TRANSFORMATION OF EASTER LILY VIA PARTICLE BOMBARDMENT

Particle bombardment was investigated as a potential transformation method for Easter lily. Bulb scale explants from Lilium longiflorum Thunb. `Nellie White' were used as target material. The uidA (or gusA) reporter gene for ß- glucuronidase (GUS) expression was used in all particle bombardments to assess efficiency of gene delivery. Parameters examined to achieve optimal levels of transient GUS expression included gene promoter, helium pressure (particle velocity), and target distance. The highest level of transient GUS expression (as measured by number of indigo-stained cells/scale explant) was observed with the rice actin 1 (Act1) promoter, a helium pressure of 1500 psi, and target distances of 9 to 12 cm. Parameters considered for recovery of stable transform ants included the choice of selective agent (phosphinothricin or hygromycin) and their respective selectable resistance genes (phosphinothricin acetyltransferase and hygromycin phosphotransferase), and preculture time of scale explants prior to bombardment. Polymerase chain reaction analysis will be used to screen putative stable transformants, and from this data it will be determined which parameters yielded the highest transformation rates.

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TRANSFORMATION OF ONION CALLUS VIA PARTICLE BOMBARDMENT

HortScience ◽

10.21273/hortsci.30.3.435b ◽

1995 ◽

Vol 30 (3) ◽

pp. 435b-435

Author(s):

Ellen B. Peffley ◽

Melanie A. Hart

Keyword(s):

Particle Bombardment ◽

Transformation Method ◽

Gus Expression ◽

Particle Dispersion ◽

Gus Activity ◽

Target Distance ◽

Helium Pressure ◽

Activity Measurements ◽

Callus Initiation ◽

Dark Incubator

Particle bombardment was investigated as a potential transformation method for onion. Seeds of Allium cepa `TG 1015' were planted onto BDS medium and placed in a dark incubator at 25C for germination. Two to 3 weeks after the seeds were germinated, meristems (1 to 2 mm) were excised and placed onto BDS medium containing 2 mg 2,4-D/liter for callus initiation. Callus was transferred monthly onto fresh BDS medium containing 2,4-D until bombardment. The reporter gene for B-glucuronidase (GUS) expression was used to assess efficiency of gene delivery in all particle bombardments. Characteristics examined were target distance and helium pressure (particle velocity). Tissues were subjected to the 5-bromo-4-chloro-3-indoyl-B-D-glucuronide (xgluc) test for detection of GUS activity. Measurements were taken on particle dispersion as affected by target distance and helium pressure. GUS expression was detected in putatively transformed tissues.

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Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132003000100001 ◽

2003 ◽

Vol 46 (1) ◽

pp. 1-6 ◽

Cited By ~ 17

Author(s):

João C. Bespalhok Filho ◽

Adilson K. Kobayashi ◽

Luiz F. P. Pereira ◽

Rafaelo M. Galvão ◽

Luiz G. E. Vieira

Keyword(s):

Gene Expression ◽

Particle Bombardment ◽

Citrus Sinensis ◽

Sweet Orange ◽

Transient Gene Expression ◽

Gus Expression ◽

Poncirus Trifoliata ◽

Helium Pressure ◽

High Osmolarity ◽

Carrizo Citrange

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis).

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Biolistic transformation of Eucalyptus grandis x E. urophylla callus

Functional Plant Biology ◽

10.1071/pp01153 ◽

2002 ◽

Vol 29 (8) ◽

pp. 917 ◽

Cited By ~ 12

Author(s):

Laudete Maria Sartoretto ◽

Luis Pedro Barrueto Cid ◽

Ana Cristina Miranda Brasileiro

Keyword(s):

