In vitro germination and viability of buckwheat (Fagopyrum esculentum Moench) pollen

An in vitro method for the germination of common buckwheat pollen was developed. Pollen grains were successfully germinated in an artificial medium consisting of 0.2 g each of MnSO₄, Ca(NO₃)2.4H₂O and KNO₃, 0.04 g H₃BO₃, 15 g sucrose and 30 g polyethylene glycol (molecular weight approximately 20,000) dissolved in 100 ml of double distilled water. The viability of pollen was assessed by in vivo and in vitro germination tests at 20 °C and 25 °C over a 38 h time period. Pollen grains were collected and germinated at 4 h intervals from freshly harvested flowers grown under 16 h day length and a constant temperature. Maximum pollen viability was found 2 h and 6 h after first light when plants were maintained at 25 °C and 20 °C, respectively. Viability, as measured by germination percentage, was similar at both temperature regimes. Some pollen remained viable for approximately 34 to 38 h in intact flowers, but all pollen lost viability in less than an hour when stored at room temperature without humidity control. This revised version was published online in July 2006 with corrections to the Cover Date.     

Creating cold resistant strawberry via interploidy hybridization between octoploid and dodecaploid

Strawberry cultivars showed limited cold resistance in the Northeast of China, while we obtained a synthetic dodecaploid strawberry hybrid ‘YH15-10’ (2n = 12x = 84) which showed sufficient cold resistance in this area. The reciprocal crosses between F. × ananassa cv. ‘Allstar’ (2n = 8x = 56) and ‘YH15-10’ (2n = 12x = 84) were carried out to select cold resistant strawberry in this study. The 134 seedlings were obtained from the cross of Allstar × YH15-10, while failed in its reciprocal cross. The 30 randomly selected seedlings were examined in terms of morphological characters, chromosome numbers and cold resistance. Most morphological characters were widely separated among F1 progeny with a high broad-sense heritability, which showed that these variations mainly resulted from genetic effect. Some hybrids exhibited heterosis, especially in growth vigor and runner production. Among the 30 tested hybrids, 28 decaploids (2n = 10x = 70), one octoploid (2n = 8x = 56) and one enneaploid (2n = 9x = 63) were observed. The 63.3% hybrids demonstrated higher cold resistance than that of ‘Allstar’ at P < 0.05. These high polyploidy strawberries have potential values in commercial production and modern cultivar improvement.     

An assessment of thein vitro germination capacity of pollen grains of five tea hybrid rose cultivars

Summary Germination trials of pollen grains of the tea hybrid rose cultivars: Ferry Porche, Bronze Masterpiece, Queen Elizabeth, John F. Kennedy and Lady X, were carried outin vitro, to determine whether these cultivars are suitable as pollen donors in a breeding programme.Pollen grains of all five cultivars germinated poorly in a medium containing sucrose only. Addition of boric acid in the medium improved the germination percentages. The highest germinability was observed in Ferry Porche and Lady X on 15% sucrose+50 ppm boric acid, and 20% sucrose+100 ppm boric acid, respectively. Addition of calcium nitrate in the medium reduced germination percentages in all cultivars.The effect of pH of the medium on pollen grain germinability depended on the cultivar and the composition of the medium. On a medium free of boric acid, the pH values between 5.5 and 7.0 caused a significant increase in the germination percentages of pollen grains of Lady X and Queen Elizabeth. The addition of boric acid decreased the responsiveness of the pollen of all cultivars to the pH changes of the medium.The germination capacity of pollen grains of Lady X and Ferry Porche was not affected by incubation temperatures ranging between 20°C and 30°C.On the basis of the above results Lady X and Ferry Porche are considered the most suitable for pollen donors.     

In vitro selection of tomato transformants at the immature embryo stage

Summary A method is described for selecting resistant transformed tomato genotypes in vitro at the stage of immature embryos. The utilization of HLH nutrient medium with the selective agent kanamycin is proposed. Normal development of seedlings from immature embryos, which do not form callus, is a good and true indicator for isolation of resistant genotypes. The immature embryos do not germinate and develop on MS selective medium.     

