Doubled Haploids in Eggplant

Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies.

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Improvement of methods of creating hybrids of cabbage

VEGETABLE CROPS OF RUSSIA ◽

10.18619/2072-9146-2019-4-3-7 ◽

2019 ◽

pp. 3-7 ◽

Cited By ~ 1

Author(s):

Anna I. Mineykina ◽

Lyudmila L. Bondareva ◽

Darya V. Shumilina ◽

Elena A. Domblides ◽

Alexey V. Soldatenko

Keyword(s):

First Generation ◽

Microspore Culture ◽

Diallel Cross ◽

High Yield ◽

Isolated Microspores ◽

Economically Valuable Traits ◽

Complete Diallel Cross ◽

Speed Up ◽

Pure Lines

Relevance One of the basic directions of the cabbage crop breeding is the creation of F1 hybrids with a complex of economically valuable traits. This process is difficult and time-consuming as to get pure lines must be within 6-12 years hold inbreeding. Herewith not every line gives the desired heterotic effect that also requires additional verification. Methods Biotechnological method culture of isolated microspores in vitro, which allows in the first generation to receive a line with 100% homozygosity, was used to speed up the breeding process. Combination ability were performed in complete diallel cross on the basic morphological signs. Results Culture medium for cultivation of isolated microspores in vitro was optimized for each genotype of cabbage for the best embryoids regeneration. Maximum amount of embryoids was received on medium with pH 6.2 using ampicillin 100 mg/l and zeatin 1 mg/l: 466.7 ± 153.2 PCs/100 buds. A new source material for breeding – doubled haploid lines of cabbage was received. Lines – the best parents for F1 hybrids with high yield, compact rosette of leaves, with optimum inside and short outside cabbage stump was created. Studies have shown that optimization of breeding process in case of creation of pure lines of cabbage in 3 years with microspore culture requires to reduce the breeding process in 2 times.

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Genotype and Environment Strongly Influence Barley Anther Culture Response Using Australian Genotypes

Australian Journal of Botany ◽

10.1071/bt9930227 ◽

1993 ◽

Vol 41 (2) ◽

pp. 227 ◽

Cited By ~ 4

Author(s):

SJ Logue ◽

LC Giles ◽

DHB Sparrow

Keyword(s):

Anther Culture ◽

Doubled Haploids ◽

Growth Conditions ◽

Green Plant ◽

Optimal Conditions ◽

Donor Plant ◽

Breeding Programs ◽

Barley Cultivars ◽

Large Numbers ◽

Anther Culture Response

A screening of several Australian barley cultivars of commercial interest has identified a number of genotypes that respond well to anther culture, with average levels of green plant regeneration between 23 and 134 plants/100 anthers cultured. Donor plant growth conditions have a large impact on anther culture response and, although optimal conditions for specific genotypes could possibly be identified, it is likely to be more effective for the production of large numbers of doubled haploids to settle for a broadly acceptable environment. Recent advances in methodology and the identification of responsive genotypes makes anther culture a feasible procedure for Australian barley breeding programs.

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Production and utilization of doubled haploids in Brassica oleracea vegetables

Horticultural Science ◽

10.17221/3804-hortsci ◽

2011 ◽

Vol 31 (No. 4) ◽

pp. 119-123 ◽

Cited By ~ 7

Author(s):

M. Klíma ◽

M. Vyvadilová ◽

V. Kučera

Keyword(s):

Plant Regeneration ◽

Brassica Oleracea ◽

Microspore Culture ◽

Doubled Haploids ◽

Doubled Haploid Line ◽

Culture Technique ◽

F1 Hybrids ◽

Line Production ◽

Mature Embryos ◽

Whole Plants

A possibility to increase the efficiency of plant regeneration from microspore-derived embryos of selected botanical varieties of Brassica oleracea was investigated from 2001 to 2004. More than 400 regenerants of R1 generation were derived in kohlrabi, cabbage and cauliflower by means of different modifications of microspore culture technique. Distinct genotype differences in embryogenic responsibility and regenerative ability of microspore embryos to whole plants were detected. The highest frequency of embryogenesis and subsequent regeneration of plants were achieved in cauliflower cultivar Siria F1, kohlrabi line P7 and some experimental F1 hybrids of cauliflower. The best production of embryos was obtained when donor plants were grown in the growth chamber under controlled light and temperature conditions. The regeneration of plantlets was considerably improved by repeated subculture of cotyledonary embryos on media with various combinations of phytohormones and excision of the cotyledons from mature embryos. The percentage of plant regeneration from subcultured embryos in kohlrabi ranged from 11.11 to 63.64%, in cauliflower from 23.53 to 46.19% and in cabbage from 5.88 to 52.00%. The utilization of regenerants for doubled haploid line production is often complicated by male sterility also in plants with the normal diploid chromosome number.

