scholarly journals Doubled-haploid Broccoli Production Using Anther Culture: Effect of Anther Source and Seed Set Characteristics of Derived Lines

1998 ◽  
Vol 123 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Mark W. Farnham
Keyword(s):  
Flow Cytometry ◽  
Plant Regeneration ◽  
Brassica Oleracea ◽  
Anther Culture ◽  
Seed Production ◽  
Ploidy Level ◽  
Doubled Haploid ◽  
Seed Set ◽  
Dna Flow Cytometry ◽  
Ploidy Levels

Using anther culture to generate doubled-haploid (DH) homozygous lines for use as parents in F1 hybrid crosses has become a common practice in breeding broccoli (Brassica oleracea L. Italica Group). During anther culture and subsequent embryogenesis and plant regeneration, polyploidization of microspore-derived embryos may not occur or it may occur accompanied by a doubling, tripling, quadrupling, octupling, or irregular polyploidization of the genome. Thus regenerants from the process can be haploids, diploids, triploids, tetraploids, octaploids, or aneuploids. The objectives of this research were to 1) conduct repeat cycles of broccoli anther culture using a group of F1 hybrids as anther donors and develop populations of regenerants; 2) analyze resulting populations using DNA flow cytometry and determine the influence of F1 source on frequency of different ploidy levels among regenerants; and 3) compare seed set in broccoli inbreds developed in a traditional selfing program compared to seed set in DH broccoli derived from anther culture. In two cycles (1994 and 1995) of anther culture, anther-derived populations of regenerants were developed using the F1 hybrids `Marathon', `Everest', `High Sierra', and `Futura' as sources of anthers. In 1994, `Everest', `High Sierra', and `Futura' yielded populations that included 2% to 7% haploids, 53% to 56% diploids, 32% to 38% tetraploids, and 5% to 6% other types. `Marathon'-derived regenerants were 5% haploid, 78% diploid, 15% tetraploid, and 2% other, showing significantly more diploids. In 1995, `Marathon' regenerants again included significantly more diploids and fewer tetraploids than those derived from other F1 sources, confirming that the genotype of the anther source affects the frequency of a particular ploidy level among regenerants derived from culture. In manual self-pollinations of 1994 regenerants, only diploids and rare tetraploids set seed. When plants that set no seed were discounted, seed production following manual self pollinations of 1995 regenerants was not significantly different from that of traditional inbreds derived from the same F1 sources.

scholarly journals Effects of Broccoli Donor Genotype on Incidence of Diploids in Populations Regenerated from Anther Culture

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 612g-613
Author(s):  
Mark W. Farnham
Keyword(s):  
Flow Cytometry ◽  
Plant Regeneration ◽  
Brassica Oleracea ◽  
Anther Culture ◽  
High Frequency ◽  
Microspore Culture ◽  
Chromosome Complement ◽  
Ploidy Levels ◽  
Previous Cycle ◽  
Donor Genotype

Broccoli (Brassica oleracea L. Italica group) breeders are increasingly using anther or microspore culture to produce dihaploid (diploid), homozygous lines for use in making hybrids. During the process of anther culture and subsequent plant regeneration, wherein embryos develop from microspores and plants are regenerated from the embryos, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement, instead of a doubling. Thus, populations may contain haploids, triploids, or tetraploids, in addition to diploids. In two cycles (1994-95 and 1995-96) of anther culture, regenerated populations from different broccoli hybrid sources were evaluated using flow cytometry to facilitate efficient identification of diploids vs. haploids, tetraploids, or others and to determine if anther donor genotype has an effect on the frequency of different ploidy levels among regenerants. In the first cycle, five broccoli hybrids had anther-derived populations in which ≈33% were haploid, 55% diploid, 37% tetraploid, and 5% aneuploid or abherent types. The hybrid, `Marathon', was different; it's regenerants were 78% diploid and only 15% tetraploid. In the second cycle, anther-derived populations had a significantly different makeup with a most hybrids giving 30% to 40% diploids and 50% to 60% tetraploids. However, consistent with the previous cycle, `Marathon' gave significantly more diploids (68%) and fewer tetraploids (25%) than other hybrids. These results indicate that anther donor genotype affects ploidy frequency among regenerants. Genotypes producing a high frequency (>60%) of diploids may be relatively uncommon.