In vitro pollen germination and tube growth of tomato (Lycopersicon esculentum Mill.) and its relation with plant growth

Summary In vitro pollen germination at 10°C, 14°C and 22°C of four groups of two pure line tomato varieties was compared with their plant growth at 19°C D/10°C N under controlled environmental conditions. Generally, pollen germination was slow at 10°C but after 6 h the percentage of germination was similar to that at 22°C. Maximum germination was obtained at 14°C already after 4 h. The longest pollen tubes occurred at 22°C. The two varieties within each group differed significantly from each other for percentage of pollen germination. For one group, this difference was greater at 10°C than at 22°C. The varieties in two groups also differed significantly for pollen tube growth. These differences in pollen tube growth rate were greater at 22°C than at 10°C. There was no clear relationship between pollen germination, pollen tube growth and plant growth in any of the four pairs of varieties. The results are discussed with regard to the possibility of pollen selection at low temperature in order to improve the efficiency of breeding for growth at low temperature.     

不同光照强度对玫瑰组织培养中初代培养物褐化的影响

玫瑰是一种重要的观赏植物,经济价值高,但玫瑰快繁体系尚未建立。本文研究了在不同田间光照强度下,玫瑰组培初代培养过程中培养物多酚氧化酶（PPO）活性、总酚含量、褐变百分数和萌芽成活率的变化规律。结果表明：遮荫处理使玫瑰培养物的褐变百分数降低而萌芽率显著提高;正常光照强度下多酚氧化酶活性与其他遮荫处理相比并无显著变化,但总酚含量显著高于遮荫处理,二者的相关分析表明多酚氧化酶活性与组织材料的总酚含量相关性不大。     

Interspecific hybridization between Vigna unguiculata (L.) Walp. and V. vexillata (L.) A. Rich. through in vitro embryo culture

Immature embryos resulting from the cross V. vexillata × V. unguiculata were cultured on MS medium supplemented with 2,4-D (2 mg/l) and resulted in embryogenic calli. Thirteen hybrid regenerants were obtained via organogenesis by subculturing the calli on MS medium supplemented with BAP (2 mg/l) + adenine sulphate (40 mg/l) + CH (500 mg/l) + cowpea tender pod extract (10%). The interspecific regenerants showed intermediate morphological traits between the parents for leaf shape, pod colour and seed coat colour. The hybrid plants inherited stem, leaf and pod hairiness of the wild species which could serve as a mechanical barrier against viral vectors. Electrophoretic studies of two isozyme systems, peroxidase and esterase, also confirms the hybrid nature of the regenerants as they expressed unique bands of both parents. Cytological study of the meiotic chromosomes revealed high frequency of univalent formation in the hybrids suggesting that the genomes of the parental species are structurally differentiated. The hybrid regenerants exhibited high enzyme activity for three enzymes viz., peroxidase, polyphenol oxidase and phenyl alanine ammonia lyase over the cultivated parent which may be useful in conferring resistance against viral pathogens. This revised version was published online in July 2006 with corrections to the Cover Date.     

The induction in vitro of adventitious shoots in Rosa

Summary Adventitious shoots were formed on excised leaves, roots and callus of Rosa persica x xanthina and on excised leaves of R. laevigata and R. wichuraiana on culture media that included BAP and NAA as growth regulators. Shoots formed freely on freshly cultured callus of R. persica x xanthina but their production declined in successive cultures and ceased after twelve weeks. Transplantation to soil was improved by rooting plantlets in cellulose plugs in vitro and transferring plantlets to soil while still in the plugs.     