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Pembentukan Genotipe Padi Berumur Sangat Genjah melalui Kultur Antera

Buletin Plasma Nutfah ◽

10.21082/blpn.v18n2.2012.p54-61 ◽

2016 ◽

Vol 18 (2) ◽

pp. 54

Author(s):

Iswari S. Dewi ◽

A. Dinar Ambarwati ◽

Aniversari Apriana ◽

Atmitri Sisharmini ◽

Ida H. Somantri ◽

...

Keyword(s):

Anther Culture ◽

Callus Induction ◽

Culture Media ◽

Oryza Sativa L ◽

Doubled Haploids ◽

Heading Date ◽

Population Increase ◽

Early Maturity ◽

Early Maturing ◽

Pure Lines

Development of Very Early Maturing Rice Genotypes through Anther Culture. Iswari S. Dewi, A. Dinar Ambarwati, Aniversari Apriana, Atmitri Sisharmini, Ida H. Somantri, Bambang Suprihatno, and Iman Ridwan. Rice is the most important food crop in Indonesia. Increase in production is needed due to population increase. Rice production in rainfed area is contributed the second after irrigated area. Rainfed condition requiring very early maturity (90-104 days) varieties. Rice anther culture can be applied to accelerate obtainment of doubled haploids (DHs) or pure lines needed in rice breeding. The experiment was aimed to obtain pure lines for developing very early maturing and high yielding rice varieties. Materials used for anther culture were F1s of Fatmawati/Kinamase, Inpari 1/Kinamase, Fatmawati/ Waseaikoku, Inpari 1/Waseaikoku, Fatmawati/IR71146, Inpari 1/IR71146, OM4495/Silugonggo, IR7146/Dodokan, and IR71730/OM1490. Anther culture media were N6 + NAA 2,0 mg/l + kinetin 0,5 mg/l for callus induction, MS+ NAA 0,5 mg/l + kinetin 2,0 mg/l for plantlet regeneration, and MS + 0,5 mg/l IBA for rooting. Putrescine 10-3 M was added to callus induction and regeneration media. The results shown that calli forming green plantlet (CFGP) were ranged from 0.25 to 83.33%. Fatmawati/Kinamase gave the highest CFGP (245 calli), followed by Inpari 1/Kinamase (78 calli) and Fatmawati/ Waseaikoku (68 calli). Total green plantlets obtained were 2.038 plantlets. After plantlet acclimatization and greenhouse grow-out, we obtained 507 DHs. The evaluation of 100 DHs at farmer field (Ciranjang District in Cianjur), based on their 50% heading date of 65 days, resulted in 33 lines cathegorized as very early maturing lines (+100 days). They were 18 lines from Fatmawati/Kinamase, 5 lines from Inpari 1/Kinamase, 8 lines from Fatmawati/Waseaikoku, and 2 lines from Inpari 1/ Waseaikoku. AbstrakPadi (Oryza sativa L.) merupakan komoditi pangan terpenting di Indonesia. Peningkatan produksi diperlukan seiring dengan peningkatan jumlah penduduk. Lahan sawah tadah hujan merupakan lumbung padi kedua setelah sawah irigasi. Kondisi lahan sawah tadah hujan memerlukan varietas-varietas padi berumur sangat genjah (90-104 hari). Teknik kultur antera dapat digunakan untuk mempercepat perolehan tanaman dihaploid (DH) atau galur murni dalam pemuliaan padi. Penelitian ini bertujuan untuk mendapatkan galur-galur murni yang akan digunakan dalam perakitan padi berdaya hasil tinggi dan berumur sangat genjah. Bahan tanaman yang digunakan untuk kultur antera adalah malai dari tanaman F1 hasil persilangan Fatmawati/Kinamase, Inpari 1/Kinamase, Fatmawati/Waseaikoku, Inpari 1/Waseaikoku, Fatmawati/IR71146, Inpari 1/ IR71146, OM4495/Silugonggo, IR7146/Dodokan, dan IR71730/OM1490. Media kultur antera adalah N6 + NAA 2,0 mg/l + kinetin 0,5 mg/l untuk media induksi kalus, MS+ NAA 0,5 mg/l + kinetin 2,0 mg/l untuk media regenerasi, dan MS + 0,5 mg/l IBA untuk media perakaran. Putresine 10-3 M ditambahkan pada media induksi kalus dan regenerasi. Hasil penelitian menunjukkan bahwa kalus yang menghasilkan tanaman hijau (KMTH) berkisar antara 0,25-83,33%. Persilangan Fatmawati/ Kinamase memberikan KMTH tertinggi (245 kalus), diikuti oleh Inpari 1/Kinamase (78 kalus) dan Fatmawati/ Waseaikoku (68 kalus). Total tanaman hijau yang diperoleh adalah 2.038 planlet dihaploid, namun diperoleh 507 tanaman setelah planlet diaklimatisasi dan tanaman ditumbuhkan di rumah kaca. Evaluasi terhadap 100 DH dilakukan di lahan petani Ciranjang, Cianjur. Berdasarkan hari berbunga 50% (65 hari setelah semai), diperoleh 33 galur yang termasuk kategori sangat genjah (dipanen +100 hari). Galur-galur tersebut adalah 18 galur dari persilangan Fatmawati/Kinamase, 5 galur dari persilangan Inpari 1/Kinamase, 8 galur dari persilangan Fatmawati/ Waseaikoku, dan 2 galur dari persilangan Inpari 1/ Waseaikoku.