scholarly journals Determining Ploidy Level and Nuclear DNA Content in Rubus by Flow Cytometry

2002 ◽  
Vol 127 (5) ◽  
pp. 767-775 ◽  
Author(s):  
Rengong Meng ◽  
Chad Finn
Keyword(s):  
Flow Cytometry ◽  
Pacific Northwest ◽  
Ploidy Level ◽  
Dna Content ◽  
Chromosome Numbers ◽  
Nuclear Dna ◽  
Diploid Species ◽  
Nuclear Dna Content ◽  
Dna Flow Cytometry ◽  
Ploidy Levels

Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).

scholarly journals Ploidy Levels of Hardy Actinidia Accessions in the U.S. Determined by Flow Cytometry

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 439D-439 ◽  
Author(s):  
Mary Ann Start ◽  
James Luby ◽  
Robert Guthrie ◽  
Debby Filler
Keyword(s):  
Flow Cytometry ◽  
Ploidy Level ◽  
Interspecific Variation ◽  
Root Tip ◽  
Interspecific Crosses ◽  
Ploidy Levels ◽  
Haploid Chromosome Number ◽  
Tip Cells ◽  
Root Tip Cells ◽  
The U.S

The hardy Actinidia species represent a source of genetic diversity for improving A. deliciosa (kiwifruit) as well as for creating new economically important cultivars through intra- and interspecific crosses. Attempts at breeding in Actinidia have been complicated by the existence of intraspecific as well as interspecific variation in ploidy. The haploid chromosome number in Actinidia is 29 and diploid (2n=2x=58), tetraploid (2n=4x=116), and hexaploid (2n=6x=174) levels have been identified. Because of the problems encountered when crossing parents differing in ploidy level, it is desirable to know the ploidy levels of plants to be used in breeding. We determined the ploidy levels of 61 Actinidia accessions currently available in the U.S., including primarily accessions of relatively winter-hardy species. The 61 accessions, representing eight species and three interspecific hybrids, were screened for ploidy using flow cytometry. Mitotic root tip cells from one plant from each putative ploidy level were examined microscopically to confirm the ploidy level derived from flow cytometry. There were 17 diploids, 40 tetraploids, and 4 hexaploids. Intraspecific variation was not found among accessions of the species arguta, callosa, deliciosa, kolomikta, melanandra, polygama, or purpurea. All kolomikta and polygama accessions were diploid. All arguta, callosa, melanandra, and purpurea accessions were tetraploid. Actinidia deliciosa was hexaploid. One chinensis accession was tetraploid. Two accessions (NGPR 0021.14 and 0021.3), acquired as chinensis, were hexaploid and may, in fact, be A. deliciosa based on their morphology. `Issai' (arguta × polygama) was hexaploid and `Ken's Red' and `Red Princess' (both melanandra × arguta) were tetraploid.

scholarly journals Regenerasi dan Aklimatisasi Kultur Antera Enam Persilangan F1 Padi Sawah

2016 ◽  
Vol 44 (2) ◽  
pp. 133
Author(s):  
Cucu Gunarsih ◽  
Bambang Sapta Purwoko ◽  
Iswari Saraswati Dewi ◽  
Dan Muhamad Syukur
Keyword(s):  
Plant Regeneration ◽  
Anther Culture ◽  
Doubled Haploid ◽  
Callus Induction ◽  
Rainfed Rice ◽  
Randomized Design ◽  
Doubled Haploid Plants ◽  
Completely Randomized Design ◽  
N6 Medium ◽  
Haploid Plants

