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.     

Embryo rescue in plants—a review

Summary The plant breeders usually rescue inherently weak, immature or hybrid embryos to prevent degeneration. The successful production of plants from the cultured embryos largely depends upon the maturation stage and the composition of the medium. Abortion of embryos at one or the other stage of development is a characteristic feature of distant hybridization. For the first time successful embryo culture to obtain an interspecific cross between Linum perenne × L. austriacum was demonstrated by Laibach (1925, 1929). Since then several refinements have been made in embryo culture/embryo rescue techniques which have been a popular approach for raising hybrids from a number of incompatible crosses. Currently embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining plants from inherently weak embryos, obtaining haploid plants as well as for shortening the breeding cycle.     

Interspecific hybridization in the genus Gossypium through embryo culture

Summary With embryo rescue and culture techniques interspecific hybrids have been obtained amongst diploid cultivated (Gossypium arboreum, G. herbaceum) and wild species (G. stocksii, G. anomalum) of cotton. The early abscission of the young bolls was prevented by repeated application of growth regulators followed by the culture of immature hybrid embryos (15 days after pollination). The best growth and development were obtained on modified Murashige and Skoog's medium with supplements. The plants were stouter when the cultures were first incubated in the dark for 15–25 days, followed by exposure to light. The hybrid plants transferred to soil developed further and matured, and were more or less intermediate between their respective parents.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Embryo-rescue in the genus Tulipa L.; successful direct transfer of T. kaufmanniana Regel germplasm into T. gesneriana L.

Summary Embryo-rescue was studied as a means to overcome post-fertilization barriers in interspecific crosses in the genus Tulipa. With compatible T. gesneriana L. cultivar crosses, ovule culture was found to be superior to isolated embryo culture. Complete plantlet formation was possible from an embryo size of about 0.5 mm onwards.In the interspecific cross T. gesneriana x T. kaufmanniana, which is hampered by embryo breakdown, successful rescue of abortive embryos was demonstrated. Optimal embryo-rescue was achieved in cultures started seven to nine weeks after pollination. With cultures initiated at a later time, the rate of success decreased. A low number of germinative seeds were obtained after normal ripening of the seed pods, but by using ovule culture the efficiency of seedling formation could be increased dramatically.The ovule culture procedure will allow novel crosses and will offer new possibilities for the introduction of desirable genes into tulip cultivars.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.     

Ovule and embryo culture to obtain hybrids from interspecific incompatible pollinations in chickpea

Many of the wild species of chickpea were not accessible to the improvement of chickpea due to cross incompatibility. In these interspecific incompatible crosses, fertilization takes place but the embryo aborts a few days later. The only way to obtain hybrid plants is by the application of growth regulators to pollinated pistils to prevent initial pod abscission and to save the aborting hybrid embryos by embryo rescue techniques. Although there are a few papers on regeneration from different explants of chickpea, information on embryo rescue techniques is not available. The paper summarises the embryo rescue techniques developed for chickpea, by the use of which hybrid plants between C. arietinum and C. pinnatifidum were produced. The paper also emphasises the effect of genotype to successfully obtain hybrids. The morphology of the hybrid plants resembled the male parent in leaf structure and growth habit. The colour of the flowers produced on the hybrid plant was pale violet, resembling the male parent whose flowers were violet in colour. The flower colour of the female parent was white. This revised version was published online in August 2006 with corrections to the Cover Date.     

Crossability and embryo rescue enhancement in wide crosses between wheat and three Agropyron species

Summary Soft winter wheat lines were crossed with Agropyron intermedium, A. elongatum and A. trichophorum using pollen from single plants of Agropyron spp. to pollinate wheat spikes. Not only species but also individual plants within varieties of Agropyron species differed in percent seed set with a wheat genotype. In two arrays of crosses between two phenotypically different plants of A. elongatum and nine wheat lines, one Agropyron plant gave higher seed set (overall=27.1%) than the other (overall=3.7%). The differences were significant in seven of the nine cross combinations. Results are consistent with the hypothesis that these two plants differ in their crossability as pollen parents with wheat, and suggest the possibility of occurrence of crossability genes in wheatgrasses. The success rate of hybrid embryo rescue was higher (87.5%) with cold treatment (4°C) than without cold treatment (75.0%) of excised embryos on culture media. Results underscore the significance of genotype of the alien species for crossing with low crossable wheats, and of the physical factors for improving embryo rescue in wide crosses.Contribution No. 11,825, Purdue Agric. Exp. Stn., West Lafayette, Indiana 47907, USA. The research was supported in part by Public Varieties of Indiana.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

A simple technique for the identification of embryo-carrying seeds from wheat × maize crosses prior to dissection

Embryo rescue is an important step in the wheat x maize system of induced haploidy in wheat. On average, only one-third of the seeds carry embryos, but they all have to be dissected because no morphological distinction is available to distinguish between seeds with and without embryos. We here report a simple technique in which immature seeds from wheat × maize crosses are placed on a transparent surface and illuminated from above. The free-floating embryo, settled at the bottom of the seed, can be seen from below or in an image reflected on a plane mirror. Using this technique 97.8% of the embryo-containing seeds could be detected prior to dissection. The technique will be useful in reducing the time and labour involved in embryo culture, thereby scaling up haploid production.     

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l⁻¹ GA₃ and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l⁻¹ GA₃ was the best one for Satsuma mandarin, and MT plus 1.0 mg l⁻¹ GA₃ for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.     

