Callus Lines Research Articles

Callus induction and chemical characterization of cell suspension cultures of jojoba (Simmondsia chinensis L.)

Jojoba (Simmondsia chinensis L.) oil is also known as liquid wax or fixed oil. It is an important metabolite of jojoba having commercial importance in cosmetics as well as a potential biofuel source. We presented an efficient system for in vitro establishment of cell suspension cultures (CSC) from proliferating friable calluses. For this purpose, cotyledon, internode, and leaf explants were cultured on MS medium + 1, 2, 4, 6, 8 or 10 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic acid (NAA) alone or in combination with 1 or 2 µM N6-benzylaminopurine (BAP) or Kinetin. Results demonstrated that 100% healthy, friable and variegated calluses were obtained on 8 µM, 10 µM 2, 4-D or 2, 4-D 10 µM + 2 µM BAP and represented as callus lines (CL) CL-1, CL-2 or CL-3, respectively, after 38 days. One-gram callus tissue per CL was then immersed in the respective liquid medium and agitated on an orbital shaker at 60-70 rpm under the growth room conditions (25 ± 2 °C, 16 h light period) for the preparation of CSC. After 15 days, CSC was sieved and large clumps were removed. Growth measurement of CSC was determined by cell counting, packed cell volume (PCV) and cell viability. The highest number of viable cells was obtained at 2.57 OD with CL-3, where PCV was highest (0.35 ml) on CL-1 of 38 days old calluses. 2,3,5-Triphenyltetrazolium chloride was a reliable approach for the determination of cell viability of CSC.

High-efficiency plastome base editing in rice with TAL cytosine deaminase

High-efficiency plastome base editing in rice with TAL cytosine deaminase

Sex-Linked Molecular Markers Identify Female Lines in Endosperm-Derived Kiwifruit Callus and in Regenerants

This is the first report of molecular markers application for the analysis of endosperm-derived callus and nonaploid kiwifruit (Actinidia chinensis var. deliciosa, formerly: Actinidia deliciosa) plants. As a source of explants, fruits of ‘Hayward’, the most popular cultivar, were used. Additionally, analyses of the nuclear DNA content and sex were conducted on the regenerated plants. Hexaploid seedlings were used as control for the flow cytometric analyses. Most of the plants (about 90%) regenerated via endosperm-derived callus possessed 2C = 9Cx DNA, which confirmed their endosperm origin and nonaploidy. Because Actinidia is a dioecious species, and female plants bearing fruits are desired by breeders, it is crucial to identify the sex of an individual at early stages of development. Analyses were conducted with ex vitro and in vitro samples. Results revealed that specific markers for a Y-chromosome applied at the callus stage allowed us to reliably predict the sex of plants regenerated from it. This is a novel application of sex-linked markers for early selection of female and male callus lines when the sex of the initial explants is still unknown, such as fresh isolated embryos and endosperm. It may have significant importance for breeding kiwifruit programs, which involve tissue culture techniques.

A piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice.

SummaryTargeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double‐stranded transfer DNA (dsT‐DNA), with subsequent excision of the transgenes by the re‐transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T‐DNA works to deliver transgenes in rice. Following positive–negative selection to exclude transgenic cells randomly transformed with T‐DNA, piggyBac‐mediated transgenesis from the extrachromosomal dsT‐DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof‐of‐concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9‐induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac‐mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9‐mediated targeted mutagenesis in vegetatively propagated crops.

Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation

Miraculin is a taste-modifying protein that interacts with human sweet-taste receptors and transforms a sour taste into sweet taste. Miraculin is extracted from the miracle fruit (Synsepalum dulcificum). Since mass production of miraculin is difficult because of regional and seasonal limitations, several attempts have been made to express miraculin in various cell systems. In this study, a binary vector containing the miraculin gene under the control of the SWPA2 promotor was introduced into carrot (Daucus carota) callus via Agrobacterium-mediated transformation to synthesize miraculin in carrot cell cultures. After 4 weeks of co-cultivation with Agrobacterium tumefaciens, 20 tentative transgenic callus (TC) lines were obtained on kanamycin selection medium. PCR analysis confirmed that 18 of these 20 lines (TC1–TC18) carried the miraculin gene, and 4 TC lines with high cell growth and gene expression (determined by RT-qPCR) were selected for further analysis. Protein analysis of these four TC lines by SDS-PAGE and Western blot showed that the miraculin protein was stably produced in TC lines. The cell growth showed no correlation with gene expression levels. The DNA content and G1 phase ratio were negatively correlated, whereas the S and G2/M phase ratios were positively correlated with gene expression. The ratio of cell cycle was determined by counting the number of cells in each step through flow cytometric analysis. These results indicate that gene expression was higher in TC lines with active cell division. Overall, our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems. This study expressed the miraculin gene in carrot callus via Agrobacterium-mediated transformation based on miraculin expression and cell cycle analyses. Our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems

LC-MS-Based Profiling Provides New Insights into Apocarotenoid Biosynthesis and Modifications in Citrus Fruits.

Apocarotenoids contribute to fruit color and aroma, which are critical quality and marketability attributes. Previously, we reported that the red peels of citrus fruits, which are characterized by higher expression levels of a carotenoid cleavage dioxygenase 4b (CitCCD4b) gene, accumulate higher levels of β-citraurin and β-citraurinene than yellow peels. Here, we identified and quantified 12 apocarotenoids, either volatile or nonvolatile, in citrus peel using liquid chromatography-mass spectrometry (LC-MS). Our results show that red peels contain also dramatically higher amounts of β-apo-8'-carotenal, crocetin dialdehyde known from saffron, β-citraurol, β-cyclocitral, and 3-OH-β-cyclocitral and up to about 17-fold higher levels of 3-OH-β-cyclocitral glucoside (picrocrocin isomer). The content of these apocarotenoids was also significantly increased in different CitCCD4b-overexpressing transgenic callus lines, compared with corresponding controls. Transient expression of CitCCD4b in Nicotiana benthamiana leaves resulted in a striking increase in the 3-OH-β-cyclocitral level and the accumulation of picrocrocin. Thus, our work reinforces the specific function of CitCCD4b in producing C10 apocarotenoid volatiles and C30 pigments in citrus peel and uncovers its involvement in the biosynthesis of picrocrocin, C20 dialdehyde, and C30 alcohol apocarotenoids, suggesting the potential of this enzyme in metabolic engineering of apocarotenoids and their derivatives.

The relationship between morphology changes and antioxidant enzymes activity during somatic embryogenesis of long-term-maintained callus of Zoysia matrella (L.) Merr.

A callus line of manila grass [Zoysia matrella (L.) Merr.] has been maintained for 8 years in our laboratory. The present study investigated changes in ultrastructure and antioxidant enzyme activity during regeneration of the callus and examined the correlation between these changes and regeneration ability. The changes in fresh weight and diameter of the callus over time could be described by a sigmoidal growth curve with different stages. Electron microscopy revealed small embryonic callus cells, isodiametric in shape, with large, obvious nuclei, and dense cytoplasm. The cellular structures and morphology changed dramatically as regeneration proceeded. Of particular note was the formation of folded scutellum-like embryos at 14 d, which might be the turning point for morphological differentiation. Catalase (CAT) and peroxidase (POD) activities were the lowest at 14 d, the same time when superoxide dismutase (SOD) activity was highest. Thus, we speculate that the formation of the scutellum-like structures is associated with higher activity of SOD and lower activities of CAT and POD.

Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca – in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.

