Improving microspore culture as a rapeseed breeding tool: the use of auxins and cytokinins in an induction medium

1988 ◽  
Vol 66 (8) ◽  
pp. 1671-1675 ◽  
Author(s):  
D. G. Charne ◽  
W. D. Beversdorf
Keyword(s):  
Microspore Culture ◽  
Microspore Embryogenesis ◽  
Growth Substance ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Brassica Napus L ◽  
Surface Design ◽  
Shoot Production ◽  
Log Linear ◽  
Composite Response

The effect of naphthaleneacetic acid (NAA), an auxin, and N6-benzyladenine (BA), a cytokinin, on microspore embryogenesis in two F1 (hybrids of rapeseed (Brassica napus L.) was investigated, using a two-factor, central-composite response surface design. Total embryo yields of both hybrids increased in a log-linear fashion with BA concentrations in the range 0.01–0.255 mg L−1; within this range, yields approximately doubled for every fivefold increase in BA concentration. NAA concentrations of 0.136–1.85 mg L−1 had no effect on embryo yields. Within the range of concentrations studied, neither growth substance had any effect on the proportion of cotyledonary embryos produced. When cotyledonary embryos were regenerated on B5 medium without hormones, no significant effects on either root development or shoot production attributable to the NAA–BA treatments could be detected. In general, higher embryo yields were associated with a slower rate of development, but a greater degree of morphological synchrony within individual cultures.

Recovery of haploid plants from asparagus microspore culture

1994 ◽  
Vol 72 (3) ◽  
pp. 296-300 ◽  
Author(s):  
X. R. Feng ◽  
D. J. Wolyn
Keyword(s):  
Yeast Extract ◽  
Microspore Culture ◽  
Casein Hydrolysate ◽  
Culture Conditions ◽  
Asparagus Officinalis ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Ms Medium ◽  
Stage Of Development ◽  
Maturation Medium

Asparagus (Asparagus officinalis L.) microspore culture was performed in an array of experiments that assessed the roles of plant growth and culture conditions. The following protocol provided the best results. Flowers with microspores at the late uninucleate stage of development were collected from greenhouse plants grown at 22:18 °C (light:dark) and stored at 5 °C for 3 days. One millilitre of MS medium plus 0.2 g/L yeast extract, 500 mg/L casein hydrolysate, 800 mg/L glutamine, 2.0 mg/L naphthaleneacetic acid, 1.0 mg/L benzyladenine, and 6% sucrose (MSFY) was conditioned with 10 anthers/mL for 1 week, after which it was filtered. One hundred anthers were added to shed their microspores (1.6 × 105 per mL) and were removed after 3 weeks when 0.5 mL of fresh medium was added. Cultures were incubated at 35 °C for 1 week, then 30 °C for 5 weeks. Microcalli were collected subsequently on a 100-μm screen and placed on induction medium (MSFY minus yeast extract, plus 3 g/L gelrite) in darkness at 35 °C for 4 weeks and then in light at 25 °C for 4 weeks. Shoots, roots, and bipolar embryos were produced. The latter were transferred to maturation medium (MS plus 0.1 mg/L naphthaleneacetic acid, 0.5 mg/L kinetin, 3% sucrose, 3 g/L gelrite, and 0.65 mg/L ancymidol) for 4 weeks, then to germination medium (MS plus 1.0 mg/L gibberellic acid, 3% sucrose, 3 mg/L gelrite). Plantlets were grown and maintained on maturation medium. Approximately 0.3% of the cultured microspores produced calli, and 85% of calli produced plantlets. Of 10 plants analyzed, 2 were haploid, 7 were diploid and, 1 was tetraploid. Key words: asparagus, haploid, microspore.

