Somaclonal variation on in vitro callus culture potato cultivars

Synthetic seeds can be an alternative for those species in which botanical seeds are not viable. One of the major problems of in vitro plant cultivation is the high level of somaclonal variation. The most common factors affecting somaclonal variation are genotype, explant source, in vitro period and cultivation conditions in which the culture is established. In this work, calli were induced using leaf and stem explants of the commercial potato cultivars Achat, Baraka, Baronesa, Bintje, and Contenda in MS culture media supplemented with 1.65 mM of picloram and 11.5 mM of 2,4-D. Seventy and 90 days after induction, DNA samples of 40 calli were compared concerning the effects of the two explant (leaf and stem) and two growth regulator sources on five potatoes cultivars. A total of 20 arbitrary sequence primers were evaluated. The RAPD pattern generated by these primers suggested a high percentage of polymorphic fragments among the five genotypes, indicating a high level of genetic variation among cultivars. Cultivar Baronesa showed the highest number of polymorphic fragments for all treatments. The cultivar Contenda showed the smallest somaclonal variation, for most of the treatments, except for the treatment which consisted of stem explants, picloram (1.65 mM) application, and a 70-day period of callus formation. 'Contenda' is, therefore, the most suitable cultivar for synthetic seed production.

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In vitro answer of Bulgarian pepper (Capsicum annuum L.)

Genetika ◽

10.2298/gensr0602129r ◽

2006 ◽

Vol 38 (2) ◽

pp. 129-136

Author(s):

Velichka Rodeva ◽

Stanislava Grozeva ◽

Velichka Todorova

Keyword(s):

Culture Medium ◽

Culture Media ◽

Casein Hydrolysate ◽

Basal Medium ◽

Regeneration Ability ◽

Stem Explants ◽

Media Composition ◽

Breeding Lines ◽

High Level

Callusogenesis and regeneration ability of cotyledon and hypocotyl explants from three Bulgarian pepper varieties in MS basal medium supplemented with l-3mg/l BAP. l.0mg/1 IAA and 0.5mg/l GA3 was studied. In the different variants of culture medium was registered high level of callusogenesis and organogenesis in both type of explants from the all varieties. The highest percentage of plant-regenerants is established in cotyledon explants (from 3.3 to 18.3) in variant 3 of the culture medium containing 3mg/l BA. In the process of micropropagation by stem explants of the same studied pepper varieties the addition of the vitamins C. B12. Casein hydrolysate and Ferulic acid had a stimulation effect on the plant growth in height and rooting. In result of anther cultivation from three pepper varieties and four breeding lines the highest percentage of embryo structure formation was registered in varieties Albena and Strjama (12.0 and 13.8 respectively). The Bulgarian peppers are recalcitrant and their in vitro answer is different depending from the explants type, genotype and the culture media composition.

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FACTORS AFFECTING THE REGENERATION OF PEPPER (CAPSICUM ANNUUM L.)

HortScience ◽

10.21273/hortsci.25.9.1120 ◽

1990 ◽

Vol 25 (9) ◽

pp. 1120G-1120

Author(s):

J. L. Jacobs ◽

C. T. Stephens

Keyword(s):

Growth Hormone ◽

Silver Nitrate ◽

Callus Formation ◽

Leaf Explants ◽

Regeneration System ◽

Factors Affecting ◽

Nitrate Concentrations ◽

Leaf Differentiation ◽

Whole Plants

Several growth hormone combinations and silver nitrate concentrations were examined for their effect on regeneration of different pepper genotypes. Primary leaf explants from in vitro seedlings were cultured on a revised Murashige and Skoog medium supplemented with auxin, cytokinin and 1.6% glucose. Combinations of different concentrations of indole-3-acetic acid (IAA), 0-5 mg/l, and 6-benzylaminopurine (BAP), 0-5 mg/l, were tested to determine the most effective medium for shoot primordium formation. Experiments with IAA and BAP did not result in a specific growth hormone combination appropriate for regeneration of all genotypes tested. Of the silver nitrate concentrations tested, 10 mg/l resulted in the best shoot and leaf differentiation and reduced callus formation. Differences in organogenic response of individual genotypes were evaluated on a single regeneration medium. Whole plants were regenerated from 11 of 63 genotypes examined. Based on these experiments, a reproducible regeneration system for pepper was developed with a total of 500 plants regenerated to date.

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P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Human Reproduction ◽

10.1093/humrep/deab130.581 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Biricik ◽

V Bianchi ◽

F Lecciso ◽

M Surdo ◽

M Manno ◽

...

