Production Of Haploid Plants Research Articles

Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture

The efficient doubled haploid (DH) plant production methods play a key role in accelerating the breeding of new varieties and hybrids in cultivated plants. Consequently, DH plant production methods are continuously improving for barley (Hordeum vulgare L.) breeding and research programs. Two plant regeneration (FHGR and K4NB) and three rooting media (MSr, N6I and ½N6I + Ca) were compared with four F1 barley cross-combinations to clarify the effect of medium on the regeneration of green and albino plantlets and acclimatization. The plant regeneration efficiency was higher using K4NB medium (74.53 green plantlets/100 anthers and 30.85 albino/100 anthers) compared to FHGR (55.77 green plantlets/100anthers and 21.32 albino/100 anthers). The percentage of acclimatization was highest when the K4NB regeneration medium was combined with the MSr rooting medium. Altogether, 61.83% of the anther culture-derived plantlets of 8 cross-combinations acclimatized to the greenhouse conditions, and 1403 acclimatized plantlets were produced from the F1 cross-combinations. Haploid (22.52%), diploid (69.37%) and tetraploid (8.11%) plantlets were identified among the 111 tested green plantlets by flow cytometric analyses. The tetraploid lines can be explored to offer new scopes for future barley research and breeding directions. Nearly one thousand DH plants have been integrated into our barley breeding program.

Gümüş Nitrat ve Ön Soğuk Uygulamalarının Çilek Anter Kültüründe Kallus Oluşumuna Etkileri

Strawberry (Fragaria x ananassa Duch.) which is one of the widely grown berry species in the world has economic and commercial importance. In commercial strawberry varieties, in order to increase yield and quality, it is necessary to obtain starting materials that are resistant/tolerant to biotic and abiotic stress factors. Biotechnological methods have an important place in strawberry breeding studies due to the long and costly process of classical breeding methods, the genetic expansion of seed production, high ploidy level and strong heterozygosity. Haploid plant production is an efficient breeding method that has been successfully applied to most plant species. However, due to the lack of sufficient haploid studies on strawberry and the fact that a specific protocol for this species has not yet been developed the necessary progress has not been made in this regard. In this study, the effectiveness of some factors determine the success in anther culture which has a significant place in obtaining haploid strawberries was investigated. For this reason, first, different sodium hypochlorite doses (NaOCl; 1%, 2%, 3%) and application durations (10, 15, 20 min) were used to determine the appropriate method for sterilisation, then cold pre-treatments (24, 36, 48, 72 hours at +4 °C) and different silver nitrate doses (AgNO3; 10, 20, 30, 40 mg l-1) were employed for callus induction in Festival strawberry variety. At the conclusion of the study it was observed that the lowest contamination rate (1%) was obtained by soaking in 1% sodium hypochlorite solution for 10 minutes. Cold pre-treatment of flower buds at +4 °C for 36 hours produced the highest callus induction rate (96%). The evaluation of the effect of AgNO3 application at different doses on the callus induction rate revealed that the highest callus induction (82%) was obtained from 20 mg l-1 AgNO3 dosage. This study showed that anther culture practices in strawberry can be improved by using cold pre-treatment, appropriate sterilization method and silver nitrate addition to the medium.

Biberde (Capsicum annuum L.) Haploid Bitki Eldesinde Anter Kültürü Yöntemi

Pepper is from the Capsicum genus of the Solonaceae family and is one of the vegetable species that is loved and grown in the world and in our country. After China and Mexico, Turkey ranks third in pepper production in the world. It is a great advantage for us that the climate structure of our country is suitable for vegetable cultivation. For this reason, it has caused a significant variety of pepper in the country, as it adapts to every region in its pepper. In this way, it has also ensured the enrichment of our gene resources. The richness of gene resources is an important factor in the success of breeding studies. One of the purposes of breeding studies is to develop varieties. It is used in breeding studies with anther culture method in pepper, and new varieties are developed in this way. Because of obtaining homozygous lines in plants, haploid plant production has an important place in plant breeding. In this study, the definition and application of anther culture and the factors affecting success, the researches made with the anther culture method in the world and in our country and the developments in recent years are discussed.