The effects of cis-trans ABA on embryo germination and seed dormancy in wheat

Pre-harvest sprouting of wheat grain can cause economic losses especially in cultivars with low levels of seed dormancy. The aim of this study was to determine genotype differences in embryo sensitivity to germination in response to exogenous (+/–) cis-trans ABA treatments at different concentrations. Six white and four red seed-colored bread wheat genotypes that differed in dormancy were grown in a field near Swift Current, Saskatchewan in 2000 as a randomized complete block design with four replicates. The seed samples from this experiment were germinated in a controlled environment at 20 °C without light. The exogenous ABA treatments were 0 μM – whole seed (control), 0 μM-embryos, 25 μM – embryos and 50 μM – embryos. The ABA experiment was a factorial design with four randomized complete blocks with four ABA treatments in all combinations with the ten genotypes. A weighted (by day) germination index (WGI) was calculated for each genotype in each ABA treatment. Genotypes differed in response to ABA. The genotypes, ABA concentration and genotype by ABA concentration interaction effects were significant (p ≤ 0.05). Excised embryos showed significantly decreased dormancy in most of the experimental genotypes. The addition of exogenous ABA enhanced embryo dormancy of most genotypes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Resistance of 12 rose genotypes to 14 isolates of Diplocarpon rosae Wolf (rose blackspot) collected from eastern North America

Blackspot disease, caused by the fungus Diplocarpon rosae Wolf, is the most serious disease problem of roses worldwide. From a breeding perspective, the documented occurrence of races in the pathosystem necessitates the characterization of the race structure for the pathogen, identification of resistance genes in rose germplasm, and determination of the nature of genetic resistance to breed stably resistant rose cultivars. The objective of this study was to characterize the diversity of eastern North American races of D. rosae and to simultaneously determine the degree to which partial resistances existed in rose hosts. Twelve rose genotypes were inoculated with 14 single‐conidial isolates collected from eastern North America. Three distinct D. rosae races were identified based on presence/absence of infection in the differential array. There were five distinct reaction patterns across pathogen isolates among the 12 rose genotypes. The partial resistance components of leaf area with symptoms, lesion length and incubation period were evaluated for all inoculated plants. Variation for expression of partial resistance was noted, and partial resistance appears to behave in a race‐specific manner for some rose genotypes. Early defoliation differed between genotypes and may prove to be another component of partial resistance.     

Interspecific hybridization between sunflower and wild perennial Helianthus species via embryo rescue

Interspecific crosses were made between the cultivated sunflower inbred line HA89 and accessions of five wild perennial Helianthus species (H. giganteus L., H. laevigatus T. & G., H. resinosus Small, H. pauciflorus Nutt. and H. decapetalus L.) resistant to broomrape ( Orobanche cernua Loefl.). Using the genetic male-sterile isogenic version of that line as female, successful reciprocal crosses were also obtained with all these wild species except with H. decapetalus. Five-day-old hybrid embryos were excised and cultured in vitro. In all cases, few mature plants were obtained from embryos in early developmental stages (early heart and globular) but up to 28% mature plants were obtained from embryos in later stages of development. Very immature embryos were difficult to excise without damage. Hybrid embryos and mature plants were obtained from all five wild species. Interspecific hybrid embryos from different wild species showed distinct developmental potentials, the proportion of hybrid embryos in different developmental stages varying among species. Differences in the proportion of hybrid embryos at the several developmental stages were also obtained for the reciprocal crosses (cultivated × wild), which showed higher proportion of fully developed embryos. Hybrids involving H. giganteus and cultivated sunflower were difficult to obtain without the use of embryo culture. Embryo culture proved to be an useful tool to overcome post-zygotic hybrid incompatibility in different Helianthus spp., and facilitated interspecific transfer of resistance to O. cernua. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro interspecific fertilization,embryo development and formation of hybrid seedlings between Gossypium hirsutum and G. arboreum

Summary In vitro hybridization between Gossypium hirsutum, and G. arboreum was carried out. Hybrid seedlings were obtained after successive use of the following five kinds of media: 1) pollen grain germination medium, 2) double-fertilization medium, 3) embryo development medium, 4) seedlings formation medium, 5) green seedlings growth medium. The factors affecting in vitro interspecific fertilization, embryogenesis and seedling formation were studied. The key factors were temperature and relative humidity (RH). The optimum RH for in vitro interspecific fertilization was 65%, and a suitable temperature range was 26–30°C. Pollen grain germination ratio decreased rapidly at a RH of 80%. When the temperature and the RH were higher than 32°C and 80%, respectively, the fertilization rate decreased to zero.Effects of petals and calyces of the maternal flowers on in vitro interspecific fertilization and ovule growth were identified. Even though the petals or calyces were excised, hybrid plantlets were also obtained after media were improved and a shake culture was used. In addition, the process of double-fertilization and embryo development were studied cytologically. The developmental characteristics of the hybrid embryos derived from in vitro interspecific fertilization were a later occurrence of the double-fertilization process and a much lesser division of endosperm cells. But the embryo development was not affected by these characteristics, and young hybrid embryos can develop to their cotyledon stage finally on artificial media.     

