不同抗生素对金发草愈伤组织生长分化的影响

Effects of different antibiotics on callus differentiation and growth of Pogonatherum paniceum

关键词:  金发草, 愈伤组织, 抗生素, 遗传转化, 抗性筛选剂, 抑菌剂

Key words:  Pogonatherum paniceum, callus, antibiotics, genetic transformation, resistance selection agent, antibacterial agent

摘要:

该研究以金发草愈伤组织为材料,通过分析比较不同抗生素种类(卡那霉素、潮霉素、头孢噻呋钠和氨苄青霉素)和浓度对金发草愈伤组织生长分化的影响,来确定适用于金发草遗传转化体系中的抗性筛选剂和抑菌剂。结果表明:(1)金发草愈伤组织对卡那霉素很敏感,且其分化率随着卡那霉素浓度的增加显著减少(P=0.01)。当卡那霉素浓度为10 mg·L^-1时,金发草愈伤组织的生长分化受到明显抑制,且有大量的白化苗形成,但分化率仍有36.56%; 当卡那霉素浓度为15 mg·L^-1时,金发草愈伤组织的分化率为11.94%,只有很少部分的愈伤分化出绿色的丛生苗; 当卡那霉素浓度为20 mg·L^-1时,金发草愈伤组织基本褐化死亡,分化率仅为2.26%。因此,浓度为15 mg·L^-1的卡那霉素适合作为金发草遗传转化体系中的抗性筛选剂。(2)金发草愈伤组织对潮霉素的敏感性要比卡那霉素弱,且潮霉素对金发草愈伤组织分化率的影响小,但毒害作用大。因此,潮霉素不适合作为金发草遗传转化体系中的抗性筛选剂。(3)300 mg·L^-1的头孢霉素和氨苄青霉素对金发草愈伤组织生长分化影响很小且能有效抑制杂菌的生长,较高浓度的氨苄青霉素对金发草愈伤组织的抑制作用不太明显。因此,300 mg·L^-1的头孢霉素和较高浓度的氨苄青霉素均可作为金发草遗传转化体系中的抑菌剂。该研究确定了适用于农杆菌介导的金发草遗传转化体系中的抗性筛选剂和抑菌剂,为金发草的遗传改良及功能性基因的研究奠定了基础。

Abstract:

In the study, Pogonatherum paniceum calluses were selected as the experimental materials, and the effects of different kinds of antibiotics(kanamycin, hygromycin, ceftiofur sodium and ampicillin)and its concentrations to the growth and differentiations of P. paniceum calluses were compared and analysed in order to find out the suitable resistance selection agent and antibacterial agent in genetic transformation system of P. paniceum, respectively. The results were as follows:(1)P. paniceum callus had a strong sensitivity to kanamycin and the differentiation rate significantly decreased as the concentration of kanamycin increased(P=0.01). When the concentration of kanamycin was 10 mg·L^-1, the growth and differentiation of P. paniceum callus were significantly suppressed and differentiated a large number of whiten seedlings, but the differentiation rate was still at 36.56%. When the concentration of kanamycin was 15 mg·L^-1, differentiation rate of P. paniceum callus was at 11.94%, and only part of calluses were differentiated into green seedlings. However, when the concentration of kanamycin was 20 mg·L^-1, P. paniceum callus almost turn brown or dead, and the differentiation rate was only at 2.26%. Hence, 15 mg·L^-1 of kanamycin was fit for serveing as the resistance selection agent in genetic transformation system of P. paniceum.(2)P. paniceum callus had a less strong sensitivity to hygromycin than kanamycin. The effects of hygromycin on differentiation rate of P. paniceum callus was very small, but the toxicity was big, so hygromycin was not suitable as the resistance selection agent in genetic transformation system of P. paniceum.(3)300 mg·L^-1 of ceftiofur sodium and ampicillin had a little effect on the growth and differentiation of P. paniceum callus, and all were able to inhibit the bacterials effectively. Morever, the inhibition of little higher level of ampicillin to the growth and differentiation of P. paniceum callus was less obvious. Therefore, 300 mg·L^-1 of ceftiofur sodium and little higher level of ampicillin could be used as the antibacterial agent in genetic transformation system of P. paniceum as well. This study found out the suitable kinds and concentration of antibiotics served as the resistance selection agent and the antibacterial agent in Agrobacterium tumefaciens mediated genetic transformation system of P. paniceum, respectively, which provides the reference for studing genetic improvement and functional genes of P. paniceum.