版纳甜龙竹愈伤组织诱导与植株再生体系的建立
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摘要
本研究以版纳甜龙竹成熟种胚为试验材料,研究了基本培养基及植物激素对种胚愈伤组织诱导、愈伤组织增殖、愈伤组织分化及再生苗壮苗生根的影响,筛选出各培养阶段适宜的培养基组分与培养条件,首次建立了高效稳定的版纳甜龙竹植株再生体系,为竹子的组织培养及转基因研究奠定了基础。试验结果具体如下:
1.种胚愈伤组织诱导的适宜培养基为MS基本培养基,添加2,4-D 3 mg·L~(-1)、脯氨酸(Pro)500 mg·L~(-1)、谷氨酰胺(Gln)500 mg·L~(-1)、水解酪蛋白(CH)500 mg·L~(-1)时愈伤组织诱导率和良好愈伤组织诱导率较高,分别达80%和60%左右。
2.添加低浓度KT(0.5 mg·L~(-1))愈伤组织增殖效果与对照相比无显著差异,而高浓度KT(1或2 mg·L~(-1))抑制愈伤组织的增殖,愈伤组织继代几次后普遍褐化严重,失去增殖与分化能力。
3.正交实验表明,愈伤组织分化的适宜培养基为MS + 2 mg·L~(-1) BA + 4 mg·L~(-1) KT + 1 mg·L~(-1) NAA+ 30 g·L~(-1)蔗糖+ 0.8% Type A agar,愈伤组织分化率高达89.5%,再生植株伸长生长良好。
4.当IBA浓度为5 mg·L~(-1)时试管苗十天左右即诱导出根,平均每植株生根数7-8条,生根率达80%以上。生根试管苗移植到泥炭:蛭石:珍珠岩=1:1:1的混合基质中,植株成活率达100%。
5.组织细胞学观察表明版纳甜龙竹种胚愈伤组织分化既有器官发生途径又有体细胞胚胎发生途径。
6.农杆菌转化的版纳甜龙竹抗性愈伤组织分化获得一批转基因白化苗,抗性愈伤组织及转基因再生苗经GUS染色显蓝色呈阳性。
In this study, using mature zygotic embryos as explants, plant growth regulators and various media were tested with the aim of selecting out the appropriate culture components and culture condition for callus initiation, callus maintenance, shoot differentiation and plantlet rooting of the bamboo species Dendrocalamus hamiltonii. We firstly developed an efficient and consistent regeneration system for plantlets of the bamboo species D. hamiltonii, providing a useful basis for developing culture protocols of the regeneration of bamboo plants and a platform for genetic transformation in bamboo species.
The main results obtained are presented as follows:
1 Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 3 mg·L~(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 500 mg·L~(-1) proline (Pro), 500 mg·L~(-1) glutamine (Gln), 500 mg·L~(-1) casein hydrolysate (CH), with the frequency of callus and granular and compact callus to 80% and 60% respectively.
2 Low concentration of kinetin (KT, 0.5 mg·L~(-1)) did not significantly improve callus growth during subculture. Meanwhile, high concentrations of KT(1or 2 mg·L~(-1))had a detrimental effect on callus growth, resulting in callus browning and loss of maintenance and differentiation potential.
3 Optimal shoot differentiation and subsequent shoot growth were obtained in MS medium supplemented with 2 mg·L~(-1) benzyladenine (BA), 4 mg·L~(-1) KT, and 1 mg·L~(-1) naphthaleneacetic acid (NAA), 30 g·L~(-1) sucrose and 8 g·L~(-1) Type A agar, with the highest differentiation rate of 89.5%.
4 Root induction was enhanced by the addition of 5 mg·L~(-1) indole-3-butyric acid (IBA) to the culture medium. 7-8 roots were induced per shoot in 10 days and the rooting rate was up to 80%. Rooted plantlets were transferred to artificial mixture (peat: vermiculite: perlite=1:1:1) with 100% shoot survival.
