Vernalization of immature embryos of winter wheat genotypes

Summary The effect of direct vernalization of immature embryos on flowering was studied in six winter wheat genotypes. Fourteen-, 17-, and 20-day-old embryos were excised and vernalized for 0–6 weeks on synthetic medium during a conditioning period. Percent germination of embryos was high (overall 96.1%), and free from genotypic effects. Genotypes differed for flowering in response to cold treatment of excised embryos. Embryo vernalization was as effective as or more than conventional vernalization (control, seedling vernalization for 6 weeks). Seventeen-day-old embryos were the most responsive to vernalization. With a 5-week vernalization of 17-day-old embryos, the percentage of plants anthesed was higher than those from 14-and 20-day-old embryos. For 17-day-old embryos vernalized for 5 weeks, the mean number of days from culture to anthesis was less than that of 6 week vernalization, less than that of 14- and 20-day-old embryos, and less than controls.Purdue Univ., Agronomy Dept., W. Lafayette, IN 47907, USA.     

The Effectiveness of Vernalization of Immature Embryos of Winter Wheat var. Grana as Related to Age and Exogenous Phytohormones

Starting from the 10th day after pollination, immature embryos of winter wheat var. Grana were isolated and then vernalized for 4 to 8 weeks on Murashige and Skoog nutrients containing BAP (0.5 or 2 mg/dm³), NAA (0.5 or 2 mg/dm³), or GA₃ (5 or 20 mg/dm³). Vernalized seedlings were cultivated in a greenhouse and the number of days to the heading of individual plants as well as the percentage of plants capable of generative development were estimated. The lower limit of size for 50 % survival of embryos strongly depended on the phytohormone used: from 0.9 mm in control, 1.1 mm in nutrient containing BAP, 1.2 mm for NAA, up to 1.7 mm in nutrient with GA₃. Exogenous GA₃ was lethal for embryos younger than 18 days but induced elongation of older embryos. Embryos isolated 2.5 to 4 weeks after pollination showed minimal requirements for the length of vernalization. BAP increased the percentage of heading plants originated from the youngest embryos. GA₃ improved partial vernalization, strongly increasing the percentage of heading plants, but did not change the time from the end of vernalization to heading. It has been postulated that GA₃ increases number of plants capable of overcoming the threshold of induction of generative development but does not accelerate the flowering process. Hormonized plants showed no deformation of generative development. As the effectiveness of vernalization increased, the heading of plants was faster but they were shorter, forming spikes with a smaller number of spikelets and producing fever lateral shoots. The very young embryos probably have in reserve sufficient amounts of auxins and gibberellins and therefore exogenous GA₃ decreases their viability or even exerts a deleterious effect. However, as the embryos' ageing, gibberellin starvation develops. This being especially pronounced during vernalization. The de novo synthesis or activation of gibberellins takes place during the second stage of vernalization. This is why exogenous hormone improves the effectiveness of partial vernalization, though it is not possible to substitute by gibberellins the vernalization requirements of immature embryos.     

Shortening vernalization of winter wheat with kinetin

Summary The addition of 5 to 100 ppm kinetin during vernalization is a substitute for a part of the cold treatment in the winter wheat cultivar Sava.A cold treatment that is not long enough in itself makes vernalization possible even if only 5 ppm kinetin is added. The effect can be gradually improved by increasing the dose from 20 to 100 ppm, but 200 ppm kinetin is harmful. The smallest quantity of kinetin added to the otherwise optimal 40-day cold treatment results in total oververnalization.A heading percentage comparable to that of the 40-day vernalization period without kinetin can be obtained in 20-day with 100 ppm kinetin. This method can reduce the vernalization time by half and accelerate winter wheat generations.     

Simplified culture method of detached ears and its application to vernalization in wheat

Summary The effects of the addition of sulfurous acid into culture solution and of cold treatment of the solution were examined to simplify the culture of detached wheat ears. In the simplified method, detached ears could be cultured at room temperature on the liquid medium containing 100 g/l sucrose and 0.075% sulfurous acid without any sterilization. The immature seeds in detached ears cultured by this method were treated with low temperature or with chemicals known to have vernalizing effect. The chemical treatment did not affect the chilling requirement of immature embryos, although photoperiodic response and narrow-sense earliness were reduced by kinetin and trypsin. The low temperature treatment drastically affected the chilling requirement, and fully vernalized mature seeds having normal germinability were obtained by treating the detached ears in culture with low temperature from 10 days after anthesis.     

