Nelson-Vasilchik, KimberlyHague, Joel P.Tilelli, MichaelKausch, Albert P....
12页
查看更多>>摘要：The standard stalwart Agrobacterium-mediated transformation protocols for sorghum use differentiating embryogenic callus induced from immature embryos in the genotype BTx430. While reliable, the standard protocols are lengthy, and both time and resource expensive. We have investigated the use of altruistic morphogenic regulator mediated transformation (MRMT) to improve sorghum transformation for use in genome editing. Here, we show a method for rapid transformation and plant regeneration of sorghum (Sorghum bicolor L. cultivar BTx430) mediated by altruistic Baby boom and Wuschel2. Confirming and extending previous recent work using altruistic MRMT, we show that the altruistic MRMT rapid protocol results in a higher transformation frequency expressed as a function of the number of independent events per immature embryos infected and requires less time in a transformation pipeline which translates to an increased number of constructs one full-time equivalent (FTE) can manage per yr. This approach is compatible with genome editing requirements and should help address the transformation bottleneck for functional genomics and genome editing studies in sorghum.
查看更多>>摘要：An Agrobacterium-mediated transformation protocol for the purple raspberry (R. occidentalis x R. idaeus) 'Amethyst' was developed. Using the system, the FER-like iron deficiency-induced transcription factor 1 gene cloned from Populus tremula (PtFIT) was expressed in transgenic raspberry plants. Effects of four inoculum densities and two co-cultivation times on 'Amethyst' transformation were tested in two separate experiments. Results showed that an average transformation frequency of 3.9% was achieved under the conditions of 25 mg L-1 kanamycin selection, 3-d co-cultivation, and OD600 0.3 to 0.55 inoculum density. A total of 12 PtFIT-transgenic lines of 'Amethyst' were verified using polymerase chain reaction (PCR) analysis. Expression of the PtFIT gene in transgenic lines was evaluated under the iron deficiency or sufficiency condition using the real-time quantitative PCR (RT-PCR); however, the expression showed an inconsistent response to iron deficiency among different transgenic lines. An established transformation system could provide a research tool used to understand gene functions and trait development in raspberry; therefore, the present research will be beneficial to breeding and germplasm improvement of raspberry or other Rubus species.
查看更多>>摘要：Cucumber mosaic virus (CMV) infects a large number of plant species including Piper nigrum L. and related species. As natural resistance to CMV is absent in Piper spp., the study was undertaken to produce transgenic P. nigrum plants harboring the complete coat protein (CP) gene of CMV via Agrobacterium-mediated transformation and their evaluation for resistance against the virus. Among one hundred and nine hardened transformed plantlets, eight revealed the presence of the transgene in PCR. The production of transcript in these plants was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and buildup of CMV CP by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). Screening of all eight transgenic lines against CMV through cleft grafting revealed that all lines except one were symptomless or showed mild or moderate symptoms. The transgenic line with the highest resistance was vegetatively propagated and integration of transgene in these clones was validated by Southern hybridization. The presence of transcript in clones was affirmed by Northern blotting- and Western blotting-ratified translation of transgene. Furthermore, relative expression studies proved manifold expression of transgene compared to actin gene as analyzed by RT-qPCR. These studies validate the stable integration and expression of transgene which might be inhibiting the movement of virus to the scions in graft inoculated plants. This is the first report on CP-mediated resistance in P. nigrum and paves the way to the production of transgenic CMV-resistant P. nigrum using CP and other desirable genes, the only effective method to combat CMV attack in the crop.
查看更多>>摘要：Lactuca indica L. (Asteraceae), a wild lettuce, is used as a vegetable and in traditional medicine. This study aims to establish in vitro propagation protocol and evaluate lactucin and antibacterial property from in vitro and natural plant tissues. Leaf blades and petioles were cultured in vitro on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) and 1.2 mg L-1 indoleacetic acid (IAA). Leaf petioles and a lower BAP concentration (0.5 mg L-1) were optimal for direct shoot induction, while the leaf blade and a higher BAP (4 mg L-1) concentration performed best for callus induction. When the callus was subcultured, 98.7% of samples regenerated plants on MS medium supplemented with 1.5 mg L-1 BAP and 0.5 mg L-1 IAA. MS medium containing 1 mg L-1 IAA was best for in vitro rooting. A high-performance liquid chromatography analysis of the in vitro samples revealed a higher amount of lactucin (sesquiterpene lactones) in the root than the callus and the leaf, whereas in naturally grown plants, higher lactucin amounts were obtained from the juvenile root followed by the root of the flowering plant and juvenile leaf as the lowest concentration. All tissue extracts showed antibacterial activity against Pseudomonas fuscovaginae (a rice pathogen) and Escherichia coli, which was directly proportional to amount of lactucin produced. This in vitro regeneration and phytochemical investigation will facilitate the further exploitation of this useful wild plant.
