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 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 Direct Organogenesis from Rhizome Explants in Marsilea quadrifolia L.: A Threatened Fern Species

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Mahipal S. Shekhawat ◽  
M. Manokari
Keyword(s):  
Multiple Shoots ◽  
Direct Organogenesis ◽  
Root Induction ◽  
Ms Medium ◽  
Full Strength ◽  
Marsilea Quadrifolia ◽  
Fern Species ◽  
Half Strength

An efficient micropropagation protocol has been developed for Marsilea quadrifolia L. through direct organogenesis. The mature rhizomes were used as explants and successfully sterilized using 0.1% HgCl2 for the establishment of cultures. The multiple shoots were differentiated from the explants on Murashige and Skoog (MS) medium augmented with 6-benzylaminopurin (BAP). Full strength MS medium was reported to be effective for the induction of sporophytes from the rhizomes after four weeks of inoculation. Maximum response (96%) with average of 6.2 shoots (2.72 cm length) was achieved on full strength of MS medium augmented with 0.5 mg/L BAP in culture initiation experiments. The cultures were further proliferated in clusters (79.0±0.37 shoots per explant) with stunted growth on half strength MS medium supplemented with 0.25 mg/L BAP after four weeks. These stunted shoots were elongated (5.30 cm long) on half MS medium devoid of growth hormones. Root induction and proliferation (3.0–4.0 cm long) were observed after 4th subculture of sporophytes on hormone-free half strength MS medium. The rooted plantlets were hardened in the fern house for 4-5 weeks and transferred to the field with 92% survival rate. There were no observable differences in between in vivo grown and in vitro propagated plantlets in the field.

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 a Cultivated Potato Variety Using Marker Genes

2020 ◽  
Vol 30 (1) ◽  
pp. 149-160
Author(s):  
Sanjida Rahman Mollika ◽  
RH Sarker ◽  
MI Hoque
Keyword(s):  
Genetic Transformation ◽  
Potato Variety ◽  
Nodal Segments ◽  
Direct Organogenesis ◽  
Pcr Analysis ◽  
Bacterial Suspension ◽  
Marker Genes ◽  
Nodal Segment ◽  
Root Induction

Agrobacterium-mediated genetic transformation was carried out for Asterix (BARI Alu- 25), a popular potato (Solanum tuberosum L.) variety cultivated in Bangladesh. For Direct organogenesis of shoots the best response was noted when nodal segments and microtuber discs of Asterix along with Diamant - another popular potato variety were cultured on MS with 4.0 mg/l BAP and 1.0 mg/l IAA. MS without plant growth regulators was most effective for root induction from the excised regenerated shoots. Following optimum root development, the in vitro regenerated plantlets were successfully established in soil. Agrobacterium tumefaciens strain LBA4404/pBI121 containing GUS and nptII genes showed maximum transformation response in nodal segment with bacterial suspension having an optical density of 0.6 at 600 nm in Asterix variety. Moreover, 30 min incubation followed by 72 hrs co-cultivation was found most effective for transformation as has been determined by transient GUS histochemical assay. Transformed shoots were selected using MS with 4.0 mg/l BAP, 1.0 mg/l IAA, 0.5 mg/l GA3, 300 mg/l carbenicillin and 200 mg/l kanamycin. Stable integration of GUS and nptII genes were confirmed by PCR analysis using the genomic DNA isolated from transformed shoots. Plant Tissue Cult. & Biotech. 30(1): 149-160, 2020 (June)

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 of Phyllanthus amarus Schum. and Thonn.: An Important Medicinal Plant

Our Nature ◽  
1970 ◽  
Vol 7 (1) ◽  
pp. 110-115 ◽  
Author(s):  
A. Sen ◽  
M.M. Sharma ◽  
D. Grover ◽  
A. Batra
Keyword(s):  
In Vitro Regeneration ◽  
Multiple Shoots ◽  
Shoot Elongation ◽  
Nodal Segments ◽  
Nodal Segment ◽  
Phyllanthus Amarus ◽  
Regenerated Plants ◽  
Half Strength ◽  
Important Medicinal Plant