From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes

Oats (Avena sativa) hold immense economic and nutritional value as a versatile crop. They have long been recognized as an exceptional choice for human consumption and animal feed. Oats’ unique components, including proteins, starches, and β-glucans, have led to its widespread use in various food products such as bread, noodles, flakes, and milk. The popularity of oat milk as a vegan alternative to dairy milk has soared due to the increasing number of vegetarians/vegans and growing environmental awareness. Oat milk offers a sustainable option with reduced greenhouse gas emissions during its production, rendering it an appropriate choice for individuals who are lactose-intolerant or have dairy allergies. To ensure improved adaptability and enhanced nutrition, the development of new oat varieties is crucial, considering factors like cultivation, climate, and growing conditions. Plant cell culture plays a crucial role in both traditional and contemporary breeding methods. In classical breeding, plant cell culture facilitates the rapid production of double haploid plants, which can be employed to accelerate the breeding process. In modern breeding methods, it enables genetic manipulation and precise genome editing at the cellular level. This review delves into the importance of oats and their diverse applications, highlighting the advantages of plant cell culture in both classical and modern breeding methods. Specifically, it provides an overview of plant tissue culture, encompassing genetic transformation, haploid technology, protoplast technology, and genome editing.

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Obtaining haploid plants by irradiated pollen culture in oil seed crops

Haploid plant production is of great importance to shorten the breeding period in plant breeding programs. Obtaining pure lines in plant growing programs require an intensive work with huge labor and time. Obtaining one hundred percent homozy-gous pure lines is a key point for the improvement and development of new cultivars. Haploid plants with a single set of homozygous chromosomes have become a valua-ble tool in plant breeding. Dihaploid plants that are homozygous at all loci with dou-bling of their chromosomes can be propagated by seed and reach full homozygosity in a single generation. Traditional methods take seven years to reach homozygosity. Dihaploidization methods provide significant advantages in terms of gaining homo-zygosity in a short period of one year and bringing pure lines into agriculture. Anther culture and irradiated pollen technique are among the most widely used techniques in this respect; where physical or chemical agents are used to induce mutated pollen grains and anthers that are subsequently employed to develop dihaploids through in vitro cultures. These techniques are a good source to facilitate gene mapping, cyto-genetic research, and evolutionary studies. Irradiated pollen culture techniques have been applied to many oilseed crops to obtain pure lines. This study highlights some salient features of producing dihaploids using irradiated pollen grains and their maintenance.

Application of Auxins in Haploid Embryo Induction in Hexaploidy Wheat

Doubled haploid (DH) plant production plays a crucial role in modern plant breeding programs, offering an efficient means to generate homozygous lines from heterozygous parents within a single generation. Different types of auxins have been utilized in wheat cross with maize DH production, with 2,4-D being the most widely used and effective hormone, followed by dicamba. Other auxins, including picloram, Indole-3-acetic acid (IAA), phenylacetic acid (PAA), silver nitrate, 1-naphthaleneacetic acid (NAA), kinetin, 6-benzyladenine (BA), and zearalenone, have also been tested for their potential role in haploid embryo induction. Various methods have been explored for the application of 2,4-D, such as spray, tiller injection, dipping, and spikelet culture methods. 2,4-D found to be most effective auxin treatment both alone and with combination with other phytohormones.

Molecular profiling of some blast resistance genes and analysis of physiological variability in rice plants produced by anther culture and hybridization

