Genetic control of somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.     

Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction

This is the first report on Scots pine (Pinus sylvestris L.) somatic embryo plants regenerated and growing in a greenhouse. The present work focused on improving somatic embryogenesis of the species by studying the factors affecting culture induction. Developmental stage of explants that were immature female gametophytes, including the zygotic embryos with suspensor tissues, was investigated in detail. The genetic background of the material, cold treatments (14 d, 1 or 2 months at +5C) of cones including explants, as well as the plant growth regulator composition of the initiation medium, were also examined. When initiation of somatic embryogenesis was successful, the zygotic embryos in the explants were either proembryos or early embryos. Cold treatment of the cones had no significant effect on induction, nor were there any differences among the treatments with different duration, thus improving the practical applicability of the culture technique. The explants in cold-stored cones probably retained their initiation capacity due to the conversion of starch to sugars. This was observed as decreased number and size of starch grains in the megagametophytes compared with the controls. The seed family and the medium significantly affected induction success, the medium with auxin (9.1 or 13.6 M 2,4-dichlorophenoxyacetic acid) and cytokinin (2.2 M 6-benzylaminopurine) being better than the medium with cytokinin (5 M 6-benzylaminopurine) alone. The significance of the genetic background of the explants and the initiation medium indicate that it might be possible to improve the initiation rates by using explants from controlled crossings between competent genotypes, and by developing more specific media for important seed families.     

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

Summary An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.     

两个苏丹草品系成熟种子的组织培养和植株再生研究

该研究以苏丹草品系S722和Sa的成熟种子为外植体、MS培养基为基础培养基,2,4-D和NAA各3个浓度共6个处理对这两个苏丹草品系成熟种子进行愈伤诱导,探讨不同品系在不同植物生长物质浓度及植物生长物质组合中诱导愈伤组织和继代培养以及分化的能力。结果表明：苏丹草S722和Sa成熟种子的愈伤诱导率差异不显著,平均诱导率为17.19％。诱导培养基中2,4-D浓度为0.5或1 mg?L-1时,诱导效果最佳,而添加NAA不能提高愈伤诱导率。在继代培养中,设定2,4-D和6-BA各两个浓度共4个处理组合,处理1(2,4-D 1 mg?L-1＋6-BA 0 mg?L-1)的继代培养效果最佳。为了解不同植物生长物质对愈伤分化的影响,设定6-BA、NAA 各两个不同浓度、KT 3个不同浓度共5个处理组合对继代培养的愈伤进行分化培养。在5个处理中,处理1(6-BA 2 mg?L-1＋NAA 0 mg?L-1＋KT 0 mg?L-1)对 S722成熟种子诱导的愈伤分化率最高,达33.3％。在这两个苏丹草品系中,S722更容易分化培养。综上结果表明,2,4-D浓度为1 mg?L-1时诱导愈伤和继代培养效果较好,6-BA浓度为2 mg?L-1时分化效果较好。另外,针对不同苏丹草品系进行组织培养和植株再生时,适当调整植物生长物质浓度能提高植株再生的成功率。     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

大豆主栽品种体细胞胚胎发生的影响因素及再生植株

Factors on in vitro somatic embryogenesis of soybean (three elite cultivars) were studied using cotyledons of 3.0-6.0 mm immature seed as explants. Not only the kinds, concentrations and combinations of plant growth regulatory substances but also immature embryo length and inoculum density have main effects on the approaches of embryogenesis. The results of two-factors analysis of variance experiments showed that immature embryo length, plant growth substance concentration and basic medium type have very significant effects on the frequency of embryogenic response, furthermore, interactions exist between the former two factors and are just very significant(at 1% level). The best combinations between 2,4-D concentration and cotyledon length are 10 mg/L 2,4-D & 4.0 mm immature embryos, 20-40 mg/L 2,4-D & 5.0 mm immature embryo. Under these combinations, the salt composition of E1 are very significantly better than that of MS. In conclusion, in the regeneration system established by us the frequency of somatic embryogenesis from the soybean immature cotyledons is greater than 50% and the frequency of conversion of normal (not fused) somatic embryos is about 52.9%-62.6%.     