An efficient in vitro plant regeneration protocol was developed for the medicinally potent plant species Phyllanthus amarus Schum. and Thonn. (Euphorbiaceae) using nodal segment as explant. Maximum multiplication of shoots (15.275±0.96) was achieved on Murashige and Skoog’s medium supplemented with BAP (0.5 mg/l) after 3-4 weeks of inoculation. The shoots were separated from cluster and subcultured for their elongation on the same medium. In vitro flowering was also observed on the elongated shoots after 3–4 weeks of sub culturing on the shoot elongation medium. In vitro rooting was obtained on half strength MS medium supplemented with IBA (0.5 mg/l).  Regenerated plants were successfully hardened and acclimatized, 80 % of plantlets survived well under natural conditions after transplantation.Key words: In vitro regeneration, multiple shoots, nodal segments, Phyllanthus amarusDOI: 10.3126/on.v7i1.2557Our Nature (2009) 7:110-115

scholarly journals An efficient in vitro regeneration protocol to generate stable transgenic lines of Black gram (Vigna mungo)

2018 ◽  
Vol 78 (3) ◽  
Author(s):  
Bidyut Kumar Sarmah ◽  
Trishna Konwar ◽  
Borsha Borah ◽  
Arun Kumar Handique ◽  
Sumita Acharjee
Keyword(s):  
Transformation Efficiency ◽  
In Vitro Regeneration ◽  
Multiple Shoots ◽  
Vigna Mungo ◽  
Ms Medium ◽  
Black Gram ◽  
Transgenic Lines ◽  
Half Strength ◽  
The Individual

An efficient and quick in vitro regeneration protocol was developed for black gram (Vigna mungo) using wounded embryonic axis with cotyledon as explant. Murashige and Skoog (MS) medium supplemented with 4.44 μM BAP and 2.32 μM Kinetin was found to be effective in producing maximum number (mean 7.80) of multiple shoots. The individual shoots elongated to 4.5 cm when MS medium was supplemented with 2.89 μM GA3 along with 0.44 μM BAP and 0.46 μM KIN. A novel in vitro rooting technique was also optimized for black gram using half-strength liquid MS medium supplemented with 1.34 μM NAA. The shoots in this medium produced the highest number (mean 7.50) of roots with root length of 6.02 cm. The plantlets were transferred to soil mixture and placed in greenhouse where more than 80% successfully grew to maturity. The same protocol was successfully used to generate transgenic black gram lines carrying Bt-Cry2Aa gene through Agrobacteriummediated transformation with a transformation efficiency of 0.42%. The rooted T0 plants grew to maturity and produced T1 seeds with the presence and expression of transgene in T1 plants. Thus, we have standardized an in vitro regeneration protocol suitable for generation of stable transgenic black gram plants.

scholarly journals In vitro regeneration of two high-yielding eggplant (Solanum melongena L.) varieties of Bangladesh

1970 ◽  
pp. 08-12
Author(s):  
Sabina Yesmin, Mst Muslima Khatun, Tanzena Tanny ◽  
Anica Tasnim Protity ◽  
Md Salimullah ◽  
Iftekhar Alam
Keyword(s):  
In Vitro Regeneration ◽  
Solanum Melongena ◽  
Multiple Shoots ◽  
Multiple Shoot ◽  
Healthy Plant ◽  
Ms Medium ◽  
Best Response ◽  
Half Strength ◽  
Multiple Shoot Regeneration

An in vitro regeneration protocol was developed for two high-yielding eggplant varieties (Solanum melongena L.) namely BARI begun-4 and BARI begun-6. Multiple shoots were regenerated from cotyledonary explants through organogenesis with growth regulators of different combinations and concentrations.  The best response towards multiple shoot regeneration was achieved from cotyledon explants on MS media complemented with 1 mg/l BAP + 0.2 mg/l IAA in both the two varieties of eggplant. Elongation of shoots was achieved on hormone free MS medium. Regenerated shoots of both the varieties produced   active in vitro root system on half strength of MS medium supplemented with 0.2 mg/l IBA.  The in vitro grown plantlets were acclimatized in soil, grew up to maturity, flowered, fruited and produced seeds as normal healthy plant like the control.

