The projected maximum temperature was calculated by adding 5°C to the current maximum . A difference of 6 °C between day and night temperatures was chosen in accordance with the current day/night temperature difference and with previous studies of C. album and C. canadensis . CO2 treatment levels were ambient and elevated , which corresponds to future climate projections and within the range of CO2 levels projected by the year 2100. Environmentally controlled growth chambers , equipped with non-dispersive infrared CO2 analyzers and valves injecting pure CO2 to the incoming air stream, were set at either the near normal ambient CO2 level or at the elevated CO2 level . Chamber CO2 concentrations were logged at 30 second intervals and averaged for each 24h period, showing that CO2 levels averaged 490±40 ppm for the ambient treatment and 720±5 ppm for the elevated CO2 treatment. Seeds from each C. album and C. canadensis population sampled were germinated in fats filled with commercial potting media . Seedlings of C. album at the two- to four-leaf stage and C. canadensis at the three- to four-leaf stage were transplanted into 5 by 5cm plastic pots filled with the same potting media and grown in a growth chamber set at 25/15 °C temperatures and 11-h photoperiod, representative of the day length for February/March in California, and a light intensity of 600 µmol m−2 s−1 provided by fuorescent and incandescent bulbs. Seedlings were watered daily. Tree days after transplanting, 20–40 healthy seedlings from each population were moved to two growth chambers that differed in the following temperature and CO2 conditions: LT/ACO2 – low temperature combined with ambient CO2 , and HT/ECO2 – high temperature combined with elevated CO2 but with the same photoperiod and light intensity as described above. Seedlings of C. album were grown to a height of 6–8 cm, clone trays whereas seedlings of C. canadensis were grown to the 8–10 rosette leaf stage , then treated with glyphosate at the labeled field rate of 867 g ae ha−1 using an automated spray chamber equipped with a fat-fan 8001E nozzle .

The sprayer was calibrated to deliver 187 L ha−1 of glyphosate solution at a pressure of 296 kPa. For each treatment, five unsprayed individual plants were designated as untreated controls. One hour after glyphosate treatment, plants were returned to their respective growth chambers. Plant survival was assessed 21 days after treatment . The experiment was repeated 2–3 times. Treatment combinations and experimental runs were rotated between the two chambers. In addition, seedlings of two populations were assessed for plant response to glyphosate under two additional temperature and CO2 combinations: LT/ECO2 – low temperature combined with elevated CO2 , and HT/ACO2 – high temperature combined with ambient CO2 . Photoperiod, light intensity, glyphosate application and data collection were the same as described above. Due to a shortage of available growth chambers in which CO2 levels could be regulated, only one population of each species could be tested at these environmental conditions.For each population of C. album and C. canadensis, 16 plants grown under LT/ACO2 and 16 plants grown under HT/ECO2 conditions [eight plants sprayed with glyphosate and eight unsprayed plants ] were measured for leaf greenness and an estimate of chlorophyll content using a portable chlorophyll meter , following the method of Yannicccari et al.. Tree independent measurements were taken at the middle section of the youngest fully expanded leaf four days following treatment with glyphosate. Chlorophyll content was calculated as the average of the three measurements and expressed for glyphosate-treated plants as a percentage of the respective values obtained for untreated control plants.Glyphosate absorption and translocation under different temperature and CO2 conditions was assessed using a completely randomized experimental design with four replicates. Seeds from C. album population CA1 and C. canadensis population CCS were germinated and seedlings grown as described above. Seedlings of C. album at the two- to four-leaf stage and C. canadensis at the three- to four-leaf stage were transplanted into 40ml vials and grown hydroponically with a dilute nutrient solution, as described in Moretti and Hanson, in the growth chambers maintained at LT/ACO2 and HT/ECO2 conditions.

