Establishment of an efficient in vitro regeneration and Agrobacterium rhizogenes-mediated genetic transformation protocol for safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.)

2017 ◽  
Vol 53 (6) ◽  
pp. 571-578 ◽  
Author(s):  
Jaafar Juju Nakasha ◽  
Uma Rani Sinniah ◽  
Noor Azmi Shaharuddin ◽  
Siti Aishah Hassan ◽  
Sreeramanan Subramaniam ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Agrobacterium Rhizogenes ◽  
In Vitro Regeneration ◽  
Transformation Protocol ◽  
Chlorophytum Borivilianum ◽  
Safed Musli

scholarly journals Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

2019 ◽  
Vol 67 (2SUPL) ◽  
pp. S83-S94 ◽  
Author(s):  
Laura-Yesenia Solís-Ramos ◽  
Julio-César Ortiz-Pavón ◽  
Antonio Andrade-Torres ◽  
Romano Porras-Murillo ◽  
Arturo Brenes-Angulo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phaseolus Vulgaris ◽  
Genetic Transformation ◽  
Common Bean ◽  
In Vitro Regeneration ◽  
Marker Genes ◽  
Transformation Protocol ◽  
Glucuronidase Activity ◽  
Rich Media

Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.

scholarly journals In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

2012 ◽  
Vol 40 (2) ◽  
pp. 140 ◽  
Author(s):  
Hafiz Mamoon REHMAN ◽  
Iqrar Ahmad RANA ◽  
Siddra IJAZ ◽  
Ghulam MUSTAFA ◽  
Faiz Ahmad JOYIA ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Genetic Transformation ◽  
Callus Induction ◽  
In Vitro Regeneration ◽  
Induction Medium ◽  
Native Forest ◽  
Ms Medium ◽  
Dalbergia Sissoo ◽  
Callus Induction Medium

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols.

scholarly journals Agrobacterium-mediated Genetic Transformation for Local Cultivars of Potato (Solanum tuberosum L.) Using Marker Genes

1970 ◽  
Vol 20 (2) ◽  
pp. 145-155 ◽  
Author(s):  
Rita Sarah Borna ◽  
M. I. Hoque ◽  
R. H. Sarker
Keyword(s):  
Solanum Tuberosum ◽  
Genetic Transformation ◽  
Solanum Tuberosum L ◽  
In Vitro Regeneration ◽  
Direct Regeneration ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Transformation Rate ◽  
Best Responses

Genetic transformation using nodal and internodal segments from three economically important potato (Solanum tuberosum L.) varieties namely, Diamant, Cardinal and Granola was conducted using an Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI12 containing the GUS and nptII genes. Node and internodal segments were used for direct regeneration as well as regeneration with the intervention of callus. best responses were  obtained for direct regeneration of shoots when the explants were cultured on MS supplemented with 4.0 mg/l BAP +1.0 mg/l IAA, 1.5 mg/l BAP  + 0.5 mg/l IAA and 5.0 mg/l BAP +1.0 mg/l IAA in Diamant, Cardinal  and  Granola, respectively. In Diamant spontaneous in vitro microtuberization was obtained from these proliferated shoots. Further culturing of these in vitro grown green microtubers regenerated a large number of shoots on MS containing 4.0 mg/l BAP +1.0 mg/l IAA. By combining the best treatments, this protocol yielded an average transformation rate of 87% of treared explants. Stable expression of GUS gene was visualized in the various parts of transformed shoots through histochemical assay. Genomic DNA was isolated from transformed shoots and stable integration of the GUS and nptII genes was confirmed by PCR analysis.   Key words:  Potato, in vitro regeneration, transformation   D.O.I. 10.3329/ptcb.v20i2.6894   Plant Tissue Cult. & Biotech. 20(2): 145-155, 2010 (December)

scholarly journals In vitro Regeneration and Agrobacterium-mediated Genetic Transformation of Tomato (Lycopersicon esculentum Mill.)

1970 ◽  
Vol 19 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Rakha Hari Sarker ◽  
Khaleda Islam ◽  
M.I. Hoque
Keyword(s):  
Lycopersicon Esculentum ◽  
Genetic Transformation ◽  
In Vitro Regeneration ◽  
Leaf Explants ◽  
Multiple Shoots ◽  
Multiple Shoot ◽  
Root Induction ◽  
Cotyledonary Leaf ◽  
Lycopersicon Esculentum Mill