Keyword(s):

Data Analysis ◽

Embryo Culture ◽

Culture Media ◽

Embryo Biopsy ◽

Non Invasive ◽

Culture Time ◽

Ngs Data Analysis ◽

High Level ◽

Ngs Data

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (>84%) of concordance rate for ploidy and sex, sensitivity (>88%), and speciﬁcity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (>5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and speciﬁcity were 92,8% and 76,7%, respectively with no signiﬁcant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

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Control of morphogenetic pathways in thin cell layers of tobacco by pH

Canadian Journal of Botany ◽

10.1139/b89-087 ◽

1989 ◽

Vol 67 (3) ◽

pp. 650-654 ◽

Cited By ~ 17

Author(s):

A. Cousson ◽

P. Toubart ◽

K. Tran Thanh Van

Keyword(s):

Butyric Acid ◽

De Novo ◽

Culture Media ◽

Callus Formation ◽

Medium Ph ◽

Vegetative Buds ◽

Cell Layers ◽

Initial Medium ◽

Vegetative Bud

Thin cell layer explants of tobacco were floated in vitro on the surface of liquid culture media. The initial exogenous concentrations of indolyl-3-butyric acid, and kinetin, the initial medium pH, and the explant density were varied. Various patterns of de novo and direct differentiation without any intermediate callus (flower, vegetative bud, root) as well as the absence of morphogenesis and callus formation without any subsequent organogenesis were separately controlled on 100% of the explants. On the same exogenous combination of glucose, indolyl-3-butyric acid, and kinetin, changes in initial medium pH changed the pattern of morphogenesis. For a given initial exogenous indolyl-3-butyric acid concentration, vegetative buds were obtained at either pH 6.1 or 7.8, whereas a mixture of flowers and vegetative buds was obtained at pH 6.8. Furthermore, changes in explant density changed the morphogenetic response. It is suggested that the effects of the initial medium pH and explant density on morphogenesis may be related partially to modifications of the physicochemical properties of the cell wall and (or) plasmalemma.

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Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽

10.1530/rep.1.00087 ◽

2004 ◽

Vol 127 (2) ◽

pp. 187-194 ◽

Cited By ~ 26

Author(s):

Y H Choi ◽

L B Love ◽

D D Varner ◽

K Hinrichs

Keyword(s):

Embryo Culture ◽

Intracytoplasmic Sperm Injection ◽

Room Temperature ◽

Culture Media ◽

Developmental Competence ◽

Glucose Medium ◽

Factors Affecting ◽

Nuclear Maturation ◽

High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

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IN VITRO CALLUS INDUCTION AND COMPARATIVE GC-MS ANALYSIS OF METHANOLIC EXTRACTS OF CALLUS AND LEAF SAMPLES OF AMPELOCISSUS LATIFOLIA (ROXB.) PLANCH

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i9.27879 ◽

2018 ◽

Vol 10 (9) ◽

pp. 68

Author(s):

Deep Chhavi Anand ◽

Rishikesh Meena ◽

Vidya Patni

Keyword(s):

Callus Induction ◽

Culture Media ◽

Biological Activities ◽

Gas Chromatography Mass Spectrometry ◽

Dodecanoic Acid ◽

Stem Explants ◽

Ms Analysis ◽

Wide Range

Objective: The aim of the present study was to develop a callus induction protocol and comparative study of therapeutic phytochemicals present in in vivo leaf and in vitro callus extracts through Gas Chromatography-Mass Spectrometry analysis.Methods: Murashige and Skoog media was used as culture media for callus induction. In vitro callus induction protocol was developed by studying the effects of various plant growth regulators like auxin, 2, 4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalic acetic acid), alone and in combination with cytokinin BAP (benzyl aminopurine), on leaf and stem explants. The GC-MS analysis of Ampelocissus latifolia was carried out on Shimadzu QP-2010 plus with thermal desorption system TD 20 to study the phytochemical profile.Results: In vitro callus induction protocol was developed for the plant and callusing was done from leaf and stem explants of Ampelocissus latifolia. The best result for callus induction was obtained using leaf explant, and callus production were maximum in Murashige and Skoog medium fortified with BAP (0.5 mg/l) and NAA (1.0 mg/l). Major compounds identified in the GC-MS analysis were Campesterol, Stigmasterol, Beta-Sitosterol, Docosanol, Dodecanoic acid, etc., in in vitro extract and Beta Sitosterol, Tocopherol, Squalene, Bergamot oil, Margarinic acid, Hexadecanoic acid, etc., in in vivo extract. The different active phytochemicals identified have been found to possess a wide range of biological activities, thus this analysis forms a basis for the biological characterization and importance of the compounds identified for human benefits.Conclusion: This is the first report on callus induction in Ampelocissus latifolia. From the results obtained through the in vitro callus induction and its comparative GCMS analysis with in vivo extract, it is revealed that Ampelocissus latifolia contains various bioactive compounds that are of importance for phytopharmaceutical uses. The GCMS analysis revealed that the amount of Beta-sitosterol and 5-Hydroxymethylfurfural (HMF) was very high in in vitro extract as compared to in vivo extract.

