Method of acquiring haynaldia villosa homozygous regeneration plant through anther culture
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A technology for regenerating plants and Trichopyrhiza, applied in the field of plant biology, can solve problems such as difficult to obtain regenerated plants, achieve the effects of improving anther culture efficiency, avoiding heterozygosity, and improving genome sequencing
Inactive Publication Date: 2018-02-23
SHANGHAI ACAD OF AGRI SCI
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Anther culture technology is a commonly used method to obtain haploid regenerated plants. After chromosome doubling, doubled haploid plants that are homozygous for all traits can be obtained at one time, but the application of this technology is largely limited by the genetic characteristics of the culture material itself. type limitation, it is difficult to obtain regenerated plants
So far, there is no report of obtaining regenerated plants of A. villosa through anther culture
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Embodiment 1
[0042] Embodiment 1. wheat tufts is planted and young ear is taken material, process
[0043] T. villosa line 91c43 (provided by the Cytogenetics Laboratory of Nanjing Agricultural University) was planted in an artificial climate chamber at 22°C with 12 hours of light and 12 hours of darkness. Young panicles whose microspore development was in the uninucleate early-middle stage were selected ( figure 1 , 2 ), put it in a fresh-keeping bag, and treat it at a low temperature of 5°C ( image 3 ) about 1 week.
Embodiment 2
[0044] Example 2. Anther pretreatment and callus induction
[0045] In Example 1, the young ears of W. villosa pretreated were treated with 10% NaClO for 10 minutes, washed with sterile water, and then the anthers were taken in a petri dish, about 50 anthers were taken from each dish, and 3 repetitions were made, and 1.5 ml of extract was added. (Containing mannitol 60g / L, adding 1.1g / L CaCl 2 , 0.976g / L MES and 20mg / L colchicine, pH5.8), 24°C, dark treatment for 2 days ( Figure 4 ), replaced with 1.5ml induction medium (N6 medium + maltose 90g / L + glutamine 1600mg / L + hydrolyzed casein 400mg / L + 20mg / L proline + KT0.5mg / L + 2,4-D 1.0mg / L, pH5.8), 24°C, cultured in dark for 28 days, induced callus formation ( Figure 5 ).
Embodiment 3
[0046] Embodiment 3. green seedling differentiation and strong seedling
[0047] In embodiment 2, callus is transferred to differentiation medium (2 / 3MS medium+30g / L maltose+0.5mg / L 6-BA+1.5mg / L KT+0.05mg / L NAA+5.5g / L agar powder , pH 5.8), cultured at 24°C, 12h light, 12h dark ( Figure 6 ). When the regenerated shoots grow to 2-4cm, transfer to strong seedling rooting medium (1 / 2MS medium+sucrose 30g / L+glutathione 100mg / L+NAA 0.6mg / L+chlormequat 0.5mg / L+5.5 g / L agar powder, pH 5.8) cultured in ( Figure 7 ), 24°C, 12h light, 12h dark.
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Abstract
The invention relates to the field of plant biotechnology, in particular to a method of acquiring a haynaldia villosa homozygous regeneration plant through anther culture. The invention also providesan improved sound seedling and rooting culture medium for the anther culture method. By adopting the method provided by the invention, the anther separation activity and the induced reaction are effectively improved from the source by determining the haynaldia villosa drawing critical period, and the problem of weak growth of a regeneration plantlet is solved through sound seedling and hardening-seedling key measures. The invention establishes a set of simple, rapid and stable regeneration system with anther in vitro, haynaldia villosa homozygous regeneration plant seeds can be acquired withinhalf an year by utilizing a program of the invention, so that the hybridization problem caused by haynaldia villosa cross pollination can be effectively solved, improvement of study on a haynaldia villosa genetic map and genome sequencing is facilitated, the anther culture efficiency of wheat distant hybrids can be improved at the same time, and application of haynaldia villosa wild baluable generesources in wheat genetic improvement is accelerated.
Description
technical field [0001] The invention relates to the field of plant biotechnology, in particular to a method for obtaining regenerated haploid plants of A. pilosa by culturing anthers. Background technique [0002] Haynaldia villosa (Haynaldia villosa, 2n=14, VV) is a diploid species of Triticum genus, which has many important agronomic traits, such as high resistance to powdery mildew, and resistance to leaf rust, stem rust, take-all, Various diseases such as eye spot disease, wheat streak virus vector gall mite, leaf blight and barley yellow dwarf. The grain content of trichomes is high in protein and lysine, and it has strong tillering ability, cold resistance, drought resistance, dense ear Many flowers and other characteristics are the rare good gene resource bank for the improvement of wheat varieties in the world. Therefore, the study of wheat clumps is of great significance to the development of wheat breeding and improvement research. [0003] Tissue culture-induced ...
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