Comparison of callus culture with embryo culture at different times of embryo rescue for primary triticale production

Summary Callus culture of immature wheat-rye hybrid embryos was compared with embryo culture in two experiments. Embryos were rescued from field grown mother plants at two day intervals 13–21 days after pollination and plated for 1) callus culture on Murashige and Skoog medium (MS) supplemented with 2 mg/12,4-dichlorophenoxy acetic acid followed by plant regeneration on hormone free MS medium with half strength mineral salts, 2) embryo culture on Taira and Larter medium (TL). Observations were made on embryo size and condition at time of rescue (Experiment 1) and embryo development directly into plants (embryo culture) or through embryogenesis (callus culture). Fewer 19 and 21 day old embryos developed into plants from callus culture than from embryo culture in Experiment 1. Callus culture was more efficient than embryo culture in promoting plant recovery from 17 day old embryos in Experiment 2. The number of plants per embryo was significantly higher from callus culture than from embryo culture. In both experiments callus culture promoted embryogenesis in more embryos than developed in embryo culture. Embryo rescue 15–17 days after pollination was optimal in both experiments.     

Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes

Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.     

Measurement of response to vernalization in Australian wheats with winter habit

Summary Development in wheat is strongly controlled by sensitivity to vernalization and photoperiod, and to a lesser degree by non-vernalizing temperature and intrinsic earliness. A method to measure effect of vernalization in wheats with winter habit is described. Twenty seven wheats with winter habit and eight with spring/facultative habit were studied, comprising breeding lines and cultivars with maturities suited to south-central New South Wales. Effect of vernalization on the development of these wheats was quantified by integrating responses to vernalizing treatments of differing duration. Intrinsic earliness was measured as time for vernalized seedlings to grow to ear emergece in an 18h photoperiod with day/night temperature of 21/16°C, and response to photoperiod as the difference in time to ear emergence between 9 and 18h daylengths. Integrated response to vernalization is sensitive to both cumulative and thresh-hold responses and is applicable to wheats of all habit type. Integrated response to vernalization and intrinsic earliness were positively associated within wheats with winter habit. Wheats were largely of restricted origin, so that there were few allelic differences at Vrn loci to disrupt this association, which suggests intrinsic earliness may modify response to vernalization. Though integrated response to vernalization was measured with artificial treatments, it was strongly associated with ear emergence for wheats with winter habit when grown at a site in New South Wales.     

Influence of Warm Intervals on the Effects of Vernalization and the Composition of Phospholipid Fatty Acids in Seedlings of Winter Wheat

Excised embryos of winter wheat were submitted to a different number of vernalization (2 °C) and devernalization (20 °C) cycles of different lengths. In all treatments sum of cold and warm intervals was 50 and 10 days, respectively. The influence of different temperature conditions of seedling growth on the effectiveness of vernalization and correlations between effectiveness of vernalization and final content of phospholipid fatty acids were analyzed.
All indexes of generative development of plants (length of vegetative phase, % of generative plants, number of side shoots) showed the most effective was uninterrupted vernalization beginning immediately after excision of embryos. Increasing the number of 2 ° C/20 °C cycles rapidly depressed effectiveness of vernalization, which reached the minimum with the 8.3 days at 2 °C and 2 days at 20 °C cycle and then improved again as cycles became shorter and more numerous.
Correlations between indexes of generative plant growth and indexes describing composition of fatty acids in seedlings showed that from 50 % to 60 % of variability in the composition of phospholipid can be associated with changes in the degree of generative induction. A higher degree of generative induction was associated with a higher share of < 16, 16: 0 and 18:2 acids and a lower share of 18: 3 acid and also with a lower value of the 16: 0/< 16 ratio of phospholipids.
The relation between the generative induction of plants and the composition of phospholipid fatty acids may however be blurred when induction proceeds under conditions of alternate short spells of vernalization and devernalization temperatures.     