ABSTRACT<br /><br />The breeding of rainfed rice tolerant to drought can be accomplished using anther culture. The objectives of this research were to determine regeneration abilities of six F1 anther culture and its acclimatization ability. The experiment was arranged in completely randomized design with 14 replications. The treatments consisted of six F1 derived from crossing:  INPARI 18 x IR83140-B-11-B (G1), INPARI 18 x B12825E-TB-1-25 (G2), INPARI 18 x IR87705-14-11-B-SKI-12 (G3), INPARI 22 x IR83140-B-11-B (G4), Bio-R81 x O18b-1 (G5), Bio-R82-2 x O18b-1 (G6). Media for callus induction was based on N6 medium + 2.0 mg L-1 NAA + 0.5 mg L-1 kinetin + 1.0 mM putresin + 60 g L-1 sucrosa, media for regeneration was based on MS + 0.5 mg L-1 NAA + 2.0 mg L-1 kinetin + 1.0 mM  putresin, and media for rooting was based on  MS + 0.5 mg L-1 IBA + 30 g L-1 sucrosa. The result indicated that all six F1 had different ability in anther culture. Bio-R82-2 x O18-b1 (G6) and  Bio-R81 x O18-b1 (G5) F1 genotype had good response both of callus induction and plant regeneration. These two F1 genotypes also gave the highest ratio of green planlet production to number of anther inoculated (GP:AI) were 5.50% and 4.65%,  respectively. In this research, there were identified doubled haploid plants were developed from 4 F1 derived cross namely G2 (2 plants), G3 (4 plants),  G5 (21 plants), and G6 (26 plants).<br /><br />Keywords: Callus induction, doubled haploid, rice<br /><br />

Nuclear DNA amount in pure species and hybrid willows (Salix): a flow cytometric investigation

1998 ◽  
Vol 76 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Jérôme Thibault
Keyword(s):  
Flow Cytometry ◽  
Ploidy Level ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Natural Hybridization ◽  
Ploidy Levels ◽  
Key Words Flow Cytometry ◽  
Dna Values ◽  
Dna Amounts

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of 11 Salix species and 5 hybrids. One hundred and sixty nine individuals were studied including 159 individuals from a sequence of 32 communities along a stretch of river in France and 10 individuals from French and English collections for comparison. Isolated nuclei were stained with propidium iodide. FCM was a significantly more practical and rapid technique than that of establishing the karyotype to survey many samples of Salix for variation in ploidy. The 2C DNA amounts for diploid species ranged from 0.76 to 0.98 pg, and tetraploid values ranged from 1.62 to 1.80 pg. The DNA values were consistent with the known ploidy levels. With the exception of a doubtful Salix xquercifolia, ploidy levels and DNA amounts of hybrids were intermediate compared with those of their parents. Intraspecific variation of nuclear DNA values including instrumental variation was low (i.e., 6-11% at the same ploidy level). FCM appeared to be an accurate tool for determination of Salix triploid hybrids. However, it remains limited concerning hybrids from crosses between species of the same ploidy level. Results suggest that natural hybridization might not be frequent in the communities studied, although they have been subject to disturbance. Previous overestimates of hybridization frequency in willows were probably due to misinterpretation of the effects of the environment on Salix spp. morphology; however, the extent and mechanisms of introgression in the genus remain to be further investigated. Key words: flow cytometry, Salix, hybridization, nuclear DNA content, riparian vegetation, disturbance.

scholarly journals Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1950
Author(s):  
Guadalupe Palomino ◽  
Javier Martínez-Ramón ◽  
Verónica Cepeda-Cornejo ◽  
Miriam Ladd-Otero ◽  
Patricia Romero ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chromosome Number ◽  
Ploidy Level ◽  
Dna Content ◽  
Chromosome Numbers ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Root Tips ◽  
Ploidy Levels ◽  
Dna Amounts

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

scholarly journals NUCLEAR DNA CONTENT IN BLUEBERRY DETERMINED BY FLOW CYTOMETRY

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 580e-580
Author(s):  
Rodomiro Ortiz ◽  
D.E. Costich ◽  
T.P. Meagher ◽  
N. Vorsa
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Haploid Genome ◽  
Dna Flow Cytometry ◽  
Dna Amount ◽  
Ploidy Levels ◽  
Polyploid Evolution ◽  
Haploid Genome Size

DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.

scholarly journals Ploidy Mosaics - Does Endopolyploidy in Explants Affects the Cytogenetic Stability of Orchids Regenerated From PLBs?