Eucalyptus Grandis ◽

Target Material ◽

Polymerase Chain Reaction Analysis ◽

Cauliflower Mosaic Virus ◽

Dichlorophenoxyacetic Acid ◽

Gus Gene ◽

Polymerase Chain ◽

Hybrid Eucalyptus ◽

2,4 Dichlorophenoxyacetic Acid ◽

Npt Ii Gene

A procedure for genetic transformation of the hybrid Eucalyptus grandis × E. urophylla using particle bombardment is described. Cotyledon- and hypocotyl-derived calli growing on SP medium supplemented with 2�m�thidiazuron or on MS modified (MSM) medium supplemented with 10 m 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5�m�6-benzylaminopurine (BAP), were used as target material for bombardment assays. Multiple preincubation and bombardment conditions were tested. Tungsten particles were coated with the plasmid pBI426 harbouring a β-glucuronidase (gus) and neomycin phosphotransferase II (npt II) gene fusion controlled by a double 35S cauliflower mosaic virus (CaMV) promoter. Four days after bombardment, the transient transformation efficiency was determined by expression of the gus gene. Fully GUS-positive calli were then obtained after 105 d in MSM medium supplemented with 2,4-D, BAP, and the selective agent kanamycin at 200 mg L-1. The presence of the gus gene in these kanamycin-resistant calli was confirmed by polymerase chain reaction analysis. Extensive experiments were performed aiming to identify conditions for the regeneration of these GUS-expressing calli. However, they were unable to regenerate transgenic shoots, suggesting that conditions suitable for regeneration are unsuitable for transformation and vice versa.

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L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis)

Frontiers in Plant Science ◽

10.3389/fpls.2021.767140 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ganeshan Sivanandhan ◽

Jiae Moon ◽

Chaemin Sung ◽

Solhee Bae ◽

Zhi Hong Yang ◽

...

Keyword(s):

Chinese Cabbage ◽

Transformation Efficiency ◽

Transformation Method ◽

Gus Expression ◽

Chain Reaction ◽

Functional Studies ◽

Cotyledon Explants ◽

Polymerase Chain ◽

Subsequent Regeneration

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

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Factors Affecting Transient Gene Expression in Mango Tissue Using Particle Bombardment

HortScience ◽

10.21273/hortsci.39.4.754e ◽

2004 ◽

Vol 39 (4) ◽

pp. 754E-755

Author(s):

Vaughan Agrez* ◽

Zora Singh ◽

Mis Gill

Keyword(s):

Gene Expression ◽

Particle Bombardment ◽

Mangifera Indica ◽

Transient Gene Expression ◽

Gus Expression ◽

35S Promoter ◽

Helium Pressure ◽

Factors Affecting ◽

Pre Treatment

Particle bombardment seems to be a promising method for genetic transformation of mango and the information on various factors affecting transient gene expression is inconclusive. Pro-embryonic masses (PEMs) of mango (Mangifera indica L. cv. Kensington Pride) used for particle bombardment were established and maintained in vitro using various liquid and semisolid media containing 2,4-D. Various factors affecting the biolistic transformation efficiency in mango and subsequent transient gene expression were optimized using β-glucuronidase (GUS) as a reporter gene driven by CaMV 35S promoter. Our results show that bombardment pressure significantly affects transient gene expression with the best results obtained from PEMs bombarded at 1200 kPa of helium pressure under vacuum. The application of osmoticum pre and post-bombardment also significantly increased the transient gene expression in the PEMs as compared to the controls. Mannitol (0.2 m) proved to be the best osmoticum in improving transient GUS expression as compared to sorbitol. The duration of exposure of PEMs to mannitol (0.2 m) both pre and post-bombardment also played a significant role in improving transient gene expression. The transient GUS expression was significantly highest with a pre-treatment of 0.2 m mannitol for 4 hours as compared to 0, 8 and 12 hours. The post-bombardment treatment of 0.2 m mannitol for 16 hours resulted in significantly highest transient gene expression as compared to 0, 4, 8, 12, 20 and 24 hours. In conclusion, PEMs of `Kensington Pride' mango bombarded at 1200 kPa, which were exposed to mannitol (0.2 m) for 4 and 16 hours pre and post bombardment respectively, resulted in the highest transient GUS expression (25.1 GUS foci/mg PEMs).

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