The molecular path to in vitro shoot regeneration

Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols.     

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

Summary The effects of high-concentration, 2-h liquid pulses of N⁶-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 M BA, and 100 and 200 M TD. Shoots developed best from buds induced with 300, 400, and 800 M BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 M BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.Abbreviations BA N⁶-benzylaminopurine - TD thidiazuron - SH Schenk and Hildebrandt (1972) - NAA 1-naphthaleneacetic acid Paper 2691 of the Forest Research Laboratory, Oregon State University, Corvallis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the authors or Oregon State University.     

Adventitious bud development in embryonic tissue ofPinus caribaea andPseudotsuga menziesii

Summary Adventitious bud formation was induced on detached cotyledons and on cotyledons attached to excised embryos ofPinus caribaea andPseudotsuga menziesii. The embryonic tissue was exposed to the cytokinin 6-benzyl amino purine contained within an agar medium. This exposure resulted in the formation of a meristematic zone, involving both epidermal and sub-epidermal cells, and then nodules on the tissue surface. Some of these nodules were induced to differentiate into bud primordia, and thence shoots, following exposure to a combination of auxin (IAA) and cytokinin. Shoots were produced over the entire surface of detached cotyledons ofPs. menziesii but predominantly on the adaxial surfaces of detached cotyledons ofPi. caribaea and the tips and adaxial surfaces of the cotyledons on the entire embryos of both species. Thus, inter-specific differences in the distribution of competent areas for adventitious bud production were detected in embryos.     

Shoot regeneration from leaves of Prunus serotina Ehrh. (black cherry) and P. avium L. (wild cherry)

Leaves excised from shoot cultures of Prunus avium cvs. F12/1 and Charger and genotype 1908, and from five genotypes of P. serotina and two hybrids of P. avium×P. sargentii developed shoots on Woody Plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Regeneration in both P. avium 1908 and a genotype of P. serotina was improved using TDZ rather than BA in the medium. Regeneration occurred more frequently in P. serotina if leaves were cultured on medium with WPM rather than modified Driver and Kuniyuki walnut medium. The proportions of leaves that regenerated varied between genotypes of the same species. Regenerated shoots of both P. avium and P. serotina developed into shoot cultures following transfer to the media used to produce the shoot cultures used as explant sources. Received: 10 July 1996 / Revision received: 11 November 1996 / Accepted: 6 January 1997     

Genetic distance and species formation in evolving populations

Summary We compare the behavior of the genetic distance between individuals in evolving populations for three stochastic models.In the first model reproduction is asexual and the distribution of genetic distances reflects the genealogical tree of the population. This distribution fluctuates greatly in time, even for very large populations.In the second model reproduction is sexual with random mating allowed between any pair of individuals. In this case, the population becomes homogeneous and the genetic distance between pairs of individuals has small fluctuations which vanish in the limit of an infinitely large population.In the third model reproduction is still sexual but instead of random mating, mating only occurs between individuals which are genetically similar to each other. In that case, the population splits spontaneously into species which are in reproductive isolation from one another and one observes a steady state with a continual appearance and extinction of species in the population. We discuss this model in relation to the biological theory of speciation and isolating mechanisms.We also point out similarities between these three models of evolving populations and the theory of disordered systems in physics. Offprint requests to: P.G. Higgs     

Higher levels of cell proliferation rate and cyclin CycD3 expression in the Arabidopsis amp1 mutant.

Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The amp1 mutant of Arabidopsis has a five-fold higher level of cytokinin than the wild type plant. The mutant has a number of phenotypes with apparently increased cell proliferation rate similar to those seen in plants transformed with an ipt gene and having higher cytokinin levels. In order to identify molecular factors responsible for this cytokinin-induced higher cell division rate in the mutant we have examined cyclin genes expression levels. While expression of the CycD1 and cycC cyclins is not altered or is slightly decreased in the mutant, cyclin CycD3, is more highly expressed. The level of the CycD3 expression decreases in a revertant of amp1, meaning that the over-expression of this particular cyclin is linked to the phenotype of the amp1 mutant. Thus the expression of a specific G1 cyclin is upregulated in a mutant which has more cytokinin than the wild type.     

