scholarly journals Agrobacterium-mediated Genetic Transformation of Rice var. BRRI Dhan 58

2021 ◽  
Vol 31 (1) ◽  
pp. 71-80
Author(s):  
Mst Sufara Akhter Banu ◽  
Bulbul Ahmed ◽  
Shahanaz Parveen ◽  
Md Harun Ur Rashid ◽  
Kazi Md Kamrul Huda
Keyword(s):  
Genetic Transformation ◽  
Rice Plant ◽  
Crop Improvement ◽  
Marker Genes ◽  
Hygromycin Resistance ◽  
Ms Medium ◽  
Gus Gene ◽  
Functional Studies ◽  
Regeneration Response ◽  
Modified Ms Medium

Agrobacterium mediated genetic transformation of BRRI Dhan 58 was conducted by using immature embryos following indirect regeneration. High percentage of callus induction at 96.5% was obtained when seeds of BRRI dhan 58 were cultured on modified MS medium supplemented with 2.5 mg/l 2, 4-D under dark condition. The maximum regeneration response was rerecorded when MS was supplemented with 3 mg/l BAP + 0.5 mg/l NAA and 1.0 mg/l Kn. Genetic transformation was performed using A. tumefaciens strain LBA4404 harboring pCAMBIA1301 plasmid carrying the marker genes for β-glucuronidase (GUS) and hygromycin resistance (hptII). Integration of the GUS gene into the genome of the rice plants was confirmed by PCR. The leaf segments of the PCR positive transformed plants showed the expression of GUS. The results of this study would be an effective tool for crop improvement and functional studies of gene on rice plant. Plant Tissue Cult. & Biotech. 31(1): 71-80, 2021 (June)

scholarly journals Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

Agronomy ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1104
Author(s):  
Ritika Bhatt ◽  
Prem Prakash Asopa ◽  
Rohit Jain ◽  
Aditi Kothari-Chajer ◽  
SL Kothari ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Transformation Efficiency ◽  
Normal Growth ◽  
Induction Medium ◽  
Ms Medium ◽  
Gus Gene ◽  
Cultivation Medium ◽  
Kodo Millet

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM EHA 105, 250 µM–LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

2021 ◽  
Author(s):  
Aparna Priyadarshini Patra ◽  
Kailash Chandra Samal ◽  
Gyana Ranjan Rout ◽  
Simachal Sahu ◽  
Prem Narayan Jagadev
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Vigna Radiata ◽  
In Vitro Regeneration ◽  
Cotyledonary Node ◽  
Green Gram ◽  
Grain Legume ◽  
Marker Genes ◽  
Soil Productivity ◽  
Ms Medium

Background: Green gram is grown in many parts of India as a source of dietary protein (21-25%). It is an important nitrogen fixing crop which fixes atmospheric nitrogen (119-140 kg/ha) to soil and enhance the soil productivity. In the present investigation, efficient Agrobacterium-mediated genetic transformation of Vigna radiata L. (Wilczek) has been achieved with VrTIP1 gene for abiotic stress resistance i.e. moisture and salinity stress. Methods: Four days old shoot tip and cotyledonary node were used for in vitro regeneration with MS medium supplemented with BAP 2.0 mg/l, kinetin 0.5 mg/l and 50 mg/l kanamycin for co-cultivation with Agrobacterium tumefaciens strains, LBA 4404. The modified binary vector pCXSN, EHA105 containing hygromycin phosphotransferase II (hpt II) marker genes and a synthetic TIP1 gene under a constitutive CaMV35S promoter were used for transformation of Vigna radiata L. cotyledonary node explants. Putative transformants selected from hygromycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg/l NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. Result: The T1 plants were produced from PCR positive T0 plants and analysed for presence and integration of transgenes in putative T1 plants were confirmed by polymerase chain reaction (PCR) amplification of 752 bp of hpt II fragment. This protocol can be effectively used for transferring new traits in greengram and other legumes for their quantitative and qualitative improvements.

scholarly journals Genetic transformation of Eucalyptus camaldulensis by agrobalistic method

2013 ◽  
Vol 37 (3) ◽  
pp. 419-429 ◽  
Author(s):  
Evânia Galvão Mendonça ◽  
Vanessa Cristina Stein ◽  
Flávia Pereira Balieiro ◽  
Carolina Delfin Fernandes Lima ◽  
Breno Régis Santos ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transient Expression ◽  
Culture Media ◽  
Tissue Injury ◽  
Callus Formation ◽  
Nitrogen Concentration ◽  
Eucalyptus Camaldulensis ◽  
Ms Medium ◽  
Gus Gene

Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1) and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2). To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun), to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS), for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes). The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained.

scholarly journals Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus

2020 ◽  
Vol 11 ◽  
Author(s):  
Yue Song ◽  
Xiaoming Bai ◽  
Shiwei Dong ◽  
Yuning Yang ◽  
Hao Dong ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Improvement ◽  
Populus Trichocarpa ◽  
Cambial Activity ◽  
Transformation Method ◽  
Gus Gene ◽  
Specific Expression ◽  
Functional Studies ◽  
Coniferous Tree Species ◽  
Short Time

Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and β-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies.