Modeling the Growth Boundary of Listeria monocytogenes in Ready-to-Eat Cooked Meat Products as a Function of the Product Salt, Moisture, Potassium Lactate, and Sodium Diacetate Concentrations

2004 ◽  
Vol 67 (10) ◽  
pp. 2195-2204 ◽  
Author(s):  
J. D. LEGAN ◽  
D. L. SEMAN ◽  
A. L. MILKOWSKI ◽  
J. A. HIRSCHEY ◽  
M. H. VANDEVEN
Keyword(s):  
Listeria Monocytogenes ◽  
Meat Products ◽  
Growth Conditions ◽  
Continuous Variables ◽  
Surface Design ◽  
Potassium Lactate ◽  
Contour Plots ◽  
Sodium Diacetate ◽  
Factor Interactions ◽  
Composite Response

A central composite response surface design was used to determine the time to growth of Listeria monocytogenes as a function of four continuous variables: added sodium chloride (0.8 to 3.6%), sodium diacetate (0 to 0.2%), potassium lactate syrup (60% [wt/wt]; 0.25 to 9.25%), and finished-product moisture (45.5 to 83.5%) in ready-to-eat cured meat products. The design was repeated for ready-to-eat uncured meat products giving a fifth categorical variable for cure status. Products were stored at 4°C. The results were modeled using a generalized regression approach. All five main effects, six two-factor interactions, and two quadratic terms were statistically significant. The model was used to show the boundary between growth and no-growth conditions at 4°C using contour plots of time to growth. It was validated using independent challenge studies of cured and uncured products. Generally, the model predicted well, particularly for cured products, where it will be useful for establishing conditions that prevent the growth of L. monocytogenes. For uncured products, there was good agreement overall between predicted and observed times to growth, but the model is less thoroughly validated than for cured products. The model should initially only be used for screening of formulations likely to prevent growth of Listeria monocytogenes in uncured products, with recommendations subject to confirmation by challenge studies.

scholarly journals Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

2021 ◽  
Vol 12 ◽  
Author(s):  
Xueyuan Li ◽  
Sjur Sandgrind ◽  
Oliver Moss ◽  
Rui Guan ◽  
Emelie Ivarson ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Shoot Regeneration ◽  
Gene Editing ◽  
Protoplast Culture ◽  
Callus Formation ◽  
Protoplast Regeneration ◽  
Induction Medium ◽  
Shoot Induction ◽  
Brassica Napus L ◽  
High Concentrations

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

scholarly journals  Effect of 2,4-D as a novel inducer of embryogenesis in microspores of Brassica napus L.

2011 ◽  
Vol 47 (No. 3) ◽  
pp. 114-122 ◽  
Author(s):  
S.H. Ardebili ◽  
M.E. Shariatpanahi ◽  
R. Amiri ◽  
M. Emamifar ◽  
M. Oroojloo ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Heat Shock ◽  
Microspore Embryogenesis ◽  
Control Treatment ◽  
Dichlorophenoxyacetic Acid ◽  
Brassica Napus L ◽  
High Concentrations ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Short Time ◽  
First Time

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) applied at high concentrations for a short time was investigated as a novel stress for induction of microspore embryogenesis for the first time. Brassica napus L. cvs. Topas and Hyola 420 were used as model plants for testing this hypothesis. Microspores were subjected to 2,4-D at 4 concentrations (15, 25, 35 and 45 mg/l) for 15–45 min while the classical heat shock was used as the control treatment. Among 2,4-D treatments in Topas, the highest yield of torpedo-stage embryos was achieved at 15 mg/l 2,4-D for 30 min while more normal plantlets were produced when 2,4-D (25 mg/l for 30 min) was applied to the microspores. In Hyola 420 the results showed a lower number of embryos and normal plantlets at all concentrations of 2,4-D. Although Hyola 420 was almost equally embryogenic as Topas after heat shock treatment, large differences between genotypes (concerning embryogenic response) occurred after 2,4-D treatment. However, the mean number of embryos and regenerants was higher in heat shock as compared to 2,4-D induced stress (one magnitude of order). According to the results obtained, 2,4-D can be introduced as a new stress for induction of embryogenesis in microspores similarly like in zygotic and somatic cells. This novel stress is very important for plant species whose microspores are extremely sensitive to classical stresses.

scholarly journals Efficiency of SSR markers for determining the origin of melon plantlets derived through unfertilized ovary culture

2011 ◽  
Vol 38 (No. 1) ◽  
pp. 27-34 ◽  
Author(s):  
A.A. Malik ◽  
Cui Li ◽  
Zhang Shuxia ◽  
Chen Jin-feng
Keyword(s):  
Ssr Markers ◽  
Doubled Haploids ◽  
Parent Material ◽  
Plant Production ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Heterozygous Parent ◽  
Pre Treatment ◽  
Homozygous Diploid

