Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

三叶半夏叶片一步成苗离体培养技术

以药用植物三叶半夏叶片为材料,通过比较直接和间接器官发生两种途径,建立了半夏一步成苗的快速繁殖技术体系。结果表明,经过愈伤组织阶段的一步成苗培养基为MS+0.5mg/L2,4-D+1.0mg/LKT,90d左右方可得到再生植株,植株分化率为74%,每个外植体上分化的块茎数为5.61±1.04。附加NAA与BA两种激素对一步成苗培养基进行优化,筛选出一步成苗最佳培养基MS+0.5mg/LNAA+0.5mg/LBA,60d后就可直接发育成完整植株,植株分化率为76%,每个外植体上分化的块茎数高达9.97±0·81,对这种培养基上的再生小植株进行移栽,1个月后,移栽成活率达100%。     

胡杨器官和体胚发生方式的植株再生

目的：为以胡杨为亲本的体细胞杂交育种奠定基础。方法：以胡杨苗叶片为外植体,通过器官和体胚两种不同发生方式建立了离体再生体系。结果：附加0．75mg/L BA、0．5mg/L NAA基本培养基及3w暗培养是愈伤组织诱导的最佳条件;附加0．25mg/L BA和0．1mg/L NAA的基本培养基上不定芽的诱导率最高;1／2大量元素的MS培养基附加0．1mg/l NAA、0．05mg／L和1．5％蔗糖对不定芽生根效果最好;诱导并筛选出的胚性愈伤组织在附加了0．5mg/L BA、0．5mg／L NAA的基本培养基上诱导获得大量胚状体,干化处理后大部分能经子叶胚期萌发成苗。结论：外植体的采集周期和培养条件影响胡杨离体叶片的形态发生途径。     

Daily temperature gradients and processes of organogenesis in apical meristem of <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

We studied the influence of daily temperature gradients on organogenesis in apical and axil shoot meristems at different developmental stages in Cucumis sativus L. The level of organogenic activity of meristems was determined according to the number of leaf primordia on the main and lateral shoots, number of 2nd order shoots, and rudiments of flowers of different levels of development. At the studied ontogenetic stages (mesotrophic seedling or juvenile state), plants were grown under the controlled conditions: photoperiod 12 h, light intensity 100 Wt/m², range of mean daily temperatures 20…30°C, and daily temperature gradients ?20…+20°C. After the temperature treatment, some plants were returned to the optimal, for growth and development, conditions for two weeks (aftereffect). Three types of organogenic activity of meristems in response to the influence of variable daily temperatures were described: stimulation, inhibition, or absence of effect. The phenomenon of stimulation includes two subtypes: optimization, when a maximum effect, observed at other constant temperatures, was attained under the influence of variable temperatures and maximization, when maximum values markedly exceeded those at constant temperatures. The patterns described are preserved on the whole in the aftereffect of daily temperatures.     

Embryo developmental disruption during organogenesis produced by CF-1 murine periconceptional alcohol consumption

The aim was to study the control females (CF)-1 mouse embryo differentiation, growth, morphology on embryonic E- and N-cadherin expression at midgestation after periconceptional moderate alcohol ingestion. Adult female mice were exposed to 10% ethanol in drinking water for 17 days previous to and up to day 10 of gestation (ethanol-exposed females, EF) and were compared with nonexposed CF. EF presented reduced quantities of E10 to E10.5 embryos, greater percentage of embryos at stages less than E7.5, reduced implantation site numbers/female, and increased resorptions compared with CF. EF-embryo growth was significantly affected as evidenced by reduced cephalic and body sizes of E10 and E10.5 embryos (scanning electron microscopy) and decreased protein content of E10.5 embryos vs. CF embryos. A significantly higher percentage of EF-E10-10.5 embryos presented abnormal neural tube (NT) closure vs. the percentage of CF. E10 embryos from EF presented elevated tissue disorganization, pyknosis and nuclear condensation in somites, mesenchymal and neuroepithelial tissue. Immunohistochemical E- and N-cadherin distribution patterns were similar in organic structures of E10 embryos between groups. However, western blot revealed that E- and N-cadherin expression levels were significantly increased in EF-derived embryos vs. controls. Perigestational ethanol consumption by CF-1 mice induced significant damage in the organogenic embryogenesis by producing delayed differentiation, growth deficiencies, and increasing the frequency of NT defects. Ethanol exposure may disrupt cell-cell adhesion leading to upregulation of E- and N-cadherin expression suggesting that deregulation of cell adhesion molecules could be involved in the disruption of embryo development at organogenesis in CF-1 mouse.     

THE ORIGIN OF AN ORGAN: PHYLOGENETIC ANALYSIS OF EVOLUTIONARY INNOVATION IN THE DIGESTIVE TRACT OF FLIES (INSECTA: DIPTERA)

The cardia, a prominent digestive tract organ consisting of several specialized cell types, occurs throughout the “higher” or muscoid flies, division Schizophora of order Diptera. Phylogenetic analysis of cellular organization in 65 insect species from 36 families indicates that this organ originated within the order Diptera from ancestrally undifferentiated tissues. “Lower” flies, suborder “Nematocera,” display little or no epithelial cell specialization at the corresponding site. Scorpionflies of the outgroup order Mecoptera are similarly unspecialized. Intermediate levels of cellular specialization occur in Tabanomorpha, Asilomorpha and Aschiza, dipteran taxa that diverge between “Nematocera” and Schizophora. The distribution of epithelial characteristics suggests that the cardia evolved through a sequence of simple tissue transformations, combining changes in epithelial configuration with local differentiation of cell structure and function. The evolution of locally specialized cell types implies the emergence of structural genes and regulatory mechanisms through the modification of an ancestral genome that had not supported such extensive differentiation. Comparison of localized gene expression in Drosophila melanogaster with that in other fly species having greater or lesser degrees of cell specialization may provide a practical model system for studying specific patterns of mutation associated with such evolutionary innovation.     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

Regeneration of a wide range of African cassava genotypes via shoot organogenesis from cotyledons of maturing somatic embryos and conformity of the field-established regenerants

Genotypic differences in the ability of immature leaf lobes and apical shoot meristems of cassava to form primary somatic embryos in P-CIM were observed (p 0.05). The mean number of apical meristems forming primary organized embryogenic structures when cultured in embryo induction medium supplemented with picloram (P-CIM) had greatest variability between genotypes (C.V.=22.70%). Maturation frequencies of primary embryos were genotype-dependent and ranged from 17 to 100%. Secondary embryo formation was also genotype-dependent and their maturation frequencies varied from 48 to 100%. Cyclic somatic embryogenesis was successfully established and maintained in 11 genotypes in P-CIM. All genotypes underwent organogenesis with significant genotypic variation (p 0.05), and organogenic potential ranging from 5.4 to 76.8%. The number of somatic cotyledons forming multiple shoot buds or more than 10 shoot buds per cluster had the greatest variability between genotypes (C.V.=36.96%) as compared with the overall embryogenic potential. Shoot regeneration ability was neither related to primary embryogenic potential nor to explant type for primary embryo induction. Plantlet regeneration per responding explant ranged from 0.1 to 12. Regenerants established in the field at the frequency ranging from 60 to 100%. DNA content of regenerants was homogeneous and similar to that of mother plants and ploidy level was unchanged (2n = 36). The potential benefits of a systematic tissue culture approach for screening agronomically superior genotypes for regeneration capability and its usefulness in selecting those suited for transgenic programs are discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.