Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca²⁺-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T₀ regenerants and sprdr6 null T₁ progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

The bamboo Bambusa edulis has a long juvenile phase in situ, but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by use of two sequencing platforms: Illumina and 454. Combination of the two datasets resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes control flower development, and the ABCDE model has been developed to explain how the genes function together to create the different whorls within a flower.

Background: Chronological age is the primary consideration when studying the physiological development, aging, and flowering of bamboo. However, it’s difficult to determine bamboo’s chronological age if the time of germination is unknown. To investigate the chronological age of bamboo from the genomic DNA methylation profile, methylation-sensitive amplification polymorphism (MSAP) was employed to analyze the genomic DNA methylation of Moso bamboo (Phyllostachys heterocycla var. pubescens) from stands of nine germination-ages, using six primer pairs which have previously been shown to yield methylation rates that reflect the age of Moso bamboo. Results: The results showed that the total genomic DNA methylation rates in Moso bamboo at different chronological ages were significantly different, and the increase in genomic DNA methylation rate was consistent with the increase of chronological age. Six primer pairs displayed different genomic DNA methylation rates in Moso bamboo of nine age’s group; however, a significantly positive correlation existed among these primer pairs. An integrated index was obtained by performing principal component analysis on the six primer pairs to represent the genomic DNA methylation levels in Moso bamboo of various chronological ages, and a quadratic curve between the chronological age and genomic DNA methylation levels was obtained. Conclusions: Such a relationship between DNA methylation and its chronological age may serve a reference for its aging study in Moso bamboo.

Chimonobambusa farcta, a new species of temperate woody bamboos from western Guangxi, China is described and illustrated. The new species is similar to C. pubescens in the solid internodes of culms, but differs in having taller culm to 7 m with longer verrucose internodes to 23.5 cm and intranodes to 4 mm, intranode usually with a ring of 7–9 root thorns below mid-culm, abaxially brown or brown-purple verrucose-setose culm leaf sheaths with indistinct transverse veins, conspicuously developed culm leaf blades to 3.2 cm long, longer foliage leaf sheaths to 5.2 cm, larger and broader foliage leaf blades to 22 × 1.4 cm. It also somewhat resembles C. convoluta, but can be easily distinguished by having solid internodes and longer intranode 2–4 mm, very prominent nodes with supranodal ridge obviously more elevated than sheath scar, usually persistent and sometimes brownish striate culm leaf sheaths, longer culm leaf blades to 3.2 cm, and abaxially glabrous foliage leaf sheaths. Based on the morphological characteristics, this new species is assigned to C. sect. Chimonobambusa. The character description of C. pubescens are revised for its culm to 2.1 m tall, 1.1 cm in diameter and glabrous foliage leaf blades. The systematic position of C. luzhiensis is discussed, and this species is proposed as a member of C. sect. Chimonobambusa.

Chimonobambusa pleiacantha, a newly-identified species of the genus Chimonobambusa Makino from southeast Sichuan, China, is meticulously described and illustrated herein. It is morphologically similar to Ch. tuberculata, but differs in having 4-angled internodes, thicker wall to 4.5–8.5 mm, more reclinate and elongated root thorns to 5–8 mm long, culm internodes with three grooves and two longitudinal ridges on the branch-bearing side, persistent culm leaves, densely brown-purple setose at the bottom of culm leaf sheaths together with sheath scar, developed foliage leaf fimbriae, 6–8 on each shoulder, ca. 3–8 mm long, abaxially white pubescent foliage leaf blades. Phenologically, new shoots usually appear in September to October. In the light of these key morphological and phenological characteristics, Ch. pleiacantha was identified as a new species of the genus Ch. Makino which is different from published species of this genus.

Wild tomatoes are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free protoplast regeneration and CRISPR-Cas9 genome editing system for Solanum peruvianum , an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 ( SpRDR6 ) and SUPPRESSOR OF GENE SILENCING 3 ( SpSGS3 ); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 ( SpPR-1 ) and PROSYSTEMIN ( SpProsys ); and fungal resistance ( MILDEW RESISTANT LOCUS O, SpMlo1 ) using diploid or tetraploid protoplasts derived from in vitro -grown shoots. The ploidy level of these regenerants was not affected by PEG-calcium-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T ₀ regenerants and sprdr6 null T ₁ progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding. One-sentence summary: DNA-free CRISPR-Cas9 genome editing in wild tomatoes creates stable and inheritable diploid and tetraploid regenerants.