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Advances in Haploid Production in Higher Plants
Abstract
The discovery of haploid Datura plants in 1964 initiated great excitement in plant breeding and genetics communities. Recent years have witnessed a resurgence of activities especially in developing protocols, identifying genes and mechanisms and large scale commercial take up.
The identification of controlling genes has driven functional genomic studies which now dovetail with studies in gene expression, metabolism and changes in cell ultra-structure. World wide take up by plant breeders has been no less impressive, and valuable haploid technologies are increasingly patent protected. The intense activity in haploid research has also resulted in unexpected findings with novel applications.
The core of this book is based on the international symposium on "Haploidy in Higher Plants III", (Vienna 2006), which attracted top international experts in the field. Other invited contributions have been included to provide a rounded view of activities. The book covers topics in: Historical Overviews; Basic Biology; Genetics; Biotechnology; Breeding and Novel Applications. It therefore will appeal to undergraduate students,researchers and small and large scale commercial biotechnology companies.
Chapters (27)
In the early 1990s, many basic protocols were developed for haploidy and doubled haploidy, but most were inefficient. During
the last decade, progress in technology has been achieved mainly by empirical, time and cost consuming testing of protocols;
as a consequence success was proportional to the number of laboratories involved. In the most frequently studied crops (barley,
wheat, triticale, maize, rice and rapeseed) improved protocols are now used routinely in breeding and although several problems
remain the benefits make doubled haploidy well worthwhile. Significant advances have also been achieved in vegetable, fruit,
ornamental, woody and medicinal species, though responses in many remain low with legumes being particularly recalcitrant.
There has been resurgence in doubled haploids over the last few years with protocols published for almost 200 plant species.
The present review aims to show the recent progress in haploid and doubled haploid technology of higher plants.
Gynogenic haploid regeneration is an alternative procedure for haploid induction used in several agronomically important species
such as sugar-beet and onion. At inoculation, developmental stage of ovules is frequently immature, but in contrast to androgenesis,
in vitro maturation of embryo sac occurs. The egg cells were found to be the predominant source of haploid embryos while antipodal
or synergid origin has been also proposed for some species. For majority of species triggering factors promoting haploid embryogenesis
are not apparent, but media constituents such as phytohormons and carbohydrates evidently have some role in reprogramming.
The majority of regenerants obtained via gynogenesis are haploid, for optimization of treatments large experimental units
are proposed and alternative to the use of antimitotic agents spontaneous duplication via adventitious regeneration is proposed.
Major characteristics of successful protocols are listed
Production of haploids in barley has been widely used for many years for breeding and breeding method research. Initially
the Bulbosum method was available and provided a good random sample of female gametes as plants. Anther culture (male gametes)
improved more gradually but has been widely used and more recently, isolated microspore culture has been perfected for barley
breeding programmes and research. Thus, barley is now considered the cereal model crop species for haploid production and
research. Doubled haploid populations have been extensively utilized in molecular marker work in barley providing detailed
chromosome maps. Barley microspores are often selected for biochemical and cytological investigations of androgenesis. With
the advancement in molecular, biochemical and cytological tools, haploid research has shifted to sequencing of DNA and transcriptome
analyses that reveals hundreds of genes are involved in induction and embryogenesis from microspores. These genes can be identified
and associated with various biochemical pathways and the proteins and enzymes involved. Transformation in barley is progressing
well using haploid systems
The production of doubled haploid (DH) plants is one of the most important developments in biotechnology. Haploids and DH
have been produced in Brassica ssp. using anther culture or isolated microspores. The microspore culture is a simple and affordable technique for production
of DH plants, and therefore is the method of choice for plant genetic research and breeding programmes in oilseed Brassicas. Microspore culture is a useful tool in producing doubled homozygous lines for breeding since lines exhibiting desired agronomic
traits can be rapidly obtained. Microspore culture is also useful in gene transfer, biochemical and physiological studies
and in the production of desired traits such as herbicide resistance and fatty acid modification through mutagenesis and selection
Factors influencing DH production as well as current applications of Brassica doubled haploidy are discussed in this work
Androgenesis is a process of redirection of normal pollen development towards the formation of haploid embryos and ultimately
doubled-haploid plants. This review gives a historical overview of different procedures used to induce androgenesis in tobacco.