In Vitro Regeneration of Valencia-type Peanut (Arachis hypogaea L.) from Cultured Petiolules, Epicotyl Sections and Other Seedling Explants1

1992 ◽  
Vol 19 (2) ◽  
pp. 82-87 ◽  
Author(s):  
Ming Cheng ◽  
David C. H. Hsi ◽  
Gregory C. Phillips
Keyword(s):  
In Vitro Regeneration ◽  
Multiple Shoots ◽  
Shoot Formation ◽  
Direct Organogenesis ◽  
Naphthaleneacetic Acid ◽  
Regeneration System ◽  
Arachis Hypogaea L ◽  
Wide Range ◽  
Cultivated Peanut

Abstract This study evaluated plant development via direct organogenesis from in vitro-cultured young seedling tissues of cultivated peanut, especially the valencia-type peanut. Complete plants were regenerated from in vitro-cultured petiolule-with-blade-attached explants, leaflet segments, and epicotyl andpetiole sections. Multiple shoots arose on Murashige and Skoog medium (MS) supplemented with 6-benzylaminopurine (BA) (5–25 mg/L) plus 1-naphthaleneacetic acid (NAA) (0.5–3 mg/L). After 30 d culture on 25 mg/L BA + 1 mg/L NAA, 1.6 buds or shoots/explant were regenerated from the petiolule-with-blade-attached explants. Comparable numbers of shoots were obtained from epicotyl sections of the first node region of the seedling after 60 d culture using 10 mg/L BA + 1 mg/L NAA. Leaflet segments and petiole sections were less responsive for shoot formation. Excised shoots developed roots in vitro upon transfer for 15 d to MS medium supplemented with NAA at 1 mg/L. Plantlets were transferred to soil and grown in a greenhouse to maturity. A wide range of cultivated peanut genotypes was evaluated for organogenic responsiveness, using the petiolule-with-blade-attached explant source. Only valencia-type cultivars, or a hybrid derivative with a Valencia background, were responsive with this regeneration system.

scholarly journals High Frequency In vitro Regeneration of Gynura procumbens (Lour.) Merr.

2017 ◽  
Vol 27 (2) ◽  
pp. 207-216
Author(s):  
Tanjina Akhtar Banu ◽  
Barna Goswami ◽  
Shahina Akter ◽  
Mousona Islam ◽  
Tammana Tanjin ◽  
...  
Keyword(s):  
In Vitro Regeneration ◽  
Leaf Explants ◽  
Multiple Shoots ◽  
Multiple Shoot ◽  
Nodal Segments ◽  
Nodal Segment ◽  
Root Induction ◽  
Petiole Explants ◽  
Number Of Shoots

An efficient rapid in vitro regeneration protocol was described from nodal segment, leaf and petiole explants. MS medium supplemented with 1.0 mg/l BAP and 0.5 mg/l IAA was found best for the multiple shoot formation from nodal segments. In this combination 99% explants produced multiple shoots and the average number of shoots per explants was 20.1 ± 1.96. For petiole and leaf explants best response was observed on MS supplemented with 2.0 mg/l BAP, 1 mg/l IAA and 0.5 mg/l Kn. Petiole explants produced highest mean number of shoots/explant (22.9 ± 1.728) among the three explants when the explants were cultured on MS with 2.0 mg/l BAP, 1 mg/l IAA and 0.5 mg/l Kn. The highest frequency of root induction (100%) and mean number of roots/plantlets (11.75) were obtained on MS. The rooted plantlets were transferred for hardening following acclimatization and finally were successfully established in the field.Plant Tissue Cult. & Biotech. 27(2): 207-216, 2017 (December)

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