As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

An efficient and reproducible in vitro method for indirect somatic embryogenesis was optimized by culturing leaf and leaf with petiole explants of Lycium barbarum L. Murashige and Skoog (MS) medium, supplemented with various concentrations of Picloram and 2,4-Dichlorophenoxyacetic acid (2,4-D), individually and in combinations, were tested. Picloram (1.0 µM) showed a better response compared to 2,4-D and results indicate it to be a better auxin for induction of somatic embryos for Goji berry. It was seen that the leaf explants were more responsive in callus and somatic embryo induction than the leaf with petiole explant when incubated in the dark for 5 weeks. Embryogenic callus, after being transferred to MS medium containing Benzyl amino purine (BAP) in 1.0 µM, 5.0 µM and 10.0 µM, began to differentiate in light after one week. MS medium with 1.0 µM Picloram + 10 µM BAP resulted as the most favorable treatment for somatic embryogenesis in Lycium barbarum L. Removal of plant growth regulators from MS medium and culturing induced calluses under 16 h photoperiod resulted in globular, heart, torpedo, cotyledons, and further development into plantlets. Well-developed plants have been obtained and are capable of acclimatizing in ex vitro conditions. In addition, the effects of desiccation treatments (0, 1, 3, 6, 9 h, and 12 h) on embryogenic callus for somatic embryo induction were found to be directly proportional to the length of desiccation treatment at room temperature. After 9 h and 12 h of desiccation treatments, 60% and 90% of plated calluses resulted in somatic embryos, respectively. In a L. barbarum callus mass, Acetocarmine and Evans blue double staining differentiated between embryogenic and non-embryogenic callus. These findings will help Goji berry improvement by elite clone production, ex situ conservation projects, scaling up plant production, and agronomy for the commercial production of this superfruit in the future.