For genetic improvement of rice plants against blast fungus disease caused by Magnaporthe oryzae, eight rice genotypes including four Egyptian and four foreign genotypes which represented a broad range of variation for blast resistance and other several traits were used in this study through line X tester mating design to produce F1 hybrids used as source of anther culture. Results of analysis of variance showed highly significant differences for all studied traits indicating overall genetic variations among parents and hybrids suggested that specific combining ability referred heavily to the expression of studied traits. Data of anther culture experiment for parents (8 parents) and hybrids (15 crosses) showed highly significant differences for all studied traits which illustrate genetic variations through the used materials. The highest callus induction percentage was found for CR7 (18.3%) and P6 (14.0%) while, the lowest callus induction percentage was found for CR5 (2.0%) and CR1 (4.0%). On the other hand, nine out of 15 hybrids recorded regeneration percentage more than the highest parental genotypes. Anther culture-derived plants and their parental genotypes were evaluated for blast disease resistance according to artificial infection with 10 blast races. The results showed that resistance percentage varied between 10% and 100%. While all anther culture-derived plants showed high level of resistance (100%) against all tested blast races. For enzyme activities for catalase, peroxidase and polyphenol oxidase in anther culture-drived plants and their parent, results showed that line TC9 derived from CR9 gave the highest value for catalase activity (301). Also, CR11 and P4 recorded the high level of peroxidase and polyphenol oxidase activities, respectively. Moreover, genetic diversity among anther culture-derived plants and their parental genotypes using five SSR primers related to Pi5(t), Pikh, Pi-b, Pi-1 and Pi-ta blast resistance genes showed heterozygous bands in all tested genotypes. The highest PIC value recorded for RM 247 and RM 206 markers which had PIC values of 0.778 and 0.761, respectively. Out of the used five SSR markers, RM 247 was the most informative marker with amplicon size ranged from 134 to 242 bp. These markers are therefore useful markers for studying polymorphism concerning blast resistance in rice and hence can be used in the marker-assisted selection for blast resistance. While development of resistant genotypes is the most effective way to control rice blast disease, anther culture technique used in this study helpful in production of resistant double haploid plants used in rice preeding programe in Egypt.

Doubled Haploids: Contributions of Poland’s Academies in Recognizing the Mechanism of Gametophyte Cell Reprogramming and Their Utilization in Breeding of Agricultural and Vegetable Species

Abstract Diverse processes leading to doubled haploid (DH) plant production, such as microspore embryogenesis, gynogenesis, and distant hybridization followed by genome elimination, are based on the unique ability of plant cells to form haploid embryos without fertilization. All of these are possible because of various in vitro culture systems that enable the growth and development of tissues or single cells outside of the parental organism. The possibility of re-directing cell development from its original pathway to embryogenesis brings several benefits to many research areas, but the most important is the possibility of its implementation in breeding programs. This review summarizes the achievements of Polish research groups in studies of the mechanisms of haploid/DH embryo development and demonstrates the practical applications of these systems in basic studies and plant breeding. It shows the results of studies on economically important crops including barley ( Hordeum vulgare L.), oilseed rape ( Brassica napus L.), triticale (× Triticosecale Wittm.), oat ( Avena sativa L.), rye ( Secale cereale L.), sugar beet ( Beta vulgaris ssp. vulgaris L.), and some vegetable species, including carrot ( Daucus carota L.), onion ( Allium cepa L.), red beet ( Beta vulgaris L.), and members of the Brassicaceae.

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

The Production of Helianthus Haploids: A Review of Its Current Status and Future Prospects.

The genus Helianthus comprises 52 species and 19 subspecies, with the cultivated sunflower (Helianthus annuus L.) representing one of the most important oilseed crops in the world, which is also of value for fodder and technical purposes. Currently, the leading direction in sunflower breeding is to produce highly effective heterosis F1 hybrids with increased resistance to biotic and abiotic stresses. The production of inbred parental lines via repeated self-pollination takes 4-8 years, and the creation of a commercial hybrid can take as long as 10 years. However, the use of doubled haploid technology allows for the obtainment of inbred lines in one generation, shortening the time needed for hybrid production. Moreover, it allows for the introgression of the valuable genes present in the wild Helianthus species into cultivated sunflowers. Additionally, this technology makes it possible to manipulate the ploidy level, thereby restoring fertility in interspecific hybridization. This review systematizes and analyzes the knowledge available thus far about the production of haploid and dihaploid Helianthus plants using male (isolated anther and microspore cultures) and female (unpollinated ovaries and ovules culture) gametophytes, as well as by induced parthenogenesis using γ-irradiated pollen and interspecific hybridization. The genetic, physiological, and physical factors influencing the efficiency of haploid plant production are considered. A special section focuses on the approaches used to double a haploid chromosome set and the direct and indirect methods for determining the ploidy level. The current analyzed data on the successful application of haploid sunflower plants in breeding are summarized.

