| The original plant of radix angelicae is Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook, f or A. dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss.) Shan et Yuan. Suining is the genuine producing area of A. dahurica. Mixed genotype is very common because of cross-pollination of A. dahurica. The purpose of this study is to obtain haploid plants by anther culture so as then to obtain the homozygous diploid plants and provide materials to variety breeding of A. dahurica. This paper used A. dahurica anther as experimental materials, and the best sampling period of anther for anther culture, the callus induction and plant regeneration, preliminary ploidy identification of regenerate plants were systematically studied. The main results are as follows:1. The results of anther culture technical system for A. dahurica line B-10showed that the anthers with no low temperature pretreatment got the highest callus induction ratio, then the pretreatment under low temperature (4℃) for two days, and the lowest callus induction ratio for four days. The anther culture under the dark condition got the highest callus induction ratio and the callus with good quality. Cultured efficiency38.89%was achieved on the medium of MS+2.0mg·L-12,4-D+1.0mg·L-16-BA. The anther calli were transferred to differential medium with different hormone contents and ratios of NAA, IAA,6-BA and KT. The calli on these medium for a period of time showed undifferentiated. But the surface morphology of anther callus was different. The callus on the medium of MS+0.5mg·L-1NAA+1.5mg·L-1KT showed compact and the callus was the best for differentiation. The anther callus under the differentiation medium with AgNO3showed differentiated. The optimum medium for differentiate anther callus was MS+0.5mg·L-1NAA+1.5mg·L-1KT+10mg·L-1AgNO3.1/2MS medium supplemented with0.5mg·L-1IBA can well promote adventitious bud to take roots, and obtained complete regeneration plants. The chromosome number of regenerate plants was studied with conventional plant root tip squash method. The result showed the haploid regenerate plant had been induced.2. The relationship between the microspore development and the morphological characteristics of the buds and anthers of three Angelica dahurica lines of CX08-2, B-2and B-6was studied. The results showed that microspore of A. dahurica developed at five stages:tetrad, early uninucleate, late-uninucleate, binucleate, trinucleate and each stage demonstrated distinctive characteristics. Microspore development also had correlation to the morphological characteristics of buds and the color of anther of the three A. dahurica lines. Among them, CX08-2had the longest flower stalk, the diameter bud and the length of anther, then B-2, B-6had the shortest. The length of flower stalk, the diameter of bud and the length of anther of late-uninucleate period had no significant difference with the adjacent period. The color of petal, dehisce or not and the color of anther could be the direct reference for anther culture.3. The anther callus induction of three A. dahurica lines of CX08-2, B-2and B-3was also studied. When the anthers at the stage of late-uninucleate of three lines were under the previous optimum medium for callus induction, all the three lines can induce calli, and it indicated that the previous anther callus induction medium of A. dahurica was feasible. It also showed the significant difference of callus induction ratio among different lines. The line of B-2got the highest callus induction ratio, and the line of CX08-2got the lowest ratio. So the induction condition should be further optimized as to different lines. |
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