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Androgenesis of Red Cabbage in Isolated Microspore Culture In Vitro

Plants ◽

10.3390/plants10091950 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1950

Author(s):

Anna Mineykina ◽

Ludmila Bondareva ◽

Alexey Soldatenko ◽

Elena Domblides

Keyword(s):

Culture Medium ◽

Microspore Culture ◽

Doubled Haploids ◽

Antioxidant Properties ◽

F1 Hybrids ◽

Anthocyanin Content ◽

Isolated Microspore Culture ◽

Culture In Vitro ◽

Red Cabbage

Red cabbage belongs to the economically important group of vegetable crops of the Brassicaceae family. A unique feature of this vegetable crop that distinguishes it from other members of the family is its unique biochemical composition characterized by high anthocyanin content, which gives it antioxidant properties. The production mainly uses F1 hybrids, which require constant parental lines, requiring 6–7 generations of inbreeding. Culture of isolated microspores in vitro is currently one of the promising methods for the accelerated production of pure lines with 100% homozygosity. The aim of this study is to investigate the factors and select optimal parameters for successful induction of red cabbage embryogenesis in isolated microspore culture in vitro and subsequent regeneration of DH plants. As a result of research, for the first time, it was possible to carry out the full cycle of obtaining DH plants of red cabbage from the induction of embryogenesis to their inclusion in the breeding process. The size of buds containing predominantly microspores at the late vacuolated stage and pollen at the early bi-cellular stage has to be selected individually for each genotype, because the embryoid yield will be determined by the interaction of these two factors. In the six samples studied, the maximum embryoid yield was obtained from buds 4.1–4.4 mm and 4.5–5.0 mm long, depending on the genotype. Cultivation of microspores was carried out on liquid NLN culture medium with 13% sucrose. The maximum number of embryoids (173.5 ± 7.5 pcs./Petri dish) was obtained on culture medium with pH 5.8 and heat shock at 32 °C for 48 h. Successful embryoid development and plant regeneration by direct germination from shoot apical meristem were achieved on MS culture medium with 2% sucrose and 0.7% agar, supplemented with 6-benzylaminopurine at a concentration of 1 mg/L. Analysis of the obtained regenerated plants, which successfully passed the stage of adaptation to ex vitro conditions by flow cytometry, showed that most of them were doubled haploids (up to 90.9%). A low number of seeds produced by self-fertilization in DH plants was observed.

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Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.

Plants ◽

10.3390/plants9020278 ◽

2020 ◽

Vol 9 (2) ◽

pp. 278 ◽

Cited By ~ 3

Author(s):

Daria Shumilina ◽

Dmitry Kornyukhin ◽

Elena Domblides ◽

Alexey Soldatenko ◽

Anna Artemyeva

Keyword(s):

Microspore Culture ◽

Doubled Haploids ◽

Genetic Disorder ◽

Cold Treatment ◽

Microspore Embryogenesis ◽

Culture Conditions ◽

Secondary Embryogenesis ◽

Isolated Microspore Culture ◽

Pure Lines

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

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Ploidy of Regenerated Broccoli Derived from Microspore Culture Versus Anther Culture

