阅读说明：本技术 一种获得白及单倍体的育种方法 (Breeding method for obtaining rhizoma bletillae haploid ) 是由 林云斌 梁志勇 黄剑雄 于 2019-12-11 设计创作，主要内容包括：本发明涉及一种获得白及单倍体的培育方法,特别涉及一种采用植物组织培养获得白及单倍体的培育方法,属于白及单倍体的选育领域。本发明选取花药进行花药培养,诱导愈伤组织再分化为单倍体植株,提高了愈伤诱导频率和愈伤组织的分化能力,本发明优化了愈伤组织诱导培养基、不定芽分化培养基以及生根培养基,进行白及花药培养,使愈伤诱导率85％以上,出苗率90％以上,应用于白及育种,为白及的育种、品质改良、遗传研究提供有力的手段。本发明方法培育的白及单倍体植株生长良好、移栽成活率高,是大规模工厂化生产优质白及种质的基础。(The invention relates to a cultivation method for obtaining a bletilla striata haploid, in particular to a cultivation method for obtaining the bletilla striata haploid by adopting plant tissue culture, and belongs to the field of breeding of the bletilla striata haploid. The method selects the anther to culture the anther, induces the callus to be redifferentiated into haploid plants, improves the callus induction frequency and the differentiation capacity of the callus, optimizes a callus induction culture medium, an adventitious bud differentiation culture medium and a rooting culture medium, cultures the bletilla striata anther, ensures that the callus induction rate is more than 85 percent and the emergence rate is more than 90 percent, is applied to bletilla striata breeding, and provides a powerful means for breeding, quality improvement and genetic research of the bletilla striata. The bletilla striata haploid plants cultivated by the method have good growth and high transplanting survival rate, and are the basis for large-scale industrialized production of high-quality bletilla striata germplasm.)

1. A cultivation method for obtaining rhizoma bletillae haploid is characterized in that: comprises the following steps of 1) selecting anthers of which microspores are in a mononuclear border stage, 2) disinfecting bletilla striata anthers, 3) inoculating the anthers to a callus induction culture medium for callus induction culture to obtain bletilla striata callus; 4) inoculating bletilla striata callus on an adventitious bud differentiation culture medium to perform differentiation culture of adventitious buds to obtain bletilla striata adventitious buds; 5) inoculating the bletilla striata adventitious bud on a rooting culture medium for rooting culture to obtain a test-tube plantlet; 6) and obtaining the bletilla striata haploid plant through ploidy identification.

2. Cultivation process for obtaining rhizoma bletillae and haploids as claimed in claim 1, characterized by the fact that: the method for selecting the anther of the microspore in the mononuclear border period comprises the following steps: fresh anthers are selected from anthers of which microspores are in the uninucleate near-edge stage, and the pollen development stage is determined to be the uninucleate near-edge stage through magenta acetic acid dyeing.

3. A breeding method for obtaining rhizoma bletillae and haploid as claimed in claim 1, wherein: the method for sterilizing the common bletilla pseudobulb anther comprises the following steps: the anther is firstly disinfected by 75% alcohol for 1 minute, disinfected and soaked by 7% sodium hypochlorite solution for 15 minutes, washed by sterile water for 3-5 times, and then the sterile filter paper absorbs water.

4. A breeding method for obtaining rhizoma bletillae and haploid as claimed in claim 1, wherein: the callus induction culture medium is MS improved culture medium, naphthylacetic acid (NAA)0.1-2.0mg/L, 6-furfuryl aminopurine (KT)0.5-2.0mg/L, sucrose 15-25g/L and agar 5-15 g/L; the callus culture method of the culture medium is that under a dark condition, the culture temperature is 25 +/-1 ℃; obtaining bletilla striata callus.

5. A breeding method for obtaining rhizoma bletillae and haploid as claimed in claim 1, wherein: the adventitious bud differentiation culture medium is MS modified culture medium, 0.2-2.0mg/L of naphthylacetic acid (NAA), 0.5-1.0mg/L of 6-benzyladenine (6-BA), 15-25g/L of sucrose and 5-15g/L of agar; the culture temperature of the culture medium for adventitious bud differentiation is 25 +/-1 ℃, the illumination is 1500-2000 lx, and the illumination time is 12 hours/day.

6. A breeding method for obtaining rhizoma bletillae and haploid as claimed in claim 1, wherein: the rooting culture medium is 1/2MS improved culture medium, 0.2-1.0mg/L of indolebutyric acid (IBA), 15-25g/L of sucrose and 5-15g/L of agar, the rooting culture temperature is 25 +/-1 ℃, the illumination is 1500-2000 lx, and the illumination time is 12 hours/day.

Technical Field

The invention relates to the technical field of plant breeding, in particular to a breeding method for obtaining rhizoma bletillae haploid.