查看更多>>摘要：Invitro culture in combination with aeroponics is observed to be an efficient means for mass propagation of Sarcostemma acidum in the present investigation. S. acidum is a rare leafless xerophytic shrub of the family Apocynaceae, commonly known as Som-lata or khir-kheep, a source of medicines and the religious drink "Somaras." Aeroponic technique was used for the induction of adventitious roots from stem cuttings of field-grown plants for the development of quality plants in the greenhouse as a source of explants for micropropagation. Among various concentrations of IBA, NAA and NOA used for induction of adventitious rooting from stem cuttings 3.0 g L-1 NAA were found to be most suitable with average 90% induction response and 14 adventitious roots. Surface-sterilized nodal shoot segments of 6 to 7 cm length were inoculated on basic and modified Murashige and Skoog's medium containing 3% (w/v) sucrose, 0.8% (w/v) agar, and various concentrations of BAP and kinetin for activation of axillary shoot buds. Shoots were mass multiplied on a modified MS (MMS) medium containing 3.5 mg L-1 BAP and 0.01 mg L-1 IAA. Invitro multiplied shoots were rooted invitro on half-strength MS medium containing 0.25 mg L-1 NAA. Ninety percent of invitro regenerated plantlets were hardened successfully on Soilrite in the greenhouse and survived on garden and field soils. This protocol will be useful for invitro propagation of S. acidum for mass plantations in the desert ecosystem for prospects.
查看更多>>摘要：Curculigo orchioides (Kali Musli) is a medicinal plant commonly used in the Indian and Chinese traditional medicine system to treat jaundice and asthma. It is also used as an aphrodisiac in several countries. C. orchioides is treated as an essential resource in many pharmaceutical industries as a source of bioactive compounds. Indiscriminate use, depletion of its natural habitat, and its seasonal availability have resulted in its rarity. As per the IUCN status, Curculigo orchioides is now listed as an endangered plant. The present micropropagation study resolves its seasonal availability by ensuring a continuous supply of plants. In vitro somatic embryos were induced on the leaf explants of C. orchioides in one-fourth-strength Murashige and Skoog (MS) media. The embryos developed into whole plants in 8 wk. The in vitro plants produced were evaluated for their phytochemical evaluation and anti-oxidant activity along with their wild counterparts as controls. Both sets, wild plants termed in vivo and tissue-cultured plants called in vitro plants, were subjected to phytochemical extraction using a solvent gradient method. The extracts were analyzed qualitatively and quantitatively for phytochemical constituents. The anti-oxidant property of the extracts was screened using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and reducing power assays. The in vivo aqueous root extract (AR) and aqueous leaf extract (AL) had similar DPPH radical scavenging activity for both in vivo and in vitro plants. In vitro methanolic root extracts showed a significantly higher DPPH radical scavenging activity than other in vitro root extracts. The study demonstrates the medicinal properties of in vitro plants conclusively. It could be effectively used as an alternative, time-saving method for sustainable, season-independent usage of endangered plants for extraction while conserving and protecting their diversity and habitat in nature.
Xiong, YupingChen, XiaohongWu, Kunlinda Silva, Jaime A. Teixeira...
7页
查看更多>>摘要：Cordia subcordata Lam. (Boraginaceae) is a woody tree that grows in coastal wasteland and mangrove edges. Its wood is used for woodcraft, and the tree, which is used in landscaping, also has medicinal and ecological value (e.g., coastal protection). In this study, a shoot proliferation and regeneration system was established for this plant. Callus was induced from terminal young leaf explants on Murashige and Skoog (MS) medium with 1.0 mg L-1 thidiazuron or 1.0 mg L-1 2,4-dichlorophenoxyacetic acid, or optimally with 0.5 or 1.0 mg L-1 of both. Callus differentiated from an average of 35.4% of explants into 3 to 5 adventitious shoots (indirect shoot organogenesis) within 45 d. Optimal shoot proliferation medium was MS medium with 0.8 mg L-1 6-benzyladenine (BA). A higher concentration of BA (1.5 mg L-1) in the presence of alpha-naphthaleneacetic acid (NAA) enhanced callus induction from the base of shoots and decreased shoot proliferation. On 1/2 MS medium with 0.5 mg L-1 NAA and/or indole-3-butyric acid, adventitious roots were induced from shoots. Within 1 mo, 95.4% of plantlets transferred to a substrate of vermiculite and peat (1:1, v/v) survived.