Agrobacterium-mediated genetic transformation system has been developed for two tomato (Lycopersicon esculentum Mill.) varieties, namely Pusa Ruby (PR) and BARI Tomato-3 (BT-3). Prior to the establishment of transformation protocol cotyledonary leaf explants from the two varieties were cultured to obtain genotype independent in vitro regeneration. Healthy multiple shoot regeneration was obtained from the cut ends of cotyledonary leaf segments for both the varieties on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. The maximum root induction from the regenerated shoots was achieved on half the strength of MS medium supplemented with 0.2 mg/l IAA. The in vitro grown plantlets were successfully transplanted into soil where they flowered and produced fruits identical to those developed by control plants. Transformation ability of cotyledonary leaf explants was tested with Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121, containing GUS and npt II genes. Transformed cotyledonary leaf explants were found to produce multiple shoots on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l since kanamycin resistant gene was used for transformation experiments. Shoots that survived under selection pressure were subjected to rooting. Transformed rooted plantlets were transferred to soil. Stable expression of GUS gene was detected in the various tissues from putatively transformed plantlets using GUS histochemical assay.  Key words: In vitro regeneration, transformation, tomato D.O.I. 10.3329/ptcb.v19i1.5004 Plant Tissue Cult. & Biotech. 19(1): 101-111, 2009 (June)

scholarly journals Genotype, Explant and Growth Regulator Effects in the Determination of Adventitious Regeneratin in Curcurbits, in Aid of Genetic Transformation

1992 ◽  
Author(s):  
Dennis Gray ◽  
Victor Gaba
Keyword(s):  
Genetic Transformation ◽  
In Vitro Regeneration ◽  
Target Tissue ◽  
Culture Systems ◽  
Original Proposal ◽  
Suitable Target ◽  
Regeneration In Vitro ◽  
Transformation Systems

The objective of this study was to gain an understanding of the in vitro regeneration process in watermelon and melon to enable the development of genetic transformation systems. The objectives were met and additional progress, unplanned during the original proposal, was made. Organogenic regeneration in vitro was studied in both melon and watermelon. Genotype played a significant role in regeneration. In melon, epidermal cells were responsible for most regeneration. Methods to obtain in vitro-derived watermelon tetraploids, needed for seedless varieties, were developed. The culture systems were refined so that they could be routinely used for transformation. Particle guns were constructed and Agrobacterium strains were obtained to study the effect of transformation procedures on culture system performance, allowing refinement of transformation protocols. The culture systems were shown to enable the stable transformation of both crops, allowing their future use for insertion of agriculturally-important genes. In addition, we showed that shoot apical meristems might be suitable target tissue for transformation and allow a wider range of genotypes to be used, which is needed for crops as diverse as cucurbits.

scholarly journals 117 An In Vitro Regeneration System for Basil (Ocimum basilicum L.)

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 461E-461
Author(s):  
Winthrop B. Phippen ◽  
James E. Simon
Keyword(s):  
In Vitro Regeneration ◽  
Morphological Characteristics ◽  
Leaf Explants ◽  
Basal Medium ◽  
Induction Medium ◽  
Shoot Induction ◽  
Regeneration System ◽  
Transformation Protocol ◽  
Regeneration Procedure

A plant regeneration protocol was successfully developed for basil (O. basilicum L.). Explants from 1-month-old seedlings yielded the highest frequency of regeneration of shoots (37%) with an average number of 3.6 shoots per explant. Calli and shoot induction were initiated on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) (4 mg/L) for ≈30 days. Shoot induction and development was achieved by refreshing the induction medium once after 14 days. The most morphogenetically responsive explants were basal leaf explants from the first fully expanded true leafs of greenhouse-grown basil seedlings. Developing shoots were then rooted on MS media in the dark without TDZ. Within 20 days, rooted plantlets were transferred and acclimatized under greenhouse conditions where they developed normal morphological characteristics. This is the first report of a successful in vitro regeneration system for basil through primary callus. The establishment of a reliable regeneration procedure is critical when developing a transformation protocol for enhancing the production of basil for insect and disease resistance and improved essential oil constituents.

scholarly journals In Planta Genetic Transformation of Mungbean (Vigna radiata (L.) Wilczek) with Marker Gene

2019 ◽  
Vol 29 (2) ◽  
pp. 245-255
Author(s):  
Mohammad Mahmood Hasan ◽  
Sujay Kumar Bhajan ◽  
M. Imdadul Hoque ◽  
R.H. Sarker ◽  
Mohammad Nurul Islam
Keyword(s):  
Genetic Transformation ◽  
Genetic Improvement ◽  
In Vitro Regeneration ◽  
Marker Gene ◽  
Gus Expression ◽  
In Planta ◽  
Leaf Tissues ◽  
Gus Gene Expression ◽  
Screenable Marker

In genetic improvement of mungbean much success has not been achieved due to its recalcitrant nature towards in vitro regeneration. An attempt was made to develop an Agrobacterium-mediated in planta genetic transformation protocol for a locally grown mungbean variety BARI Mung-3 using a screenable marker gene. Two minutes of vacuum infiltration followed by 60 minutes of incubation period in Agrobacterium suspension of Winans’ AB medium containing wounded tobacco leaf extract was found most suitable towards genetic transformation in pricked de-coated half seed explants. An optical density (OD600) of 0.7 was found most effective for transient gus gene expression. Chimeric GUS expression was observed in the root and leaf tissues from the successfully transformed plantlets obtained through in planta transformation. This methodology of genetic transformation was found more suitable, easier and less time consuming than tissue culture based genetic transformation, which may be used for the genetic improvement of mungbean.

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