5 Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus differentiation and the development of plantlets from the mature zygotic embryos.
6. Transgenic albino plantlets were obtained by Agrobacterium-mediated transformation. Transient GUS expression was observed in resistant calli and regenerated plantlets.
1.种胚愈伤组织诱导的适宜培养基为MS基本培养基,添加2,4-D 3 mg·L~(-1)、脯氨酸(Pro)500 mg·L~(-1)、谷氨酰胺(Gln)500 mg·L~(-1)、水解酪蛋白(CH)500 mg·L~(-1)时愈伤组织诱导率和良好愈伤组织诱导率较高,分别达80%和60%左右。
2.添加低浓度KT(0.5 mg·L~(-1))愈伤组织增殖效果与对照相比无显著差异,而高浓度KT(1或2 mg·L~(-1))抑制愈伤组织的增殖,愈伤组织继代几次后普遍褐化严重,失去增殖与分化能力。
3.正交实验表明,愈伤组织分化的适宜培养基为MS + 2 mg·L~(-1) BA + 4 mg·L~(-1) KT + 1 mg·L~(-1) NAA+ 30 g·L~(-1)蔗糖+ 0.8% Type A agar,愈伤组织分化率高达89.5%,再生植株伸长生长良好。
4.当IBA浓度为5 mg·L~(-1)时试管苗十天左右即诱导出根,平均每植株生根数7-8条,生根率达80%以上。生根试管苗移植到泥炭:蛭石:珍珠岩=1:1:1的混合基质中,植株成活率达100%。
5.组织细胞学观察表明版纳甜龙竹种胚愈伤组织分化既有器官发生途径又有体细胞胚胎发生途径。
6.农杆菌转化的版纳甜龙竹抗性愈伤组织分化获得一批转基因白化苗,抗性愈伤组织及转基因再生苗经GUS染色显蓝色呈阳性。
In this study, using mature zygotic embryos as explants, plant growth regulators and various media were tested with the aim of selecting out the appropriate culture components and culture condition for callus initiation, callus maintenance, shoot differentiation and plantlet rooting of the bamboo species Dendrocalamus hamiltonii. We firstly developed an efficient and consistent regeneration system for plantlets of the bamboo species D. hamiltonii, providing a useful basis for developing culture protocols of the regeneration of bamboo plants and a platform for genetic transformation in bamboo species.
The main results obtained are presented as follows:
1 Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 3 mg·L~(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 500 mg·L~(-1) proline (Pro), 500 mg·L~(-1) glutamine (Gln), 500 mg·L~(-1) casein hydrolysate (CH), with the frequency of callus and granular and compact callus to 80% and 60% respectively.
2 Low concentration of kinetin (KT, 0.5 mg·L~(-1)) did not significantly improve callus growth during subculture. Meanwhile, high concentrations of KT(1or 2 mg·L~(-1))had a detrimental effect on callus growth, resulting in callus browning and loss of maintenance and differentiation potential.
3 Optimal shoot differentiation and subsequent shoot growth were obtained in MS medium supplemented with 2 mg·L~(-1) benzyladenine (BA), 4 mg·L~(-1) KT, and 1 mg·L~(-1) naphthaleneacetic acid (NAA), 30 g·L~(-1) sucrose and 8 g·L~(-1) Type A agar, with the highest differentiation rate of 89.5%.
4 Root induction was enhanced by the addition of 5 mg·L~(-1) indole-3-butyric acid (IBA) to the culture medium. 7-8 roots were induced per shoot in 10 days and the rooting rate was up to 80%. Rooted plantlets were transferred to artificial mixture (peat: vermiculite: perlite=1:1:1) with 100% shoot survival.
5 Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus differentiation and the development of plantlets from the mature zygotic embryos.
6. Transgenic albino plantlets were obtained by Agrobacterium-mediated transformation. Transient GUS expression was observed in resistant calli and regenerated plantlets.
引文
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