Genetic Architecture of Two Fitness-related Traits in Drosophila melanogaster: Ovariole Number and Thorax Length

In Drosophila melanogaster, ovariole number and thorax length are morphological characters thought to be associated with fitness. Maximum daily egg production in females is positively correlated with ovariole number, while thorax length is correlated with male reproductive success and female fecundity. Though both traits are related to fitness, ovariole number is likely to be under stabilizing selection, while thorax length appears to be under directional selection. Current research has focused on examining the sources of variation for ovariole number in relation to fitness, with a view towards elucidating how segregating variation is maintained in natural populations. Here, we utilize a diallel design to explore the genetic architecture of ovariole number and thorax length in nine isogenic lines derived from a natural population. The full diallel design allows the estimation of general combining ability (GCA), specific combining ability (SCA), and also describes variation due to reciprocal effects (RGCA and RSCA). Ovariole number and thorax length differed with respect to their genetic architecture, reflective of the independent selective forces acting on the traits. For ovariole number, GCA accounted for the majority (67.3%) of variation segregating between the lines, with no evidence of reciprocal effects or inbreeding depression; SCA accounted for a small percentage (3.9%) of the variance, suggesting dominance variation; no reciprocal effects were observed. In contrast, for thorax length, the majority of the non-error variance was accounted for by SCA (17.9%), with only one third as much variance (6.2%) due to GCA. Interestingly, RSCA (nuclear–extranuclear interactions) accounted for slightly more variation (7.5%) than GCA in these data. Thus, genetic variation for thorax length is largely in accord with predictions for a fitness trait under directional selection: little additive genetic variation and substantial dominance variation (including a suggestion of inbreeding depression); while the mechanisms underlying the maintenance of variation for ovariole number are more complex.     

Asexual embryogenesis and plant regeneration in Quercus

Red oaks (Quercus rubra L.) were regenerated via direct and indirect asexual embryogenesis from immature zygotic embryo tissues. Late heart and early cotyledonary explants cultured in light on modified MS medium proved to be most embryogenic. Embryoids arose from explants cultured on various combinations of 2,4-D and BA. However, the highest percentages of normal polar embryoids were produced by explants cultured on growth-regulator-free media. Epicotyl dormancy of embryoids was overcome by desiccation (air drying and use of an osmoticum) and rehydration treatments. Asexual plantlet development paralleled developmental changes associated with seed germination. White oak (Quercus alba L.) embryoids were also regenerated, but failed to germinate.     

Comparative analysis of the differential regeneration response of various genotypes of <Emphasis Type="Italic">Triticum aestivum</Emphasis>, <Emphasis Type="Italic">Triticum durum</Emphasis> and <Emphasis Type="Italic">Triticum dicoccum</Emphasis>

An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant) different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall 80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks. This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits in the three species of wheat.     

General and specific combining ability from partial diallels of radiata pine: implications for utility of SCA in breeding and deployment populations

Variances for general combining ability (GCA) and specific combining ability (SCA) and the relationship between mid-parental GCA and SCA effects were estimated for tree diameter (DBH) from a series of 20 sets of 6×6 half-diallel mating experiments in radiata pine, planted at ten sites across Australia. Significant SCA variance for DBH was almost equal to GCA variance for the combined analysis of all ten sites. The importance of SCA variance varied among sites, from non-significant to SCA variance accounting for all genetic variation among full-sib families. Significant SCA × site interaction was detected among the ten sites. A significant and positive correlation between mid-parental breeding values and best linear unbiased predictions of the SCA effects was observed. About a quarter of extra genetic gain is achievable through use of SCA variance if selection is based on the best breeding values. To fully exploit genetic gain from SCA variance in a deployment population, positive assortative matings are required for the best parents. It is estimated that the additional deployment gain of 46.0% for ten sites combined, or 52.9% for four sites combined that had significant GCA as well as SCA effects, were achievable relative to gain from GCA only, if all SCA variance within this breeding population was exploited. For a breeding population, selection for breeding values may be sufficient due to positive correlations between breeding values and SCA values. For a deployment population to capture more SCA genetic gain, it is preferable to make more pair-wise mating for parents with higher breeding values.Communicated by O. Savolainen     

Optimized somatic embryogenesis in Pinus strobus L.

