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伴矿景天Sedum plumbizincicola是景天科Crassulaceae景天属Sedum的新种,多年生肉质草本,以无性繁殖为主;适应性广,生物量大,生长速度快,在中国大部分地区均可正常生长[1]。作为一种新的锌、镉超富集植物[2],目前对伴矿景天重金属耐受和解毒的研究较多,主要集中在植株对重金属的响应和耐性机制及细胞生理等方面[3-4];对于重金属对蛋白质表达的影响、重金属胁迫下基因的调控及其与信号转导途径的关系等分子机制的研究甚少,目前,对伴矿景天再生体系的研究鲜有报道,伴矿景天再生体系的建立是基因枪法、农杆菌介导法遗传转化的基础,更是研究其重金属耐受和解毒分子机制、培育优良新品种的重要途径。现以伴矿景天幼嫩叶片为材料,研究了不同植物生长调节剂组合对伴矿景天再生培养的影响,以期为建立稳定的遗传转化体系提供良好的受体材料。
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2, 4-D在诱导愈伤组织中起着重要作用。培养基中只添加0.30 mg·L-1 6-BA,结果发现无愈伤组织产生;在添加2, 4-D的培养基中,叶片生长10 d左右即开始膨大,30 d左右开始产生愈伤组织。2, 4-D质量浓度对愈伤组织诱导率有影响,当2, 4-D质量浓度从1.00 mg·L-1升高到2.00 mg·L-1时,愈伤组织诱导率从92.01%增加到94.91%,但差异不显著(P>0.05);当达4.00 mg·L-1时,愈伤组织诱导率为81.58%,显著下降(表 1)。TDZ可以促进愈伤组织的诱导,只添加3.00 mg·L-1NAA的培养基中,叶片多数分化出根,愈伤组织诱导率仅为4.67%;同时添加0.50~1.00 mg·L-1 TDZ可以显著提高愈伤组织的诱导率(P<0.05),TDZ质量浓度为0.50 mg·L-1时诱导率最高,为66.82%(表 1)。
表 1 不同植物生长调节剂组合对愈伤组织诱导的影响
Table 1. Effects of different concentrations of plant growth regulators on callus induction from leaf
ρ2, 4-D/(mg·L-1) ρ6-BA/(mg·L-1) 诱导率/% ρTDZ/(mg·L-1) ρNAA/(mg·L-1) 诱导率/% 0.00 0.30 0.00 ± 0.00 a 0.00 3.00 4.67 ± 2.51 a 1.00 0.30 92.01 ± 2.90 c 0.50 3.00 66.82 ± 17.68 c 2.00 0.30 94.91 ± 2.80 c 0.70 3.00 50.70 ± 1.47 bc 4.00 0.30 81.58 ± 2.41 b 1.00 3.00 46.74 ± 1.63 b 说明:数值后相同字母表示差异不显著(P>0.05) 新生的愈伤组织通常有2种形态,一种是淡黄色透明型,一种为白色致密型;有研究认为,淡黄色透明状愈伤组织更有利于分化[6]。本研究发现,在添加0.30 mg·L-1 6-BA和1.00 mg·L-1 2, 4-D的培养基中,叶片边缘多产生块状的淡黄色透明愈伤组织,体积大,生长快(图 1A,图 1B);而在添加0.30 mg·L-1 6-BA和2.00~ 4.00 mg·L-1 2, 4-D,3.00 mg·L-1NAA和0.50~1.00 mg·L-1 TDZ的培养基中叶片边缘多产生白色致密愈伤组织,体积小,生长慢。这说明添加0.30 mg·L-1 6-BA和1.00 mg·L-1 2, 4-D的培养基对诱导伴矿景天叶片愈伤组织的质量更佳。
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增殖培养20 d后,挑选淡黄色透明状愈伤组织转接于分化培养基上。观察发现愈伤组织在光下逐渐由淡黄色变成绿色,表面长出突起,逐渐形成黄绿色的芽丛(图lC,图 1D)。未添加6-BA,只添加0.10 mg·L-1 2, 4-D的培养基中,愈伤组织不能分化(表 2);随着6-BA质量浓度的升高,愈伤组织分化率也升高,6-BA质量浓度为1.00 mg·L-1和2.00 mg·L-1时分化率为27.44%和21.85%,两者无显著差异。6-BA的质量浓度影响愈伤组织的分化,伴矿景天愈伤组织分化最佳培养基为1.00 mg·L-1 6-BA+0.10 mg·L-1 2, 4-D。
表 2 不同质量浓度的6-BA对愈伤组织分化的影响
Table 2. Effects of different concentrations of 6-BA on bud formation of callus
ρ6-BA/(mg·L-1) ρ2, 4-D/(mg·L-1) 分化率/% 0 0.10 0.00 ± 0.00 a 0.10 0.10 2.27 ± 0.34 ab 0.50 0.10 6.42 ± 0.32 b 1.00 0.10 27.44 ± 6.73 c 2.00 0.10 21.85 ± 1.84 c 说明:数值后相同字母表示差异不显著(P>0.05) -
出芽的愈伤组织转接于增殖培养基中,添加0.10 mg·L-1 2, 4-D和不同质量浓度(0.50,1.00 mg·L-1) 6-BA。培养30 d后,芽丛膨大,2种培养基中,不定芽的繁殖系数都在8.0以上;6-BA质量浓度为0.50 mg·L-1时,芽生长健壮、叶片鲜绿(图 1E),6-BA质量浓度为1.00 mg·L-1时增殖效果更加明显,但是芽细小,生长较弱(图 1F)。因此,伴矿景天最佳不定芽增殖培养基为0.10 mg·L-1 2, 4-D+0.50 mg·L-1 6-BA。
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伴矿景天易生根,在添加不同质量浓度(0,0.10,0.50,1.00,2.00 mg·L-1)IBA的培养基中根诱导率都能达100%,但影响根和植株的生长状态。在添加1.00,2.00 mg·L-1 IBA的培养基中,根较粗壮,植株生长快且健壮(图 1G,图 1H);在IBA质量浓度小于0.50 mg·L-1的培养基中,根较细小,植株矮小,生长势差。添加1.00~ 2.00 mg·L-1 IBA的培养基是适宜伴矿景天愈伤组织的生根培养基(图 1I)。