Induction of Generative Development of Winter Rape (Brassica napus L. var. oleifera) in Relation to Vernalization Conditions and Age of Vernalized Plants

Germinating seeds and young winter rape plants were vernalized 56–63 days at 5 or 2°C under nine-hour days or in darkness. The highest percentage of generative plants and the most rapid flowering were obtained following the vernalization of young seedlings and germinating seed under conditions of nine-hour day, at 5°C. The least effective induction of generative development followed the vernalization at 2°C in continuous darkness. The vegetation period from the end of vernalization till the beginning of flowering was the shortest when four-week-old plants were vernalized under conditions of nine-hour day, yet the vegetation period from the beginning of germination to flowering was the shortest when seed germinating under conditions of nine-hour day were vernalized. The period was extending as older plants were being vernalized. Data indicating that the optimal temperature for vernalization of older plants is higher than for germinating seed and young seedlings were obtained.     

Influence of selected wheat and rye genotypes on the direct synthesis of hexaploid triticale

Summary In the synthesis of primary hexaploid triticale, a cross-incompatibility barrier exists when tetraploid wheat (4X) is crossed with diploid (2X) rye. Fertilization may occur, however, abnormal endosperm development usually leads to premature embryo death. Four selected tetraploid wheat lines were crossed as females with seven open-pollinated rye lines and the resulting embryos were rescued in vitro 13–16 days after pollination. The wheat genotypes showed a major influence on crossability (seed set), embryo development and plant recovery. The highest efficiency of amphihaploid plant recovery (18.3 plants per 100 pollinated florets) was obtained from one 4X wheat line originally selected from the cross T. carthlicum × T. dicoccoides. Some of the 3X amphihaploid plants (ABR) derived from two wheat lines showed relatively high level of partial fertility presumably as a result of meiotic restitution. Correlation analysis showed that crossability (seed set), normal hybrid embryo development in vivo and embryo culturability were independent of each other.     

Effect of vernalization and photoperiod on flax flowering time

Vernalization and photoperiodism are two important physiological processes related to yield of many cool-season annual crops. The flowering response of 20 flax (Linum usitatissimum L.) genotypes to two vernalization regimes (vernalized and unvernalized) and two photoperiod treatments (10 and 14 h) was evaluated in a growth chamber study in 2010 and 2011. The results suggest that photoperiod, vernalization, and genotype all had an effect on earliness as measured by days to anthesis. Unlike flax grown in the Upper US Midwest and Canada, Texas flax is grown in the fall due to high spring and summer temperatures. Genotype interaction was observed with both vernalization and photoperiod. Specifically, flax genotypes from Texas (winter type) were sensitive to both vernalization and photoperiods for flowering. Texas genotypes delayed anthesis for 7 days or more in unvernalized seedlings, whereas flowering time of most other spring grown flax genotypes was unaffected by the vernalization treatments. Texas genotypes also delayed anthesis for 12 days or more under vernalized and short day conditions, whereas most other genotypes were not influenced by photoperiodism in vernalized seedlings. The selection for vernalization and photoperiodic sensitivity in Texas genotypes and introgression of these traits into recently adapted spring grown genotypes is needed for development of high yielding flax genotypes for southern Great Plains production areas.     

Relationship between cold resistance,heading traits and ear primordia development of wheat cultivars

Summary For breeding early heading wheat cultivars with resistance to frost damage which are well adapted to dry areas of West Asia and North Africa, the relationships between winter hardiness, ear primordia development and heading traits, i.e. veernalization requirement, photoperiodic response and narrow-sense earliness, were assessed using a total of 30 genotypes of wheat (Triticum aestivum L.) grown in an experiment in Syria. The results of artificial freezing tests indicated that cultivars with good winter hardiness were to be found only in the winter wheat cultivars which required 50 or more days of vernalization treatment. These winter wheat cultivars did not initiate internode elongation without vernalization even at 95 days after planting. Thus their ear primordia were still underground and were protected from frost injury at this stage. Photoperiodic response and narrow-sense earliness were not associated with winter hardiness and earliness of internode elongation, but were related to the number of days to heading after planting. This indicated the possibility for breeding early heading cultivars with winter hardiness and tiller frost avoidance by combining high vernalization requirement, short narrow-sense earliness and neutral response to photoperiod.     

Analysis of hybrid lethality in F<Subscript>1</Subscript> wheat-rye hybrid embryos

The effect of the Embryo lethality mutant (Eml) of rye was studied in crosses between hexaploid wheat and corresponding inbred rye line (L2). Histological analysis of hybrid embryos revealed morphological differences 16 days after pollination. Eml was found to arrest the formation of shoot meristem but had no influence on root meristem formation. The effect of Eml cannot be overcome by in vitro embryo rescue via direct regeneration on Kruse medium. The possibility of complementary interactions between wheat and rye genes and of changes in gene expression through increased variation in dosage-regulated gene expression during hybrid formation is discussed.     

