Effect of biotic factors on the isolation of Lupinus albus protoplasts

2003 ◽  
Vol 51 (1) ◽  
pp. 103 ◽  
Author(s):  
Anupam Sinha ◽  
Andrew C. Wetten ◽  
P. D. S. Caligari
Keyword(s):  
Lupinus Albus ◽  
Protoplast Isolation ◽  
Biotic Factors ◽  
Average Yield ◽  
Fresh Tissue ◽  
Progressive Decline ◽  
Protoplast Yield ◽  
Protoplast Production ◽  
Protoplast Viability

Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.

scholarly journals Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

2012 ◽  
Vol 40 (2) ◽  
pp. 123 ◽  
Author(s):  
Esmaeil CHAMANI ◽  
Seyyed Karim TAHAMI ◽  
Nasser ZARE ◽  
Rasool Asghari-ZAKARIA ◽  
Mehdi MOHEBODINI ◽  
...  
Keyword(s):  
Analysis Of Variance ◽  
Treatment Time ◽  
Leaf Explants ◽  
Protoplast Isolation ◽  
Combination Method ◽  
Interspecific Incompatibility ◽  
Protoplast Yield ◽  
Protoplast Preparation ◽  
Protoplast Production ◽  
Protoplast Viability

For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant withdesired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluatevarious combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Liliumledebeourii Bioss. The results of experiment revealed that the protoplast yield was significantly affected by different treatment levels.Cellulase at 4% gave the highest numbers of protoplasts at 3.71×105 protoplast/g FW. Pectinase at 1% gave the highest numbers ofprotoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated thatconcentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at0.2% for 24 h gave the highest viability. Interactions of cellulase × pectinase, cellulase × time, pectinase × time and cellulase × pectinase× treatment time were significant at P≤0.05 for protoplast number. The highest and lowest protoplast numbers were produced in mediacontaining 4% cellulase and 1% pectinase for 24 h (6.65×105 protoplast/g FW) and 1% cellulase and 0.2% pectinase for 12 h, respectively.It’s concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.

scholarly journals Effects of environmental preconditioning, donor tissue and isolation conditions on tomato (Lycopersicon esculentum Mill.) protoplast yield

2013 ◽  
Vol 48 (2) ◽  
pp. 113-120
Author(s):  
Elżbieta Kuźniak ◽  
Marzena Wielanek ◽  
Urszula Małolepsza ◽  
Henryk Urbaniak
Keyword(s):  
Bovine Serum Albumin ◽  
Protoplast Isolation ◽  
Isolation Procedure ◽  
Low Light ◽  
Cold Pretreatment ◽  
Isolation Medium ◽  
Donor Tissue ◽  
Protoplast Yield ◽  
Pretreatment Conditions

The effects of soil or <em>in vitro</em> grown plants, pretreatment conditions, donor tissue and isolation procedure on protoplast yield from cotyledons and leaves of tomato cv. 'Perkoz' and 'Zorza' were studied. The highest protoplast yield of 1.5 x 10<sup>7</sup>/g FW was obtained from leaves of <em>in vitro</em> grown plants. Low light intensity during donor plants <em>in vitro</em> culture and dark pretreatment were essential for successful protoplast isolation while cold pretreatment was not. Tissue preplasmolysis prior to transfer to enzyme mixture increased 4-fold the number of isolated protoplasts. Glycine and bovine serum albumin in the isolation medium did not significantly influence the protoplast yield.

Aspects of isolation underpinning mitotic behaviour in lupin protoplasts

2004 ◽  
Vol 52 (5) ◽  
pp. 669 ◽  
Author(s):  
Anupam Sinha ◽  
Peter D. S. Caligari
Keyword(s):  
Protoplast Culture ◽  
Lupinus Albus ◽  
Maturity Level ◽  
Developmental Age ◽  
Early Maturing ◽  
Division Frequency ◽  
Tissue Maceration ◽  
Protoplast Division ◽  
Protoplast Production

This study reports on the influence of critical isolation factors on the subsequent culture of protoplasts of Lupinus albus L. Protoplasts were isolated from in vitro seedling cotyledons of five early maturing accessions in which protoplast yields and division frequencies appeared to be correlated as a high protoplast yield corresponded with a high division frequency. The overall difference among the accessions for mitosis was non-significant, although the highest yield and division frequency were observed in accession LA132, with Alban giving a significantly lower level. Accession Lucrop produced the lowest number of protoplasts, all of which collapsed during culture. Of the enzyme types used for tissue maceration, Pectolyase Y23, was significantly inferior to Macerase in terms of giving way to mitosis. The extent of division in Macerase-isolated protoplast population was 266% higher than that in the Pectolyase Y23-isolated one. The physiological maturity level of the explant, expressed in terms of developmental age, was optimal when 14–18-day-old seedling cotyledons were used for protoplast production and culture, rather than more mature ones, despite higher protoplast yields in the latter. On K8p medium, the protoplast division frequency was 129% greater when 18-day-old seedling cotyledons were used, than that with any other treatment. This work on protoplast culture of the potentially important lupin species, which is a pulse rich in dietary protein, oil and fibre, allows a further understanding of the biology, with an aim to advance lupin biotechnology.

