scholarly journals Agrobacterium-mediated Genetic Transformation in Lentil (Lens culinaris Medik.) followed by In vitro Flowering and Seed Formation

2012 ◽  
Vol 22 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Subroto K. Das ◽  
Kishwar Jahan Shethi ◽  
M. I. Hoque ◽  
R. H. Sarker
Keyword(s):  
Genetic Transformation ◽  
Lens Culinaris ◽  
Multiple Shoots ◽  
Cotyledonary Node ◽  
In Vitro Flowering ◽  
Pcr Analysis ◽  
Seed Formation ◽  
Half Strength ◽  
Genetic Transformation System

Genetic transformation system was developed for two microsperma varieties of lentil (Lens culinaris Medik.), namely Bari Masur-4 (BM-4) and Bari Masur-5 (BM-5) using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing GUS and nptII genes. Three different types of embryo explants, namely cotyledonary node (CN), decapitated embryo (DE) and cotyledone attached decapitated embryo (CADE) were used. Highest GUS positive expression was found in DE followed by CADE as detected by transient assays. Following Agrobacterium infection CADE showed better response in developing multiple shoots on MS supplemented with 2.22 µM BAP, 2.32 µM Kn, 0.29 µM GA3 and 30.35 µM tyrosine. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin up to 200 mg/l. Transgenic lentil shoots were produced with an overall frequency of 1.009%. In vitro rooting appeared to have a limitation in obtaining complete plantlets in lentil, therefore in vitro flowering and seed formation were induced in transformed shoots of lentil with a view to recovering of the transgenic progenies.  GUS positive shoots were found to produce in vitro flowers and pods on half-strength MS containing 98.4 µM IBA and 2.69 µM NAA. Expression of gene was detected in various tissues of the transformed shoots. Stable integration of GUS gene was also confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 22(1): 13-26, 2012 (June) DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11243 

scholarly journals In vitro Regeneration and Agrobacterium-mediated Genetic Transformation of Local Varieties of Mungbean (Vigna radiata (L). Wilczek)

2019 ◽  
Vol 29 (1) ◽  
pp. 81-97
Author(s):  
Sujay Kumar Bhajan ◽  
Setara Begum ◽  
Mohammad Nurul Islam ◽  
M Imdadul Hoque ◽  
Rakha Hari Sarker
Keyword(s):  
Genetic Transformation ◽  
Vigna Radiata ◽  
Zygotic Embryo ◽  
In Vitro Regeneration ◽  
Multiple Shoots ◽  
Direct Organogenesis ◽  
Pcr Analysis ◽  
Root Induction ◽  
Half Strength

An efficient Agrobacterium-mediated transformation compatible in vitro regeneration protocol was developed for two important varieties of mungbean (Vigna radiata (L.) Wilczek) cultivated in Bangladesh, namely Binamoog-5 and BARI Mung-6. Two different zygotic embryo derived explants, such as cotyledonary node (CN) and cotyledon attached decapitated embryo (CADE) were used for direct organogenesis of shoot. MS supplemented with 4.0 μM BAP was found to be the best for the development of highest number of multiple shoots from CADE in both the varieties of mungbean. While in case CN the best shoot formation was achieved on MS containing 4.0 μM BAP and 0.5 μM NAA in both varieties. Half strength of MS with 2.0 μM IBA was found to be most effective for producing healthy root from regenerated shoots. Following root induction, the in vitro raised plantlets were successfully transplanted to soil for their establishment. Considering overall responses, genetic transformation efficiency was found to be better with CADE explant using Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 conferring GUS and nptII genes. Different factors influencing transformation was optimized during this study. Selection of transformed shoots was carried out by gradually increasing the concentration of kanamycin and such transformed shoots were eventually selected using 200 mg/l kanamycin. Stable expression of the GUS gene was detected in various parts of regenerated transformed plantlets. Transformed shoots were rooted on half strength MS containing 2.0 μM IBA and 100 mg/l ticarcillin. Rooted transformed plantlets were successfully transferred to soil. Stable integration of GUS and nptII genes in the putative transformed shoots was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 29(1): 81-97, 2019 (June)

scholarly journals Agrobacterium-mediated Genetic Transformation of Lentil (Lens culinaris Medik.) with Chitinase Gene followed by In vitro Flower and Pod Formation

