Optimization of Seed Sterilization and Rooting Medium for Regeneration of Brassica napus
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摘要:目的 植物的种子灭菌和生根培养是组织培养的重要步骤。目前已报道的用于甘蓝型油菜种子灭菌的次氯酸钠(NaClO)浓度、灭菌时间和生根培养基的植物生长调节剂种类及其浓度均存在较大的差异,因此,本研究旨在筛选出甘蓝型油菜种子的最佳NaClO灭菌处理方法和生根培养基最适的植物生长调节剂及其浓度。方法 比较了甘蓝型油菜的种子经不同浓度(浓度范围为0.1% ~ 30.0%)NaClO灭菌后的发芽率、健康芽率和菌落率,筛选出最适的种子灭菌NaClO浓度,再在该最适NaClO浓度基础上找出了最适的种子灭菌时间;通过比较不同质量浓度(0.1 ~ 2.0 mg·L−1)的植物生长调节剂萘乙酸(NAA)或吲哚丁酸(IBA)对甘蓝型油菜再生根的促进作用,找出油菜生根培养最适的植物生长调节剂种类及其浓度。结果 甘蓝型油菜种子的最佳灭菌NaClO浓度为2.0%或3.0%,在3.0% NaClO下的最适灭菌时间为15 min,种子经该灭菌方法处理后的发芽率和健康芽率分别可达97.4%和85.9%,且菌落发生率最少。该油菜种子灭菌的方法也适用于其他9个植物品种,尤其适合于甘蓝型油菜、大白菜和拟南芥的种子灭菌。在生根培养基中添加0.1 mg·L−1 NAA对甘蓝型油菜再生根的促进作用最强,油菜培养7 d后即有92.5%的累积生根率,再生根从切点处生长,且再生的须根数最多。结论 本研究优化了甘蓝型油菜种子的NaClO灭菌方法和生根培养基的植物生长调节剂及其浓度,为高效的油菜组织培养研究奠定了基础。Abstract:Objective Seed sterilization and rooting culture are important steps in plant tissue culture. At present, there is considerable variation in the concentration of sodium hypochlorite (NaClO) and seed sterilization time of Brassica napus, as well as the type and concentration of hormone in the rooting media. The purpose of this study was to optimize the method of seed sterilization with NaClO and the formula of rooting medium for B. napus regeneration.Method The optimum concentration of NaClO for seed sterilization was screened by comparing the growth status (such as germination rate, healthy shoot rate and rate of colonies) of seeds treated with different concentrations (0.1%–30.0%) of NaClO, and the appropriate sterilization time was optimized based on the growth status of seeds treated with the optimal concentration of NaClO for 10—30 min. Moreover, the optimum formula of rooting media was screened by comparing the rooting regeneration of seedlings in the media containing 0.1–2.0 mg·L−1 naphthylacetic acid (NAA) / indolebutyric acid (IBA).Result The optimum concentration of NaClO for seed sterilization was 2.0% or 3.0%, and the best time for seed sterilization with 3.0% NaClO was 15 min, causing the 97.4% of germination rate and 85.9% of healthy shoot rate with least rate of colonies. Moreover, this method of seed sterilization is applicable to other 9 plants, especially for B. napus, B. pekinensis and Arabidopsis thaliana. The root regenerated best in the rooting medium containing 0.1 mg·L−1 NAA, in which 92.5% of root regenerated from the cutting point on the 7th day with the most lateral roots.Conclusion This study optimized the method of seed sterilization and the formula of rooting medium, which laid a foundation for efficient plant tissue culture of B. napus.