Regulation of hepatic triiodothyronine production in the streptozotocin-induced diabetic rat

1984 ◽  
Vol 247 (4) ◽  
pp. E526-E533
Author(s):  
A. S. Jennings
Keyword(s):  
Diabetic Rats ◽  
Diabetic Rat ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Insulin Administration ◽  
Progressive Decline ◽  
Serum T4 ◽  
Fasted Rats

The effect of diabetes on 3,5,3'-triiodothyronine (T3) production was determined in the isolated perfused rat liver. Induction of diabetes with streptozotocin resulted in decreased serum thyroxine (T4) and T3 levels and a progressive decline in hepatic T3 production over 5 days. The decline in T3 production resulted from decreased conversion of T4 to T3, whereas T4 uptake was unchanged. Insulin administration restored serum T4 and T3, hepatic conversion of T4 to T3, and T3 production to normal levels. When serum T4 levels in diabetic rats were maintained by T4 administration, the conversion of T4 to T3 and T3 production returned to control levels. However, restoration of serum T4 levels in fasted rats failed to correct the decrease in hepatic T4 uptake or T3 production. Glucagon, at supraphysiological concentrations in vitro and in vivo, slightly decreased T4 uptake and T3 production without altering the conversion of T4 to T3. These data suggest that the fall in serum T4 levels observed in diabetic rats is important in mediating the decreased hepatic conversion of T4 to T3 and T3 production.

scholarly journals Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

2022 ◽  
Vol 23 (2) ◽  
pp. 837
Author(s):  
Sudip Biswas ◽  
Nancy J. Wahl ◽  
Michael J. Thomson ◽  
John M. Cason ◽  
Bill F. McCutchen ◽  
...  
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Peanut Butter ◽  
Processing System ◽  
Protoplast Isolation ◽  
Sequencing Analysis ◽  
Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

scholarly journals Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

2021 ◽  
Vol 12 ◽  
Author(s):  
Gabriela Conti ◽  
Beatriz Xoconostle-Cázares ◽  
Gabriel Marcelino-Pérez ◽  
Horacio Esteban Hopp ◽  
Carina A. Reyes
Keyword(s):  
Genetic Transformation ◽  
New Technologies ◽  
Negative Impact ◽  
Protoplast Isolation ◽  
Future Perspective ◽  
Juvenile Period ◽  
Breeding Programs ◽  
Regulatory Processes ◽  
Marker Free

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

Ontogeny of fetal hepatic and placental growth and metabolism in sheep

1992 ◽  
Vol 263 (3) ◽  
pp. R619-R623
Author(s):  
I. Vatnick ◽  
A. W. Bell
Keyword(s):  
Oxygen Consumption ◽  
Fetal Liver ◽  
Relative Size ◽  
Relative Weight ◽  
Dry Weight ◽  
Maximum Growth ◽  
Late Gestation ◽  
Progressive Decline ◽  
Highly Correlated

Ontogeny of fetal hepatic and placental growth and in vitro oxygen consumption (VO2) was investigated in fetal lambs at 75, 100, and 136 days postconception. Fetal hepatic relative weight and placental absolute and relative weights declined during this period. Oxygen consumption per gram dry weight of fetal liver and maternal placenta declined between mid and late gestation while fetal placental VO2 was unchanged. Estimated VO2 of the whole placenta did not change while the estimated total hepatic VO2 increased more than threefold between 75 and 136 days. Total hepatic VO2 was highly correlated with total placental VO2 at 136 days (r = 0.84). The results suggest that the placenta reaches its maximum growth and metabolic capacity before 100 days and possibly at or before midgestation. Changes in hepatic weight-specific total VO2, in addition to the declining relative size of the fetal liver, must contribute to the progressive decline in metabolic rate of the whole fetus during the second half of pregnancy. Correlations between placental and fetal liver weights and metabolic rates suggest the possibility of placental regulation of fetal hepatic growth and metabolism.