Metabolic Engineering of Isoflavonoid Biosynthesis by Expressing Glycine max Isoflavone Synthase in Allium cepa L. for Genistein Production.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for the production of various secondary metabolites. In our study, we attempted to produce the isoflavone genistein, a well-known health-promoting metabolite, in Allium cepa L. (onion) by introducing Glycine max Isoflavone synthase (GmIFS). The GmIFS gene was cloned into the pEarleyGate 102 HA vector and transformed into onion by Agrobacterium-mediated and biolistic methods. The presence of GmIFS in transgenic onion was confirmed by PCR, dot blot, and Southern hybridization. Analysis of the transgenic onion calli lines demonstrated that the expression of the GmIFS gene led to the production of isoflavone genistein in in vitro tissues. The biolistic stable transformed calli with transformation efficiency of 73% (62.65 nM/g FW) accumulated more genistein than the Agrobacterium stable transformed calli with transformation efficiency of 56% (42.5 nM/g FW). Overall, heterologous gene expression of GmIFS was demonstrated by modifying the secondary metabolite pathway in onion tissues for the production of isoflavone genistein that can boost up human health with its health-promoting properties.

Correlating mRUST scoring with fracture healing in the mouse model

Background/Objective: Long bone fractures are an expensive and frequent cause of disability in humans. Research seeking to accelerate and improve the healing process is more essential than ever. Animal models, mice especially, provide an inexpensive and reproducible model of in vivo fracture healing. However, many measures of murine fracture healing outcomes are either expensive or destructive, limiting their ability to be translated to clinical studies. We seek to determine how these measures such as biomechanics, µCT, and histology correlate to the relatively new, inexpensive, and non-destructive method of mRUST scoring in a mouse model. 
 Methods: One hundred and thirty-five, 12-week old male C57BL6/J mice were divided into nine groups of 15 mice. Mice underwent a surgically created, femoral fracture. At biweekly timepoints, anteroposterior and lateral radiographs were taken, and 15 mice were sacrificed at each time point (7, 10, 14, 17, 21, 24, 28, 35, and 42 days post-surgery) for biomechanical, µCT, and histological analyses. The modified Radiographic Union Scale for Tibial fractures (mRUST scoring) provides a score based on the visualization of a callus and fracture line in four cortices on the radiographs. Data analysis will be performed to determine the degree of correlation between mRUST scoring and other fracture healing outcomes. 
 Results/Conclusion: Data collection in this experiment is still forthcoming. Upon successful completion of this project, we will have established numerical correlations between mRUST scoring and other fracture healing outcomes, such as biomechanics, µCT microarchitecture, and histology. These correlations will provide a powerful tool in future mouse fracture healing studies, as data on the state and strength of fracture repair could be determined by simple radiograph. 
 Scientific/Clinical Policy Impact and Implications: This study will both provide future murine fracture studies with an inexpensive and non-destructive method of assessment that is more directly translatable to human fracture studies.

Efficient protoplast isolation from ovule-derived embryogenic callus inCitrusvolkameriana

The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana (Citrus volkameriana L.), which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis (WAA),were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium (MT basal medium + 0.5 gL–1 malt extract), with the addition of phytohormones [kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP)] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media (P ≤ 0.01) on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D (1.0 mg L–1) + BAP (0.5 mg L–1). Callus induction frequency was the highest (90.00%) in the same culture medium when the ovules wereculturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis (24%). Embryogenic callus initiation was the highest (25.56%) using EME + KIN (1.0 mg L–1) and ovules cultured at 8 WAA (14%) resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 × 105protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C. volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.

Micropropagation of Tulip via Somatic Embryogenesis

An effective method of tulip regeneration via somatic embryogenesis (SE) was developed. Explants, flower stem slices excised from cooled bulbs were incubated in darkness on MS modified media containing auxins alone (2,4-dichlorophenoxyacetic acid—2,4-D, 1-naphthalene acetic acid—NAA and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid—picloram) or combined with thidiazuron (TDZ) at 0.1 and 0.5 mg L−1. Yellowish-white callus with a granular structure was developed in the presence of all auxins on the cut surface from the tissues of the vascular bundles. From this, lines of embryogenic calli were derived. The addition of TDZ to the medium with auxins significantly stimulated somatic embryo formation. Cyclic and the most intensive proliferation of embryogenic callus as well as embryo formation was obtained in the presence of 2,4-D at 0.1 mg L−1 combined with TDZ at 0.5 mg L−1. Addition of proline enhanced either callus proliferation rate or frequency of embryo formation. The best quality embryos with cotyledons longer than 10 mm able to form bulbs were recorded when TDZ was replaced with 6-benzylaminopurine (BAP) at the concentration of 0.1 mg L−1. Histomorphology showed that the development of somatic embryos could have either external or internal origins. Embryos of external origin were initiated by cell division on the edge of embryogenic calli. Embryos of internal origin resulted from the division of parenchyma cells inside the tissue. Embryonic cells were characterized by their small volume, regular shape, dense cytoplasm and large nuclei. The globular embryos were covered by a distinct layer of periderm. Then, the embryos developed into structures having leaf-shaped cotyledons with a procambial strand and a sideward-orientated meristem of the vegetative apex (stolon). Cotyledon embryos did not show vascular connections with the parent tissue, and they did not develop embryonic roots.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

