ABSTRACT

In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO₄, AgNO₃ and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO₄, AgNO₃ and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO₄ or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO₄, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO₄. In another study, the basal induction and regeneration medium were fortified with AgNO₃ or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO₃ or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO₃ or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.

Abbreviations: 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), IAA (Indole-3-acetic acid), AgNPs (silver nanoparticles), CuNPs (copper nanoparticles)

摘要

小麦对再生的体外抗逆性是通过基于愈伤组织的遗传转化对其进行改良的主要瓶颈。纳米技术是最具活力的研究领域之一，它可以改变农业和生物技术，在可持续的基础上确保粮食安全。本研究旨在研究 CuSO ₄ , AgNO _3的影响及其纳米颗粒对小麦基因型成熟胚培养的组织培养反应（AS-2002 和 Wafaq-2001）。最初，基于 MS 的愈伤组织诱导和再生培养基使用不同浓度的生长素（2,4-D，IAA）和细胞分裂素（BAP，激动素）针对两种基因型进行了优化。胚胎发生愈伤组织诱导和再生潜力的基因型不同。基因型 AS-2002 对 3.0 mg/l 2,4-D 的反应产生了最大的胚性愈伤组织，而 Wafaq-2001 对在诱导培养基中补充的 3.5 mg/l 2,4-D 提供了最高的胚性愈伤组织。基因型 AS-2002 在响应包含 0.5 mg/l IAA、0.3 mg/l BAP 和 1.0 mg/l Kin 的再生方案时表现出最大再生 (59.33%)，而 Wafaq-2001 在响应 0.5 mg/l IAA 时表现最佳， 0.3 mg/l BAP 和 1.5 mg/l Kin，再生效率为 55.33%。₄、AgNO ₃及其纳米粒子采用独立实验。用不同浓度的 CuSO ₄或 CuNPs 强化的优化诱导培养基证实了对胚性愈伤组织频率的显着影响。添加 0.020 mg/l 或 0.025 mg/l CuSO ₄或 0.015 mg/l CNP 显示出可比较的胚胎发生愈伤组织诱导结果，并且在统计学上分别与 74.00%、75.67% 和 76.83% 的胚胎发生愈伤组织诱导相当。_{与标准的 0.025 mg/l CuSO 4}相比，添加 0.015 mg/l 或 0.020 mg/l CuNPs 的基于 MS 的再生培养基实现了显着更高的再生。在另一项研究中，基础诱导和再生培养基中添加了 AgNO ₃或范围为 1 至 7 mg/l 的 AgNPs 以及不含 AgNO ₃或 AgNPs 的基础再生培养基（对照）。与对照和其余处理相比，从添加 3.0 mg/l 或 4.0 mg/l AgNPs 的培养基中观察到最大的胚性愈伤组织。用 5.0 mg/l AgNO _{3强化的标准化再生培养基}或 3.0 mg/l AgNPs 对小麦基因型的再生有显着影响，并且与对照相比提供了最大的再生。还测试了 Cu 和 Ag 纳米颗粒以及对照（每种基因型的基础再生介质）的单独和组合效应。令人惊讶的是，金属纳米粒子的共同应用显示基因型的胚胎发生愈伤组织形成显着增加。与对照和其他铜和银纳米颗粒组合相比，添加 0.015 mg/l CuNPs + 4.0 mg/l AgNPs 或 0.020 mg/l CuNPs + 2.0 mg/l AgNPs 的诱导培养基显示出出色的结果。最大再生是通过共同应用 0.015 mg/l CuNP 和 4.0 mg/l AgNPs 实现的，再生增量比对照增加 21%。

缩写：2,4-D（2,4-二氯苯氧乙酸）、BAP（6-苄基氨基嘌呤）、IAA（吲哚-3-乙酸）、AgNPs（银纳米粒子）、CuNPs（铜纳米粒子）