Noble Research Institute, LLC, Ardmore, OK, 73401, USA.
BMC Plant Biol. 2021 Mar 4;21(1):128. doi: 10.1186/s12870-021-02903-z.
Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS).
Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments.
In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.
柳枝稷（Panicum virgatum L.）是一种重要的生物能源和饲料作物。柳枝稷的异花授粉特性使其无法通过有性繁殖来维持一个基因型。目前柳枝稷的无性繁殖协议存在各种局限性。需要一种简单而高效的营养繁殖方法来繁殖大量的柳枝稷基因型，以进行全基因组关联研究（GWAS）。
通过节点培养进行微繁殖被发现是柳枝稷营养繁殖的快速方法。在节点培养过程中，细菌和真菌的污染是培养失败的主要原因。在表面消毒后和培养基中添加杀菌剂混合物（PPM，0.2%）和苯菌灵（5mg/l），可以显著降低细菌和真菌的污染。此外，在继代培养前“修剪芽”对大多数测试基因型的芽增殖有积极影响。使用优化的节点培养程序，我们在三个单独的实验中成功地繁殖了 330 个柳枝稷 GWAS 面板的基因型。在基因型之间观察到芽诱导效率和芽生长的巨大差异。另外，我们通过刺激气生腋芽直接在母株上生长成芽，开发了一种体内节点培养方法，通过这种方法，在 6 周内可以生成生根植物。通过绕过组织培养步骤和避免应用外部激素，体内节点培养方法具有劳动和成本效益高、易于掌握、成功率高的特点。通过体内节点培养方法生成的植物与幼苗相似，可以直接用于各种实验。
在这项研究中，我们通过最小化细菌和真菌污染并增加芽增殖来优化柳枝稷节点培养方案。使用改进的方案，我们成功地繁殖了多样化的柳枝稷 GWAS 面板中的四分之三的基因型。此外，我们建立了一种新颖的高通量体内节点培养方法。这些方法共同为研究人员提供了更好的选择，以加速柳枝稷的营养繁殖。