A solution containing glyphosate at a fnal concentration that approximated an 867 g ae ha−1 spray solution at 187 L ha−1 carrier volume was prepared by mixing 14C-glyphosate and a commercial formulation of glyphosate . When plants reached a height of 6–7 cm for C. album and the 6–8 rosette leaf stage for C. canadensis, a 2µL volume of the solution was applied to the middle adaxial surface of the youngest fully expanded leaf of each plant using a blunt-edged syringe in the form of two droplets, as described in Nandula and Vencill. Plants were harvested at 6, 12, 24, 48 and 72hours after treatment . At each harvest time, the treated leaf was removed and gently rinsed for 30 seconds in vials containing 10mL of distilled water to remove the unabsorbed 14C-glyphosate from the leaf surface. After removing the treated leaf, plants were dissected into shoots and roots. Phosphor image analysis was used to visualize herbicide translocation. 14C-glyphosate treated and dissected plant parts were pressed between two layers of paper and dried at 60 °C for 72h. After cooling to room temperature, each sample was placed in a 20×40 cm exposure cassette and brought into contact with a standard storage phosphor screen for 24h. Glyphosate translocation was visualized using the Storm 860 PhosphorImager system . Image analysis was conducted using the ImageQuant 5.0 sofware . Following phosphor image analysis, 14C-glyphosate translocation was quantified at three harvest time points, 12, 24 and 48 HAT, for both species. To measure the amount of non-absorbed glyphosate, rinsate was evaporated to dryness and resuspended in 10 ml of scintillation cocktail . Rinsate radioactivity was quantified using a liquid scintillation spectrometer device . The oven-dried plant samples used for phosphor image analysis were also used to assess the distribution of 14C-glyphosate. Treated leaves and roots were combusted with no further dissection whereas shoots were divided into several subsections as illustrated in Supplementary Fig. S1. For C. album, each shoot was divided into three parts: 1) shoot apical meristems including young undeveloped leaves, 2) leaves+petioles below the treated leaf, and 3) stem. For C. canadensis, each shoot was divided into two parts: 1) shoot meristems including young undeveloped leaves and 2) the remaining rosette leaves.

Different plant parts were placed separately into a combustion cone and dried at 60 °C for 96h. Each cone was combusted in a biological oxidizer . The evolved 14CO2 was trapped in 10ml of a carbon dioxide adsorbent solvent and mixed with 10ml of scintillation cocktail . Radioactivity was quantified using the LSS device described above.The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system has emerged as a powerful genome-engineering technology with success in diverse organisms. Cas9-mediated genome editing technology provides enormous advantages over other classical methods in crop improvement and plant research by generating desired modifications at a specific target sequence. In some cases, CRISPR/Cas9 permits the direct introduction of mutations conferring resistance in crop plants, without traditional back crosses or plant breeding. Cas9-DNA scissors makes site-specific double-strand cut in the genome, inducing modifications at targeted locus through homologous recombination or non-homologous end-joining repair mechanisms while Cas9 base editor have ability to alter a specific nucleotide into another. Te most frequently used CRISPR/Cas9 system, type II, has three components: Cas9 endonuclease, CRISPR RNA and trans activating crRNA . Cas9-mediated DNA cleavage is guided by a tracrRNA:crRNA duplex that is complementary to the target DNA. Recently the tracrRNA:crRNA complex is fused into a single chimeric RNA known as single guide RNA containing an 18 to 20-nucleotide sequence which determines the target DNA sequence. The NGG protospacer adjacent motif that is present at 3′-end of the target sequence was recognized by the CRISPR/Cas9 system. Use of CRISPR/Cas9 has been reported as a most effective tool for nucleotide sequence modification or editing in numerous crop species, including Arabidopsis, wheat, rice, sorghum, cotton, maize, soybean and tobacco. The genera of parasitic weeds, Orobanche and Phelipanche , the broomrapes, cannabis drying room consist of over 100 species and represent one of the most destructive and great challenge in agricultural production. These are obligate plant parasites that attack through the host roots of almost all economically important crops in the Solanaceae, Fabaceae, Asteraceae, Brassicaceae and Apiaceae plant families. The life cycle of P. aegyptiaca is divided into two stages, preparasitic and parasitic.