影响棉花幼胚(珠)离体培养及植株建成的因素分析

幼胚(珠)离体培养是棉花远缘杂交胚挽救的重要手段,也是研究棉花胚胎发育和纤维发育机理的基础技术。自1935年以来,棉花离体幼胚(珠)培养取得了一定的进展,但目前形成的幼胚培养技术较为复杂,需要在不同胚胎发育阶段变换使用不同的培养基,才能实现幼胚发育成熟和植株形态建成。常用培养基为BT和MS培养基,幼胚的基因型、多种激素(IAA,KT,GA)、碳源和氮源及不同的NO3-/NH4 比例等诸多因素均对培养效果有着重要影响。     

Embryo rescue,for the production of F1 hybrids,in English rose

Summary The ability to recover embryos from achenes resulting from two sexual crosses of English rose varieties was investigated. Functional germination of recovered (rescued) embryos was considerably higher than for seed material when germinated conventionally. This technique also enabled the germination process to be fully controlled, and the aseptic techniques employed, avoided death of the germinating embryo due to fungal or bacterial infection. Seedlings, produced from recovered embryos, were successfully transferred to glasshouse conditions and matured to flowering plants. Many such plants exhibited combined floral characteristics of the two parent lines. The effects of media type, carbohydrate source and specifically the growth regulator BAP on the germination of recovered embryos was also assessed. The low percentage germination observed for achenes as used in this study, was due to both physical and chemical restrictions on the embryo. It is now therefore possible, to use embryo rescue techniques on English roses to recover flowering plants, of potential commercial value, that would normally be lost after sexual crossing.Abbreviations BAP 6-benzylaminopurine     

Interspecific Hybridization in the Genus Lens Applying in Vitro Embryo Rescue

Summary Four different hybridization experiments were carried out to obtain interspecific hybrids with Spanish cultivars of lentil (Lens culinaris M.). In hybridization experiments I and II, undertaken with only pollination and pollination with the addition of gibberellic acid after fertilization, respectively, no lentil hybrids were recovered. A single interspecific hybrid with L. odemensis was obtained in experiment III using the embryo rescue protocol of Cohen et al. (1984), in this case, a crossing efficiency of 0.11% and a rescue efficiency of 2.5% were obtained. Hybridization experiment IV used a specific embryo rescue protocol developed in this study. In this experiment, ovule-embryos of 18 DAP were cultured on MS salts with 1% sucrose and 1 μ M IAA + 0.8 μ M KN; after two weeks, embryos were released from the ovular integuments and cultured on the same medium for another two weeks in upright position. Afterwards, the embryos were transferred to test tubes containing the same medium and one month later plantlets were obtained. Using the above protocol, out of a total of 1707 pollinations, 6 interspecific hybrids with L. odemensis, 2 with L. nigricans and one with L. ervoides were recovered, yielding on average a crossing efficiency of 0.53% and an average rescue efficiency of 8.26%. Taking into consideration only the interspecific crossing blocks in which hybrids were recovered, the crossing efficiency with L. odemensis was 9%, while with both L. nigircans and L. ervoides the crossing efficiencies were 3%. Rescue efficiencies based on hybrids recovered per number of ovules cultured ranged between 50–100%.     

棉花组织培养中畸形胚的发生和转化

在棉花组织培养中，时常有畸形胚的发生。根据畸形胚的表型，可分为九类：联体胚、子叶畸形胚、胚轴畸形胚、单极胚、多极胚、无极胚、重新愈伤化胚、玻璃化胚和白化胚。畸形胚的发生频率很高，可达９４．０％。培养时间和培养基组成等均影响畸形胚的发生。通过调整培养基中激素含量可有效地使畸形胚萌发转化为正常苗。     

胚挽救技术创造无核抗病葡萄新种质

为提高无核抗病、优质葡萄新种质的选育效率,以无核欧洲葡萄品种作母本,中国野生刺葡萄‘塘尾’、‘雪峰’、山葡萄‘双优’及欧山杂交品种‘北醇’做父本,利用胚挽救技术对取样时间和培养基进行优化;用分子标记筛选后代的无核与抗病性状。结果表明：（1）15个杂交组合中共获得杂交果实13341个,接种幼胚珠8589枚,幼胚发育胚1338枚,平均胚发育率15.59%,正常成苗234株,平均成苗率2.73%,‘红宝石无核’ב塘尾’组合胚挽救效果最好,胚发育率为40.17%、成苗率7.76%和成苗植株数178株。（2）‘火焰无核’×北醇’授粉后42天取样,胚发育率(11.37%)和萌发率(3.52%)为最高。‘京可晶’ב北醇’授粉后38天取样,胚发育率(4.3%)和萌发率(1.08%)为最高。（3）以MM3为基础培养基,外源添加3 mmol/L的腐胺,‘红宝石无核’ב塘尾’获得胚发育率(55.83%)和成苗率(20%)为最高。（4）4个自然授粉的品种（‘昆香无核’、‘美丽无核’、‘红宝石无核’、‘赫什无核’）,其胚珠在E20A培养基中更有利于幼胚发育率提升。（5）使用葡萄无核标记SCF27-2000和GSLP1-569、抗霜霉病标记S294-369和S382-615及抗白粉病标记OPY13-661进行筛选,杂种后代中有16株同时携带无核且抗霜霉病与抗白粉病特异性条带。该试验为无核葡萄抗病育种提供了新材料,将为进一步开展中国野生葡萄育种相关研究奠定基础。