Efficiency Of Haploid Production Research Articles

Use of mannitol for cold pretreatment of isolated spikes and as a component of nutrient medium for spring barley anther culture in vitro

Aim. The investigation was aimed to compare the efficiency of mannitol as a component of spike cold pretreatment solution and of medium for spring barley haploid production via anther culture in vitro. Methods. Field-grown plants of two genotypes with a contrast androgenic capacity were used as donors. Cut tillers were pretreated in water at 4⁰C for 5 days, while aseptically isolated spikes were kept in 0.3 M mannitol for 10 days at the same temperature. Isolated anthers were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0%); the media with and without 0.1M mannitol differed in solidifying agents. Results. In highly-responsive line DH00-126, there was an almost two-fold increase in mmgreen plant regeneration frequency (from 48 to 58 plants per 100 anthers) in all experimental variants in comparison to the control. In recalcitrant variety Mebere, the combination of tiller pretreatment in water with anther cultivation on inductive starch-gelling media supplemented with 0.1 M mannitol resulted in the highest green plant yield. Conclusions. Both methodical approaches were proved to be applicable to increase the efficiency of haploid production in spring barley anther culture in vitro.

<i>In vitro</i> fabric culture methods to develop the initial material for triticale breeding in the Volga region

The current paper presents the estimation efficiency results of in vitro fabric culture methods used to develop the initial material for triticale breeding in the Povolzhie. A culture of immature embryos (embryo rescue) was used to obtain wheatrye amphigaploids when cross breeding durum wheat with rye. The hybrid kernels formation varied from 10.4 to 47.7%. When polluting 2223 flowers of durum wheat there were formed 768 kernels, 604 of which had embryos with different degrees of differentiation. The largest frequency of hybrid kernels formation was found in the hybrids where the mother forms were the durum wheat varieties “Amazonka” (47.7%), “Diona” (37.5%), and “Aksinit” (37.6%). Fertile amphidiploids (primary hexaploid triticale) were obtained after doubling the number of chromosomes. In vitro immature embryo cultivation was also used to obtain triticale hybrids with soft wheat in those cases when it served as the maternal parent in cross breeding. The efficiency of haploid production in the triticale anthers depended on the genotype. The frequency of embryogenic anthers formation varied from 6.7 to 17.4%, plant regeneration from the new forms was 11.4–31.4%. The 68 obtained of the 338 plants were green and 270 were albinos. In vitro microclonal propagation using segments of young heads as explants allowed preserving and propagating sterile plants for further work to restore their fertility.

Mapping of QTL for kernel abortion caused by in vivo haploid induction in maize (Zea mays L.).

Kernel abortion is common phenomenon in vivo haploid induction and closely linked with haploid induction rate, but little information of kernel abortion is available and its genetic basis still unclear. We used two mapping populations including 186 and 263 F2.3 family lines to analyze the different degree of kernel abortion and identify quantitative trait loci (QTL) responsible for kernel abortion during haploid induction. In total 62 putative QTL, accounting for 3.27–14.70% of the phenotypic variation in kernel abortion traits, were detected across all 10 chromosomes. Ten QTL with over 10% contribution to phenotypic variation were affecting the fifth level of endosperm abortion (EnA5th), endosperm abortion (EnA) and total abortion (TA). Co-localization among kernel abortion traits QTL was observed in both populations and among different kernel abortion types. Five overlaps were indentified in the QTL for kernel abortion traits and HIR traits. Maize chromosome bins 3.01–3.02, 3.04–3.06, 4.05–4.06, 5.03–5.04, 8.06 were QTL hotspots for three or four traits related to the kernel abortion during haploid induction. Total kernel abortion rate (TAR) and HIR showed highly significant positive correlation. These findings may help to reveal haploid induction mechanisms and improve haploid production efficiency.

Improving in vivo maize doubled haploid production efficiency through early detection of false positives

With 1 figure and 2 tablesAbstractIn vivo doubled haploid (DH) technology provides a means of creating new maize inbred lines relatively quickly; however, productivity is limited by false‐positive (FP) plants for haploidy and for dihaploidy, which consume resources of space and labour until detected. This work examines the potential for using stomata guard cell length measurement as a means for early detection of FP plants. We found that the true haploid and DH plants could be differentiated from FP and untreated diploid controls as early as Leaf 2 stage by stomata guard cell length measurement. Furthermore, DH plants were distinguishable from haploid and other diploid plants by the Leaf 7 growth stage. Results suggest that, when used together with screening through the anthocyanin colour marker system and flower fertility, stomata guard cell measurement is an easy, non‐destructive, early screening method that may lead to a greater efficiency in DH production systems and optimization of resource allocation for space and labour.

