.
4 Development prospect of haploid breeding
The application prospect of haploid technology in maize breeding is very attractive, which has the following advantages: (1) obtaining pure lines quickly; In haploid, gene interaction can be simplified, superdominance effect can be eliminated, and favorable additive and additive epistatic effects can be retained. Harmful, lethal and semi-lethal recessive genes can also be eliminated. However, haploid breeding technology also has shortcomings: it can not effectively break the linkage of bad genes; The probability of recombination between genes is low; Haploid induction frequency is not high. In addition, it is difficult to double chromosomes during diploid recombination. At present, the diploid frequency can reach 15%-25%. In addition, haploids have been used in maize breeding in some countries, and some high-frequency inducers have been patented, which limits the international exchange of materials. If China wants to use this technology in maize breeding, it must create its own haploid induction line.
Haploid breeding is an organic combination of germplasm amplification and breeding material improvement. Firstly, the target inducing material is effectively recombined and improved, and enough favorable gene loci are accumulated, and then the pure line is obtained by haploid technology, which will greatly give play to the advantages of haploid breeding and greatly improve the breeding efficiency. In addition, double haploid population has important research value in genetic research, which can be used to construct genetic map, gene mapping and cloning [2]. In addition to further improving the haploid induction system and increasing the haploid induction rate, the future work should also focus on haploid doubling technology. Du Juan et al. (1999) used sexual hybridization to recombine favorable genotypes and then conducted anther culture to quickly obtain pure maize lines [1 1], and established a new maize haploid breeding system, which not only has theoretical significance, but also has great production potential.
refer to
1. Guo Chunsheng, Guo Zhichun, Gui Ying. Anther culture and haploid breeding of maize in China [J]. In: Y. P. S. Bajaj (ed.), Biotechnology in Agriculture and Forestry, 1994, Volume 25: Maize, Spinghel Press, Berlin.
2. Liang, Qi Tingxiang, Xu. Application and new progress of haploid technology in maize breeding [J]. Journal of journal of maize sciences, 2004,12 (3):13-15, 18)
3. Liu Zhizeng, Song. Research Progress on Parthenogenetic Haploid Induced by Maize Hybridization [J]. Journal of journal of maize sciences,1999,7 (2):16-19.
4. Shi Taiyuan. Studies on Parthenogenesis of Maize Induced by Drugs [J]. Miscellaneous Crops, 20,2000 (1):17-20
5. Guo Yiming, Yang Yinggen and Guo Zhongcan. New progress in anther culture and haploid breeding of maize [J]. chinese bulletin of botany, China, 200 1, 18 (1): 23-30.
6. Jiang Yu, Wang Yumin, Wang Zhongwei, et al. Research progress of maize haploid breeding technology in China [J]. Journal of journal of maize sciences, 2008, 16 (6): 48-57.
7. Han Xueli, Tang Qilin, Cao Moju, et al. Preliminary study on identification method of haploid induced by Stock6 hybridization [J]. Journal of journal of maize sciences, 2006, 14 (1): 64-66.
8. Zhao Yanming, Dong and Zhang Suoliang. Research and application progress of maize haploid breeding technology [J]. Journal of journal of maize sciences, 2007, 15 (5): 60-64.
9. Tang Feiyu, Faye Wong, Wang Guoying. Detection of diploid in parthenogenesis of maize by SSR markers [J]. Journal of Jiangxi Agricultural University, 2004,26 (6): 859-862
10. Cai Zhuo, Xu, Tang Changming, et al. Research progress of maize haploid breeding [J]. Journal of journal of maize sciences, 2008, 16 (1): 1-5.
1 1. Du Juan, Mu Qiuhua, Jia Yufeng, et al. Study on improving the induction rate of maize anther culture by bridge combination transfer method [J]. Journal of journal of maize sciences,1999,7 (3):16-18.