2019 ◽  
Vol 29 (1) ◽  
pp. 99-109
Author(s):  
Subroto K Das ◽  
Kishwar Jahan Shethi ◽  
MI Hoque ◽  
RH Sarker
Keyword(s):  
Genetic Transformation ◽  
Lens Culinaris ◽  
Target Gene ◽  
Gene Selection ◽  
Chitinase Gene ◽  
Pcr Analysis ◽  
Agrobacterium Strain ◽  
Half Strength ◽  
Selection Of

To investigate the integration of chitinase gene in lentil (Lens culinaris Medik.) namely, BARI masur-4 (BM-4), BARI masur-5 (BM-5) and BARI masur-6 (BM-6) through Agrobacterium-mediated genetic transformation was performed using Agrobacterium strain EHA 105 harboring bar (resistant to phosphinotrycin) and chitinase (gene of interest) gene. Selection of transformed shoots was carried out by gradually increasing the concentration of phosphinotrycin (PPT) up to 2.0 mg/l. Transgenic lentil shoots were produced with an overall frequency of 0.36 in case of BM-4 and BM-6 and 0.34 in case of BM-5, respectively. Most of the selected shoots developed in vitro flowers and pods following their sub-culture on half strength of MS supplemented with 20 mg/l IBA, 0.5 mg/l NAA with 50 mg/l ticarcillin. Seedlings germinated from the seeds were successfully transferred to soil for the development of further progeny. Stable integration of target gene was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 29(1): 99-109, 2019 (June)

scholarly journals Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 860
Author(s):  
Saba Haider ◽  
Yaohui Gao ◽  
Yike Gao
Keyword(s):  
Transgenic Plants ◽  
Genetic Transformation ◽  
Model Organism ◽  
Plant Morphology ◽  
Pcr Analysis ◽  
Transformation System ◽  
Transformation Protocol ◽  
Genetic Transformation System ◽  
Chrysanthemum X Morifolium

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

In vitro flowering and seed formation in lentil (Lens culinaris Medik.)

2012 ◽  
Vol 48 (5) ◽  
pp. 446-452 ◽  
Author(s):  
R. H. Sarker ◽  
Subroto K. Das ◽  
M. I. Hoque
Keyword(s):  
Lens Culinaris ◽  
In Vitro Flowering ◽  
Seed Formation

scholarly journals In vitro shoot regeneration of Stereospermum suaveolens DC. using cotyledonary node and nodal explants

2015 ◽  
Vol 24 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Darshini R Trivedi ◽  
Aruna G Joshi
Keyword(s):  
Woody Plant ◽  
Shoot Multiplication ◽  
Multiple Shoots ◽  
Cotyledonary Node ◽  
Nodal Explants ◽  
Shoot Number ◽  
Proliferation Response ◽  
Half Strength ◽  
In Vitro Shoot Regeneration

The cotyledonary node and nodal explants Stereospermum suaveolens DC. were cultured on Woody Plant medium (WPM) supplemented with individual BAP 2?25 ?M), Kn (2?25 ?M) and TDZ (0.1?2 ?M. Observations revealed that in both cotyledonary node and nodal explants, lower concentrations of cytokinins (BAP/Kn/TDZ) were effective in inducing proliferation response. The axillary bud of both the explants proliferated into single shoots which were further subcultured on fresh medium with respective concentrations for shoot multiplication. It was observed that multiple shoots developed only in presence of Kn (8 ?M) in both the explants but the shoots developed from nodal explants were strong and healthy with average shoot number reaching to 3.8 ± 0.6 and length 5.4 ± 0.4 cm by the end of third passage. Rooting (100%) was achieved in microshoots when transferred to half strength WPM liquid medium supplemented with IBA (2 ?M). The plantlets were transferred to different substrates for acclimatization.Plant Tissue Cult. & Biotech. 24(2): 235-246, 2014 (December

scholarly journals An Improved Protocol for Multiple Shoot Regeneration from Seedlings and Mature Explants of Citrus macroptera Mont.