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Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

Australian Journal of Experimental Agriculture ◽

10.1071/ea02199 ◽

2004 ◽

Vol 44 (1) ◽

pp. 27 ◽

Cited By ~ 16

Author(s):

M. Bajji ◽

P. Bertin ◽

S. Lutts ◽

J-M. Kinet

Keyword(s):

Durum Wheat ◽

Somaclonal Variation ◽

Drought Resistance ◽

In Vitro Selection ◽

Culture Media ◽

Field Studies ◽

Resistance Mechanisms ◽

Wide Range ◽

Triticum Durum Desf

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4�generations. Differences in electrolyte leakage, chlorophyll fluorescence (Fv/Fm), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4�parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

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Virus and fungal resistance: from laboratory to field

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0157 ◽

1993 ◽

Vol 342 (1301) ◽

pp. 271-278 ◽

Cited By ~ 33

Keyword(s):

Potato Virus X ◽

Field Trials ◽

Potato Cultivars ◽

Fungal Resistance ◽

Horticultural Crops ◽

Antifungal Proteins ◽

Commercial Potato ◽

Genes Encoding ◽

High Level

Virus and fungal resistance traits are important targets in the genetic engineering of agricultural and horticultural crops. We have engineered resistance against potato virus X in important commercial potato cultivars. Four years of field trials with resistant potatoes have demonstrated the commercial feasibility of improving potato cultivars by selectively adding new traits while preserving intrinsic properties. In our pursuit for a broad resistance against fungi we have focused on the exploitation of genes encoding antifungal proteins. We present results demonstrating the antifungal effect of some of these proteins in vitro , as well as the synergy between specific chitinases and β-1,3-glucanases. We also report high level resistance against Fusarium oxysporum in transgenic tomato plants expressing a specific combination of genes encoding these enzymes.

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FACTORS AFFECTING THE REGENERATION OF PEPPER (CAPSICUM ANNUUM L.)

HortScience ◽

10.21273/hortsci.25.9.1120g ◽

1990 ◽

Vol 25 (9) ◽

pp. 1120g-1120 ◽

Cited By ~ 3

Author(s):

J. L. Jacobs ◽

C. T. Stephens

Keyword(s):

Growth Hormone ◽

Silver Nitrate ◽

Callus Formation ◽

Leaf Explants ◽

Regeneration System ◽

Factors Affecting ◽

Nitrate Concentrations ◽

Leaf Differentiation ◽

Whole Plants

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Low Cost Tissue Culture Technologies in Vegetables: A Review

International Journal of Biochemistry Research & Review ◽

10.9734/ijbcrr/2020/v29i930226 ◽

2020 ◽

pp. 66-78

Author(s):

Reshav Naik ◽

Anil Bhushan ◽

R. K. Gupta ◽

Anamika Walia ◽

Anshika Gaur

Keyword(s):

Tissue Culture ◽

Culture Media ◽

Callus Formation ◽

Low Cost ◽

Direct Regeneration ◽

Clonal Variation ◽

In Vitro Mutagenesis ◽

Vegetable Crops ◽

Brown Sugar

The demand of vegetable crops is increasing day by day due to changes in consumption patterns, so the need of the hour is to develop technologies that enhance the vegetable production at a rapid rate. Plant Tissue culture is one such remarkable biotechnological tool that has its application in vegetable propagation and improvement, disease elimination, herbicide resistance, salinity tolerance, incorporation of high nutrient content, genetically improved plants and conservation of endangered plant species and in the near future usage of this technology is going to increase further manifold. It is used for production of disease free quality planting material and development of varieties through direct regeneration, anther/ovule culture, somatic embryogenesis etc. or for creation of new variation (organogenesis via callus formation, soma-clonal variation and in vitro mutagenesis). In spite of being a very important and viable non-conventional biotechnological tool, high cost of production of seedlings in vitro remains a major impediment in popularization of this technology. High cost of producing seedlings is due to availability of limited resources, high recurrent costs of consumables for media and lack of awareness, which limits its application only to a few institutions and rich farmers especially in developing countries. Therefore, in order to make this technology a successful and viable option for the farmers, future thrust must be on cost reduction of in vitro seedlings. The components of tissue culture technology such as culture media components, glassware, lighting and water for media preparation can be replaced with low cost alternatives to reduce the overall cost of tissue culture. The usage of alternatives for gelling agent’s like isabgol (potato, tomato, cassava, turmeric, ginger), sago (potato, tomato, turmeric, ginger) cassava starch (potato, cassava, sweet potato) barley starch, phytagel etc. and for carbon sources like table sugar (potato, turmeric, ginger), jaggery, sugarcane juice, cube sugar (bittergord), brown sugar etc have already been documented worldwide. The present paper reviews the work done by researchers around the globe in developing various low cost alternative technologies with focus on vegetable crops.

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