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Keywords:
- Brassica napus /
- seed sterilization /
- sodium hypochlorite /
- rooting /
- NAA /
- IBA
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0. 引言
【研究意义】种子灭菌和生根培养是植物组织培养的重要步骤。种子灭菌的目的是获得无菌的愈伤组织,避免后续的细胞再生过程受到污染,同时也要尽量减少对种子自身发芽能力的损伤;生根培养是细胞再生芽形成植株的最后一步,只有成功生根的再生芽才能成为一颗完整的植株,因此种子灭菌和生根培养对植物组织培养的研究具有重要的意义[1-3]。【前人研究进展】目前,甘蓝型油菜种子灭菌使用最普遍的是NaClO灭菌法[4],但已报道使用的NaClO浓度和灭菌时间存在着较大的差异,例如有报道使用以下的浓度和灭菌时间:0.1% NaClO灭菌10 min[5]、1.0% NaClO灭菌45 min[6]、2.0% NaClO灭菌20 min[7]、3.0% NaClO灭菌30 min[8]、5.0% NaClO灭菌20 min[9]、12.5% NaClO灭菌20 min[4]、15.0% NaClO灭菌6 min[10]和50.0% NaClO灭菌10 min[11]等。甘蓝型油菜生根的植物生长调节剂种类主要有萘乙酸NAA、吲哚丁酸IBA和吲哚乙酸IAA等[12],其中常使用的是NAA和IBA 2种,但其使用的浓度差异较大,例如NAA的使用质量浓度主要为0.1 ~ 2.0 mg·L−1[7, 9, 13],IBA的使用质量浓度主要为0.1 ~5.0 mg·L−1[4, 14-18]。此外,郝梦宇等[3]对甘蓝型油菜生根培养基的凝固剂、基本培养基和蔗糖浓度进行了优化,但并未对生根培养基的植物生长调节剂种类及其浓度进行过优化。甘蓝型油菜商业化品种浙大622为2014年浙江省审定推广的杂交新品种,具有稳定高产的优点,适宜作为油菜背景材料构建转基因油菜新品种。【本研究切入点】甘蓝型油菜种子无菌化的效果与使用的NaClO浓度和灭菌时间之间的关系,油菜的再生根效果与生根培养基中的植物生长调节剂NAA或IBA浓度之间的联系均未见系统性的研究报道,有待于进一步的优化。【拟解决的关键问题】筛选出甘蓝型油菜种子最佳的NaClO灭菌方法和最适的生根培养基植物生长调节剂种类及其浓度,为高效的油菜细胞再生植株和基因转化研究奠定基础。
1. 材料与方法
1.1 试验材料和环境
甘蓝型油菜商业化品种浙大622和其他8种农作物品种的种子均购买自福州市的种子零售店,拟南芥种子由本实验室提供。75%酒精和NaClO原液(有效氯含量 ≥ 8%)购买自福州伯乐林生物技术有限公司,MS固体培养基粉末、硝酸银(AgNO3)、蔗糖、琼脂粉、NAA和IBA等试剂购买自索莱宝生物科技有限公司。试验环境保持在光照度为2 000 lx、温度(25 ±1) ℃、相对湿度50% ~ 70%和光周期为16 L∶8 D。
1.2 油菜种子灭菌的NaClO浓度和时间的筛选
1.2.1 0.1% ~ 30.0%大范围浓度NaClO的种子灭菌效果比较
挑选饱满且无伤痕的种子放在水中浸泡1 h,挑选沉在水底的种子,自来水冲洗1遍。将种子转移至200 mL的组培瓶(直径6 cm,高12 cm)内,75%酒精消毒1 min,无菌水冲洗2遍,再转移至超净工作台内操作。用不同浓度(V/V)NaClO(0.0%、0.1%、1.0%、5.0%、10.0%、20.0%和30.0%)100 mL浸泡种子10 min,之后用无菌水漂洗6遍,再转移至无菌干燥的滤纸上晾干待播种。另设一全程仅用无菌水处理种子作为对照组,检测种子的原始发芽能力。灭菌后的种子播种于种子萌发培养基(2.22 g·L−1 MS + 30 g·L−1蔗糖 + 8 g·L−1琼脂粉),5 d后记录种子发芽数、健康芽数(指茎秆高于5 cm、子叶正常展开且未染菌的健康苗)和菌落数,每组处理32颗种子(每8颗种子播种于一个组培瓶内),共4个重复128颗种子。
1.2.2 1.0% ~ 5.0%小范围浓度NaClO的种子灭菌效果比较
从NaClO大范围浓度灭菌效果比较的结果发现,1.0% ~ 5.0%浓度间可能存在一个更加适合的浓度用于种子灭菌。为了验证这一假设,设计了1.0%、2.0%、3.0%、4.0%和5.0%共5组浓度NaClO用于进一步的种子灭菌试验,试验条件、操作过程和结果分析均与上述一致。