2021 ◽  
Author(s):  
Yohan Fritsche ◽  
Thiago Sanches Ornellas ◽  
Valdir Marcos Stefenon ◽  
Miguel Pedro Guerra
Keyword(s):  
Flow Cytometry ◽  
Plant Regeneration ◽  
Natural Question ◽  
Root Tips ◽  
Ploidy Levels ◽  
Model Plant ◽  
Somatic Tissues ◽  
High Oxidation ◽  
Regenerated Plantlets ◽  
Endopolyploid Cells

Abstract The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry (FC) was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, protocorms bases and protocorms apexes, which were subsequently used as explants for PLB induction and plant regeneration. The ploidy screening showed contrasting ploidy patterns in the samples. Endopolyploidy was detected in all tissues, with C-values ranging from 1C to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apexes showed the lowest proportion. Flower parts presented high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate was observed at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants.

A comparison of the persistence of Medicago truncatula cv. Paraggio with other annual medics in the Victorian Mallee

1993 ◽  
Vol 33 (4) ◽  
pp. 443 ◽  
Author(s):  
RA Latta ◽  
PE Quigley
Keyword(s):  
Plant Regeneration ◽  
Seed Production ◽  
Medicago Truncatula ◽  
Seed Set ◽  
Agricultural Productivity ◽  
The Other ◽  
Plant Densities ◽  
Seed Reserves ◽  
Annual Medics ◽  
Better Than

The annual medic Medicago truncatula cv. Paraggio has been widely sown in pastures throughout the Victorian Mallee region; however, its ability to persist in cereal pasture rotations is not known. Seed permeability and production, and plant regeneration of Paraggio, were studied in the field over 4 years, and the changes in its seed reserve were examined under 3 different cereal-pasture rotations. When compared with medic cvv. Parabinga, Harbinger, and Jemalong, Paraggio had up to twice the level of permeable seed over the summer-autumn period in 2 years (7-36% v. 2.5-19%). Paraggio produced 336-928 kg/ha of seed over 3 years, and after seed production was halted, it regenerated at densities of 150-1438 plants/m2 over the next 4 years. These results were generally the same as, or better than, the other cultivars. When seed set occurred in 1 and 2 years in the pasture phase of a 2- and 3-year rotation, respectively, Paraggio seed reserves were maintained at >4000 seeds/m2. This study demonstrated that Paraggio resulted in superior plant densities when grown in a number of typical rotations. It persisted satisfactorily and is expected to improve agricultural productivity in shor-tterm cereal-pasture rotations.

Ploidy of Regenerated Broccoli Derived from Microspore Culture Versus Anther Culture

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 533g-534
Author(s):  
Min Wang ◽  
Mark W. Farnham
Keyword(s):  
Anther Culture ◽  
Doubled Haploid ◽  
Direct Method ◽  
Microspore Culture ◽  
Doubled Haploids ◽  
Chromosome Complement ◽  
Culture Method ◽  
Lower Percentage ◽  
Ploidy Levels ◽  
Culture Techniques

Anther and microspore culture are commonly utilized to produce doubled-haploid (diploid), homozygous lines in broccoli (Brassica oleracea L. Italica Group). It is well-documented that doubled-haploid regenerants are produced by means of polyploidization during anther culture. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. As a consequence, regenerated populations from anther culture contain diploids, but also haploids, triploids, and tetraploids. Microspore culture represents a simpler and more direct method for producing doubled-haploids. Although a similar mix of ploidy types is likely to be observed among regenerants derived from microspore culture, the actual ploidy levels of such regenerants have not been documented for broccoli. Thus, the objectives of this study were to compare ploidy levels of regenerants developed using both anther and microspore culture in broccoli, and to examine phenotypic variation in ploidy makeup of populations developed from both anther and microspore culture using different F1 hybrids. Broccoli regenerants were derived simultaneously from both anther and microspore cultures using the same four F1 hybrids, including Everest, Patriot, Greenbelt and Major. Ploidy level was determined by flow cytometry. A majority of regenerants derived from both anther and microspore culture, were determined to be diploids or tetraploids. Significant differences in ploidy makeup of populations were observed among hybrid varieties for both culture techniques. Regardless of the culture method used, `Everest' produced a greater percentage of diploids and a lower percentage of tetraploids than `Patriot' did. Haploids were observed more frequently from microspore culture than from anther culture when `Everest' and `Major' served as parents.

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