The germination of parasite seeds is triggered by a highly specialized detection system for strigolactones exuded by host roots. Te parasitic stage initiates with the parasite developing a special intrusive organ the haustorium- that connects directly to the vascular system of the host. Following successful attachment and invasion of the host root, the broomrape seedling grows into a structure known as tubercle and after 4–5 weeks of tubercle growth, a foral meristem is produced, which emerges above the ground to produce flower and seeds. SL is a plant hormone required for shoot branching and used as a signaling molecule for the rhizosphere microflora. SLs occur in all green lineages of the plant kingdom and its synthesis start with the all trans β-carotene, a carotenoid molecule which produce 9-cis-β-carotene by the activity of Dwarf 27 , after that Carotenoid Cleavage Dioxygenases 7 convert it into 9 cis β–apo 10′-carotenal and fnally Carotenoid Cleavage Dioxygenases 8 leads to the production of carlactone, then cytochrome P450 enzymes, More Axillary Growth 1 convert it into various SLs. Existence of homologs CCD7 and CCD8 have been reported in P. ramosa and P. aegyptiaca. In the rhizosphere, SL acts as a host-detection cue for symbiotic arbuscular mycorrhizal fungi and stimulates seed germination of parasitic plants. Different types of SLs, e.g., strigol, 5-deoxystrigol, sorgolactone, solanacol, didehydroorobanchol, orobanchol and others, are known as germination stimulants for root parasites. The altered SL production conferred resistance in the host by reducing the germination of parasite seeds. Host resistance to the Orobanchaceae root parasite Striga has been observed in crops with altered SL production. In addition, previous studies found that the tomato SL-ORT1 mutagenized by fast-neutron display highly resistant to Phelipanche and Orobanche spp, the resistance results from its inability to produce and secrete SLs regarded as natural germination stimulants to the rhizosphere. Diferent methods of parasitic weed-control have been applied in attempts to control broomrape, but difficulties are encountered in targeting specific plant–plant systems. Moreover, most control strategies are less effective and have considerable limitations. Here we report the development of tomato plants that are resistant to the parasitic weed P. aegyptiaca upon mutation of the SL-biosynthesis gene CCD8 using CRISPR/Cas9.To investigate the efficacy of using CRISPR/Cas9 to create host resistance in tomato plants against parasitic weeds, we chose to disrupt the SL-biosynthesis gene CCD8 in tomato. SLs are synthesized from plant carotenoids via a pathway involving CCD7 and CCD8. Te CCD8sgRNA construct was designed using the single sgRNA cassette in the pENTR vector, which was then recombined into the pDest vector to target the second exon of CCD8 with a BsrI restriction site located upstream to the protospacer adjacent motif . Fourteen independent T0 transgenic tomato lines were generated by Agrobacterium-mediated transformation at the Plant Transformation Facility of the University of California, Davis, USA. However, 10 out of 14 of the CCD8Cas9 transgenic plants showed mutation at SlCCD8 locus, as determined by PCR and restriction analysis . Mutants showed similar kind of mutation at genome level considered as one single line in our study. We chose to continue our study with only those lines that showed different kind of genome editing in the T0 generation such as line 1, 2, 5 and 11 and the presence of transgene Npt-II was confirmed by PCR .To detect mutations induced by the Cas9 nuclease in T0 plants, we assessed loss of the BsrI restriction enzyme site that might arise due to imprecise non-homologous end-joining repair. For each of the T0 plants, we PCR-amplifed the CCD8Cas9 target region using genomic DNA from the T0 lines and then digested it with BsrI and examined products on a 2% agarose gel. Digestion of the wild type PCR fragment with BsrI results in three products of 147 bp, 298 bp and 7 bp, while Cas9 editing of the target site will generally eliminate the BsrI site that is adjacent to the PAM, leading to a 445 bp fragment.