1.2.3 3.0% NaClO灭菌种子10 ~ 30 min的效果比较
在筛选到种子灭菌的最佳NaClO浓度(2.0%或3.0%)后,为了进一步找出种子在该浓度NaClO下灭菌的最适时间,试验比较了种子在3.0% NaClO处理种子不同时间(10、15、20、25和30 min)后的灭菌效果。每组处理为64颗种子,共3个重复192颗种子,其他试验条件、操作过程和结果分析均与上述一致。
1.3 种子灭菌方法在芸苔属作物中的通用性
为了验证已筛选出的甘蓝型油菜种子灭菌方法对其他芸苔属作物的通用性,试验使用这一种子灭菌方法(即3.0% NaClO处理种子15 min)对其他9种植物种子进行了种子灭菌效果分析。包括7个芸苔属作物:甘蓝(B. oleracea)京丰一号(JF)、白菜型油菜(B. compestris)红菜苔(HC)、小白菜(B. campestris)夏尊(XZ)、甘蓝型油菜(B. napus)亚科油68(YK)、甘蓝型油菜(B. napus)高油605(GY)、甘蓝型油菜(B. napus)德丰油(DF)和大白菜(B. pekinensis)F1早熟五号(ZS),1个莴苣属作物莴苣(Lactuca sativa)意大利全年生菜王(SC)和模式植物拟南芥(Arabidopsis thaliana, AT)。试验条件、操作过程和结果分析均与种子灭菌的NaClO浓度筛选试验一致。
1.4 NAA/IBA对油菜生根效果的比较
甘蓝型油菜种子经3.0% NaClO灭菌15 min,播种于种子萌发培养基5 d后,切取高为8 ~ 10 cm苗的根上部分用于再生根实验。在生根培养基(4.43 g·L−1 MS + 30 g·L−1蔗糖 + 8 g·L−1琼脂粉 + 5 mg·L−1 AgNO3)内分别添加不同质量浓度(0.0、0.1 、0.5 、1.0 、2.0 mg·L−1)的植物生长调节剂NAA或IBA。将切去根后的苗转移至生根培养基内生根培养,每个处理组8株,5次重复,共40株。每日统计生根情况至24 d后,洗去再生根根部的培养基,测量再生根的主根长、生根部位(再生根位置与切点间的距离),并统计再生的须根数。
1.5 数据分析
应用软件SPSS 22.0分析不同处理组数据间的差异。本文涉及的百分率数据均先进行反正弦平方根(arcsin
√x )数值的转换后,再用单因素方差分析(one-way ANOVA)的Duncan检验分析多个处理间的显著性差异水平(P = 0.05)。其中,不同处理组间再生根的累积生根率比较采用卡方检验分析显著性差异水平(P = 0.05)。发芽率/% =(发芽的种子数 / 总的种子数)× 100;健康芽率/% =(正常生长的芽数 / 总的种子数)× 100;菌落率/% =(染菌的芽数 / 总的种子数)× 100;累积生根率/% =(生根的苗数 / 总苗数)× 100。2. 结果与分析
2.1 油菜种子灭菌的NaClO浓度和处理时间的优化
2.1.1 种子灭菌的NaClO大范围浓度的初步筛选
油菜种子经0.0%、0.1%、1.0%、5.0%、10.0%、20.0%和30.0% NaClO灭菌后的发芽率结果表明,不同处理组间存在着显著性的差异(F = 203.734, df1,2 = 6, 21, P = 0.000),其中种子经0.0%、0.1%和1.0% NaClO灭菌后的发芽率为93.8% ~ 99.2%,显著地高于其他浓度处理组的种子发芽率(图1-A),但与种子的原始发芽率(95.8%)无显著性差异(F = 3.458, df1,2 = 3, 12, P = 0.051),这表明0.0%、0.1%和1.0% NaClO处理组不会损伤油菜种子的发芽能力。不同浓度NaClO处理组的种子健康芽率存在着显著性差异(F = 98.742, df1,2 = 6, 21, P = 0.000),其中种子经0.0%、0.1%和1.0% NaClO灭菌后的健康芽率 ≥ 75.8%,显著地高于其他浓度处理组的种子健康芽率(图1-B)。同样,不同处理组间的菌落率也存在着显著性差异(F = 12.020, df1, 2 = 6, 21, P = 0.000),其中种子经浓度 ≥ 1.0% NaClO灭菌后的菌落率显著地低于0.0%和0.1% NaClO处理组(图1-C)。这些结果表明种子经1.0% NaClO处理后的发芽率和健康芽率最高,同时其菌落率最少,值得注意的是NaClO浓度在1.0% ~ 5.0%间可能还存在一个更适合种子灭菌的浓度。