The effects of temperature pre-treatment, thidiazuron, naphthaleneacetic acid, and 6-benzylaminopurine on in vitro gynogenic plant production from un-pollinated melon (Cucumis melo L.) ovaries were investigated. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. The temperature pre-treatment (4°C) for 4 days increased embryo formation frequency (63.3%) significantly. Addition of thidiazuron (0.04 and 0.02 mg/l) in the induction medium significantly increased the number of responding ovaries (46.6%, 65.83%), respectively. The maximum number of plantlet regeneration (22.5%) was achieved by culturing the ovary derived embryos on Murashigue and Skoog medium (MS medium) supplement with 0.6 mg/l 6-benzylaminopurine. Spontaneous doubled haploids originated directly through embryogenesis were subjected to genetic analysis using SSR molecular marker with 23 primers pair for homozygosity. SSR markers with microsatellite CMGA172, confirmed that the alleles in the parental material were also present in the gynogenic plantlets, but amplified only two alleles as compared to four alleles of the heterozygous parent material at same locus. Therefore these regenerated plantlets were consider homozygous and produced through a process of gametophytic embryogenesis.

scholarly journals The Influence of pH on Microspore Embryogenesis of White Cabbage (Brassica oleracea L.)

2013 ◽  
Vol 5 (4) ◽  
pp. 485-489 ◽  
Author(s):  
Tina Oana CRISTEA
Keyword(s):  
Brassica Oleracea ◽  
Microspore Culture ◽  
Microspore Embryogenesis ◽  
Critical Factor ◽  
Cellular Level ◽  
Strong Base ◽  
White Cabbage ◽  
In Vitro Microspore Culture ◽  
Influence Of Ph

In vitro microspore culture is one of the top techniques utilised now-a-days for the obtaining of double haploid plants in many plant species, including Brassica. The pH of the medium is a critical factor for the success of In vitro microspore culture as it influences the invertase enzyme activity, translated at cellular level through an acceleration or reduction of sucrose cleavage. The results published until now shows rather contradictory findings, as the response of microspores have been proved to be highly depending on genotypes, most of them being focused on Brassica napus. Thus, in the present study, the effect of different NLN liquid medium pH, ranging between 5.0 to 7.0 were tested in order to establish the most suitable pH for the expression of embryogenic competences of microspores cultivated on medium In vitro and ultimately for the obtaining of microspore-derived embryos. Among the 11 values of pH tested, the best results were obtained on variants with pH 5.8 and 6.0, both in what concern the maintaining of microspores viability and the number of microspore-derived embryos. The findings of the present study provide a strong base for the establishment of an efficient protocol for the In vitro culture of microspore at Brassica oleracea L. genotypes with Romanian origin.

scholarly journals Impact of Ionic Liquids on Induction of Wheat Microspore Embryogenesis and Plant Regeneration

Agronomy ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 839
Author(s):  
Dorota Weigt ◽  
Idzi Siatkowski ◽  
Magdalena Magaj ◽  
Agnieszka Tomkowiak ◽  
Jerzy Nawracała
Keyword(s):  
Ionic Liquids ◽  
Plant Regeneration ◽  
Positive Impact ◽  
Microspore Embryogenesis ◽  
Green Plant ◽  
Induction Medium ◽  
Dichlorophenoxyacetic Acid ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
The Impact

Ionic liquids are novel compounds with unique chemical and physical properties. They can be received based on synthetic auxins like 2,4-dichlorophenoxyacetic acid or dicamba, which are commonly used hormones in microspore embryogenesis. Nevertheless, ionic liquids have not been adapted in plant in vitro culture thus far. Therefore, we studied the impact of ionic liquids on the ability to undergo microspore embryogenesis in anther cultures of wheat. Two embryogenic and two recalcitrant genotypes were used for this study. Ten combinations of ionic liquids and 2,4-dichlorophenoxyacetic acid were added to the induction medium. In most cases, they stimulated induction of microspore embryogenesis and green plant regeneration more than a control medium supplemented with only 2,4-dichlorophenoxyacetic acid. Two treatments were the most favorable, resulting in over two times greater efficiency of microspore embryogenesis induction in comparison to the control. The effect of breaking down the genotype recalcitrance (manifested by green plant formation) was observed under the influence of 5 ionic liquids treatments. Summing up, ionic liquids had a positive impact on microspore embryogenesis induction and green plant regeneration, increasing the efficiency of these phenomena in both embryogenic and recalcitrant genotypes. Herbicidal ionic liquids can be successfully used in in vitro cultures.

scholarly journals A Simple Procedure for Mesophyll Protoplast Culture and Plant Regeneration in Brassica oleracea L. and Brassica napus L.