Summary Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.     

Somatic embryogenesis in white spruce (Picea glauca): genetic control in somatic embryos exposed to storage,maturation treatments,germination, and cryopreservation

Genetic controls for growth of embryogenic cultures, storage, maturation treatments, germination and cryopreservation in white spruce somatic embryogenesis (SE) were examined. These SE processes were under genetic control but less strongly so than the initiation phase. For all the SE characters examined, variance due to clones within families was significant and often the largest genetic component of variance. This was further partitioned using an additive-dominance-epistasis model. A relatively-large proportion of the total genetic variance was due to epistatic variance in the maturation and germination of somatic embryos. Embryogenic lines were cryopreserved easily without a distinct genetic influence being noticed.     

兴安落叶松胚性愈伤组织诱导影响因子的研究

以成熟和未成熟合子胚为外植体,研究影响兴安落叶松（Larix gmelinii）胚性愈伤组织诱导的几种主要因子。结果表明兴安落叶松合子胚带胚乳培养有利于胚性愈伤组织的诱导;内蒙沙地种源成熟合子胚的诱导率显著（p<0.05）高于加格达奇山地种源;冷藏处理可以提高成熟合子胚胚性愈伤组织的诱导率;不同发育时期的未成熟合子胚的诱导率存在显著差别（p<0.05）,其中以子叶初期合子胚（7月5日）诱导率最高;2,4-D对胚性愈伤组织诱导的影响较大,且与BA、KT存在一定的协同作用;S培养基比DCR和MS培养基更有利于胚性愈伤组织的诱导;培养基中琼脂含量为4 g·L^-1时,诱导率较高。     

Plant regeneration in Stone pine (<Emphasis Type="Italic">Pinus pinea</Emphasis> L.) by somatic embryogenesis

Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.     

Efficacy of administration of praziquantel on 2 days 2 weeks apart against Schistosoma japonicum eggs in mice

We compared a group of mice given multiple oral doses of praziquantel (PZQ) on 1 day 7 weeks after infection with Schistosoma japonicum and another group given multiple doses of PZQ over 2 days (7 and 9 weeks after infection) by examining the number of schistosome eggs and oograms in their tissues and feces. In the 1-day-protocol group (4 x 100 mg/kg x 1 day), calcified dead eggs or shells accounted for 77.4 and 70.1% of the total EPG (the total number of immature eggs, mature eggs, and calcified eggs and shells per 1 g tissue) in the liver and small intestine, respectively, 11 weeks after infection. Shells accounted for nearly half of the total EPG (54.3% liver and 46.6% small intestine). Mature eggs accounted for 8.4% (liver) and 5.1% (small intestine) of the total EPG. Only shells were found in the feces. In the 2-day-protocol group (4 x 100 mg/kg x 2 days, 2 weeks apart), dead eggs accounted for 87.2 and 89.5% of the total EPG in the liver and small intestine, respectively, 11 weeks after infection. Shells accounted for 62.5% (liver) and 75.8% (small intestine) of the total EPG. In the 2-day group, mature eggs accounted for 0.9% (liver) and 0.6% (small intestine) of the total EPG. In liver and small intestine, the EPG of immature and mature eggs in the 2-day group was significantly smaller than in the 1-day group. Especially, the tendency was clear in the case of the EPG of mature eggs. There were no schistosome eggs in the feces of the 2-day group. We found that administration of PZQ over 2 days separated by a 2-week interval was effective against S. japonicum.     

Somatic embryogenesis in Araucaria angustifolia

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.     

