Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Analysis of Morphological Traits, Cannabinoid Profiles, THCAS Gene Sequences, and Photosynthesis in Wide and Narrow Leaflet High-Cannabidiol Breeding Populations of Medical Cannabis.

Cannabis sativa L. is one of the oldest cultivated crops, used in medicine for millennia due to therapeutic characteristics of the phytocannabinoids it contains. Its medicinal properties are highly influenced by the chemotype, that is, the ratio of the two main cannabinoids cannabidiol (CBD) and Δ-9-tetrahydrocannabinol (THC). Based on published data, the chemotype should correlate with plant morphology, genetics, and photosynthetic properties. In this work, we investigated leaf morphology, plant growth characteristics, cannabinoid profiles, THCAS gene sequences, and plant photosynthetic traits in two breeding populations of medical cannabis (MX-CBD-11 and MX-CBD-707). The populations differed significantly in morphological traits. The MX-CBD-11 plants were taller, less branched, and their leaves had narrower leaflets than the bushier, wideleaved MX-CBD-707 plants, and there were significant differences between populations in the dry biomass of different plant parts. Based on these morphological differences, MX-CBD-11 was designated as a narrow leaflet drug type or vernacular "Sativa" type, while MX-CBD-707 was classified as wide leaflet drug type or "Indica" type. Chemical characterisation revealed a discrepancy between the expected chemotypes based on plant morphology; although both populations have high CBD, within each Type II (CBD/THC intermediate) and Type III (CBD dominant) plants were detected. The THCAS gene sequence analysis clustered the plants based on their chemotypes and showed high similarity to the THCAS sequences deposited in NCBI. In silico complementary analysis, using published molecular markers for chemotype determination, showed their low discrimination power in our two populations, demonstrating the genotype dependence of the molecular markers. Basic photosynthetic traits derived from light and CO2 response curves were similar in the populations. However, measurements of gas exchange under chamber conditions revealed higher stomatal conductivity and photosynthesis in MX-CBD-707 plants, which were also characterised by higher day respiration. The results of this study showed that based on visual appearance and some morphological measurements, it is not possible to determine a plant's chemotype. Visually homogenous plants had different cannabinoid profiles and, vice versa, morphologically distinct plants contained similar CBD and THC content. The two chemotypes identified in our experimental plants therefore did not correlate with plant visual appearance, leaf morphometry, and photosynthetic properties of the populations studied. Correlation was only demonstrated with the respect to THCAS sequences, which showed great discrimination power between the chemotypes.

Production of Feminized Seeds of High CBD Cannabis sativa L. by Manipulation of Sex Expression and Its Application to Breeding.

The use of the cannabis plant as a source of therapeutic compounds is gaining great importance since restrictions on its growth and use are gradually reduced throughout the world. Intensification of medical (drug type) cannabis production stimulated breeding activities aimed at developing new, improved cultivars with precisely defined, and stable cannabinoid profiles. The effects of several exogenous substances, known to be involved in sex expressions, such as silver thiosulfate (STS), gibberellic acid (GA), and colloidal silver, were analyzed in this study. Various concentrations were tested within 23 different treatments on two high cannabidiol (CBD) breeding populations. Our results showed that spraying whole plants with STS once is more efficient than the application of STS on shoot tips while spraying plants with 0.01% GA and intensive cutting is ineffective in stimulating the production of male flowers. Additionally, spraying whole plants with colloidal silver was also shown to be effective in the induction of male flowers on female plants, since it produced up to 379 male flowers per plant. The viability and fertility of the induced male flowers were confirmed by fluorescein diacetate (FDA) staining of pollen grains, in vitro and in vivo germination tests of pollen, counting the number of seeds developed after hybridization, and evaluating germination rates of developed seeds. Finally, one established protocol was implemented for crossing selected female plants. The cannabinoid profile of the progeny was compared with the profile of the parental population and an improvement in the biochemical profile of the breeding population was confirmed. The progeny had a higher and more uniform total CBD (tCBD) to total tetrahydrocannabinol (tTHC) ratio (up to 29.6; average 21.33 ± 0.39) compared with the original population (up to 18.8; average 7.83 ± 1.03). This is the first comprehensive report on the induction of fertile male flowers on female plants from dioecious medical cannabis (Cannabis sativa L.).

Proposal of a New Hybrid Breeding Method Based on Genotyping, Inter-Pollination, Phenotyping and Paternity Testing of Selected Elite F1 Hybrids.