2009 ◽  
Vol 18 (1) ◽  
pp. 17-24
Author(s):  
Md. Nesawar Miah ◽  
Shahina Islam ◽  
Syed Hadiuzzaman
Keyword(s):  
Shoot Regeneration ◽  
Multiple Shoots ◽  
Multiple Shoot ◽  
Nodal Explants ◽  
Cow Dung ◽  
Half Strength ◽  
Multiple Shoot Regeneration ◽  
Shoot Tip Explants ◽  
In Vitro Shoot Regeneration

Efforts have been made to establish a protocol for direct multiple shoot regeneration from both in vitro grown seedlings and mature plants of Citrus macroptera. Both nodal and shoot tip explants taken from in vitro grown seedlings were cultured in MS supplemented with different concentrations of BAP and Kn either singly or in combinations. Both these explants are capable to regenerate and produce in vitro multiple shoots. Maximum number of shoots were obtained from nodal explants in MS supplemented with 1.0 mg/l BAP. BAP alone was found superior to Kn. On the other hand, only nodal explants from mature plants were used and 1.0 mg/1 BAP was also found best suitable for shoot induction and multiplication. Ex vitro rooting in pot soil (mixed with biogas slurry derived from cow-dung) was most successful compared to in vitro rooting in half strength of MS supplemented with different concentrations of NAA and IBA. Key words: In vitro, Shoot regeneration, Citrus macroptera D.O.I. 10.3329/ptcb.v18i1.3246 Plant Tissue Cult. & Biotech. 18(1): 17-24, 2008 (June)

scholarly journals An Efficient Protocol for High-frequency Direct Multiple Shoot Regeneration from Internodes of Peppermint (Mentha x piperita)

2010 ◽  
Vol 5 (12) ◽  
pp. 1934578X1000501
Author(s):  
Sanjog T. Thul ◽  
Arun K. Kukreja
Keyword(s):  
Shoot Regeneration ◽  
Multiple Shoots ◽  
Shoot Elongation ◽  
Shoot Formation ◽  
Multiple Shoot ◽  
Ms Medium ◽  
Mentha X Piperita ◽  
Half Strength ◽  
Multiple Shoot Regeneration

A simple, repeatable and efficient protocol for direct multiple shoot regeneration from internodal explants has been defined in peppermint ( Mentha x piperita var. Indus). In vitro regenerated shoots of peppermint were excised into 4 to 8 mm long internodes and cultured on Murashige and Skoog's medium supplemented with different cytokinins. In the hormonal assay, 3.0 mg L-l zeatin or 6-isopentenyl adenine independently supplemented to half strength MS medium exhibited multiple shoot regeneration, while thiaduzorn (0.1-3.0 mg L−1) showed no morphogenetic effect. A maximum of 85% in vitro cultured explants showed multiple shoot formation with an average of 7 shoots per explant on MS medium supplemented with zeatin. Multiple shoots were initiated within three weeks of cultivation. Internodes with regenerated multiple shoots were transferred to half - strength MS medium without supplementing with any plant growth hormone for shoot elongation and rhizogenesis. Rooted plants acclimatized and grew to maturity under glasshouse conditions. The plantlets developed were phenotypically identical to the parent plant and exhibited 96 % survival.

scholarly journals In vitro Flowering of Shoots Regenerated from Cultured Nodal Explants

2011 ◽  
Vol 39 (1) ◽  
pp. 84 ◽  
Author(s):  
Kantamaht KANCHANAPOOM ◽  
Suttinee JINGJIT ◽  
Kamnoon KANCHANAPOOM
Keyword(s):  
Multiple Shoots ◽  
Shoot Formation ◽  
In Vitro Flowering ◽  
Nodal Explants ◽  
Callus Growth ◽  
Ms Medium ◽  
Old Field ◽  
Gypsophila Paniculata ◽  
Initial Culture

A protocol for the regeneration of Gypsophila paniculata L. using nodal explants from 2-month-old field grown plants was established. The induction of multiple shoots was best obtained on Murashige and Skoog (MS) medium supplemented with 13.3 μM BA. Callus growth was observed on MS medium containing 44.3 μM BA. Calluses were transferred to MS medium supplemented with 2, 4-D (4.5, 13.5, 22.6 μM), NAA (5.3, 16.1, 26.8 μM) or BA (4.4, 13.3, 22.1 μM) for 2 months to induce shoot formation. After 6 weeks of initial culture, multiple shoots were regenerated from calluses cultured on MS medium supplemented with 13.3 μM BA. All regenerated shoots produced roots on 16.1 μM NAA containing MS medium within 4 weeks. Rooted plantlets were hardened and established in pots at 100% survival. For induction of in vitro flowering, regenerated shoots could be induced to flower efficiently when cultured on MS medium containing 13.3 μM BA and 50 g/l sucrose.

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)

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