HortScience ◽

10.21273/hortsci.33.3.533g ◽

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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INDUCTION OF SPOROPHYTIC DIVISION IN ORCHIDS MICROSPORES BY STRESS

KnE Life Sciences ◽

10.18502/kls.v2i1.181 ◽

2015 ◽

Vol 2 (1) ◽

pp. 390

Author(s):

Ari Indrianto ◽

Chairani Siregar ◽

Sutikno Linuhung ◽

Mekartinita _ ◽

Tri Sartikoningsih

Keyword(s):

Doubled Haploid ◽

Male Gametophyte ◽

Microspore Culture ◽

Doubled Haploids ◽

Microspore Embryogenesis ◽

Flower Bud ◽

Culture Methods ◽

Cold Temperatures ◽

Media Formulation ◽

Isolated Microspores

Orchid is one of the important ornamental plants in Indonesia this plant generally propagated by seed. Enhancing quality of this plant through breeding technology by various plant tissue culture methods and biotechnology, including doubled haploid technology are necessary. The most efficient method in creating doubled haploids plant is via microspore embryogenesis. We have develop new, innovative doubled haploid technology using the technique of isolated microspore culture. The goals are to obtain data on the male gametophyte development, viable embryogenic microspores, microspores derived embryos and double haploid plants of Orchid. Development of male gametophytes were analysed by isolation of microspores and pollen at various stages and staining with DAPI. Isolated orchid buds of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata were subjected to cold temperatures (4oC) for 7 days, microspores were then isolated by crushing the pollinia using glass rod and cultured them in embryogenesis A2, NP, MS and VW medium, viability of the microspores were determine by using Flourescein diacetate (FDA). Isolated Orchid pollinia were cultured in starvation medium B at various temperatures and duration of time to evaluate embryogenic response, isolated microspores then were cultured further in the basic embryogenesis medium and incubated at 25 oC in the darkness. The result showed that floral characteristics for the late-uninucleate stage of the microspores were different for every orchid spesies. Ovulum lenght was used for Vanda, while in Dendrobium, Phalaenopsis, Arachnis, Spathoglottis plicata and Cattleya, varied length of flower bud was used. Isolated microspores of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata at 7th days of culture in different media formulation showing different respond of viability. Medium A2 keeping viability of Dendrobium hybrid 1 microspores better than any other medium, while in Vanda tricolor and Spathoglotis plicata embryogenesis NP medium was superior. Incubation of orchid pollinia at 4 and 25 oC were successfully maintain viability of the microspores during starvation periods but not able to block gametophytic development. In contrast starved pollinia at 33oC were succesfully block gametophytic development, percentage of embryogenic microspores after starvation of isolated pollinia at 33°C for 4 days was superior compare to any other treatments. Symmetrical divisions and some multicellular structures were observed, which were clear indication for the sporophytic development of microspore-derived embryos, they had developed and after a few weeks they degenerated and died. Keywords: flower bud-pollinia-microspore-stress-embriogenic-embryo-Orchid

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High production of wheat double haploids via anther culture

Genetika ◽

10.2298/gensr0703405k ◽

2007 ◽

Vol 39 (3) ◽

pp. 405-411

Author(s):

Ankica Kondic-Sipka ◽

Boris Kobiljski ◽

Nikola Hristov

Keyword(s):

Anther Culture ◽

Triticum Aestivum L ◽

Green Plant ◽

F1 Hybrids ◽

Regeneration Ability ◽

Breeding Programs ◽

High Production ◽

Dh Lines ◽

Wheat Lines

Androgenous and regeneration abilities of 14 randomly selected F1 hybrids of wheat (Triticum aestivum L.) were analyzed. Anthers were grown in vitro on a modified Potato-2 inductive medium. The hybrid NS111-95/Ana had the highest average values for androgenous capacity (33%) and callus yield (119%), while the hybrid NS 92-250/Tiha had the lowest values for these traits (9 and 21%, respectively). Seven genotypes (50%) had a frequency of green plants relative to the number of isolated anthers of over 10%, with the highest frequency of 21.3% (NS111-95/Sremica). This hybrid produced 12.8 doubled haploid (DH) lines per spike used for isolation. In the other genotypes, the number of produced DH lines per spike ranged from 1 (30?Sc.Smoc.88-89/Hays-2) to 11.2 (NS111-95/Ana). As half of the randomly selected genotypes exhibited high green plant regeneration ability and a high production of DH lines per spike, it can be concluded that in vitro anther culture can be successfully used in breeding programs for rapid production of homozygous wheat lines.

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