Establishment of Cajanus cajan (Linn.) Millsp. cell suspension cultures as an effective in vitro platform for the production of pharmacologically active phenolic compounds

Phenolic compounds are validated to be the pharmacologically active phytochemicals in Cajanus cajan (Linn.) Millsp. leaves (CCLs) and the medicinal product (Tongluo Shenggu capsules, TSCs) thereof. For the first time, C. cajan cell suspension cultures (CCSCs) were established as an effective in vitro platform for the production of phenolic compounds. Callus was induced from C. cajan leaves by 2.0 mg/L 2,4-D, 5.0 mg/L α-NAA, and 0.1 mg/L Kn, and then subcultured on MS solid medium supplemented with 1.5 mg/L 2,4-D, 3.0 mg/L α-NAA, and 0.25 mg/L Kn. Culture conditions of a selected high-productive callus line (CCCL VII) were systematically optimized in terms of biomass productivity and phenolic yield, and eleven target compounds were identified and quantified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Under the optimal culture conditions (MS liquid medium, sucrose concentration 3.5 %, temperature 26 °C, inoculum size 3.0 %, and time 49 day), CCSCs produced the highest level of total phenolics (3462.99 μg/g DW) along with the maximum biomass productivity (10.03 g/L), which was higher than the phenolic yield in field-grown CCLs (3288.81 μg/g DW). Notably, CCSCs exhibited superiority in the content of the most valuable compound cajaninstilbene acid (2858.76 μg/g DW) as compared with CCLs (2114.60 μg/g DW). Also, CCSCs were discovered to be more abundant in genistin and genistein as against both CCLs and TSCs. This work concluded that CCSCs might be attractive alternative sources for the production of phenolic compounds with potential uses in pharmaceutical/nutraceutical industries.

Poison ivy hairy root cultures enable a stable transformation system suitable for detailed investigation of urushiol metabolism.

Poison ivy (Toxicodendron radicans) is best known for causing exasperating allergenic delayed‐contact dermatitis symptoms that last for weeks on persons who have contacted the plant. Urushiols are alkylcatechols produced by poison ivy responsible for causing this dermatitis. While urushiol chemical structures are well known, the metabolic intermediates and genes responsible for their biosynthesis have not been experimentally validated. A molecular genetic characterization of urushiol biosynthesis in poison ivy will require stable genetic transformation and subsequent regeneration of organs that retain the capacity synthesize urushiol. To this end, Agrobacterium rhizogenes was used to generate hormone‐independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick‐inoculated with A. rhizogenes, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for A. rhizogenes prick‐inoculation did not affect the initial formation of calli/hairy root primordia, but rather significantly influenced the establishment of long‐term hormone‐independent hairy root growth. A. rhizogenes harboring a recombinant T‐DNA binary plasmid with an intron‐containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady‐state urushiol levels relative to wild‐type roots, but higher urushiol levels than a poison ivy undifferentiated callus line with undetectable urushiol levels, suggesting that urushiol biosynthesis requires intact poison ivy organs. The lower urushiol levels in poison ivy hairy root lines facilitated the first identification of anacardic acid metabolites initially in hairy roots, and subsequently in wild‐type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse‐genetic studies of urushiol biosynthesis in poison ivy hairy roots.