Missing pieces in protein deposition and mobilization inside legume seed storage vacuoles: calcium and magnesium ions

2012 ◽  
Vol 22 (4) ◽  
pp. 249-258 ◽  
Author(s):  
Cláudia N. Santos ◽  
Marta M. Alves ◽  
Isabel T. Bento ◽  
Ricardo B. Ferreira
Keyword(s):  
Alkaline Earth ◽  
Storage Proteins ◽  
Lupinus Albus ◽  
Physiological Role ◽  
Experimental Conditions ◽  
Protein Storage Vacuoles ◽  
Aggregation And Disaggregation ◽  
Alkaline Earth Cations

AbstractDuring the maturation of dicotyledonous seeds, organic carbon, nitrogen and sulphur are stored in protein storage vacuoles (PSVs) as storage globulins. Several studies point to the coexistence of storage proteins with proteases responsible for their degradation inside PSVs. Different mechanisms have been proposed to explain why there is no proteolysis during this period. Protein aggregation to form large supramolecular structures resistant to proteolytic attack could be the reason. However, during germination, and particularly following its completion, the globulin aggregates must undergo disintegration to allow protease attack for protein reserve mobilization. Based on the well-described concentration-dependent ability of Ca2+ and Mg2+ to promote in vitro aggregation and disaggregation of globulins, we explored a possible role for these alkaline earth cations in globulin packaging and mobilization. Ca2+ and Mg2+ measurements in purified PSVs [6.37 μmol and 43.9 μmol g− 1 dry weight (DW) of cotyledons, respectively] showed the presence of these two alkaline earth cations within this compartment. To our knowledge, this is the first time that Ca2+ and Mg2+ have been quantified in purified PSVs from Lupinus albus seeds. Considering the importance of these two alkaline earth cations inside PSVs, which represent 14.6% and 60.7% of the total seed Mg2+and Ca2+, respectively, globulin aggregation and disaggregation profiles were assayed using experimental conditions closer to those that are physiologically present (proportion of Ca2+ and Mg2+, and acidic pH). Based on: (1) the high in vivo abundance of Ca2+ and Mg2+ inside PSVs; and (2) globulin aggregation and disaggregation profiles, together with structural and physiological evidence already reported in the literature, an important physiological role for Ca2+ and Mg2+ in globulin packaging and mobilization inside PSVs is suggested.

scholarly journals Germinated and Ungerminated Seeds Extract from TwoLupinusSpecies: Biological Compounds Characterization and In Vitro and In Vivo Evaluations

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Bogdan Andor ◽  
Corina Danciu ◽  
Ersilia Alexa ◽  
Istvan Zupko ◽  
Elena Hogea ◽  
...  
Keyword(s):  
Cinnamic Acid ◽  
Therapeutic Potential ◽  
Lupinus Albus ◽  
Biological Evaluation ◽  
Lupinus Angustifolius ◽  
Cinnamic Acid Derivatives ◽  
Natural Agents ◽  
Germinated Seeds

In recent years, nutraceuticals attracted a great amount of attention in the biomedical research due to their significant contribution as natural agents for prevention of various health issues. Ethanolic extracts from the ungerminated and germinated seeds ofLupinus albusL. andLupinus angustifoliusL. were analyzed for the content in isoflavones (genistein) and cinnamic acid derivatives. Additionally, the extracts were evaluated for antimicrobial, antiproliferative, and anti-inflammatory properties, using in vitro and in vivo tests. Germination proved to be a method of choice in increasing the amount of genistein and cinnamic acid derivatives in bothLupinus albusL. andLupinus angustifolius L.seeds. Biological evaluation of all vegetal extracts revealed a weak therapeutic potential for both ungerminated and germinated seeds.

An extensin peroxidase is associated with white-light inhibition of lupin (Lupinus albus) hypocotyl growth

1999 ◽  
Vol 26 (1) ◽  
pp. 29 ◽  
Author(s):  
P. Jackson ◽  
S. Paulo ◽  
C. P. P. Ricardo ◽  
M. Brownleader ◽  
P. O. Freire
Keyword(s):  
Cell Wall ◽  
White Light ◽  
Cell Expansion ◽  
Structural Changes ◽  
Lupinus Albus ◽  
Extension Rate ◽  
Hypocotyl Growth ◽  
Cross Links ◽  
Quantitative Changes

The spatial distribution of the major basic (B2; pI 8.8) peroxidase of the intercellular fluid has an inverse relation with extension rate in etiolated hypocotyls of Lupinus albus L., suggesting its possible role in the control of cell expansion. White-light irradiation of etiolated hypocotyls resulted in growth inhibition and the induction of B2 and acidic (A2, pI 4.7–5.2) isoperoxidases (EC 1.1.11.7) to higher physiological activities. However, only the activities of the B2 isoperoxidases underwent quantitative changes in both space and time which suggested their role in growth-retardation. We have purified the B2 and A2 (pI 5.2) peroxidases to apparent electrophoretic homogeneity. To corroborate evidence obtained elsewhere that growth cessation coincides with cell wall structural changes and cell wall rigidification, we have shown that the B2 peroxidase, and not A2 peroxidase, cross-links tomato extensin in vitro. The B2 peroxidase may therefore catalyse the developmentally and light regulated formation of a covalently cross-linked cell wall extensin matrix in lupin hypocotyls. The cell wall would be more rigid or more recalcitrant to wall-loosening and subsequently contribute to the control of cell expansion.

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