图 1 甘蓝型油菜种子灭菌的NaClO大范围浓度初步筛选注:A, 发芽率; B, 健康芽率; C, 菌落率。同一图中的不同字母表示处理间具有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。图2~3同。Figure 1. Preliminary screening of large-scale concentration of NaClO for seed sterilization in B. napusNote: A, germination rate; B, healthy shoot rate; C, rate of colonies. Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”. The same as Fig.2~3.2.1.2 种子灭菌的NaClO浓度小范围筛选
种子经1.0%、2.0%、3.0%、4.0%和5.0% NaClO灭菌后的发芽率为85.2% ~ 96.1%,不同处理组的发芽率无显著性差异(F = 2.190, df1,2 = 4, 15, P = 0.120; 图2-A)。不同浓度NaClO灭菌后的种子健康芽率存在显著性差异(F = 21.877, df1, 2 = 4, 15, P = 0.000),其中种子经1.0%、2.0%和3.0% NaClO灭菌后的健康芽率 ≥ 82.5%,显著地高于4.0%和5.0% NaClO处理组的种子健康芽率(图2-B)。种子经NaClO灭菌后的菌落率也存在着显著性差异(F = 4.977, df1, 2 = 4, 15, P = 0.009),其中2.0%、3.0%、4.0%和5.0% NaClO处理组的种子菌落率为0.78% ~ 1.56%,显著地低于1.0% NaClO灭菌种子后的菌落率(图2-C)。综上可知,油菜种子在2.0%或3.0% NaClO处理下的灭菌效果最好。
2.1.3 种子灭菌的时间筛选
种子在3.0% NaClO下灭菌10、15、20、25和30 min后的发芽率(92.2% ~ 97.4%)无显著性差异(F = 2.018, df1,2 = 4, 10, P = 0.168; 图3-A)。3.0% NaClO处理种子10 min后的健康芽率显著地低于其他处理组(F = 3.875, df1, 2 = 4, 10, P = 0.037; 图3-B)。此外,不同处理组的菌落率无显著性差异(F = 1.272, df1, 2 = 4, 10, P = 0.344; 图3-C)。由此可知,种子经3.0% NaClO灭菌15、20、25和30 min后的效果优于灭菌10 min的效果。
2.2 种子灭菌方法在芸苔属作物中的通用性
9个植物品种的种子经3.0% NaClO灭菌15 min后的发芽率为80.0% ~ 100.0%,其中的6个芸苔属植物品种(包括小白菜XZ、大白菜ZS、拟南芥AT、甘蓝型油菜YK、甘蓝型油菜GY和甘蓝型油菜DF)的发芽率(≥ 94.4%)显著地高于其他3个植物品种(F = 30.306, df1, 2 = 8,18, P ≤ 0.000;图4-A、D、E)。这6个发芽率较高的芸苔属植物中的5个(不包括小白菜XZ)的种子健康芽率(≥ 96.7%)显著地高于其他4个植物品种(F = 30.306, df1, 2 = 8,18, P ≤ 0.000;图4-B)。此外,7个植物品种(不包括小白菜XZ和大白菜ZS)的菌落率(≤ 1.11%)显著地低于其他2个植物品种(F = 3.969, df1, 2 = 8,18, P = 0.007;图4-C)。这些结果表明本文筛选出的甘蓝型油菜种子灭菌的方法也适用于其他植物品种,尤其适合于甘蓝型油菜品种、大白菜品种和模式植物拟南芥。