Phenylethanoids, phenylpropanoids, and phenolic acids quantification vis-à-vis gene expression profiling in leaf and root derived callus lines of Rhodiola imbricata (Edgew.)

Salidroside and rosavins have been found as the most potent ingredients used in Rhodiola based herbal formulations. Rhodiola imbricata is a medicinal herb of the trans-Himalayan Ladakh region of India. Since, the natural supply of this herb is rapidly decreasing, due to its over-exploitation, high altitude region, and traditional usage in the Amchi system of medicine. In the present study, friable callus culture was developed for the first time from leaf and root explants of R. imbricata. Furthermore, callus cell lines were also evaluated for rapid growth rate and high metabolite accumulation. The results revealed that callus induction frequency observed in juvenile leaves (100 %) and roots (87.50 %) in MS medium enriched with 0.5 mg/L TDZ and 1 mg/L NAA. Scanning electron microscopy (SEM) analysis was done for histological observations of the callus surface. A selective and efficient ultra-performance liquid chromatography with PDA detector (UPLC-PDA) method was developed and validated for the quantification of phenylethanoids, phenylpropanoids, and phenolic acids. The maximum salidroside was detected in leaf derived friable green calli (3.59 mg/g DW) subsequently followed by leaf derived friable white calli (2.31 mg/g DW). While rosavin and rosarin were detected maximum in root derived compact green calli (0.15 mg/g DW) and root derived friable green calli (0.07 mg/g DW). Genes encoding enzymes involved in salidroside and rosavins biosynthesis were also investigated for transcript abundance in wild as well as in vitro cultures using the RT-qPCR approach. The present study explained the friable callus culture as a potential alternative approach to obtain higher metabolite yield in R. imbricata. Further, it can be used for sequential scale-up of metabolite at bioreactor level to meet the industrial demand.

Characterization of allotetraploids derived from protoplast fusion between navel orange and kumquat

In this study, we aimed to produce a citrus fruit with no seed and with edible peel. Therefore, the seedless Yoshida navel oranges (Citrus sinensis ‘Yoshida navel’) and Jangsil kumquat (Fortunella japonica) with edible peel were used as the starting materials. Since two varieties of different genera and different flowering periods would be difficult to crossbreed, protoplast fusion was conducted using the polyethylene glycol method. Yoshida navel orange and Jangsil kumquat were used as an embryogenic callus line and a mesophyll line, respectively. The regenerated plants were analyzed by flow cytometry to identify tetraploids, which were further evaluated by polymerase chain reaction, using simple sequence repeat markers specific for the nuclear and cytoplasmic organellar DNAs of the two parents, to confirm allotetraploids and cybrids. Sixteen allotetraploids were finally produced and characterized phenotypically for leaf morphology and fruit quality. All allotetraploids contained mitochondria originating from Yoshida navel orange; 12 contained chloroplasts derived from navel orange whereas four contained chloroplasts of kumquat origin. We investigated both plant and fruit characteristics of allotetraploids derived from the protoplast fusion of navel orange and kumquat. The sizes of the allotetraploid leaves were intermediate between those of both parents. However, in contrast to the spindle-shaped leaves of both parents, those of the allotetraploids were obovate. Petiole wings were absent in the allotetraploids, a known kumquat trait. Similar to navel oranges, flowering time was mid-May, and pollen was sterile, whereas fruit size, external shape, soluble solids content, and acidity were all intermediate between the two parents. Peel thickness and the number of segments were similar to those of kumquat, and flesh weight was similar to that of navel orange. Collectively, these results indicated that the intergeneric allotetraploids derived from navel orange and kumquat inherited favorable traits of both parents and could be produced and selected despite a lack of extensive parental variation. This is one of the first reports showing fruiting results of protoplast-fused citrus plants. The study reported characteristics of the fruits from both the original plants as well as from the protoplast-fused plant. Detailed characteristics of the allotetraploid fruit produced by protoplast fusion would be very helpful for future polyploid studies.

