scholarly journals Developing a rapid and highly efficient cowpea regeneration and transformation system using embryonic axis explants

2019 ◽  
Author(s):  
Ping Che ◽  
Shujun Chang ◽  
Marissa K. Simon ◽  
Zhifen Zhang ◽  
Ahmed Shaharyar ◽  
...  
Keyword(s):  
Cotyledonary Node ◽  
Multiple Shoot ◽  
Embryonic Axis ◽  
Sub Saharan Africa ◽  
Vector System ◽  
Transformation System ◽  
Highly Efficient ◽  
Cotyledonary Nodes ◽  
Wide Range

SummaryCowpea is one of the most important legume crops planted worldwide, especially in Sub-Saharan Africa and Asia. Despite decades of effort, genetic engineering of cowpea is still challenging due to inefficient in vitro shoot regeneration, Agrobacterium-mediated T-DNA delivery and transgenic selection. Here, we report a rapid and highly efficient cowpea transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem by cutting through the middle of the epicotyl stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. Furthermore, the application of a ternary transformation vector system using an optimized pVIR accessory plasmid provided high levels of Agrobacterium-mediated gene delivery. The utilization of spectinomycin as the selection agent enabled more efficient transgenic selection and plant recovery. Transgenic cowpea shoots developed exclusively from the cotyledonary nodes at high frequencies of 4.5 to 37% across a wide range of cowpea genotypes. We believe that the transformation principles established in this study could also be applied to other legumes to increase transformation efficiencies.

scholarly journals Regeneration and In Vitro Flowering in Brassica Campestris (L.) Var. Bhavani

Our Nature ◽  
1970 ◽  
Vol 5 (1) ◽  
pp. 21-24 ◽  
Author(s):  
Rinki Verma ◽  
RR Singh
Keyword(s):  
Shoot Apex ◽  
Brassica Campestris ◽  
Cotyledonary Node ◽  
Shoot Formation ◽  
Multiple Shoot ◽  
In Vitro Flowering ◽  
Cotyledonary Nodes ◽  
Improvement Programs ◽  
Shoot Apex Explants

Multiple shoot formation and in vitro flowering was found in Brassica campestris (L.) var. Bhavani. Maximum numbers of shoots were produced in both cotyledonary node and shoot apex explants on MS-media supplemented with BA (2.5 mg/l) + IAA (1.0 mg/l) + Kn (0.5 mg/l). Maximum flowering (50%) was noted at IBA (1.5 mg/l) + IAA (1.0 mg/l) + Kn (0.5 mg/l) in the shoots from cotyledonary nodes. In vitro flowering may contribute in many ways to Brassica Improvement Programs. The shoots rooted well in the half and full strength media each with IBA (1.0 mg/l) and NAA (1.0 mg/l) and the plantlets have been maintained. Keywords: Brassica campestris, In vitro flowering, Regeneration.doi:10.3126/on.v5i1.793Our Nature (2007)5:21-24

scholarly journals In vitro propagation of muskmelon (Cucumis melo L.) from nodal segments, shoot tips and cotyledonary nodes

2015 ◽  
Vol 41 ◽  
pp. 71-77
Author(s):  
S. Parvin ◽  
M. Kausar ◽  
M. Enamul Haque ◽  
M. Khalekuzzaman ◽  
B. Sikdar ◽  
...  
Keyword(s):  
Cucumis Melo ◽  
In Vitro Propagation ◽  
Multiple Shoots ◽  
Multiple Shoot ◽  
Shoot Tips ◽  
Nodal Segments ◽  
Ms Medium ◽  
Cotyledonary Nodes ◽  
Cucumis Melo L

A rapid and efficient protocol is outlined for in vitro propagation of muskmelon(Cucumis melo L.) Shoot tips, nodal segments and cotyledonary nodes from invitro grown seedlings were used as explants. The explants were inoculated on MS medium fortified with different combinations and concentrations of growthregulators viz., BAP, NAA, GA3 and IBA for multiple shoot regeneration.Effective result was found on MS medium supplemented with 2.0 mg/l BAP, inwhich 90% and 70% cultures induced multiple shoots from nodal segments andshoot tip explants, respectively. Whereas, 70% cultures of cotyledonary nodeswere found to induced shoots on MS medium with 1.5 mg/l BAP + 0.1 mg/l GA3. In vitro regenerated shoots were subcultured on half strength MS mediumsupplemented with different concentrations of IBA and NAA for successful rootinduction and the effective result (up to 70%) was found in medium with 1 mg/lIBA. Well rooted in vitro grown plantlets were acclimatized in sandy soil, whereas 70% plantlets survived

scholarly journals CLIPB10 is a Terminal Protease in the Regulatory Network That Controls Melanization in the African Malaria Mosquito Anopheles gambiae

2021 ◽  
Vol 10 ◽  
Author(s):  
Xin Zhang ◽  
Miao Li ◽  
Layla El Moussawi ◽  
Sally Saab ◽  
Shasha Zhang ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Serine Proteases ◽  
Ex Vivo ◽  
Protein A ◽  
Tumor Formation ◽  
Sub Saharan Africa ◽  
Humoral Immune ◽  
Wide Range ◽  
Sub Saharan

Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.

scholarly journals In vitro Micropropagation of Plumbago indica L. Through Induction of Direct and Indirect Organogenesis

1970 ◽  
Vol 19 (2) ◽  
pp. 169-175 ◽  
Author(s):  
S. K. Bhadra ◽  
T. Akhter ◽  
M. M. Hossain
Keyword(s):  
Multiple Shoot ◽  
Nodal Segments ◽  
Old Field ◽  
Indirect Organogenesis ◽  
Shoot Buds ◽  
Wide Range ◽  
Plumbago Indica ◽  
Multiple Shoot Buds ◽  
The Media