Plant regeneration from immature and mature embryo derived calli of Hordeum marinum

Callus cultures were obtained from immature and mature embryos of Hordeum marnium on MS media containing 0.5 mg l^-1 parachlorophenoxyacetic acid or 2 mg l^-1 2,4-dichlorophenoxyacetic acid. Regeneration occurred after transferring calli to MS either devoid of hormones or supplemented with 1 mg l^-1 indole-3-acetic acid and 1 mg l^-1 zeatin. The regeneration capacity of the immature embryo derived calli (94%) was about 5 times higher than that of mature embryo derived calli (17%). A total of 30 and 964 plantlets were obtained from 21 mature and 59 immature embryo derived calli, respectively. Low frequency (less than 1%) of albino plantlets was obtained from both explants after 3–9 months in culture. Plants expressing transient chlorophyll deficiency were produced from immature embryo derived cultures at a frequency of 10%. However, when transferred to soil, these plantlets became green.     

Soybean somatic embryogenesis: interactions between sucrose and auxin

The interaction between sucrose and auxin in soybean (Glycine max L. Merr.) somatic embryogenesis was investigated by culturing immature cotyledon explants on factorial combinations of NAA (6.25, 12.5, 25 and 50 mg/l) and sucrose (0.5, 1, 2 and 4%). A significant interaction between sugar and auxin was observed; balanced concentrations of the two components were required for optimal embryo production and normality. The highest numbers of normal somatic embryos were produced on media which contained combinations of low to intermediate levels of sucrose (1 or 2%) and NAA (6.25 or 12.5 mg/l). Cotyledon explants from induction media having a low (0.5%) sucrose content showed the most efficient embryogenesis in secondary culture. The highest frequencies of germination (32 to 41%) were seen among embryos induced on media containing 0.5% sucrose.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid This paper (No. 88-3-161) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Somatic embryogenesis induction in leaves and petioles of a mature wild olive

We describe a protocol for somatic embryogenesis (SE) induction from an adult wild olive tree (Olea europaea ssp. europaea var. sylvestris. The protocol used confirms for the first time that there is no need to use juvenile or rejuvenated material for SE induction. For SE induction, petiole and leaf (proximal, intermediary and distal zones) explants were grown on Murashige and Skoog (MS) or Olive Medium (OM) media with different combinations of plant growth regulators (PGR): α- naphthaleneacetic acid (NAA), Zeatin (Zea), indole-3-butyric acid (IBA), 2-isopentyl adenine (2iP), thidiazuron (TDZ) and 6-benzylaminopurine (BAP). All media had 30 g/l sucrose and 7 g/l agar, and the pH was adjusted to 5.8. Cultures were incubated in the dark and, after 3 months, they were transferred to MS medium without PGR for expression. Petiole explants gave the highest callus production, while for SE induction and expression distal blade leaf and petiole explants gave the highest rates. The best medium for SE induction was MS with 12.25 μM IBA plus 4.56 μM Zea. Histological analyses confirmed the individuality of globular somatic embryos. This is the first report of SE expression in explants without rejuvenation in Olea genus, and opens perspectives for using this strategy in SE protocols both for this wild genotype and for commercial genotypes.     

Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro

Pig embryos at the 1- or 2-cell stage (before the 'block' to development in vitro) were cultured in 8 different media derived from Krebs'-Ringer-bicarbonate medium. A 2 x 2 x 2 factorial arrangement was used for the treatments, with glucose, glutamine and phosphate being the major effects tested. Embryos were obtained from sows approximately 44-48 h after the observation of oestrus, with the majority being at the 1-cell stage. Embryos from each female were randomly assigned to each treatment. After in-vitro culture, all embryos were scored for the stage of development attained and stained to determine final cell number. Significant effects were evident due to female, glucose, glutamine, a phosphate x glucose interaction and a glutamine x glucose interaction. None of the media components tested was inhibitory to embryo development. The greatest development (45-60% morula or blastocyst) was achieved with glucose and glutamine (both alone and in combination) in the media, demonstrating that an amino acid can serve as the sole energy source for complete preimplantation embryonic development in vitro.