The various factors that influence this developmental switch including growth of donor plants, pre-treatments, culture media,
as well as cytological aspects of cell and tissue changes that occur during the induction and development of the microspore-derived
embryos are described. Finally, this review will cover recent molecular data on mechanisms of embryogenic induction in cultured
tobacco microspores
This review report on the progress made to develop doubled haploid methods in triticale. Wide hybridization, anther culture
and isolated microspores methods have been well established in triticale, and adopted in breeding and germplasm development
programs for diverse genotypes. The development of these methods and culture media has co-evolved with those for its wheat
counterpart. Essentially the critical factors for success remain the same: genotype, growing condition of mother plants, stress-like
pretreatment and the culture medium composition. Today, C17, N6 and NPB99 induction medium for microspore culture are preferred.
They carry maltose, sucrose or a combination of both, amino acids such as glutamine or proline, a low concentration of auxin
and/or cytokinin. Spontaneous doubling has been reported at a low frequency in microspore derived plants making chromosome
doubling by colchicine treatment an absolute need, as for wide hybridization derived plants. Isolated microspore culture is
a method particularly attractive that will open new opportunities for gene transfer in triticale
Abstract One of the important themes in any discussion concerning the application of haploids in agricultural biotechnology
or elsewhere is the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential
expertise, usually related to methodology and referred to as “Trade Secrets”. This review will explain the concepts behind
patent protection, and will use the international patent databases to analyse the content of these patents and trends over
the last 20 years. This analysis from regions including North America, Europe, and Asia reveals a total of more than 30 granted
patents and a larger number of applications. The first of these patents dates from 1986, and although the peak of activity
was in the late 1990s, there has been continuous interest to the present day. The subject matter of these patents and applications
covers methods for anther and pollen culture, ovule culture, the use of specific haploid-inducing genes, the use of haploids
as transformation targets, and the exploitation of genes that regulate embryo development. The species mentioned include cereals,
vegetables, flowers, spices and trees.
Isolated microspores from selected cultivars of Brassica napus readily form embryos in culture after mild heat stress treatments (32°C for 1–3 days). Transcript profiling methods were
used to identify differentially-expressed genes as well as shifts in metabolism during the early stages of microspore embryogen-esis.
Approximately 20,000 expressed sequence tags (ESTs) from cDNA libraries representing freshly-isolated microspores (0 hours)
and microspores cultured for 3, 5 or 7 days under embryogenesis-inducing conditions were prepared. In silico analyses of ESTs and semi-quantitative and real time reverse transcription-polymerase chain reaction (RT-PCR) based profiling
identified differentially-regulated gene clusters and 16 genes that could be used as specific markers for microspore embryogenesis.
These molecular marker genes also were expressed during zygotic embryogenesis, underscoring the common developmental path
ways that function during zygotic and gametic embryogenesis. Future studies will focus on characterization of embryogenesis-related
genes and development of fluorescently-labeled gene/protein probes to precisely mark and isolate early stages of microspore
embryogenesis.
In barley, microarray technology has allowed to study the gene expression profiling associated with the stress pretreatment
phase of microspore embryogenesis, where the reprogramming of microspores takes place. Transcriptome analysis of anthers before
and after 4 days of mannitol pretreatment, revealed changes in the expression of 2,673 genes. A thorough study of these differentially
expressed genes indicated that microspores maintained their cell cycle machinery in a steady state during stress pretreatment
and underwent a major reorganization of metabolic pathways accompanied by a multi-dimensional stress response. Up-regulation
of transcription factors related to stress responses and changes in developmental programmes took place during the pretreatment.
Preliminary studies have indicated that YABBY5, ZML2, CURLY LEAF and ICE1 transcription factors have a stress pretreatment
specific induction and, therefore, could play a direct role in microspore reprogramming.
This chapter reviews the proteomics research performed to elucidate the molecular processes associated with embryo initiation
and development in Brassica napus (rapeseed) microspore-derived embryo cultures. Classical biochemical methods such as combined [35S]-methionine labelling and 2-D gel electrophoresis (2-DE) have been used for the large-scale study of proteins differentially
expressed during the inductive phase of embryogenesis. Immuno-cytological experiments have been carried out to study the (sub)cellular
localization of putative marker proteins for embryo induction. Changes in post-translational modification of proteins (phosphorylation)
have been detected during the induction phase, as have changes in the profile of proteins en peptides secreted by developing
embryos. We also discuss recent developments in quantitative proteomics technologies such as fluorescence labelling of protein
samples and stable isotope labelling by amino acids in cell cultures that enable more detailed expression analyses and the
identification of low abundant proteins in complex mixtures.
Programmed cell death (PCD) occurs in the anther during in vivo micro-spore/pollen development. The first hallmarks are detected in the tapetum at meiosis and consist in progressive degeneration
of organelles such as mitochondria, as well as DNA degradation into multiple of 180 kpb fragments. This phenomenon radially
extends in the anther sporophytic tissues and finally affects microspore/pollen at various stages of development depending
upon species. PCD may infer with the process of microspore embryogenesis. Indeed, the inducing pre-treatment is per formed
using the whole anther during the process of microspore vacuolation. Thus the stress of pre-treatment may influence the triggering
of PCD in both the anther tissues and the microspores and is likely involved in the competence of microspore to the process
of microspore embryogenesis. These data are also discussed consid ering the regeneration of albino microspore derived plants
in cereals.
Frequent formation of albino plants from in vitro cultured microspores is a particular problem for chromosome doubled haploid production in cereals and grasses. The phenomenon
was first thought to be associated with maternal inheritance of plastids visualized by large deletions and rearrangements
of plastid genomes in albino plants. Subsequently interests have changed to inactivation of plastid ribosomes, which has been
shown to create albino phenotypes in vitro. A considerable knowledge on genetic regulation of the trait is used in this chapter to forward a hypothesis that the stressful
in vitro conditions in these cultures make the plants fight their own plastids with antibiotic like compounds.
The doubled haploid (DH) technique is routinely applied in winter rapeseed breeding programmes for the generation of completely
homozygous lines. Although the method is quite old and perfected, there are still some problems that prevent a universal application
of the technique in winter rapeseed breeding. Key problems remain the insufficient diploidisation rate, the low seed yield
of primary haploids and the time-consuming and inadequate plantlet regeneration from the embryos. This contribution summarizes
current applications and approaches to tackle above problems. Methods of early in vitro selection of microspore derived embryo genotypes are presented that appear promising for a further optimisation of the DH
technology in rapeseed.
In oat (Avena sativa L.) low production rates have limited the use of doubled haploids (DH). Alternative techniques for oat DH production are
anther culture and wide hybridization. In this paper, progress in anther culture is summarized, concentrating specially on
the work done in MTT Agrifood Research Finland. Up to 30 green plants per 100 isolated anthers have been regenerated from
cultivated oat, but common recoveries are much below that. Genotype-dependency is also evident. Oat anther culture can be
used to produce DH populations for genetic mapping, as well as material for other genetic research purposes. Application to
the practical cultivar breeding may still demand improved DH production efficiency, as well as overcome of the genotypic restrictions.
The application of doubled haploids is a routine in barley and wheat breeding today. While in barley anther and isolated microspore
culture are efficient technologies in wheat wide crosses with maize and subsequent phytohormone treatment and embryo rescue
are better established due to genotype dependency of wheat in androgenic technologies. Although a quite significant number
of DH lines can be produced in breeding programmes there are still some limiting factors which are related to technical and
genetic factors. The actual status of DH lines in barley and wheat breeding and some of the limiting factors are listed and
described below. Furthermore some aspects of R- programmes to circumvent those limitations are described, followed by an outlook.
Cell- and tissue culture methods in combination with conventional breeding process were suitable way to produce new varieties.
These applications gave new breeding alternatives to release competitive genotypes in comparison with traditional ones. To
the breeding of ‘Risabell’ (1997) DH lines were produced via anther culture from F2 population of a single cross combination. The new variety was improved for resistance to blast disease, high milling and
cooking quality and long grain type. In case of ‘Janka’ (2003) haploid cell cultures were developed and their vigorous regenerants
were colchicine treated. One of the best fertile lines was released as ‘Janka’ has vigorous seedling growth, drought tolerance
and good grain quality. The variety ‘Ábel’ (2005) was improved through somatic tissue culture regeneration followed by anther
culture. Main characteristics of this variety are earliness, early stage cold tolerance and good performance in aerobic conditions.
The breeding-processes of these State approved rice varieties demonstrate the successful integration of biotechnological methods
and pedigree-breeding.
Potato (Solanum tuberosum L. ssp. tuberosum) was one of the first crop plants in which haploid techniques were used to improve cultivar breeding programmes. These new
breeding tools were introduced towards the end of the 1950s but have not totally replaced the conventional breeding of potato
at the tetraploid (2n = 4x) level. Generally (di)haploid (2n = 2x) lines are produced by pollination of cultivated potato
and related Solanum species with specific haploid inducer clones of S. phureja or alternatively by anther culture in vitro. The resultant clones provide excellent material for the subsequent reconstitution of the polyploid hybrids having maximized
heterozygosity levels. Therefore the haploids have a considerably significant role in the potato breeding programmes of quite
a few companies. Certain valuable haploid techniques, such as anther culture and somatic hybridization, are quite complex
and highly genotype dependent and thus are less readily put into practise. There is an important application for use of haploids
in interspecific hybridization to overcome incompatibility barriers caused by the differences in ploidy levels and endosperm
balance numbers. Thus, the gene pool of the potato can be broadened and certain valuable traits such as disease resistance
characters from the wild solanaceous species can be more efficiently introgressed into cultivated potato.
Although herbs, spices, medicinal, and nutraceutical plants have been used in human health for millennia, there has been renewed
interest over the past number of years. In the past, people have relied on landraces or “wild” plants as there has been very
little breeding or genetics done on these species. This is changing as consumers are demanding scientific evidence for the
medical claims (clinical trials) as well as uniformity in the products. Doubled haploidy would be one way in which to achieve
homozygous, true-breeding lines. Haploidy response, i.e. callus, embryos, regenerated haploid/doubled haploid plants, have
been reported in a number of medicinal/nutraceutical species, however the frequency of response is low compared to other species.
This review will focus on a few of the major plant families with medicinal properties.
Isolated microspore culture experiments were carried out to induce microspore embryogenesis in Chamomilla recutita, Solidago virgaurea, Sanvitalia procumbens of the Asteracea, and Valeriana officinalis of the Valerianaceae. The Asteracea is one the largest plant families of commercial significance for medicinal, aromatic, food and ornamental
use. Availability of protocols for an efficient production of doubled haploids via microspore embryogenesis would facilitate
breeding efforts in this family. Following the establishment of microspore culture protocols uninucleate microspores divided
symmetrically in all the species studied. Additionally, in Sanvitalia multi-nucleate structures were observed, without further development. Swelling of the microspores was routinely achieved,
and viability was maintained up to 2 weeks. Results were encouraging for further studies in microspore embryogenesis in the
Asteraceae.
Anther culture of over 20 carrot cultivars was studied. The uni-nucleate stage proved optimal for embryogenesis for which
bud length was used as a surrogate. Large genotypic variation in culture response was found. Anther culture at 27°C in the
dark without sub-culturing had a significant beneficial impact on embryogenesis. Embryos were obtained regardless of donor
plant culture; however, plants grown in glasshouse conditions produced more embryos than those from the field. Secondary embryogenesis
was induced in some cultures and in one case, 102 plants were obtained from one embryo (on B5 medium without hormones) over a 12 week period. Regenerated plants were planted into peat and acclimatized to glasshouse
and growth room conditions. Cytological and cytometric studies revealed that over 90% of regenerated plants possessed a doubled
chromosome complement and isozyme analysis showed that 96–100% were homozygous. Anatomical studies confirmed that embryos
had formed from microspores.
Haploid and doubled haploid (DH) production provides a particularly attractive biotechnological tool in breeding of perennial
crop species, such as fruit trees. Haploids (plants with a gametophytic chromosome number) and doubled haploids (haploids
that have undergone chromosome duplication) reduce the time needed to produce homozygous lines compared to conventional breeding.
In fruit crops, characterized by high heterozygosity, long generation cycle times, large size, and (often) self-incompatibility,
there is no other way to produce homozygous breeding lines through conventional methods that involve several generations of
selfing. Gametic embryogenesis, enables the development of haploids and double haploids from heterozygous parents in a single
step, and is therefore increasingly the object of research for fruit breeders. This chapter provides the current status of
research on doubled haploid production in many fruit crops: Actinidia deliciosa, Annona squamosa, Eriobotrya japonica, Carica papaya, Citrus, Feijoa sellowiana, Malus domestica, Morus
alba, [Musa balbisiana (BB)], Prunus armeniaca, Prunus avium, Prunus domestica, Prunus persica, Pyrus communis, Pyrus pyrifolia, Olea europaea, Opuntia
ficus-indica, Vitis vinifera.
Newly developed gynogenesis and androgenesis methods are described for tef. The diminutive floral structures of tef are problematic
and have necessitated the development of spikelet culture and more effective treatments. Media composition has been optimised.
In the case of anther culture, radiation treatments of seed of donor plants enhances the level of androgenesis. Various ploidy
levels are found in the regenerants: haploids, doubled haploids, tetraploids and octoploids. The regenerated plants show variation
for useful agronomic traits. Doubled haploidy in tef offers opportunities for improved genetic studies, accelerated breeding,
the development of useful mutants and the development of new ploidy levels for agricultural exploitation.
F1 hybrid seeds are produced by controlled hybridization of homozygous inbred lines. Male sterility avoids tedious emasculation
procedures of female parental plants but must be reversible easily in order to propagate and maintain the male-sterile lines.
Doubled haploid plants provide complete homozygosity and therefore may be used as parental lines to breed F1 hybrids. An environment-friendly F1 hybrid breeding technology combining reversible male sterility and doubled haploid production was established through controlled
starvation from glutamine to developing pollen. To this end, cytosolic glutamine synthetase was inactivated in tobacco anthers
and microspores by a dominant-negative mutant approach, resulting in male sterility. Fertility was restored by exogenous application
of glutamine. Pollen in transgenic tobacco plants aborted after the first pollen mitosis, which allowed to culture viable
microspores in vitro to produce a non-segregating population of male sterile doubled haploid plants. The design of this novel system virtually
precluded the release of transgenic pollen.
As the immediate product of meiosis, the microspore represents the first stage of pollen development. The haploid chromosome
set in addition to embryogenic competence renders microspores one of the most attractive cellular targets for the transfer
and stable integration of recombinant DNA into plant genomes. However, the development of methods of plant genetic transformation
through gene transfer to embryogenic pollen has turned out to be extremely difficult. In this chapter, diverse gene transfer
approaches based upon embryogenic pollen cultures are critically reviewed with regard to target cell type, cell cycle stage,
method of gene transfer as well as experimental evidence of transgene integration and homozygosity. Typical constraints and
potential pitfalls are discussed and possible strategies to resolve these problems are suggested. Recent demonstrations of
the successful generation of instantly true-breeding transgenic plants via gene transfer to embryogenic pollen cultures in
both dicotyledonous and monocotyledonous species may encourage further development and broad applications of such methods
in research and biotechnology.
Immature pollen grains were used for gene targeting (GT) to evaluate the potential of higher plant male gametophyte as a target
for gene targeting experiments. The artificial B18/4 target locus inserted to tobacco genome has been used to assess gene
targeting in tobacco mid-bi-cellular pollen. In this system, a neomycin-phosphotransferase (nptII) gene, exclusively expressed in seeds, was converted into a constitutive nptII gene by insertion of the CaMV 35S promotor between the HMW seeds-specific promotor and a functional restored nptII gene at the target locus. The tobacco mid-bi-cellular pollen isolated from the B18/4 target locus plants was transformed
by the biolistic approach with the repair construct. GT experiments led to the recovery of seven kanamycin resistant plants.
Southern analysis confirmed that in one transgenic line an ectopic GT event occurred arising by modification of the repair
construct by the target locus and subsequent integration elsewhere in the tobacco genome.
Doubled haploid production has become an important tool in cereal breeding and has helped in accelerating the development
of improved cultivars. Genetic variation for salt tolerance is rare in contemporary semi-dwarf (sd-1) rice germplasm (cultivars and breeding lines). Some rice landraces exhibit greater tolerance to salt, but are agronomically
unacceptable because of their tall stature. One such landrace, Pokalli was subjected to anther culture in an attempt to induce
gametoclonal variation for agronomically important traits such as plant height. Over 100 green plants were regenerated from
2,000 cultured anthers. Among these, two doubled haploid lines (DHs) exhibited semi-dwarf stature. Mutations (induced during
the culture procedure) in the semi-dwarfing gene sd-1 were confirmed by PCR using locus specific primers. The semi-dwarf DH lines were multiplied and checked for response to salinity
in hydroponics (10 dS m−1) and field tests at IRRI, Philippines. The work demonstrates the effectiveness of combining doubled haploid protocols with
mutation detection.
The development of efficient chromosome doubling protocols is essential for the useful application of doubled haploid (DH)
plants in breeding programs, since frequency of spontaneous doubling is most of the time too low. Chromosome doubling has
been traditionally applied to plantlets, being the colchicine the most widely anti-microtubule agent used in vivo and in vitro. However during the last 15 years, protocols have been developed for the incorporation of different anti-microtubule agents
during the early stages of androgenesis or gynogenesis. Factors affecting frequencies of spontaneous and induced chromosome
doubling are summarized. For a successful chemical induction, a compromise between toxicity (which can result from high concentration
and/or long time of application) and genome doubling efficiency should be adopted in order to obtain the highest number of
green DH plants.
... The first nonnatural DH system regenerated haploid embryos through tissue culture from anthers (androgenesis) of Datura (Guha & Maheshwari, 1964). In vitro HI strategies rely on specific tissue culture conditions to reprogram haploid gametophytic tissues into developing haploid embryos or plantlets, as do gynogenesis systems using the female gametophyte (Touraev et al., 2009;Jacquier et al., 2020). Despite efforts to improve the efficiency of in vitro HI, these protocols still require significant labour and skill, and are usually restricted to certain plant species or genotypes, with bespoke protocols needing to be developed empirically (Touraev et al., 2009;Jacquier et al., 2020). ...
... In vitro HI strategies rely on specific tissue culture conditions to reprogram haploid gametophytic tissues into developing haploid embryos or plantlets, as do gynogenesis systems using the female gametophyte (Touraev et al., 2009;Jacquier et al., 2020). Despite efforts to improve the efficiency of in vitro HI, these protocols still require significant labour and skill, and are usually restricted to certain plant species or genotypes, with bespoke protocols needing to be developed empirically (Touraev et al., 2009;Jacquier et al., 2020). ...
In planta haploid induction (HI), which reduces the chromosome number in the progeny after fertilization, has garnered increasing attention for its significant potential in crop breeding and genetic research. Despite the identification of several natural and synthetic HI systems in different plant species, the molecular and cellular mechanisms underlying these HI systems remain largely unknown. This review synthesizes the current understanding of HI systems in plants (with a focus on genes and molecular mechanisms involved), including the molecular and cellular interactions which orchestrate the HI process. As most HI systems can function across taxonomic boundaries, we particularly discuss the evidence for conserved mechanisms underlying the process. These include mechanisms involved in preserving chromosomal integrity, centromere function, gamete communication and/or fusion, and maintenance of karyogamy. While significant discoveries and advances on haploid inducer systems have arisen over the past decades, we underscore gaps in understanding and deliberate on directions for further research for a more comprehensive understanding of in vivo HI processes in plants.
... Complete homozygosity of cells simplifies genome assemblies after sequencing, plant breeding, hybrid seed production, quantitative genetic research and mutation discovery. However, although efficient for some plant species, its success is still highly dependent on the genotype of the plant, and it is not yet suitable for all plant genotypes (Maluszynski et al., 2003;Umehara et al., 2005;Szarejko and Forster, 2007;Touraev et al., 2010). ...
Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.
... Doubled haploids provide a number of advantages, including a shorter breeding cycle due to fast homozygosity fixation, high selection efficiency, increased genetic variety via gametoclonal variations, and early expression of recessive genes appropriate for breeding (Devaux and Pickering 2005). There are numerous reviews that explain how androgenesis produces double haploids and how they are used (Germana, 2011;Touraev et al., 2009;Seguí-Simarro 2010). According to Kim et al. (1991) the best response to anther culture came from a hybrid of Japonica × Japonica, Indica × Japonica, and finally Indica × Indica rice cultivars. ...
... Wang et al. (1973) and George and Narayanaswamy (1973), reported the first successful plant regeneration from C. annuum anthers. This was followed by developing a number of efficient and reproducible protocols for a number of species including crop plants (Touraev et al., 2009). Ferrie and Caswell (2011) investigated the effects of different factors that influence the induction of embryos and regenerating plants. ...
Capsicum annum L. genotypes were used for the double haploid production from F1 hybrid anthers obtained from the field and growth room of Indus & Burpee seeds Pvt. Ltd, Bangalore. In the present study, the effectiveness of induced androgenesis in in vitro anther culture of 6 pepper genotypes was investigated. Inoculation in C media supplemented with 2 mg/L kinetin and 0.1 mg/L 2,4-D followed by subculturing into R media supplemented with 0.1 mg/L kinetin was found to be most suitable for androgenesis. Among the genotypes tested, Caps 10-15 genotype shown the response with embryo formation and developed into double haploid plants, whereas Rizwan red shown response with callus formation. The genotyping of hybrid donor plants of Caps 10-15 and DH plants produced banding pattern differing from that of hybrid banding pattern thereby confirming its haploid origin. Further, analysis of the calluses of Rizwan red produced banding pattern same as that of hybrid banding pattern revealing the heterozygous condition. Hence concluded that these markers could be useful in future for differentiating haploids from diploids in Capsicum annum L. genotypes.
... Many reviews have been published for the production of the haploids (Magoon and Khanna, 1963;Kasha, 1974;Maluszynski et al., 2003;Touraev et al., 2009). Since date, haploids have been produced in many plant species (Dunwell, 2010). ...
Suboptimal temperatures at sowing and emergence affect the early development of maize, with potentially irreversible effects later in the growing season. We studied recovery from cold stress of an inbred line (B73) and 13 Doubled Haploid lines derived from a European flint maize landrace. After a cold treatment (20–12 °C, day–night) from sowing to seedling establishment, seedlings were transplanted and grown in the greenhouse until the V8 stage (eight leaves fully developed), when we measured agronomically relevant plant traits and spectral indices of mature leaves. Survival rates of transplanted seedlings after cold treatment ranged from 10% to 100%. After a strong delay in early development due to cold, the surviving plants were able to compensate for this delay at later stages of recovery. They reached the V8 stage after only five more growing degree days than plants grown under the control treatment (25–18 °C, day–night). Plants from the most cold-tolerant genotypes (PE0401 and PE0100) accumulated more root and shoot biomass at the end of the recovery phase compared with the same genotypes exposed to the control treatment. The genotypes with the most plastic leaf morphological traits (PE0161 and PE0072) had little reduction in leaf biomass at the end of the recovery phase in comparison with less responsive genotypes such as PE0171. We conclude that genotypes that survived cold treatment with minimal cold damage of seedling leaves can be candidates for further cold recovery studies and breeding. Nevertheless, such studies must take trait acclimation for other suboptimal conditions into consideration.
A resurrection has started in haploid and double haploid research in the twenty-first century. The haploid and double
haploid could be achieved through in vivo and in vitro anther and microspore culture techniques. Fixing the homozygosity is the most striking benefit of androgenesis. Various factors like genotypic dependency, growth condition, developmental stage of the microspore, pre-treatment, culture media, regeneration media, growth hormones, and various chemicals have a direct effect. Wheat, rice, Brassica, and tobacco are the notable crops where anther and microspore culture has been utilized. These haploidy and double haploidy through anther culture served many purposes of basic and applied research. Especially, double haploid cultivars have been cultivating around the globe. In addition, for chromosome mapping, QTL mapping, marker-assisted selection, marker-assisted backcrossing, mutation breeding, genome-wide association study, genomic engineering, and genome editing, androgenesis based haploid and double haploid plants have been exploited due to the effectiveness. Recently, researchers are trying to explain albinism that happens during anther culture from an epigenetic perspective. Further prospects of haploid and doubled haploid research through anther culture have been described in this review.
Anther culture is an efficient biotechnological tool in modern plant breeding programs to produce new varieties and parental lines in hybrid seed productions. However, some bottlenecks—low induction rate, genotype dependency, albinism—restrict the widespread utilization of in vitro anther culture in rice breeding, especially in Oryza sativa ssp. indica (indica) genotypes, while an improved efficient protocol can shorten the process of breeding. Three different induction media (N6NDK, N6NDZ, Ali-1) and four plant regeneration media (mMSNBK1, MSNBK3, MSNBKZ1, MSNBKZ2) were tested with five indica rice genotypes to increase the efficiency of in vitro androgenesis (number of calli and regenerated green plantlets). The production of calli was more efficient on the N6NDK medium with an average 88.26 calli/100 anthers and N6NDZ medium with an average of 103.88 calli/100 anthers as compared to Ali-1 with an average of 6.96 calli/100 anthers. The production of green plantlets was greater when calli was produced on N6NDK medium (2.15 green plantlets/100 anthers) compared when produced on to N6NDZ medium (1.18 green plantlets/100 anthers). Highest green plantlets production (4.7 green plantlets/100 anthers) was achieved when mMSNBK1 plant regeneration medium was used on calli produced utilizing N6NDK induction medium. In the best overall treatment (N6NDK induction medium and mMSNBK1 plant regeneration medium), four tested genotypes produced green plantlets. However, the genotype influenced the efficiency, and the green plantlets production ranged from 0.4 green plantlets/100 anthers to 8.4 green plantlets/100 anthers. The ploidy level of 106 acclimatized indica rice plantlets were characterized with flow cytometric analyses to calculate the percentage of spontaneous chromosome doubling. Altogether, 48 haploid-, 55 diploid-, 2 tetraploid- and 1 mixoploid plantlets were identified among the regenerant plantlets, and the spontaneous chromosome doubling percentage was 51.89%. Utilization of DH plants have been integrated as a routine method in the Hungarian rice breeding program. The tetraploid lines can be explored for their potential to offer new scopes for rice research and breeding directions in the future.
The induction of haploid cell development into normal plants enables the production of doubled haploid lines, which are homozygous and can be used in breeding programs as an alternative to conventionally derived inbred lines. In this paper, we present the historical background and current status of the attempts of haploid induction in carrot (Daucus carota L.). Economically, carrot is one of the most important vegetables. It is an outcrossing diploid (2n = 2x = 18) species. Nowadays, the seeds of hybrid cultivars constitute the majority of the carrot seeds sold in the world. Hybrid cultivars of carrot are produced using inbred populations. Inbreeding in this species is difficult due to an inbreeding depression and is also time-consuming, as it is a biennial crop. Therefore, the implementation of the haploidization technology into the breeding programs of carrot is of high interest. Androgenesis, gynogenesis and induced parthenogenesis are the methods that have been used for haploid induction, and their potential in haploidization of carrot is discussed. The centromere-specific histone 3 variant (CENH3) and its manipulation in carrot is also acknowledged.
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.
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