图 4 种子灭菌方法对其他芸苔属作物种子的灭菌效果注:A, 发芽率;B,健康芽率;C,菌落率;D,9种植物品种的种子;E,9种植物品种的种子灭菌7 d后的苗生长情况。JF,甘蓝B. oleracea ‘京丰一号’;HC,白菜型油菜B. compestris ‘红菜苔’;XZ,小白菜B. campestris ‘夏尊’;YK,甘蓝型油菜B. napus ‘亚科油68’;GY,甘蓝型油菜B. napus ‘高油605’;DF,甘蓝型油菜B. napus ‘德丰油’;ZS,大白菜B. pekinensis ‘F1早熟五号’;SC,莴苣Lactuca sativa ‘意大利全年生菜王’;AT,拟南芥Arabidopsis thaliana。同一小图中的不同字母表示处理间有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。Figure 4. The effects of seed sterilization for other Brassica crops with the screened optimal methodNote: A, germination rate; B, healthy shoot rate; B, rate of colonies; D, seeds of 9 plant species; E, seedling growth status of 9 plant species after seed sterilization and sowing for 7 days. JF (B. oleracea ‘Jing Feng 1’), HC (B. compestris ‘Hong Cai Tai’), XZ (B. campestris ‘Xia Zun’), YK (B. napus ‘Ya Ke’), GY (B. napus ‘Gao You’), DF (B. napus ‘De Feng’), ZS (B. pekinensis ‘Zao Shou 5’), SC (Lactuca sativa) and AT (Arabidopsis thaliana). Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”.2.3 油菜生根培养基的NAA/IBA浓度筛选
油菜在含不同浓度NAA/IBA培养基下再生根的累积生根率有显著性的差异(χ2 = 41.062, df = 8, P<0.001),其中:油菜在0.1 mg·L−1 NAA下的累积生根率最高,在第5日开始生根,第7日达到92.5%的累积生根率;其次为0.5 mg·L−1 NAA、1.0 mg·L−1 NAA和1.0 mg·L−1 IBA(图5-A)。同时,油菜在含0.1 mg·L−1 NAA培养基下的再生根部位最好(0 cm,表示再生根直接从切点处生长),显著地优于其他处理组(F = 28.249, df1, 2 = 8, 204, P<0.001;图5-B);再生的须根数也显著地多于其他处理组(F = 7.919, df1, 2 = 8, 204, P<0.001;图5-C)。但值得注意的是,油菜在含0.1 mg·L−1 NAA培养基下再生的主根长并不是最高的,其主根长低于1.0 mg·L−1 IBA或CK处理组的主根长(F = 11.265, df1, 2 = 8, 204, P<0.001;图5-D)。综上可知,0.1 mg·L−1 NAA对油菜再生根的累积生根率、生根部位和须根数的促进作用最强,在油菜再生根的研究中应优先使用0.1 mg·L−1 NAA用于根的再生培养。
图 5 不同质量浓度NAA/IBA对油菜再生根的累积生根率、生根部位、主根长和须根数的影响注:A、累积生根率;B、生根部位(再生根位置与切点处之间的距离);C、主根长;D、须根数。CK、培养基内未添加植物植物生长调节剂; 0.1 NAA, 0.1 mg·L−1 NAA; 0.5 NAA, 0.5 mg·L−1 NAA; 1.0 NAA, 1.0 mg·L−1 NAA; 2.0 NAA, 2.0 mg·L−1 NAA; 0.1 IBA, 0.1 mg·L−1 IBA; 0.5 IBA, 0.5 mg·L−1 IBA; 1.0 IBA, 1.0 mg·L−1 IBA; 2.0 IBA, 2.0 mg·L−1 IBA。图中的不同字母表示处理间具有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。