Evaluation of teosinte lines as pollen parents for wheat haploid production

Nine teosinte lines were evaluated as pollen sources for haploid wheat production together with elite maize lines. Evaluation was performed based on the percentages of seed set, embryos / seed and germination. Pollinated wheat spikes were grown under two environments; as detached spike culture (SC) or on the plants (OP). The overall efficiency of OP was 5.5% higher than that of SC. However, such difference may be compensated by the elimination of labor for 2,4-D treatment in SC. No differences in seed set between the teosinte and maize lines were detected, with a mean of 80.8%, but percentage of embryos / seed varied significantly, ranging from 13.0% to 55.5%. There was also a significant difference in germination rate, ranging from 33.6% to 89.6%. Four teosinte lines showed a significantly higher efficiency of haploid production (haploid plants / 100 florets pollinated) than the best maize line, 3 of these showed an efficiency as high as 30%. It is considered that the use of the elite teosinte lines may be advantageous for producing wheat haploids, although most of the teosinte lines had late flowering, as compared with the maize lines.

Doubled haploids of wheat from wheat x maize crosses:genotypic influence, fertility and inheritance of the 1BL-1RS chromosome

The wheat x maize cross as a technique for haploid induction in wheat was evaluated in a replicated block design comprising 18 wheat F1 hybrids and five Zea mays L. parents. Haploid plants were regenerated at an average of 9.1 (4.4-14.7) plants per 100 florets processed. Genotypic differences for haploid production efficiency were recorded for both wheat and Zea mays L. Interaction between parents was significant for number of plants/100 florets. All 610 of the 1,703 regenerated plantlets that were analyzed by flow cytometry were haploid. At maturity, 70% (60-81 %) of the colchicinetreated haploid plants were fertile, but the frequency of fertile and sterile plants was not consistent over the wheat hybrids from which they were derived. Flow cytometry performed using the first tiller which arose following colchicine treatment enabled prediction of fertility. The 1BL-1RS chromosome was found at the expected ratios in the F2 and in the haploid progenies produced through the wheat x maize cross but deviated from the 1∶1 ratio in the haploid progenies produced by anther culture.

Hybrids of Crosses between Oat and Andropogoneae or Paniceae Species

Plant breeders are interested in enlarging the genetic variability within a species of interest and in improving breeding strategies. A wide hybridization program was conducted from 1990 to 1994 in Germany to determine the prospects for sexual gene transfer or haploid production in oat (Avena sativa L.). Five cultivars of oat were pollinated with pearl millet [Pennisetum americanum (L.) Leeke], eastern gamagrass [Tripsacum dactyloides (L.) L.], and maize (Zea mays L.; 2x and 4x) in the greenhouse. Exogenous auxin application, embryo rescue, and early colchicine‐mediated doubling of chromosomes were used to overcome postzygotic barriers. The recovered plants were analyzed cytologically and phenotypically under greenhouse and field conditions. Depending on the pollinator species, the embryo frequencies varied from 0.4% (Z. mays 4x) to 9.8% (pearl millet). In embryo rescue, initial growth occurred frequently, but many plantlets died. Chromosomes of the pollinator species were lost late in embryo and plant development. One to four chromosomes of the pollinator species were found at the tillering stage in root tip cells. Overall, four viable plants were produced, among them hybrids with pearl millet and eastern gamagrass for the first time. The efficiency of haploid production (<0.1%) was too low in all combinations for application in plant breeding programs; however, the transfer of genes or chromosomes appears promising in crosses of oat with maize and pearl millet.

The genetic basis of seed set in barley genotypes varying in compatibility with Hordeum bulbosum

Low seed set, owing to partial incompatibility, can limit sexual gene transfer and haploid production efficiency in wide crosses. The inheritance of partial incompatibility in barley Hordeum vulgare L. × H. bulbosum L. crosses and its effect on gamete sampling in doubled haploid production were studied by doubled haploid progeny analysis. The dominant, monogenic control of partial incompatibility in 'Vada' was confirmed. Partial incompatibility in 'Harrington' is also monogenic but appears to be controlled by a different gene. An association between the Inc gene and a deficiency in a stigma–stylodium specific high pI protein was found in the co-segregation analysis of doubled haploid progeny. Segregation analysis of Mendelian markers in doubled haploid progeny showed that there is no evidence that the compatibility status of the parents has an effect on gamete sampling by the bulbosum technique.Key words: barley, haploids, Hordeum bulbosum, incompatibility.

