Wheat ear in-vitro culture method through wheat and corn hybridization and haploidembryo induction

Abstract

The invention discloses a wheat ear in-vitro culture method through wheat and corn hybridization and haploidembryo induction. The method comprises the steps of clipping the wheat ear after pollination by corn pollen for 24h from the field or the pot, spraying 100mg / L 2,4-D solution on the wheat ear, inserting into a container containing ear culture solution, placing the container in a manual climatic box for continuous culture through day and night, replacing the ear culture solution for the first time after three days, replacing the ear culture solution for the second time after three days, then replacing the ear culture solution for the third time after the next four days until the culture is completed, wherein in the 13th-14th days from the culture starting to the culture ending, the ear culture solution is replaced to submerge 2-3cm of the stem of the wheat ear each time, and the stem and the leaves of the wheat ear need to be treated when the ear culture solution is replaced each time, at least 2-3cm of the base stem of the wheat ear is cut into a plain end by scissors, and then the leaves which do not grow on the nodes of the base part of the stem are removed. With the adoption of the method, the haploidembryo inductivity can reach more than 70% and the embryos grow well.

Description

technical field [0001] The invention belongs to the technical field of agriculture, and relates to a method for culturing wheat ears in vitro for inducing haploid embryos by hybridization of wheat and corn. Background technique [0002] Obtaining wheat haploids through wheat×maize hybridization and the disappearance of maize chromosomes after hybridization is one of the most efficient ways to produce wheat haploids at home and abroad. Wheat haploid and its chromosome doubled doubled haploid (also known as "double haploid") have important application value in related basic research and breeding, especially can accelerate the process of wheat breeding and shorten the cultivation of new wheat varieties time. [0003] Obtaining a high and stable induction rate of wheat haploid embryos is the first key link in the application of wheat×maize hybrid haploid induction technology. Haploid embryo induction rate, also known as embryo formation rate and embryo yield rate, refers to th...

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Problems solved by technology

However, when growing in the field, the induction, development and growth of haploid embryos are easily affected by complex and variable natural temperature, light, rain (water) and other conditions, resulting in an extremely unstable induction rate of haploid embryos in wheat. The growth on the plant also takes up more space. Later, further research confirmed that the in vitro culture of wheat ears after crossing wheat × corn is the most effective technical measure to stabilize the induction rate of haploid embryos.

Initially, Laurie (1986) adopted spikelet culture, that is, 15-20 spikelets on each ear were peeled off and cultivated in a culture medium under artificial control conditions, but the specific operation was labor-intensive, time-consuming and costly. , it is difficult to process hundreds of thousands of wheat ears; later Riera-Lizarazu (1992), Suenaga (1997), Najia Saidi (1997), Inagaki (1998), Mujeeb-Kazi (2006), Muhammad Ahsan Khan (2011 ), Makhdoom Hussain (2012) used the in vitro culture of cutting ear (or "cutting ear") in the hybridization of common wheat and corn to induce common wheat to produce haploid. (Main ear and tiller ear) are cut off from the plant in batches, and the cutting length is 35-50cm, retaining the flag leaves, removing excess leaves, then placing them in tap water for cultivation, detasseling, and pollinating with corn pollen after 1-2 days, Then put it into a container containing ear culture solution (2,4-D100mg / L+sucrose 40-50g / L+sulfurous acid 6-8ml / L) for 3 days, and then move to ear culture without 2,4-D culture medium (40-50g / L sucrose + 6-8ml sulfurous acid) for 10-12 days, replenish and top up the culture medium in the container every 2-3 days, the culture temperature is 20-22°C, and the light length is 14-16 hours, relative humidity is 60-65%, light intensity is all not reported except that Muhammad Ahsan Khan reports that other studies are 10000Lux; Ears were not cut before emasculation and pollination, and ears were cut off from the plant along the base after pollination, and placed in ear culture solution (2,4-D100mg / L+40g / L sucrose+10ml / L ethanol) for 10 days. -18 days; Najia Saidi (1997) cut back the ear from the third internode (counting from the top) after 4 days of pollination, scrubbed it with 70% alcohol, and then inserted it into the culture medium ( Hoagland's nutrient solution + 2,4-D100mg / L+40g / L sucrose+10ml / L ethanol) in the container, replace the culture medium every 2 days, the culture conditions are: 16 hours of light, day / night The temperature was 22°C / 18°C, the relative humidity was 80%, and the culture period was 15 days; Cai Hua et al. (2005) cut off the pollinated wheat ears together with the stems, and inserted them into the plants containing 2,4-D100mg / L+ sucrose 40- In vitro culture in a large beaker of 50g / L + sulfurous acid 8ml / L nutrient solution, culture temperature 20°C, light time 12 hours, intensity 3000Lux, relative humidity 70%, in vitro culture for 14 days

[0004] The induction rate of haploid embryos reported in the study of isolated wheat ear culture mentioned above was 24.1-35.8% reported by Suenaga (1997), 25% by Mujeeb-Kazi (2006), and 26.3-35.8% by Cai Hua et al. (2005). 34.0% is higher, the rest range is 0-29%, and most of them are lower than 20%. Although Najia Saidi (1997) used the most complex and nutrient-rich nutrient solution, the induction rate of haploid embryos Only 0-11.7% on average

Analyzing the reasons, in addition to the differences in wheat and corn genotypes, cutting back wheat ears before detasseling is convenient for detasselling and bagging, but growing wheat ears in nutrient-deficient tap water for 1-2 days will affect the function of the plant More importantly, all the research reports have been cultivating after the ear cutting from the mother plant for the first time until the ears are taken and the embryos are peeled off, and there is no report on the second or third ear stem cutting in the middle According to our years of observation, since the ears of wheat cultured in vitro have no roots, the nutrient absorption depends entirely on the stalk. After 3-4 days of cultivation in the culture medium, the base of the stalk will be corroded by the culture medium to varying degrees. If it is not cut off in time Corroded basal stalks, as time goes on, the basal stalks become more and more corroded, and it becomes more and more difficult for wheat ears to absorb nutrients and water from the nutrient solution, which causes the leaves to turn yellow in the middle stage of cultivation, and then soon Fast withering, photosynthesis and respiration of leaves are damaged or suspended, and the water and nutrients in the culture medium are difficult to transport to the ear, so that the caryopsis (an organ similar to wheat grain formed after wheat and corn hybridization, wheat haploid embryo All the nutrients needed for growth and development come from caryopsis) thin, shriveled and yellow, which directly affects the development and vitality of haploid embryos, and some embryos even die prematurely, resulting in a low haploid induction rate

In addition, except for the research of Najia Saidi (1997), the rest of the research did not change the culture medium during wheat ear culture, but just added it continuously, which had a negative impact on the quality of the culture medium, osmotic pressure, etc., and at the same time enhanced the culture medium. Corrosion or toxicity of the liquid to the stem

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