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 Application of Chondroitin Sulfate on Organogenesis of Two Cymbidium spp. under Different Sources of Lights

2016 ◽  
Vol 8 (2) ◽  
pp. 156-160 ◽  
Author(s):  
Syeda Jabun NAHAR ◽  
Syed M. HAQUE ◽  
Shimasaki KAZUHIKO
Keyword(s):  
Plant Growth ◽  
Chondroitin Sulfate ◽  
Growth Regulator ◽  
Plant Growth Regulator ◽  
Ms Medium ◽  
Food Ingredients ◽  
Blue Led ◽  
Red Led ◽  
Green Led ◽  
Modified Ms Medium

The aim of this study was to present chondroitin sulfate as a plant growth regulator and to give an overview about light effects on PLBs (protocorm like bodies) culture of Cymbidium dayanum and Cymbidium finlaysonianum cultured in vitro. Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars N-acetylgalactosamine and glucuronic acid. It is widely used as a material for food ingredients, cosmetics and medicine. PLBs were cultured on modified MS medium containing different concentration of chondroitin sulfate (0, 0.1, 1 and 10 mg/l), under four sources of lights: conventional white fluorescent tube, red LED, green LED and blue LED. In C. dayanum, 100% PLBs formation rate was observed at 0.1 mg/l chondroitin sulfate with modified MS medium under green LED and 1 mg/l chondroitin sulfate under blue LED; the maximum shoots and roots formation were observed under green LEDs (93% and 80% respectively) when media contained 0.1 mg/l chondroitin sulfate. In C. finlaysonianum, every concentrations of chondroitin sulfate enhanced the growth rate of PLBs when compared to control treatment, under all four sources of lights. The highest values were recorded with 0.1 mg/l chondroitin sulfate which induced 100% PLBs formation under blue LED, while 10 mg/l chondroitin sulfate had induced 100% PLBs formation under green LED. The highest percentage of shoots (73%) was initiated in the medium containing 10 mg/l chondroitin sulfate under green LED. Plant development was strongly influenced by the light quality and plant growth regulator functions as chemical messengers for intercellular communication of plant. The results demonstrated that low concentrations of chondroitin sulfate could promote PLBs, shoots and roots formation of Cymbidium spp. under green and blue LED.

scholarly journals Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Agronomy ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 179
Author(s):  
Tanika Thakur ◽  
Kshitija Sinha ◽  
Tushpinder Kaur ◽  
Ritu Kapoor ◽  
Gulshan Kumar ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Crop Improvement ◽  
Rice Cultivar ◽  
Climatic Conditions ◽  
Rice Transformation ◽  
Transformation Protocol ◽  
Galactinol Synthase ◽  
Biotic Stresses ◽  
Putative Transformants ◽  
Efficient Genetic Transformation

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

scholarly journals Genetic Transformation of Apomictic Grasses: Progress and Constraints

2021 ◽  
Vol 12 ◽  
Author(s):  
Andrés M. Bellido ◽  
Eduado D. Souza Canadá ◽  
Hugo R. Permingeat ◽  
Viviana Echenique
Keyword(s):  
Genetic Transformation ◽  
Plant Transformation ◽  
Large Scale ◽  
Crop Improvement ◽  
Current Status ◽  
Grass Species ◽  
Brachiaria Brizantha ◽  
Apomictic Species ◽  
Transformation Methods

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

scholarly journals Agrobacterium tumefaciens-mediated Transformation of Embryogenic Callus and Somatic Embryos of the Banana cv “Ambon Lumut” (Musa acuminata)

2017 ◽  
Vol 2 (6) ◽  
pp. 599 ◽  
Author(s):  
Tifa R. Kusumastuti ◽  
Rizkita R. Esyantia ◽  
Fenny M. Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Crop Improvement ◽  
Pcr Analysis ◽  
Abiotic Factor ◽  
Gfp Gene ◽  
Culture Transformation ◽  
Biotechnological Approach

Banana is one of the major fruit crops, though its conventional breeding has limitations, such as sterility and high polyploidy  levels.  Biotechnological  approach  using genetic  transformation  crop for improvement  offers  an alternative  solution.  In  this  study  a  protocol  was developed  for  establishing genetic  transformation  from embryogenic callus and somatic embryos of the banana cv Ambon Lumut . Embryogenic callus was obtained in ID4 medium (MS-based medium) supplemented with 1 mg L-1 IAA, 4 mg L-1 2,4D, and 0.03 g L-1 active charcoal. Embryogenic callus was transferred into liquid mediu m to establish somatic embryos. Embryogenic callus and somatic embryos were used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain A GL1, containing pART-TEST7 p lasmid with gfp gene as a reporter and CaM V35S as a promoter, was used for transformations. The embryogenic callus and somatic embryos were transformed using heat-shock method followed by centrifugation  (2000 rpm) and co-cult ivation in liquid medium containing acetosyringone (100 M) for 3 days. Results of the GFP analysis showed transient expression from gfp gene reporter in transformed embryogenic callus and somatic embryos. Transformation efficiency in somatic embryos (85,9%) was higher than  that in embryogenic callus (32.09%). PCR analysis using CaMV primer showed bands that compatible with CaMV35S promoter at 507 bp. This is a report showing establisment of embryogenic callus and somatic embryo culture transformation by using A. tumefaciens-mediated transformation protocol of the local banana cv Ambon Lumut. This study proved  the huge potential for genetic transformation of banana cv Ambon Lumut for crop improvement, such as pest or disease  resistance and abiotic factor stress tolerance. Keywords: banana; embryogenic callus; somatic embryos.

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