Triticale haploidy <i>in vitro</i> (literature review)

Triticale (× Triticosecale Wittmack) is a hybridized grain crop developed from wheat and rye crossings. Today, triticale is a multipurpose commercial grain crop with great potential as a human food and animal feed. The sown area of the new grain crop in the world reached 4 million hectares in 2018, grain production was about 14 million tons. The current climate change, the rapid evolution of pathogens, as well as the requirements of the modern market dictate the necessity for accelerated development of varieties while reducing the cost of their development. The production of double haploids makes it possible to reduce the time required for the development of homozygous lines by an average of 5–7 years. For the mass production of haploid triticale plants in vitro, there are used two methods, namely anther/microspore culture and distant hybridization followed by selective chromosome elimination of the pollinator. The most critical factors for the success of developing haploids in anther culture are a genotype, growing conditions of donor plants, a microspore development stage, stress effects on heads or anthers, and a nutrient media. Among the unresolved problems of the method are a genotypic dependence, a high incidence of albinism and a presence of aneuploids in the androgenic plant progeny. The rye genome is more often involved in chromosomal transformations than the wheat genome. Most aneuploids are nullisomics, most often on the 2R and 5R chromosomes. Nullisomic plants for 2R and 5R chromosomes have fewer number of spikelets per head and fewer number of kernels per head. In order to develop haploids by the method of selective chromosome elimination during distant hybridization, there have been successfully used such grain crops whose pollen is insensitive to Kr-genes, as maize (Zea mays L.) and wild cereal grass ‘Imperata Cylindrical’ (Imperata cylindrical L.). The advantages of the method are less genotypic dependence, absence of albino plants, genetic stability of regenerants, and reduced costs for developing haploid plants. The length of flowering period of ‘Imperata Cylindrical’ and the absence of the need to combine the timing of flowering period of the parents ensure the economy and efficiency of using this species when developing haploid triticale plants. The purpose of the current review was to characterize the methods of mass development of haploid triticale plants, to describe their advantages and disadvantages when being used in the breeding process.

Application of Vacuum Infiltration for the Production of Haploid Plants of Cabbage Broccoli (Brassica oleracea L. Convar. Botrytis (L.) Alef. var cymosa Duch.)

Modern Concepts & Developments in Agronomy Application of Vacuum Infiltration for the Production of Haploid Plants of Cabbage Broccoli (Brassica oleracea L. Convar. Botrytis (L.) Alef. var cymosa Duch.) SK Temirbekova1*, RN Kirakosyan2, EA Kalashnikova2, YUV Afanasyeva3, EV Maslova4, OV Meleshina1, and MM Tareeva5 1All-Russian Research Institute of Phytopathology, Russia 2Russian State Agrarian University-Timiryazev Moscow Agricultural Academy, Russia 3Federal Horticultural Research Center for Breeding, Agrotechnology and Nursery, Russia 4Belgorod State University, Russia 5Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center, Russia *Corresponding author: SK Temirbekova, All-Russian Research Institute of Phytopathology, Russia Submission: February 01, 2022 Published: February 10, 2022 DOI: 10.31031/MCDA.2022.10.000735 ISSN 2637-7659Volume10 Issue 2

The effect of 2,4-dichlorophenoxyacetic acid on the production of oat (Avena sativa L.) doubled haploid lines through wide hybridization.

BackgroundDevelopment of new cultivars is one of the vital options for adapting agriculture to climate change, and the production of doubled haploid (DH) plants can make a significant contribution to accelerating the breeding process. Oat is one of the cereals with particular health benefits, but it unfortunately still remains recalcitrant to haploidization. Our previous studies have clearly demonstrated that post-pollination with hormone treatment is a key step in haploid production through wide hybridization and indicated it as the most effective method for this species. Therefore, we subsequently addressed the problem of the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration on consecutive stages of DH production.MethodsTwenty-nine genotypes were tested, 9,465 florets were pollinated with maize pollen 2 days after emasculation and then treated with 2,4-D at 50 mg/L and 100 mg/L.ResultsThe applied treatments did not reveal any differences in the number of obtained haploid embryos. However, almost twice as many haploid plants formed on MS medium after applying a higher auxin concentration and 20% more successfully acclimatized. Moreover, 100 mg/L 2,4-D treatment resulted in twice as many DH lines that produced almost three times more seeds compared to 50 mg/L treatment. Nevertheless, the results have confirmed the existence of strong genotypic variation, which may significantly limit the development of an effective and economically feasible method that could be incorporated into breeding programs.