Leaf and nodal segments of two months old field grown seedlings of Plumbago indica L. were cultured on agar solidified MS supplemented with different concentrations and combinations of NAA, IAA, 2,4-D and picloram, and BAP and Kn. The nodal segments produced either multiple shoot buds (MSBs) or callus of different nature depending on the combinations of plant growth regulators (PGRs). The callus of light green and nodular shape, on further subculture on wide range of PGRs supplemented media, differentiated into MSBs. These MSBs underwent rapid elongation on different PGRs supplemented media. The elongated shoot buds were rooted on transferring in rooting medium and were acclimated in field with 90% survivability. The leaf segments, however, produced only white and friable calluses failed to undergo any differentiation in some of the media combinations.  Key words: Callus induction, Organogenesis, Plumbago indica D.O.I. 10.3329/ptcb.v19i2.5434 Plant Tissue Cult. & Biotech. 19(2): 169-175, 2009 (December)

scholarly journals An efficient shoot regeneration system for medicinally important Elephantopus scaber Linn.

2015 ◽  
Vol 15 (2) ◽  
pp. 94-99 ◽  
Author(s):  
Jyothi Abraham ◽  
T. Dennis Thomas
Keyword(s):  
Age Groups ◽  
Cotyledonary Node ◽  
Multiple Shoot ◽  
Ms Medium ◽  
Shoot Induction ◽  
Regeneration System ◽  
Multiple Shoot Induction ◽  
Elephantopus Scaber ◽  
Cotyledonary Node Explants

An efficient protocol for the rapid micropropagation of medicinally important Elephantopus scaber has been standardized using cotyledonary node explants. Direct multiple shoot induction was observed when the cotyledonary node explants at various age groups were cultured on MS medium supplemented with various plant growth regulators. The highest shoot induction was obtained when the cotyledonary node explants from 20-day-old seedlings were cultured on MS medium supplemented with 1.5 mg L-1 TDZ and 0.5 mg L-1 NAA. On this medium, 98% of the cultures responded, with an average number of 33.7 shoots per explant. The highest frequency of rooting (100%) and mean number of roots (3.3 per shoot) were observed when the shoots were transferred to MS medium supplemented with 1.0 mg L-1 IBA. The plantlets raised in vitro were acclimatized and transferred to soil with a 92% success rate. The protocol described here may be utilized for multiplication and conservation of elite clones of E. scaber.

scholarly journals Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyung Min Jung ◽  
Young Min Kim ◽  
Jin Lee Kim ◽  
Jae Yong Han
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Zebra Finch ◽  
High Efficiency ◽  
Primordial Germ Cell ◽  
Vector System ◽  
Efficient Gene ◽  
Wide Range

AbstractZebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.

scholarly journals SUPERIOR REGENERATION AND AGROBACTERIUM INFECTABILITY OF BROCCOLI AND CAULIFLOWER TISSUES AND THE IDENTIFICATION OF A PROCEDURE FOR THE GENERATION OF TRANSGENIC PLANTS.

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 620f-621 ◽  
Author(s):  
Wendy J. Wagoner ◽  
Jill A. Kellogg ◽  
Richard K. Bestwick ◽  
James A Stamp
Keyword(s):  
Transgenic Plants ◽  
High Frequency ◽  
Binary Vector ◽  
Ethylene Biosynthesis ◽  
Vector System ◽  
Wild Type ◽  
Gene Encoding ◽  
Wide Range ◽  
In Vitro Response

Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head. Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.

scholarly journals A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries

2010 ◽  
Vol 84 (14) ◽  
pp. 7360-7368 ◽  
Author(s):  
Darren Wolfe ◽  
April M. Craft ◽  
Justus B. Cohen ◽  
Joseph C. Glorioso
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Vector ◽  
Vector System ◽  
Cellular Functions ◽  
Recombination System ◽  
Wide Range ◽  
Simplex Virus ◽  
Expression Libraries

ABSTRACT Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.

scholarly journals Utilization of Aseptic Seedling Explants for In vitro Propagation of Indian Red Wood

2013 ◽  
Vol 5 (4) ◽  
pp. 518-523 ◽  
Author(s):  
Kishore Kumar CHIRUVELLA ◽  
Arifullah MOHAMMED ◽  
Rama Gopal GHANTA
Keyword(s):  
Large Scale ◽  
Ex Situ Conservation ◽  
Cotyledonary Node ◽  
Multiple Shoot ◽  
Shoot Tip ◽  
Ex Situ ◽  
Nodal Segments ◽  
Ms Medium ◽  
Shoot Differentiation

Micropropagation has been advocated as one of the most viable biotechnological tool for ex situ conservation of rare, endangered endemic medicinal plants germplasm. Rapid clonal micropropagation protocol for large-scale multiplication of an endemic medicinal plant Soymida febrifuga (Meliaceae) was established from 15-day aseptic seedling cotyledonary node and shoot tip explants. High frequency of sprouting and shoot differentiation was observed from cotyledonary node explants compared to shoot tip, on Murashige and Skoog (MS) medium fortified with BA, KN, 2-iP and CM. Of the cytokinins used, BA (3.0 mgl-1) supported highest average number and maximum multiple shoot differentiation (16.6). In vitro proliferated shoots were multiplied rapidly by culturing nodal segments as microcuttings, further subcultured on the same media for elongation. Elongated shoots upon transfer to MS medium fortified with IBA showed rooting within two weeks of culture. Rooted plantlets were successfully hardened and 75% of rooted shoots successfully survived on establishment to the soil. Plants looked healthy with no visually detectable phenotypic variations. This protocol provides a successful and rapid technique that can be used for ex situ conservation minimizing the pressure on wild populations and contributes to the conservation of this endemic medicinally potent flora.

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