2011 ◽  
Vol 42 (No. 3) ◽  
pp. 103-110 ◽  
Author(s):  
N.D. Kaur ◽  
M. Vyvadilová ◽  
M. Klíma ◽  
M. Bechyně
Keyword(s):  
Brassica Napus ◽  
Plant Regeneration ◽  
Brassica Oleracea ◽  
Growth Regulators ◽  
Protoplast Culture ◽  
Treatment Time ◽  
Simple Procedure ◽  
Enzyme Treatment ◽  
Induction Medium ◽  
Brassica Napus L

An improved protocol for Brassica protoplast culture and plant regeneration was developed. Isolated protoplasts from four-weeks-old in vitro shoot tip culture of Brassica oleracea var. botrytis cv. Siria F1 and Brassica napus doubled haploid of breeding line OP-1 were cultured at a density of 9.8&ndash;11.2 &times; 10<sup>4 </sup>protoplasts/ml in darkness at 25&deg;C in a modified medium containing 2% glucose, 0.25 mg/l 2,4-D, 1 mg/l BAP and 1 mg/l NAA. The first divisions of protoplasts were observed on the third day of culture in B. oleracea and on the fourth day in B. napus. The protoplast cultures were diluted with low osmotic medium on 7<sup>th</sup> and 11<sup>th</sup> day. The frequency of dividing cells was about 80% in B. oleracea and 50% in B. napus. After one month, the microcalli of approximately 0.5&ndash;1 mm in size were transferred into an induction medium with various combinations of growth regulators. Minimum duration of enzyme treatment time and extended dark period in the initial phase of culture increased the survival rate of protoplasts. Organogenesis started when the calli enlarged in size on an induction medium (1 mg/l NAA, 0.02 mg/l GA<sub>3</sub>, 1 mg/l 2iP) with 2% sucrose and 0.8% agar. Regeneration frequency of calli was found to be 69&ndash;75% in B. oleracea and 2&ndash;3% in B. napus. Well-developed shoots were transferred for rooting to a half-strength MS medium without growth regulators. More than 100 B. oleracea regenerants were transferred into soil, and they produced normal heads and set seeds. This very simple procedure is efficient and suitable mainly for B. oleracea var. botrytis and represents a background for fusion experiments. &nbsp;

Plant tissue cultures from four Tuscan globe artichoke cultivars

2012 ◽  
Vol 7 (4) ◽  
pp. 680-689 ◽  
Author(s):  
Laura Bedini ◽  
Mariella Lucchesini ◽  
Francesco Bertozzi ◽  
Alberto Graifenberg
Keyword(s):  
Central Italy ◽  
Basal Medium ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Globe Artichoke ◽  
Ms Medium ◽  
Indole Butyric Acid ◽  
Double Phase ◽  
Rooting Medium ◽  
Nitrate Concentrations

AbstractThe aim of the study was to examine the possibility of propagating in vitro four of the most common cultivars in Tuscany (central Italy): Terom, Violetto di Toscana, Chiusure and Empolese. The first three belong to the “Violetti” group, while cv Empolese belongs to the “Romaneschi” group. Explants were cultured on an induction medium (IM), which is a modified MS medium consisting of nitrate concentrations reduced by one quarter, 0.8 mg L−1 6-benzylaminopurine (BA) and 0.2 mg L−1 3-indole butyric acid (IBA). Explants were then transferred to a proliferation medium (PM) consisting of the same basal medium together with 0.03 mg L−1 BA and 0.05 mg L−1 gibberellic acid (GA3). A rooting double-phase was then established. The pre-rooting medium (PRM), consisting of a basal MS medium with half strength nitrate concentrations, 0.5 mg L−1 indole-3-acetic acid (IAA) and 1 mg L−1 paclobutrazol (PBZ) was used for two weeks. Over the next four weeks, a rooting medium (MR) was used, consisting of a basal MS medium with 2 mg L−1 β-cyclodextrin and 2 mg L−1 α-naphthaleneacetic acid sodium salt (NAA). The cv Empolese provided the highest number of proliferated explants and rooted plantlets using the method described.

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