Cell lines and plants obtained after protoplast fusions of Rosa+Rosa,Rosa+Prunus and Rosa+Rubus

Summary Protoplasts of three Rosa cultivars were fused with each other, with protoplasts of Prunus `Colt' and with protoplasts of Rubus laciniatus, using polyethylene glycol 4000 as a fusogen. Protoplasts of Prunus were incapable of cell division and those of Rosa and Rubus were disabled by treatments with metabolic inhibitors, either iodoacetate (IOA) or rhodamine 6-G (R6G). Parental protoplasts were then fused in combinations that required complementation for their survival. RAPD analysis of 41 fusion-derived cell lines showed that two lines resulting from fusions of Rosa + Rosa and one from a fusion of Rosa + Prunus, contained some DNA markers from both fusion partners. The others contained markers of only one fusion parent. This showed that after protoplast fusion, the heterokaryons did not develop into cell lines with stable hybrid nuclei. Plants regenerated from cell lines derived from Rosa + Prunus and Rosa + Rubus fusions contained DNA markers of only Rosa and their DNA amounts were no greater than that of the Rosa parent. However, they differed morphologically from the Rosa parent to a remarkable degree, possibly because they inherited undetected genes of Prunus or Rubus, or because they were somaclonal variants of the Rosa parent. Alternative strategies for the production of somatic hybrids are discussed.     

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

鸢尾种子休眠与萌发特性研究

鸢尾（Iris tectorum Maxim．）种子自然萌发缓慢，周期长，发芽率低。为了探索鸢尾种子的最适萌发温度和抑制发芽物质存在的原因，对鸢尾种子进行了3组处理：（1）去种皮与不去种皮种子做发芽对照实验；（2）去种皮的鸢尾种子置于培养箱发芽，每天加入胚乳浸提液、种皮浸提液和清水；（3）去皮种子置于不同温度的培养箱中发芽，其中4个恒温15、22、26、30℃；3个变温5／15、15,／25、20／30℃（8／16h）。结果表明：22℃是最适合鸢尾种子发芽的温度；种皮对鸢尾只在发芽早期有抑制作用，在后期无明显抑制作用；胚乳和种皮浸提液在种子萌发初期有抑制作用，但在发芽的中后期则对种子发芽有促进作用。     

Tracing of 2n egg occurrence in perennial ryegrass (Lolium perenne L.) using interploidy crosses

Several genotypes of Lolium perenne (2n = 2x = 14) were screenedfor the production of 2n eggs. In each cross a diploid genotype, used asfemale parent, was pollinated with a tetraploid genotype. All interploidycrosses were made in isolation cages. Flow cytometrical analysis of theprogeny of 154 diploid plants revealed 28 tetraploid descendants. Theseoffspring plants were harvested on 15 different diploid mother plants.Amplified Fragment Length Polymorphism (AFLP) markers were used tocheck the parenthood of these tetraploids. Two seedlings died, theremaining plants could, after AFLP fingerprinting, be identified as meiotictetraploids. The results obtained allowed to conclude that the tetraploidprogeny plants harvested on 15 diploid mother plants were the result ofthe fertilisation of an unreduced egg by a reduced pollen grain of thetetraploid plant.     

In vitro culture of tissues,cells and protoplasts of Trifolium alexandrinum L. (Egyptian clover)

Summary Callus induction derived from root, hypocotyl and cotyledon explants were investigated for four cultivars of Trifolium alexandrinum L. The Murashige & Skoog (1962) medium containing 2.0 mg/L NAA and 0.5 mg/L BAP was able to induce callus from different explants. A wide range of culture media were tested to determine the morphogenetic potential of different callus types derived from different explant types. Shoot morphogenetic development was observed with the cultivars Sakha 4 and Giza 10. Cell suspension cultures were established from hypocotyl derived callus. Methods for isolation and culture of protoplasts from cotyledons are described.