High-throughput cell analysis based protocol for ploidy determination in anther-derived rice callus

Sahoo, Sripati Abhiram; Jha, Zenu; Verulkar, Satish; Srivastava, Ashish Kumar; Suprasanna, Penna

doi:10.1007/s11240-019-01561-2

Cited by 10 publications

(4 citation statements)

References 18 publications

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“…Morphogenic calli were light, opaque, compact and had green areas containing chlorophyll, which were zones of morphogenesis. This is confirmed in the works of other authors [30].…”

Section: Resultssupporting

confidence: 88%

Creation of Rice Doubled Haploids Resistant to Prolonged Flooding Using Anther Culture

Kostylev,

Kalinina,

Vozhzhova

et al. 2023

Plants

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Flood resistance in rice is very important in weed control, as weeds cannot overcome deep water. At present, there are no released varieties in Russia that would meet these requirements. The creation of such varieties will reduce production costs and pesticide load on the ecosystem. The object of the study was second-generation rice hybrids obtained by crossing the best varieties for economically valuable traits with samples carrying genes for resistance to prolonged flooding with water. To create double rice haploids resistant to prolonged flooding, the anther culture method was used, followed by molecular genetic evaluation of dihaploids for the presence of genes for resistance to prolonged flooding. An estimate of the growth energy under deep flooding was carried out according to our own method. As a result of the cultivation of anthers, 130 androgenic regenerated plants were obtained in 14 hybrid combinations. In terms of responsiveness to neoplasms, 60% of the panicles showed a positive result, while the rest 40% did not demonstrate callus formation. In total, 30 green regenerative lines were obtained from four rice hybrids, differing in visual morphological assessment. Large genotypic differences between the samples were revealed. These lines carry long-term flood resistance genes and can be used in rice breeding programs using dihaploids. As a result of the assessment of the growth energy in a number of obtained samples, the potential for rapid elongation of the first leaves, overcoming a large layer of water and accumulation of vegetative mass, was revealed.

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“…Morphogenic calli were light, opaque, compact and had green areas containing chlorophyll, which were zones of morphogenesis. This is confirmed in the works of other authors [30].…”

Section: Resultssupporting

confidence: 88%

Creation of Rice Doubled Haploids Resistant to Prolonged Flooding Using Anther Culture

Kostylev,

Kalinina,

Vozhzhova

et al. 2023

Plants

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“…The ploidy status of anther-derived callus was checked using flow cytometry, and the results show that the ploidy of the calli was haploid in nature, confirming the involvement of microspores in developing callus. Similar observations have also been reported for other plant species [7,10,39]. FCM is the widely used approach for determining the ploidy of plants developed through callus, somatic embryos and other in vitro-regenerated pathways [40].…”

Section: Discussionsupporting

confidence: 80%

“…In vitro anther culture has been attempted in various plants such as Actinidia arguta Planch [8] and Triticum aestivum L. [9]. Various factors such as stage of anther, culture conditions, plant growth regulators (PGRs) and genotypic and ploidy status determine the success of DH generation [10]. These factors necessitate ascertaining the ploidy status of anther-derived callus to generate true-to-type DH lines, which can be performed with a flow cytometric technique.…”

Section: Introductionmentioning

confidence: 99%

Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L.

Bansal

Mujib

Mamgain

et al. 2023

Plants

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Catharanthus roseus L. (G.) Don is the most widely studied plant because of its high pharmacological value. In vitro culture uses various plant parts such as leaves, nodes, internodes and roots for inducing callus and subsequent plant regeneration in C. roseus. However, till now, little work has been conducted on anther tissue using plant tissue culture techniques. Therefore, the aim of this work is to establish a protocol for in vitro induction of callus by utilizing anthers as explants in MS (Murashige and Skoog) medium fortified with different concentrations and combinations of PGRs. The best callusing medium contains high α-naphthalene acetic acid (NAA) and low kinetin (Kn) concentrations showing a callusing frequency of 86.6%. SEM–EDX analysis was carried out to compare the elemental distribution on the surfaces of anther and anther-derived calli, and the two were noted to be nearly identical in their elemental composition. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of anther and anther-derived calli was conducted, which revealed the presence of a wide range of phytocompounds. Some of them are ajmalicine, vindolinine, coronaridine, squalene, pleiocarpamine, stigmasterol, etc. More importantly, about 17 compounds are exclusively present in anther-derived callus (not in anther) of Catharanthus. The ploidy status of anther-derived callus was examined via flow cytometry (FCM), and it was estimated to be 0.76 pg, showing the haploid nature of callus. The present work therefore represents an efficient way to produce high-value medicinal compounds from anther callus in a lesser period of time on a larger scale.

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“…In this study, morphological evaluation was found to be reasonably reliable in distinguishing diploids from other ploids, and was performed quickly and easily. Although flow cytometry is an attractive approach for assessing the ploidy level of regenerants, its use is still limited in many laboratories due to the high cost of equipment and the higher cost of each analysis [27,29]. In addition, this method cannot distinguish homozygous doubled haploids from heterozygous diploid plants grown in vitro [30].…”

Section: Resultsmentioning

confidence: 99%

An in vitro anther culture method for creating rice dihaploids resistant to prolonged flooding

Kalinina,

Kostylev

2023

E3S Web of Conf.

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An assessment was made of the possibility of obtaining dihaploids by the method of anther culture in vitro to accelerate breeding for resistance to prolonged flooding of rice with water. The object of the study is F2 rice hybrids of the Federal State Budgetary Scientific Institution, Agrarian Research Center “Donskoy” (ARC “Donskoy”) rice breeding and seed laboratory, obtained by crossing the best varieties in terms of economically valuable traits with samples carrying genes for resistance to prolonged flooding with water. Basic nutrient media with the optimal composition of nutrients and growth hormones that stimulate callus and morphogenesis were used. Cultivation of anthers revealed large genotypic differences between the samples. In terms of responsiveness to neoplasms, 1/3 of the number of plants showed a positive result, the rest did not give calli. The most responsive to the formation of calli were hybrid combinations: 5009/2 – 84 pcs., 5010/2 – 94 pcs., 4565/3 –85 pcs., 4641/2 – 69 pcs. They also showed the ability to morphogenesis. Androgenic plants were obtained from 13 hybrid combinations, their share was 1.03% of the total number of inoculated anthers. 30 green regenerated lines were obtained from four rice hybrids, differing in visual morphological assessment: 5009/2 – 5 pcs., 5010/2 – 5 pcs., 4565/3 – 2 pcs., 4641/2 – 18 pcs. The isolated lines are characterized by good responsiveness in anther culture in vitro, carry genes for resistance to prolonged flooding, and can be used in rice breeding programs using DG technologies.

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High-throughput cell analysis based protocol for ploidy determination in anther-derived rice callus

Cited by 10 publications

References 18 publications

Creation of Rice Doubled Haploids Resistant to Prolonged Flooding Using Anther Culture

Creation of Rice Doubled Haploids Resistant to Prolonged Flooding Using Anther Culture

Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L.

An in vitro anther culture method for creating rice dihaploids resistant to prolonged flooding

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