Haploid Induction and Plant Production in Bottle Gourd by Pollination with Gamma Irradiated Pollen

Haploid Induction and Plant Production in Bottle Gourd by Pollination with Gamma Irradiated Pollen

Creation of an interspecific hybrid of Capsicum annuum L. and C. frutescens L. using biotechnological approaches

Relevance. Pepper is a common crop both for fresh consumption and for the preparation of spices. Recently, along with the increasing popularity of C. annuum L. pepper, there is increasing interest in other species of this genus, which have a number of breeding and important properties. The most important method of enriching the gene pool of cultivated plants is distant hybridization, through which valuable traits are transferred from wild species to cultivated ones. The development of a new variety is a lengthy process, stretching over several years. In this regard, breeders have faced the challenge of obtaining pure lines to create a pepper hybrid with desired properties by applying modern biotechnological methods that will accelerate this process. One of them is the method of microspore culture, which allows mass production of haploid plants, reducing the time for creating constant parental lines.Material and methods. The aim of the work was to create an interspecific hybrid of hot pepper (C. annuum L. x C. frutescens L.) with high ornamental properties, a complex of economically valuable traits, with good taste qualities. The research was carried out in the film greenhouse of FSBSI FSVC in the Moscow region. The research material was a variety population of hot pepper Capsicum frutescens Cz-544-14, used as a paternal line, which was heterogeneous, and a pure line of C. annuum L. (Pb-551) created by classical breeding.Results. The pepper hybrid F1 Christmas bouquet was created as a result of hybridization of species parental forms obtained by different methods (biotechnological and classical). To accelerate the production of an aligned paternal form of C. frutescens L., the technology of doubled haploids through microspore culture was used. As a result, doubled haploid plants meeting the planned model (compact low habit, purple fruit colouring in technical ripeness and red in biological ripeness) were obtained. The resulting hybrid combined all the necessary economic features: high ornamentality, compactness, bouquet arrangement of fruits, high taste and aroma. Thus, the use of remote interspecific hybridization in the breeding process in combination with biotechnological approaches can accelerate the production of new forms of hot peppers that meet the demands of the market.

Parthenogenetic Haploid Plant Production in Styrian Pumpkin by Gamma Irradiated Pollen

Doubled haploid method via parthenogenesis induction still remained prominent in Cucurbitaceae breeding due to drastic reduction in time and cost of newly released lines. In this study, different doses of Gamma ray (25, 50, 75, 100, and 200 Gy) were used to irradiate pollen grains for induction of parthenogenetic haploid embryos in oilseed pumpkin (Cucurbita pepo var. ‘Styriaca’). Parthenogenetic embryos at different developmental stages were rescued in vitro and 348 plants were obtained, of which 134 were recognized as haploid by ploidy analysis. The highest rate of haploid plants was obtained from globular (25.3%) and torpedo (23.8% plants) embryos followed by arrow-tip (13.4%), torpedo (10.5%), stick (10.5%), heart (9%), and cotyledonary (7.5%), respectively. All doses, except 200 Gy, were effective for induction of embryos and haploid plants; in a way that the highest number of haploids was obtained by 100 Gy. Our results indicated that parthenogenetic haploid embryos could be efficiently induced in C. pepo if proper Gamma ray dose and developmental stage of embryos are selected.

Doubled Haploids in Eggplant.

Simple SummaryThis review compiles the most relevant advances made in the production of doubled haploid plants in eggplant, the application of doubled haploid lines in breeding programs, and the future perspectives for the development of alternative technologies for doubled haploid generation in this species.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.

Effect of Parental Genotypes and Their Reciprocal Crosses on Haploid Plant Production by Anther Culture and Confirmation of Double Haploids by Flow Cytometry in Bread Wheat

Effect of Parental Genotypes and Their Reciprocal Crosses on Haploid Plant Production by Anther Culture and Confirmation of Double Haploids by Flow Cytometry in Bread Wheat

Haploid and Doubled Haploid Plant Production in Brassica rapa L. subsp. pekinensis Via Microspore Culture.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.