细胞分裂素信号转导研究进展

对应用突变体研究细胞分裂素信号转导、细胞分裂素受体以及参与细胞分裂素信号转导相关的蛋白质作了简要介绍 ;并且对细胞分裂素信号途径进行了推测 :细胞分裂素被受体 (CKI1、IBC6或者GCR1 )接受后 ,经传导系统转化形成特定的信息 ,一方面可能调节基因的表达 ,从而可能调节受体水平 ,导致细胞对细胞分裂素浓度应答范围发生改变 ,另外 ,基因表达使细胞产生相关的生理反应 ;另一方面形成的特定信息可能激活MAPK级联途径 (mitogen_activatedproteinkinasecascade) ,导致细胞产生相关的生理反应。     

Genetic distance analysis of twenty-two South American Indian populations.

Extent of genic variability among 22 tribal groups of South American Indians in terms of net codon differences per locus has been studied on the basis of determinations on ten genetic systems. The possibility of more than one (other than the north-south route, considered by others) migrational pattern is discussed in the light of the existing genetic variability among the Andean highland and the jungle populations. Genic similarities (as measured by gene identity) are related with geographic proximities which reflect the importance of random genetic drift in creating the present genic divergence among these population groups. Although the materials used in this paper form probably only a non-random sample of structural genome, intralocus variance (sampling) is found to contribute only a little in the variabilities of heterozygosity or genetic distance.     

突变体——侏儒小鼠(dw~t)与津白3(TA3)不同日龄骨骼、体重及体长的比较

侏儒小鼠与津白3体重的比值为1:2.5。它们的骨骼、休重、休长及尾长的测量均具非常显著或显著意义。经均衡性检验,不同日龄、不同性别间TA3/dw~t小鼠的不同骨骼、体重、体长及尾长的比值变化是均匀一致的。证明侏儒小鼠骨骼、体重、体长及尾长均系均匀缩小,是身体各部分匀称的侏儒小鼠。     

Metabolism of benzyladenine by excised embryos of Picea abies

Isolated embryos of Picea abies (L.) Karst. were pulsed with either distilled water, cold benzyladenine (BA) or [8-¹⁴C]-BA for 3 h under sterile conditions, whereafter they were transferred to cytokinin-free media for a further 24 h. Embryos of each treatment were then analysed for cytokinin activity. The embryos themselves yielded very little cytokinin activity. Analysis of the [¹⁴C]-BA-treated material revealed that some residual BA was present even after 27 h of treatment but that the total radioactivity recovered from the embryos at this time was less than 10% of that recovered after the 3 h treatment. Apart from BA, the embryos contained other radioactive peaks, the major ones apparently being of a nucleotide nature. The BA-containing medium in which the embryos were incubated initially was also analysed and again, apart from residual BA, the major metabolite(s) appeared to be BA-nucleotide. However, two radioactive peaks were found in the medium which were absent from the embryos themselves and chemical tests indicated that these could be 7- and 9-glucosides of BA. As the major metabolite resulting from the BA-pulse was of a nucleotide nature and was found to be biologically active, it is postulated that the action of the BA may be mediated via the nucleotide.     

唐菖蒲花色突变株开花后子房组织培养与植株再生的研究

以唐菖蒲花色突变株开花后的子房为外植体,接种于MS+1.0mg/L BA+0.01-0.1mg/L NAA或MS+1.0mg/LBA+1.0mg/L KT+0.01-0.1mg/LNAA的培养基中,从膨大的组织表面直接诱导不定芽.膨大的组织或带不定芽的组织每隔周转入1/2MS_1.0mg/L BA+0.01-0.1mg/L ANN培养基中,不断地诱导产生不定芽,分化的不定芽转入不含激素的1/2MS培养基中,形成幼苗、然后生根,最后可形成小球茎.幼苗转入1/2MS+0.1mg/L NAA培养基中,幼苗生根,再可形成小球茎.1个唐葛蒲突变株子房,经6—7个月培养,获得了上千株试管苗.     

Genetic distance in inversion polymorphism among natural populations of Drosophila ananassae

Based on the differences in the gene arrangement frequencies average genetic distance has been estimated among natural populations of Drosophila ananassae. Genetic distance obtained by pairwise comparison ranges from 0.002 to 0.371. The most differentiated populations are those from the Andaman and Nicobar Islands. Although the populations from localities separated by a small geographic distance show less genetic distance, the relation between genetic distance and geographic distance does not seem to be positive.     

Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

Summary Diploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 10⁷ suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.Abbreviation LT lysine + threonine in equimolar concentration     

Genetic analysis of anther-culture response in 6x triticale

A quantitative genetic analysis was conducted to determine the inheritance of androgenetic response in hexaploid triticale. One highly-responsive genotype (Do 1 triticale) and three low-responding advanced CIMMYT lines (Rhino, Juanillo 97 and Ira Drira) were used as parents to produce a complete set of reciprocal F₁, F₂ and backcross generations. Estimates for genetic effects were determined using a generation-mean analysis following the method of Mather and Jinks. Both embryo induction and plant regeneration potential fitted well with the simple three-parameter additive-dominance (AD) model indicating the absence of any significant epistatic effects. Highly significant additive effects were detected for embryo induction, suggesting that breeding and selection can be effective in improving the induction response of triticale. The high [d]/[h] ratio indicates dominance of the alleles causing high embryo induction. The production of regenerant plants from embryos appeared to be a more complex trait because of its high sensitivity to environmental factors.     

Genetic relationships between Amerindian populations of Argentina

A total of 495 individuals from five different Argentinian tribes was examined for variation in 23 blood group and protein genetic systems, and the results were integrated with previous data on some of these systems. These tribes generally present RH * R1, PGM1 * 1, and ACP * A frequencies lower and RH * R2, ESD * 1, and GLO * 1 prevalences higher than those observed in other South American Indian groups. Earlier studies with mitochondrial DNA showed that haplogroup A was present in low frequencies in these tribes, but haplogroup B showed a high prevalence among the Mataco. Average heterozygosities are very similar in the five tribes, while estimates of non-Indian ancestry are generally low. Both the blood group and protein, as well as the mtDNA data sets, divide the five tribes into two groups, and the relationships obtained with the blood group and protein systems are exactly those expected on the basis of geography and language. However, the topology obtained with the mtDNA results was different, possibly due to sampling effects or diverse patterns of exchange between the groups related to sex.     

Genetic similarities within and between human populations

The proportion of human genetic variation due to differences between populations is modest, and individuals from different populations can be genetically more similar than individuals from the same population. Yet sufficient genetic data can permit accurate classification of individuals into populations. Both findings can be obtained from the same data set, using the same number of polymorphic loci. This article explains why. Our analysis focuses on the frequency, omega, with which a pair of random individuals from two different populations is genetically more similar than a pair of individuals randomly selected from any single population. We compare omega to the error rates of several classification methods, using data sets that vary in number of loci, average allele frequency, populations sampled, and polymorphism ascertainment strategy. We demonstrate that classification methods achieve higher discriminatory power than omega because of their use of aggregate properties of populations. The number of loci analyzed is the most critical variable: with 100 polymorphisms, accurate classification is possible, but omega remains sizable, even when using populations as distinct as sub-Saharan Africans and Europeans. Phenotypes controlled by a dozen or fewer loci can therefore be expected to show substantial overlap between human populations. This provides empirical justification for caution when using population labels in biomedical settings, with broad implications for personalized medicine, pharmacogenetics, and the meaning of race.     

观赏兰科植物组培快繁及遗传转化的研究进展

兰花作为一种高档花卉,近年来其组培快繁和基因工程研究取得了比较大的进展。综述的观赏兰科植物组织培养内容包括外植体、培养基及培养方式等,遗传转化内容包括靶材料、选择标记基因、报告基因、启动子和转化方法等并总结了兰科植物基因工程研究的成果、最新进展及存在的问题 。     

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.Abbreviations BA 6-benzylaminopurine - CaMV cauliflower mosaic virus - 2,4-D 2, 4-dichlorophenoxyacetic acid - GUS ß glucuronidase - NAA naphthaleneaceticacid - nos nopaline synthase gene - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962) - MS-CP MS cell proliferation medium     

Genetic distance and electrophoretic identity of proteins between taxa

Summary The relationship between amino acid substitution and charge change of proteins in the evolutionary process is studied by using a stochastic model. A mathematical formula is developed for the electrophoretic identity of proteins between two different taxa for a given number of average codon differences per protein locus. Using this formula, a reference figure is constructed for estimating the average number of codon differences per locus between taxa.