A Comparison of Chemical Profiles of Callus, Plant and Agarwood Extracts of Gyrinops walla using Thin Layer Chromatography

Agarwood produced by Gyrinops walla Gaertner, an endemic plant species of Sri Lanka, arguably the costliest wood, is used as a major constituent in cosmetic industries. Application of plant cell culture technique serves as the best alternative for the production of fragrance compounds of G. walla while ensuring the sustainability of the species. Using the advantages of plant cell culture and Thin Layer Chromatography (TLC), a preliminary study was carried out to compare the phytochemical constituents of callus and plant extracts (i.e stem, leaves, bark and agarwood) of G. walla. Different callus lines were developed on MS and modified MS media with reduced level of NH4NO3, supplemented with different combinations and concentrations of plant growth regulators and multiplied. Freeze dried samples were extracted with hexane, ethyl acetate and methanol and run on a TLC plate under different eluting solvent systems. A higher number of TLC spots were observed in the ethyl acetate extracts of all samples, under 15 percent methanol: chloroform solvent system. Based on TLC chemical profiles of all callus lines, MS media added with 1.0 mg/L BAP, 0.5 mg/L kinetin and 0.1 mg/L 2,4-D and ½ MS contained 1.0 mg/L BAP and 0.1 mg/L 2,4-D were selected as best lines. The extracts of the selected two callus lines matched with the fingerprint profiles of best plant samples i.e stem, bark and agarwood. Compounds present in the extracts of callus samples, plant samples and agarwood, appearing at Rf=0.56, 0.74 and 0.78, could be used as possible markers for G. walla. The chromatography profiling by TLC has proven the presence of similar phytochemical constituents in callus, plant samples and agarwood. Therefore, it can be concluded that plant cell culture technique guarantees the possibility to produce same compounds that are present in plant and agarwood of G. walla.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Inducement and cultivation of novel red Cyclocarya paliurus callus and its unique morphological and metabolic characteristics

Cyclocarya paliurus (C. paliurus) is a unique plant growing in China with many bioactive components, but is endangered due to the great difficulties in seed germination and asexual propagation. Plant tissue culture can be used to overcome the resource shortage which severely limits the development and utilization of this plant. In the present paper, five typical types of C. paliurus callus lines with different morphologies, including a novel red callus line (CR) and a common light yellow-green callus line (CYG), were obtained after a long time induction and cultivation. Three triterpenoids (betulinic acid, oleanolic acid and ursolic acid) and two polyphenols ((+)-catechin and procyanidin B1) were all detected in both CR and CYG, but differed greatly in content. The contents of total triterpenoids and total polyphenols in CR were all significantly higher than that in CYG. However, anthocyanins (cyanidin-3-O-galactoside and cyanidin-3-O-glucoside) were only found in CR. Both the growth rate and biomass increment of CYG were slightly higher than that of CR. The results of high throughput RNA sequencing indicated that the expression of the triterpenoid biosynthesis pathway genes, such as HMGS (hydroxymethylglutaryl-CoA synthase), HMGR (hydroxymethylglutaryl-CoA reductase), FPS (farnesyl diphosphate synthase), SS (squalene synthase), SE (squalene epoxidase) and β-AS (beta-incense resin synthase), significantly increased in CR compared with CYG, which led to a marked rising of triterpenoid contents. Similarly, the up-regulated expression of polyphenol biosynthesis pathway genes, including CHI (chalcone isomerase), N3D (naringenin 3-dioxygenase), F3′H (flavonoid 3′-monooxygenase), DFR (dihydroflavonol 4-reductase), and 3GalT (galactosyltransferase), improved the polyphenol contents in CR, especially anthocyanin content. The decreased transcription factor expression of MYC2 and ERF003 resulted in a high triterpenoid content by increasing the expression of pathway genes in the CR. Meanwhile, the up-regulated MYB7, MYB1, bHLH122, WD40, ERF105 and down-regulated MYB44, MYC2, ERF003 enhanced the polyphenol content of the CR by stimulating the expression of the related pathway genes. RT-qPCR (quantitative real-time PCR) determination further validated the above-mentioned differentially expressed genes analysis of RNA-sequencing.