Background

Bletilla striata (Bletilla striata), also named as Bletilla striata, is a perennial herb of Bletilla of orchidaceae, belongs to the second-level endangered plant of China, has the effects of astringing to stop bleeding, reducing swelling and promoting granulation when dried rhizome is used as a medicament, and is used for treating diseases such as phthisis hemoptysis, bronchiectasis hemoptysis, gastric ulcer, hematuria, hematochezia, traumatic hemorrhage and the like; in addition, the rhizoma bletillae tubers are rich in rhizoma bletillae gum and rhizoma bletillae polysaccharide, and are widely applied to the food industry and the daily chemical industry. The economic value is very high. Wild bletilla striata resources are reduced day by day due to wild disorderly mining in recent years, but bletilla striata germplasm resources are disordered due to the fact that special physiological characteristics and ecological environment of bletilla striata are seriously damaged, the resources are almost exhausted, unreasonable introduction and cultivation of artificial planting cause the germplasm resources of bletilla striata to be disordered, and therefore optimization breeding of wild bletilla striata germplasm resources is particularly important.

The development of biological technical means such as haploid breeding technology, genetic engineering breeding and the like in recent years improves the breeding efficiency and opens up a new way of breeding. Obtaining a homozygous variety by conventional cross breeding requires a long breeding cycle.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a breeding method for obtaining rhizoma bletillae haploid. The haploid plant technology can be obtained by utilizing anther pollen culture, and has the advantages of quickly obtaining homozygous diploid, shortening breeding period, improving selection efficiency and the like.

The technical scheme of the invention is as follows:

a breeding method for obtaining rhizoma bletillae haploid is characterized by comprising the following steps:

step 1, selecting fresh anthers: selecting anthers of microspores in the mononuclear border period, and determining the pollen development period as the mononuclear border period through DAPI dyeing;

step 2, anther disinfection: sterilizing anther with 75% alcohol for 1 min, sterilizing and soaking in 7% sodium hypochlorite solution for 15 min, washing with sterile water for 3-5 times, and sucking water with sterile filter paper;

step 3, inoculating the sterilized anther on a callus induction culture medium to perform callus induction culture; performing dark treatment for 2-3 days at 4 ℃ in a dark condition, performing treatment for 5-7 days at 37 ℃, and then performing illumination for 1500-2000 lx for 12 hours/day at a culture temperature of 25 +/-1 ℃ to obtain bletilla striata callus;

step 4, inoculating the callus on an adventitious bud differentiation culture medium to perform differentiation culture of adventitious buds to obtain bletilla striata adventitious buds; the culture temperature is 25 +/-1 ℃, the illumination is 1500-2000 lx, and the illumination time is 12 hours/day;

step 5, inoculating the adventitious buds to a rooting culture medium for culture to obtain test-tube plantlets;

and 6, obtaining a bletilla striata haploid plant through ploidy identification.

In order to better achieve the culture effect, the anther of which the microspore development is in the mononuclear border stage is taken out for the induction culture of the callus, so that the induction culture rate of the callus can be effectively improved; wherein, the development stage of the bletilla striata microspore can be observed by dyeing, and the methods or means are all known by the technical personnel in the field; the invention determines the development period of the bletilla striata microspores through the dyeing observation of the carmine acetate.

The callus induction culture medium is preferably MS modified culture medium + naphthylacetic acid (NAA)0.1-2.0mg/L + 6-furfurylaminopurine (KT)0.5-2.0mg/L + sucrose 15-25g/L + agar 5-15 g/L;

the adventitious bud differentiation medium is preferably MS modified medium + naphthylacetic acid (NAA)0.2-2.0mg/L + 6-benzyladenine (6-BA)0.5-1.0mg/L + sucrose 15-25g/L + agar 5-15 g/L;

the rooting medium is preferably 1/2MS modified medium + indolebutyric acid (IBA)0.2-1.0mg/L + sucrose 15-25g/L + agar 5-15 g/L.

The invention has the beneficial effects that: the method adopts a solid culture medium to co-culture the microspores and the anther wall, and can improve the callus induction frequency and the differentiation capacity of callus by low-temperature and heat shock pretreatment, callus induction and differentiation culture medium components and culture conditions regulation. In the haploid, gene interaction can be simplified, favorable high-quality germplasm genes are reserved, and harmful recessive genes are eliminated. It can be combined with the currently adopted methods of crossbreeding, artificial mutation breeding and the like to form high-quality germplasm resources. The invention improves the callus induction frequency and the differentiation capacity of the callus by selecting anther, culturing the anther and inducing the callus to be redifferentiated into haploid plants. When the method is used for bletilla striata anther culture, the callus induction rate is over 80 percent, the emergence rate is over 90 percent, and the method can be applied to breeding practice, can accelerate breeding pace and provides a powerful means for new variety breeding.

Detailed Description

The following is further described in conjunction with the detailed description: