Irradiated Pollen Research Articles

Improvement of embryo rescue efficiency in haploid melon (Cucumis melo L.) via irradiated pollen pollination

Improvement of embryo rescue efficiency in haploid melon (Cucumis melo L.) via irradiated pollen pollination

An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development

BackgroundCucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop.ResultsPollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods.ConclusionThrough parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique’s power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

Amaranthus palmeri S. Watson reproduction system: Implications for distribution and management strategies

AbstractAmaranthus palmeri S. Watson is one of the most troublesome weed species worldwide, and is considered one of the most devastating weeds in agricultural fields in the United States. Recently, A. palmeri populations have spread beyond their native range, into the Mediterranean region, and have been reported in several European countries. Resistance to 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) and acetolactate synthase (ALS) inhibitors have been found in European populations, exacerbating the management problems with this species in the Mediterranean region. While the biology, ecology, herbicide resistance mechanisms, and agricultural significance of A. palmeri have been studied, there remains a need to discuss how A. palmeri's unique reproductive traits influence its ability to adapt to various environments, especially as an invasive species spreading into new habitats. We review how the genetic and genomic characteristics of A. palmeri interact with aspects of its reproductive biology, including its breeding system, and alter its ability to hybridize and develop herbicide resistance. Finally, we discuss the breeding system of A. palmeri in the context of weed management, and explore different approaches such as irradiated pollens, genetic manipulations, and RNA interference (RNAi).

Use of irradiated pollen technique to recover haploids in bitter gourd

Haploids play a crucial role in plant breeding as they contain only one set of chromosomes, facilitating the rapid development of homozygous lines. Parthenogenetic haploids have been successfully produced using the irradiated pollen technique. Embryo rescue facilitates the development of an underdeveloped or lethal embryo into a viable plant. Inbred development through embryo culture has yet to be utilized in bitter gourd (Momordica charantia L.). An investigation was undertaken in a bitter gourd to recover haploids through the 60Co gamma-irradiated pollen technique, and an efficient protocol for embryo culture was standardized. Male flowers of bitter gourd genotype MC-139 irradiated at different gamma-ray doses were used for artificially pollinating female flowers. Intact seeds and excised embryos were cultured in E20A medium supplemented with 0.01 mg l-1 IAA, which was found as the most suitable medium for embryo induction and subsequent culture. The use of intact seeds extracted 15 days after pollinating with 60Co gamma irradiated (90 Gy) pollen resulted in a mutant plant. A significant difference was observed in the size of the guard cell, pollen grain diameter and chloroplast number per guard cell for the plant under T9 (90 Gy) compared to the plants under other treatments. The plant (T9) produced only sterile pollen, and the seed set was not observed when self-pollinated

Exploring sterile pollen technique as a novel tool for management of Palmer amaranth (Amaranthus palmeri)

AbstractThe success of the insect sterile technique (IST) in managing insect pests raised the hypothesis that a similar approach could be employed to control weed populations. Here, we investigated the feasibility of employing irradiated sterile pollen as a means to disrupt seed production in dioecious weeds, specifically focusing on Palmer amaranth (Amaranthus palmeri S. Watson). Our goal was to determine the optimal irradiation dose that strikes a balance between inducing sterility and preserving competitiveness, as excessive doses could result in pollen mortality, while low doses may retain fertility. Plants were grown in a greenhouse during the summer of 2020 and spring of 2021. Once they reached the flowering stage, male and female individuals were isolated. Mature pollen samples were collected and exposed to varying dosages (0, 100, 200, 300, 400, and 500 Gy) of gamma rays. These irradiated and non-irradiated pollen samples were used in pollen viability assessments and hand-pollination experiments. In the hand-pollination study conducted in 2020, we employed six pollination treatments using different irradiation doses. The results showed that 300 Gy was the most effective dose, resulting in a maximum reduction of 30% in seed set compared with open pollination when irradiated pollen had prior access to the stigma through artificial pollination before open pollination. In 2021, to simulate real field conditions, three additional treatments were introduced into the study, further confirming the effectiveness of the optimal 300 Gy dose. Our findings indicate that the sterile pollen technique (SPT) using irradiated pollen can be a valuable approach for reducing weed seed production. SPT also holds potential for broad-spectrum weed control by mixing sterile pollen from multiple weed species in a single application. Additionally, it could aid in managing herbicide-resistant weeds that have survived in-season control efforts. This research contributes to the development of novel and sustainable weed management strategies.

Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam

Key messageIn Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations.Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

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.

Induction of haploid plants for speed-up breedıng in sunflower (Helianthus annuus L.) by pollen irradiation

The creation of homozygous plants is essential for both genetic studies and the production of hybrid seeds, particularly in plants that were pollinated by an alien species. Dihaploid (DH) technology is an important application in obtaining homozygous lines used to plant speed breeding programs. The generation of haploid embryos by pollen irradiation and pollination of female flowers with irradiated pollen has been successfully applied. The influence of irradiated pollen on the induction of haploids in sunflower (Helianthus annuus L.) has been evaluated on 16 different sunflower lines from Turkey’s National Sunflower Breeding Program. In this study, pollen grains were irradiated with varying dosages of Gamma rays (500-1000 Gy) to generate parthenogenetic haploid embryos in oilseed sunflowers. At a period of 12–20 days following the process of pollination, embryos were plated into modified MS culture media under a 16/8 h photoperiod and a temperature of 25◦C. Parthenogenetic 18457 embryos at different developmental stages were rescued in vitro and 650 were germinated. The ploidy analysis of regenerants was made with chromosome count and flow cytometry. Except for 500 Gy, all dosages were effective for inducing embryos and haploid plants. Obtained results indicate that parthenogenetic haploid embryos can be produced in K3AD SN:8, IMI 069, and IMI 044 sunflower genotypes by selecting the appropriate Gamma ray dose and embryonic developmental stage. This study was focused to recover sunflower doubled haploids to accelerate the production of non-segregating lines for breeding.

Impact of gamma irradiated pollen on sexual compatibility, seed setting, and fruit attributes in guava (Psidium guajava L.) cultivars

Impact of gamma irradiated pollen on sexual compatibility, seed setting, and fruit attributes in guava (Psidium guajava L.) cultivars

Effect of Pollination with Gamma Irradiated Pollen on In Vitro Regeneration of Ovule Culture in Cyclamen

The obtention of homozygous lines through in situ parthenogenesis via gamma irradiation method is a comprehensive application in vegetables. However, there are a limited number of studies on in situ parthenogenesis in ornamentals. Therefore, in situ parthenogenetic capacity of more species needs to be examined. For this purpose, the effects of pollination with gamma-irradiated pollen on in vitro ovule cultured in Cyclamen persicum L. were evaluated in this study. Flower buds were collected before anthesis and irradiated at different doses 50, 100, 150, 200, 300, and 450 Gy of gamma-ray using a Co-60 source. The control group was pollinated with non-irradiated pollen. Fruits were harvested 30 days after pollination, and isolated ovule explants were cultured on four different mediums in in vitro. M0 was control group containing half-strength MS basal media; M1 media additionally has 10 g/L maltose, 1.0 g/L proline, 2.0 g/L peptone, 200.0 mg/L spermidine and 0.5 mg/L kinetin; in addition to basal medium, M2 media additionally contains 10 g/L maltose, 1.0 g/L proline, 2.0 g/L peptone, 200.0 mg/L spermidine, 0.4 mg/L gibberellic acid (GA3) and 0.4 mg/L N6-benzyl adenine (BA). M3 media additionally contains 2.0 mg/L 2,4-D and 0.8 mg/L 6-( γ, γ-dimethylallylamino) purine (2iP). Plantlets started to form 10-12 weeks after the beginning of culture. The effects of nutrient media, irradiation dose, and their interactions on plant formation were statistically significant. The lowest plantlet regeneration (0.33%) was obtained from ovule explants exposed to 50 Gy gamma rays and cultured on M3 media while the highest plantlet regeneration (2.66%) was obtained from ovule explants cultured on M1 media 30 days after pollination with non-irradiated pollen grains. According to stomatal observations, there were no statistical differences between donor plant and in vitro regenerated plantlets.

Haploid induction and plant production in bottle gourd by pollination with gamma irradiated pollen

Bottle gourd [Lagenaria siceraria (Molina) Standley] has been used exclusively as rootstocks for Cucurbit crops. Haploid technique increases the selection efficiency for developing resistant bottle gourd varieties. This work focused on haploid induction in bottle gourd through in situ parthenogenesis by pollination with gamma-irradiated pollen. Pollination were carried out for six genotypes of bottle gourd with 50, 75, 100, 125 and 150 Gy (Gy) doses of gamma rays. Production of in vitro haploid plants was influenced by irradiation dose, genotype and embyo stage. Seeds at 17 to 21 days after pollination was found to be optimum for embryo rescue. Pollination with irradiated pollen at 100 to 125 Gy was effective for haploid induction. The ploidy level of the 11 parthenogenetic plantlets was confirmed by flow cytometry and 36.3% haploid and 9% triploid plantlets were obtained. This work provides valuable germplasm resources for bottle gourd genetic analysis and breeding programs. Haploid plantlets through in situ parthenogenesis were obtained in bottle gourd. Irradiation doses between 100-150 Gy were best for parthenogenesis induction in bottle gourd.

Double Haploids for Vegetable Crop Improvement: A Review

Vegetables are the richest source of vitamins and minerals and play a major role in nutritional security. The primary objective of vegetable breeding is enhancing production and productivity. Breeders use various conventional and non-conventional breeding approaches for genetic advance. Recent advances in biotechnology helps to enhance the efficiency and shorten the time required to achieve the fixed goals in a breeding program, as well as to address the economic and ecological goals. Among these, haploid (H) and doubled haploid (DH) production through gametic embryogenesis has long been recognized as a valuable tool to help crop improvement. Haploid plants are the sporophytes with a gametophytic chromosome number and doubled haploids (DH) are haploids that have undergone chromosome duplication. Haploids and double haploids occur spontaneously in nature. Haploids can also be induced artificially by various techniques such as wide hybridization, pollination with irradiated pollens androgenesis, gynogenesis and finally further chromosome doubling results in double haploids. Production of double haploids in vegetables will shorten the breeding cycle and also DH technology is useful in reverse breeding, CMS line production, gene stacking and various other genetic studies.

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

Anatomical studies of In vitro raised haploid and diploid citrus plants generated through gamma irradiated pollen

Histological studies of stem, root and leaf of ovule and embryo cultured haploid and diploid plantlets of Citrus grandis × C. sinensis and Citrus grandis × C. limetta crosses were carried out. Area of xylem and phloem in stem increased gradually with increasing age of explants in both cross combinations; however, it was measured minimum in haploid plantlets (1.95 and 1.62 mm2, respectively) as compared to diploid ones. Similar trend was also observed for the area occupied by xylem and phloem in root section of those plantlets. A negative relation was obtained between the ploidy level and per cent area of xylem and phloem within the stem and root of those plantlets. No cuticular and epicuticular wax was observed on the leaf surface in any plantlets irrespective of ploidy level and age of cultured explants, although the leaf thickness increased gradually with the increasing age of the explants with minimum in haploid one. Although, the area of all the parameters evaluated in stem, root and leaf under this experiment were reduced in haploid plantlets but they were well developed and similar to diploid ones which confirmed that these haploid plantlets can also grow normally under open field condition, if intensive care is taken during acclimatization.

Obtaining Individuals Derived from Gynogenesis Using γ-Ray Irradiated Pollen in Apple

雌性側の単為生殖を誘導する方法としてγ線をはじめとする放射線を照射した花粉を授粉に用いる方法が実施されている．本研究では種子親に ‘ふじ’，‘紅玉’，‘印度’ を用いて，500 Gyのγ線を照射した ‘Golden Delicious’ の花粉を授粉に用いた場合の結果率や種子の状態を調査し，SSRマーカーを用いて獲得した種子から育成した実生の由来について調査した．試験の結果，γ線照射花粉の授粉によって獲得した実生から雌性側単為発生個体は獲得できなかったが，γ線照射花粉によって胚発生が誘導され成育途中の胚および種子のほとんどが退化することが推察された．多くの胚および種子の発育が停止する時期は6月下旬（交配後約50日）以前であり，単為発生由来個体を獲得するためにはそれ以前の時期に胚を救出し胚培養を行う必要があると推察された．雌性側単為発生個体を獲得するためにはγ線照射花粉の授粉によって結実する必要がある．今回用いた品種では ‘印度’ が高い結果率を示しており実験に適した品種と考えられた．一方，‘紅玉’ は結果率が低く，この実験には適さない可能性が考えられた．除雄操作によって自殖個体は確実に除かれたが，γ線照射花粉無除雄区で得られた自殖個体は100交配中1個体以下と少ないことから，除雄操作の手間を考慮した場合，無除雄で交配果数を多くし，実生育成後に遺伝子マーカーで選抜する手順が単為発生個体を獲得する方法として効率的と考えられた．γ線の線量が500 Gyでは一部の花粉は稔性を維持することが示されたが ‘GD’ のγ線照射花粉由来と考えられる個体は最大でも700交配中5個体とわずかであり，照射線量は500 Gyが妥当と考えられた．今後はγ花粉の花粉管伸長，受精および胚発生，胚発育の停止過程を明らかにし，胚培養による半数体由来個体獲得の可能性を探る必要がある．

Cytogenetic identification and molecular marker development for the novel stripe rust-resistant wheat–Thinopyrum intermedium translocation line WTT11

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Xiaoyan 78829, a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium, is immune to wheat stripe rust. To transfer the resistance gene of this excellent germplasm resource to wheat, the translocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32, CYR33 and CYR34. A novel stripe rust-resistance locus derived from Th. intermedium was confirmed by linkage and diagnostic marker analyses. Molecular cytogenetic analyses revealed that WTT11 carries a TTh·2DL translocation. The breakpoint of 1B was located at 95.5 MB, and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism (SNP) array analysis. Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11, and 20 kompetitive allele-specific PCR (KASP) markers were developed to enable genotyping of Th. intermedium and common wheat. Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11, such as lower plant height and longer main panicles, that may be applicable to wheat improvement. As a novel genetic resource, the new resistance locus may be useful for wheat disease-resistance breeding.

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.

Nitelikli Yazlık Kabak (Cucurbita pepo L.) Genotiplerinde Işınlanmış Polen Tekniği ile Dihaploidizasyon

Summer squash (Cucurbita pepo L.) is one of the important vegetable species preferred by the people of Turkey in terms of their nutritional value. F1 hybrid summer squash varieties are widely used both in the open field and protected cultivation. The majority of these varieties come from abroad, and our dependence on abroad continues. In this study, the effectiveness of the dihaploidization method, which has an important place in variety breeding studies, was investigated to reduce our import dependency and to produce new F1 hybrid varieties that are high yielded, quality, resistant to diseases and pests. In this context, 14 summer squash genotypes were used as plant material. Dihaploidization studies were carried out using the irradiated pollen technique. In total, 165 flowers were pollinated and 64 fruits, 7034 seeds, 521 embryos, and 144 plants were obtained. 111 pants were successfully acclimatized and cultivated in controlled conditions. As a result of ploidy analysis, 28 plants were haploid (2n=x=20), 77 were diploid (2n=2x=40) and 6 were mixoploid (containing diploid and haploid cells). Haploid plants were doubled with 1% olchicines treatment, grown in the greenhouse, selfed, and seeds were obtained.

Doubled Haploid Production in Watermelon.

Doubled haploid (DH) technology is very advantageous in plant breeding. This technique is beneficial for reducing the time required to obtain pure lines and contributes to the selection efficiency. Using this technique, 100% homozygosity can be achieved in a single generation, while the development of stable lines using the traditional self-pollination method takes from 6 to 8years. It has long been used in diverse crops including cucurbits. DHs can be obtained via parthenogenesis (pollination mostly with irradiated pollen), gynogenesis (in vitro culture of ovules and ovaries), and androgenesis (in vitro culture of microspores and anthers). All these methods have been used for over 30years to develop haploid and DH lines in cucurbit crops. Nowadays, many researchers benefit from these techniques routinely. However, there are still many limits for using DH technology in watermelon breeding programmes. The number of developed DH lines is still very low.In this chapter, we present a protocol based on the different studies on haploids and DHs induced in watermelon through irradiated pollen technique, unfertilized ovule/ovary culture and anther/microspore culture. According to the results of all these studies, it is crucial to develop an efficient protocol for haploid embryo induction to enhance the frequency of obtaining haploid embryos in watermelon.

Doubled Haploid Parthenogenetic Production of Melon 'Piel de Sapo'.

Parthenogenesis is the main technique to produce doubled haploid lines in melon. Although parthenogenesis is a genotype-dependent process and melon has a huge genotypic diversity, we developed a successful protocol for haploid embryo production via pollination with irradiated pollen and a protocol for chromosome doubling of haploid plants of 'Piel de Sapo' genotypes. 'Piel de sapo' genotype has lower efficiencies during the process in comparison with other genotypes, for instance, of the agrestis subspecies. Nevertheless, the doubled haploid lines produced have a great potential as pure parentals for hybrid seed production.