Figure 5. Effects of different concentrations of NAA / IBA on cumulative rooting rate, rooting position, taproot length and number of lateral roots of regenerative roots of B. napusNote: A, cumulative rooting rate; B, rooting position (the distance between root regenerated position and cut point); C, number of lateral roots; D, taproot length. CK, media without plant hormone; 0.1 NAA, 0.1 mg·L−1 NAA; 0.5 NAA, 0.5 mg·L−1 NAA; 1.0 NAA, 1 mg·L−1 NAA; 2.0 NAA, 2.0 mg·L−1 NAA; 0.1 IBA, 0.1 mg·L−1 IBA; 0.5 IBA, 0.5 mg·L−1 IBA; 1.0 IBA, 1.0 mg·L−1 IBA; 2.0 IBA, 2.0 mg·L−1 IBA. Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”.3. 讨论与结论
本研究以甘蓝型油菜种子为材料,筛选出了油菜种子灭菌的最适NaClO浓度为2.0%或3.0%,且在3.0% NaClO基础上找到了最适的种子灭菌时间为15 min,其种子灭菌后的发芽率达到了96.9%。值得注意的是甘蓝型油菜种子经高浓度(30.0%)NaClO灭菌处理后依然有菌落生长,这可能是由于种子体内携带的内生菌污染[19]。在NaClO大范围浓度的初步筛选和小范围浓度筛选的2次使用5.0% NaClO灭菌种子的试验中,第一次使用5.0% NaClO处理后的种子发芽率显著的低于第二次使用该浓度后的种子发芽率,这可能是由于2次使用的种子的生产批次不同导致的,第一次试验使用的是前一年剩余的种子(市场上未销售当年最新种子),而第二次使用的是当年最新批次的种子。杜燕等也证实过期油菜种子的发芽能力会降低,种子的灭菌方法需要做进一步的改进和优化[20]。因此,在做植物组织培养再生实验时,应优先选择最新的植物种子作为原材料。
本研究对甘蓝型油菜生根培养基的植物生长调节剂种类及其浓度进行了筛选,结果表明0.1 mg·L−1 NAA最有利于油菜生根,培养7 d后即可达到92.5%的生根率,高于其他报道的甘蓝型油菜的生根率[21-22]。在生根培养基中使用适宜的NAA/IBA浓度可促进再生根在切点处生根,提高主根长和须根数,但不恰当的植物生长调节剂浓度则会影响油菜再生根的效果。本研究筛选出的甘蓝型油菜最适生根培养基的植物生长调节剂浓度与其他报道的采用0.1 mg·L−1 NAA用于油菜转基因抗性再生芽的生根培养植物生长调节剂浓度一致[9, 23],但该植物生长调节剂浓度是否也能有效促进其他植物品种再生根还有待于进一步验证。
本研究筛选出的甘蓝型油菜种子最适灭菌方法也适用于其他9种植物品种,尤其适合于甘蓝型油菜品种、大白菜品种和模式植物拟南芥,灭菌后的种子发芽率和健康芽率均 ≥ 95.0%,其种子发芽率均高于前人报道的相关植物品种的种子发芽率[24-29]。但值得注意的是,不同植物品种的种子经灭菌后的发芽率存在着显著性差异,这可能与不同物种间的差异有关[30],也可能与不同种子储存的外界环境条件有关[31]。此外,本研究筛选出的最适种子灭菌方法还可以与其他的种子灭菌方法联合使用,进一步提高种子灭菌的效果,例如与其他物理灭菌方法、照射灭菌方法和生物抑制灭菌方法等联合使用[32]。
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图 1 甘蓝型油菜种子灭菌的NaClO大范围浓度初步筛选
注:A, 发芽率; B, 健康芽率; C, 菌落率。同一图中的不同字母表示处理间具有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。图2~3同。
Figure 1. Preliminary screening of large-scale concentration of NaClO for seed sterilization in B. napus
Note: A, germination rate; B, healthy shoot rate; C, rate of colonies. Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”. The same as Fig.2~3.