Qarachoboogh, Ayoub FathollahiAlijanpour, AhmadHosseini, BahmanShafiei, Abbas Banj...
8页
查看更多>>摘要：Foetid juniper (Juniperus foetidissima Willd.) is one of the most important coniferous species faced with multiple threats and it is difficult to propagate it with sexual reproduction. Therefore, the main goal of this study was finding appropriate salt concentrations and growth regulators for shoot regeneration and rooting of J. foetidissima under in vitro conditions and also shortening time of mass production. The effects of 6 different basal culture media (Olive Medium (OM), Murashige and Skoog (MS), Driver and Kuniyuki (DKW), Woody Plant Medium (WPM), 1/2 MS, and 1/4 MS) on shoot proliferation and two different methods of root induction on explants were investigated. Results revealed that OM culture medium supplemented with 6-benzylaminopurine (BAP) (1 mg L-1) and indole-3-acetic acid (IAA) (0.1 mg L-1) was the best medium for shoot proliferation off. foetidissima (P < 0.05). The highest percentage of shoot regeneration (100%), maximum number of shoots (6.38 shoots per explant), and the maximum shoot length (6.74 mm) were observed in this culture medium. The lowest percentage of shoot regeneration (50%) was obtained in 1/4 MS medium (P < 0.05). In addition, the minimum number of shoots (3.44 shoots per explant) and shoot length (4.46 mm) were observed in WPM medium. The highest root induction rate (88.9%) was obtained in OM medium containing 0.5 mg L-1 of indole-3-butyric acid (IBA). In conclusion, type of basal culture medium considerably influenced proliferation, shoot growth, and root induction in J. foetidissima explants.
查看更多>>摘要：The global food crisis is an issue affecting 1 billion people worldwide-it is critical that a solution be developed to provide the world's citizens with alternative sources of food that are less energy-intensive. Both nanoparticles, molecules < 100 nm used in drug delivery, and antioxidants, molecules that inhibit oxidation reactions, have been individually studied to enhance plant growth; however, the combined effect has not been investigated. Using hydroponic, tissue culture, and hydrogel experiments, this project investigates whether a combined nanoparticle-antioxidant treatment of carbon nanoparticles and ascorbic acid (CNP-AOX) has a synergistic effect in enhancing plant growth. When tested using hydroponic experiments, NP-AOX showed significant increases in chlorophyll levels, media consumption, and plant weight compared to the other treatments. Tissue culture experiments showed that nanoparticles resulted in long roots, antioxidants resulted in dense roots, while CNP-AOX resulted in a combination of both. TEM revealed that antioxidants enabled the delivery of nanoparticles into cell organelles which may have contributed to their enhanced growth. CNP-AOX also increased soil moisture levels in the hydrogel experiments. Using CNP-AOX is economically feasible, costing less than $0.03 per L while conventional fertilizers will cost over a dollar for the same effects. The NP-AOX treatment can boost food production, decreasing the need for imports, and reducing the environmental impact of fertilizers, with particular benefits in northern communities combatting crop loss due to permafrost and drought.
查看更多>>摘要：A high-efficiency regeneration and genetic transformation system is indispensable for generating desirable traits in important trees such as Eucalyptus. However, lower regeneration efficiency is common for most varieties because of the recalcitrance of this genus. Here, a stable and highly efficient in vitro organogenesis protocol and Agrobacterium-mediated genetic transformation system of Eucalyptus were developed, and transgenic plants were obtained. In this protocol, the preferred explants were the top and middle stem internodes from in vitro micro-shoots of the E. urophylla x E. tereticornis hybrid. Modified Woody Plant Medium (mWPM) containing 0.025 mg center dot L-1 thidiazuron (TDZ) and 0.10 mg center dot L-1 indole-3-butyric acid (IBA) was used to induce multiple adventitious buds that allowed 85.6% shoot formation. The binary vector pBI121 carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes was applied for transformation. The preferred internodes were precultured for 0 to 3 d and infected with A. tumefaciens strain GV3101 grown to a bacterial density of 0.5 (OD600). Then, they were transferred to a co-culture medium supplemented with 50 mu M acetosyringone (AS) and co-cultured for 2 d in the dark. The transgenic adventitious buds formed in regeneration medium, which was replaced by the same medium with a 2-wk subculture interval through kanamycin selection. Using the aforementioned method, transgenic plantlets can be obtained within 3 mo with a transformation frequency of 3.8%, which was verified by polymerase chain reaction amplification (PCR) and histochemical analysis of GUS activity. The constructed genetic transformation system will lay a foundation for mining gene functions and further molecular breeding of Eucalyptus.
NSTL
Springer Nature