Testing inbred lines for their combining ability is, due to high numbers of line to line testing needed for determination of hybrid performance, the most limiting factor in the F1 hybrid breeding procedure. We propose a novel method of F1 hybrid breeding that enables evaluation of large number of line to line crosses for their hybrid performance. Inbred lines (preferably doubled haploid - DH) are produced from heterozygous populations, genotyped and maintained. A group of lines is inter-pollinated randomly and their progeny examined. To identify elite F1 hybrids, these individual plants are selected by their superior phenotypic characteristics. Finally using paternity testing only of selected hybrids, the origin of paternal lines is revealed. To predict the number of F1 offspring needed in relation to the number of inbred lines being inter-pollinated, a mathematical formula was developed. For instance, using this formula for the inter-pollination of 60 distinct lines, the probability of obtaining all descendants of paternal-parent lines in a maternal-parent row represented at least once is achieved with 420 F1 plants in a row (p = 0.95). In a practical experiment with white cabbage, DH lines were produced using microspore culture; plants were grown to maturity and genotyped at eight polymorphic SSR loci. Two groups of lines (36 and 33 lines per group) were inter-pollinated by two methods, either using cage pollination with bumblebees or using open pollination in isolated field. A total of 9,858 F1 plants were planted and based on their phenotypic characteristics 213 were selected as elite phenotypes. 99 of them were genetically diverse and 5 of them were selected as super elite. Selected plants were analysed by the same SSR markers and the paternal origin of selected F1 plants was determined. Out of 213 selected elite plants 48 were reciprocals thus exhibiting power of selection based on single plant. We demonstrate that this new approach to hybrid development is efficient in white cabbage and we propose breeders to test it in various vegetable and crop species. Moreover, some other aspects of the proposed technique need to be tested and verified both for practical and economic criteria.

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes.

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 µg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

New variants of CRISPR RNA-guided genome editing enzymes.

CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high-throughput tools for genetic modification by the specific targeting of double-strand breaks in the genome of hosts. Plant biotechnologists have extensively used the S. pyogenes Cas9-based system since its inception in 2013. However, there are still some limitations to its even broader usage in plants. Major restrictions, especially in agricultural biotechnology, are the currently unclear regulatory status of plants modified with CRISPR/Cas9 and the lack of suitable delivery methods for some plant species. Solutions to these limitations could come in the form of new variants of genome editing enzymes that have recently been discovered and have already proved comparable to or even better in performance than S. pyogenes CRISPR/Cas9 in terms of precision and ease of delivery in mammal cells. Although some of them have already been tested in plants, most of them are less well known in the plant science community. In this review, we describe the following new enzyme systems engineered for genome editing, transcriptional regulation and cellular imaging-C2c2 from L. shahii; Cas9 from F. novicida, S. aureus, S. thermophiles, N. meningitidis; Cpf1 from F. novicida, Acidaminococcus and Lachnospiraceae; nickase, split, enhanced and other Cas9 variants from S. pyogenes; catalytically inactive SpCas9 linked to various nuclease or gene-regulating domains-with an emphasis on their advantages in comparison with the broadly used SpCas9. In addition, we discuss new possibilities they offer in plant biotechnology.

DNA labelling of varieties covered by patent protection: a new solution for managing intellectual property rights in the seed industry.

Plant breeders' rights are undergoing dramatic changes due to changes in patent rights in terms of plant variety rights protection. Although differences in the interpretation of ¼breeder's exemption«, termed research exemption in the 1991 UPOV, did exist in the past in some countries, allowing breeders to use protected varieties as parents in the creation of new varieties of plants, current developments brought about by patenting conventionally bred varieties with the European Patent Office (such as EP2140023B1) have opened new challenges. Legal restrictions on germplasm availability are therefore imposed on breeders while, at the same time, no practical information on how to distinguish protected from non-protected varieties is given. We propose here a novel approach that would solve this problem by the insertion of short DNA stretches (labels) into protected plant varieties by genetic transformation. This information will then be available to breeders by a simple and standardized procedure. We propose that such a procedure should consist of using a pair of universal primers that will generate a sequence in a PCR reaction, which can be read and translated into ordinary text by a computer application. To demonstrate the feasibility of such approach, we conducted a case study. Using the Agrobacterium tumefaciens transformation protocol, we inserted a stretch of DNA code into Nicotiana benthamiana. We also developed an on-line application that enables coding of any text message into DNA nucleotide code and, on sequencing, decoding it back into text. In the presented case study, a short command line coding the phrase ¼Hello world« was transformed into a DNA sequence that was inserted in the plant genome. The encoded message was reconstructed from the resulting T1 seedlings with 100 % accuracy. The feasibility and possible other applications of this approach are discussed.