图 4 种子灭菌方法对其他芸苔属作物种子的灭菌效果
注:A, 发芽率;B,健康芽率;C,菌落率;D,9种植物品种的种子;E,9种植物品种的种子灭菌7 d后的苗生长情况。JF,甘蓝B. oleracea ‘京丰一号’;HC,白菜型油菜B. compestris ‘红菜苔’;XZ,小白菜B. campestris ‘夏尊’;YK,甘蓝型油菜B. napus ‘亚科油68’;GY,甘蓝型油菜B. napus ‘高油605’;DF,甘蓝型油菜B. napus ‘德丰油’;ZS,大白菜B. pekinensis ‘F1早熟五号’;SC,莴苣Lactuca sativa ‘意大利全年生菜王’;AT,拟南芥Arabidopsis thaliana。同一小图中的不同字母表示处理间有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。
Figure 4. The effects of seed sterilization for other Brassica crops with the screened optimal method
Note: A, germination rate; B, healthy shoot rate; B, rate of colonies; D, seeds of 9 plant species; E, seedling growth status of 9 plant species after seed sterilization and sowing for 7 days. JF (B. oleracea ‘Jing Feng 1’), HC (B. compestris ‘Hong Cai Tai’), XZ (B. campestris ‘Xia Zun’), YK (B. napus ‘Ya Ke’), GY (B. napus ‘Gao You’), DF (B. napus ‘De Feng’), ZS (B. pekinensis ‘Zao Shou 5’), SC (Lactuca sativa) and AT (Arabidopsis thaliana). Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”.
图 5 不同质量浓度NAA/IBA对油菜再生根的累积生根率、生根部位、主根长和须根数的影响
注:A、累积生根率;B、生根部位(再生根位置与切点处之间的距离);C、主根长;D、须根数。CK、培养基内未添加植物植物生长调节剂; 0.1 NAA, 0.1 mg·L−1 NAA; 0.5 NAA, 0.5 mg·L−1 NAA; 1.0 NAA, 1.0 mg·L−1 NAA; 2.0 NAA, 2.0 mg·L−1 NAA; 0.1 IBA, 0.1 mg·L−1 IBA; 0.5 IBA, 0.5 mg·L−1 IBA; 1.0 IBA, 1.0 mg·L−1 IBA; 2.0 IBA, 2.0 mg·L−1 IBA。图中的不同字母表示处理间具有显著性差异(P ≤ 0.05),数值表示“平均值 ± 标准误”。
Figure 5. Effects of different concentrations of NAA / IBA on cumulative rooting rate, rooting position, taproot length and number of lateral roots of regenerative roots of B. napus
Note: A, cumulative rooting rate; B, rooting position (the distance between root regenerated position and cut point); C, number of lateral roots; D, taproot length. CK, media without plant hormone; 0.1 NAA, 0.1 mg·L−1 NAA; 0.5 NAA, 0.5 mg·L−1 NAA; 1.0 NAA, 1 mg·L−1 NAA; 2.0 NAA, 2.0 mg·L−1 NAA; 0.1 IBA, 0.1 mg·L−1 IBA; 0.5 IBA, 0.5 mg·L−1 IBA; 1.0 IBA, 1.0 mg·L−1 IBA; 2.0 IBA, 2.0 mg·L−1 IBA. Different lowercase letters represent significant difference between treatments (P ≤ 0.05), the value means “mean ± standard error”.
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