Callus induction from leaf and plant regeneration of Sedum plumbizincicola
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摘要: 以伴矿景天Sedum plumbizincicola幼嫩叶片为外植体,以添加了300 mg·L-1水解酪蛋白的MS(Murashige and Skoog)培养基为基本培养基,研究不同植物生长调节剂组合对伴矿景天愈伤组织诱导及分化的影响,并建立再生体系。结果表明:1.00 mg·L-1 2,4-二氯苯氧乙酸(2,4-D)+0.30 mg·L-1 6-苄基腺嘌呤(6-BA)组合下,愈伤组织诱导效果最佳(P < 0.05),诱导率为92.01%;0.10 mg·L-1 2,4-D+1.00 mg·L-1 6-BA组合下,分化效果最优(P < 0.05),分化率为27.44%。适宜的芽增殖培养基为0.10 mg·L-1 2,4-D+(0.50~1.00)mg·L-1 6-BA;适宜的生根培养基为1.00~2.00 mg·L-1吲哚丁酸(IBA)。本研究为伴矿景天转基因技术体系的建立奠定了组织培养基础。Abstract: To establish the efficient regeneration system and lay foundation for transgenic system of Sedum plumbizincicola in this research, the young leaves of S. plumbizincicola as explants, several factors affecting callus induction, bud differentiation, and plant regeneration were studied, and then a regeneration system was established. An MS medium supplemented with 300 mg·L-1 hydrolyzed casein was used as the basic medium. Different concentrations of hormone combinations was designed. Results showed that the best medium for leaf callus induction was the basic medium with 1.00 mg·L-1 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 0.30 mg·L-1 6-Benzylaminopurine (6-BA) added having a callus induction rate of 92.01%. The best medium for bud differentiation was the basic medium supplemented with 0.10 mg·L-1 2, 4-D and 1.00 mg·L-1 6-BA having a callus differentiation rate of 27.44%. A suitable medium for bud proliferation was the basic medium with 0.10 mg·L-1 2, 4-D and (0.50-1.00) mg·L-1 6-BA added. Also, a suitable rooting medium was the basic medium with 1.00-2.00 mg·L-1 3-indolebutyric acid (IBA) added. These findings laid a tissue culture foundation for the establishment of a transgenic system with S. plumbizincicola.
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Key words:
- botany /
- tissue culture /
- Sedum plumbizincicola /
- leaf /
- callus /
- regeneration system
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表 1 不同植物生长调节剂组合对愈伤组织诱导的影响
Table 1. Effects of different concentrations of plant growth regulators on callus induction from leaf
ρ2, 4-D/(mg·L-1) ρ6-BA/(mg·L-1) 诱导率/% ρTDZ/(mg·L-1) ρNAA/(mg·L-1) 诱导率/% 0.00 0.30 0.00 ± 0.00 a 0.00 3.00 4.67 ± 2.51 a 1.00 0.30 92.01 ± 2.90 c 0.50 3.00 66.82 ± 17.68 c 2.00 0.30 94.91 ± 2.80 c 0.70 3.00 50.70 ± 1.47 bc 4.00 0.30 81.58 ± 2.41 b 1.00 3.00 46.74 ± 1.63 b 说明:数值后相同字母表示差异不显著(P>0.05) 表 2 不同质量浓度的6-BA对愈伤组织分化的影响
Table 2. Effects of different concentrations of 6-BA on bud formation of callus
ρ6-BA/(mg·L-1) ρ2, 4-D/(mg·L-1) 分化率/% 0 0.10 0.00 ± 0.00 a 0.10 0.10 2.27 ± 0.34 ab 0.50 0.10 6.42 ± 0.32 b 1.00 0.10 27.44 ± 6.73 c 2.00 0.10 21.85 ± 1.84 c 说明:数值后相同字母表示差异不显著(P>0.05) -
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2018.03.024