Inheritance of Seed Set in Crosses of Spring Barley and Hordeum bulbosum L

Seed set limits use of certain barley (Hordeum vulgare L.) genotypes in H. bulbosum L. doubled‐haploid production. This study was conducted to determine the genetic basis of low seed set in the spring barley cultivar Apex crossed with H. bulbosum. Two hundred F2 plants, all having Apex as one parent and either Havila, Krystal or Roland as the other parent, were crossed with one clone of H. bulbosum. Parameters of haploid production efficiency (seeds/100 florets, embryos/100 seeds, and plants/100 embryos), were measured. Segregation ratios in the three F2 populations suggest that reduced seed set in Apex is governed by a single codominant gene. Haploid production efficiencies were intermediate between the two parents and were acceptable for applied plant breeding.

Effect of exogenous plant growth regulators on in vitro seed growth, embryo development, and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.)

Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA3 and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar ‘Klages’ × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA3 alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA3 produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.

Genotypic Variation for Hordeum bulbosum L.‐Mediated Haploid Production in Winter and Facultative Barley

The use of Hordeum bulbosum L.‐mediated haploid production in barley (Hordeum vulgare L.) germplasm enhancement, gene mapping, and marker‐assisted selection is limited by low efficiencies of haploid production with certain barley genotypes. The primary objective of this study was to assess genotypic variation for haploid production efficiency in an array of 10 winter and facultative barley cultivars of diverse geographic origin. Another objective was to compare in vitro floret and tiller culture as aids to haploid production by the bulbosum method. There was significant genotypic variation for haploid production efficiency (HPE), computed as the number of haploid plants regenerated/100 florets pollinated with both techniques. The highest HPE (48) was achieved with ‘Schuyler’ using floret culture, a technique in which barley florets pollinated with H. bulbosum L. are cultured on modified N6 media containing 0.5 mg L−1 kinetin and 1.2 mg 2,4‐D L−1. Mean HPEs achieved with floret and tiller culture were 35 and 15, respectively. Cultivars exhibited differential HPE response. Of the components of haploid production efficiency (seeds/100 florets, embryos/100 seeds, and plants/100 embryos), a significant genotype ✕ culture technique interaction was found only for embryos/100 seeds. The use of floret culture as an aid to H. bulbosum L.‐mediated haploid production should facilitate barley germplasm enhancement, mapping, and selection efforts requiring routine doubled haploid synthesis from genetically broadbased germplasm arrays.

A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N6 media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F1 hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F1 from a winter x winter cross was inferior in haploid production compared to F1s from winter x spring crosses. No genotype x technique interaction was observed.

Comparison of haploid production efficiency through anther culture and intergeneric crossing in three wheat varieties and their F1 hybrids.

Frequencies of haploid production in wheat were compared between the two methods of anther culture and intergeneric crossing with Hordeum bulbosum L. using three varieties and their reciprocal F1 hybrids. Average frequencies of embryo and plant production per 100 florets were 5. 02 embryoids and 1.27 plants (including 0.76 albino plants) through the anther culture technique, 55.0 embryos and 39. 4 green plants through the bulbosum technique, indicating that the bulbosum technique was obviously much more efficient. Problems to be solved for applying both methods to all the wheat genotypes were discussed.

Pollen-Derived Haploids ofNicotiana Knightiana, N. raimondii,andN. attenuata

Haploid plantlets were obtained in large numbers in three diploid, 24-chromosome species of Nicotiana by culture of anthers at or just past the first pollen mitosis. The three species were N. Knightiana, N. raimondii, and N. attentiata. Efficiency of haploid production varied from about 10 per cent in N. attenuata cultures to 30 and 38 per cent respectively in cultures of N. raimondii and N. Knightiana. H-medium without hormones and standard cultural conditions were used. N. Knightiana appeared to be especially suitable for haploid studies on account of its high plantlet productivity, low chromosome number, and distinctive karyotype.