冬小麦幼胚一步成苗技术优化研究

为实现冬小麦幼胚一步苗移栽于夏季大田加代,本试验以‘济麦20’、‘烟优361’、‘泰农18’、‘中优9507’以及（‘济麦20’ב烟优361’）F1的幼胚为材料,分别选用11~13天、14~16天和17~19天3种胚龄的幼胚接种在MB、A和B三种培养基上,经离体培养一步成苗。研究结果表明,在9种接种试验处理中,11~13天接种于A培养基上出苗率最高,平均出苗率为98.30%,14~16天幼胚接种于MB培养基上,获得较高的出苗率（平均95.80%）,17~19天幼胚接种于B培养基上,出苗率最高,平均出苗率为95.20%;17~19天幼胚接种于MB培养基上,出苗率最低（平均16.20%）。14~16天幼胚接种于MB培养基上,不仅出苗率较高,而且胚苗根较长,侧根较多,表明14~16天幼胚适宜接种于MB培养基。     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

Production of interspecific hybrids between Glycine max and G. tomentella through embryo culture

Summary Interspecific hybrids have been obtained in an incompatible cross between Glycine max and G. tomentella through the in vitro culture of hybrid embryos. The percentage of successful pod setting in the crosses averaged 12.8% but there were marked differences depending on the soybean cultivar used as the female parent. Hybrid embryos at globular to heart stages were extracted from the embryo sac 15–25 days after pollination and cultured in vitro. Hybrid plants were successfully obtained by culturing the embryos on B5 medium supplemented with 0.1 mg/l IBA followed by culture on B5 medium supplemented with 0.1 mg/l TBA plus 0.25 mg/l 2-iP. The F1 plants resembled the wild male parent in growth form, but had an intermediate leaf shape between that of the parents.     

Observations of Effects of Partial Vernalization in Winter Rape Plants Grafted Onto Generative Stock

Winter rape plants vernalized under natural conditions were used as stock for grafting apical and subapical parts of rape seedlings vernalized at 5 °C in light during different periods ranging from 0 to 10 weeks. Acceleration of generative development in both kinds of grafts occurred already after 3 weeks of their vernalization, i.e. after cold treatment of insufficient length to induce flowering in plants developing independently. Effectiveness of vernalization — measured by acceleration of flowering in grafts was highest from 3rd to 5th week of cold treatment and then it declined. Processes occurring during an early stage of vernalization, though insufficient to allow independent generative development, become manifest in grafts made on vernalized stock. This allows investigations of very early stages of vernalization processes inaccessible to observation by other methods.     

Development and characterization of interspecific hybrids of Trifolium alexandrinum×T. apertum using embryo rescue

This is the first report on the development of interspecific hybrids between Trifolium alexandrinum and T. apertum using embryo rescue and characterization of F₁ plants. T. apertum was used as the male parent and T. alexandrinum as the female parent. Development of interspecific hybrids under natural conditions is not successful and so embryo rescue was attempted. Of the several combinations tried, pollination 2 days after emasculation and embryos rescued 11 days after pollination was found to be the best. For embryo culture, EC3 medium consisting of MS basal supplemented with 2.3 μM kinetin and 3% sucrose was used. Germinated embryos were transferred to LSP3 medium 25 days after inoculation wherein most of the cultures showed multiple shoots that were split and subcultured on RL1 medium for rooting. After hardening, about 75% of hybrids were successfully transferred to the field. The hybrids, in general, showed morphological traits intermediate between the two parents; however, a few hybrids showed better growth than either parent. Some F₁ plants were almost 3 weeks later in flowering than the female parent. Pollen fertility among these plants ranged from 78 to approximately 100%. Chromosomal associations at diakinesis and isozyme banding patterns for acid phosphatase (ACP), superoxide dismutase (SOD) and peroxidase also confirmed the hybrid nature of the plants.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

Embryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of lowand high-copy-number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (<0.3mm) germinated on MT medium containing 150 mM sucrose and 14.4 μM gibberellic acid. Heart stage embryos (0.3-0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 μM gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8-2.0mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.