Zygotic Seedlings Research Articles

Expressão protéica diferencial entre plântulas apomíticas e zigóticas de citros

Existem evidências do envolvimento de um ou poucos genes controlando o processo apomítico em citros; entretanto, a identidade destes genes, seus produtos e suas formas de atuação ainda não foram elucidados. Sendo as proteínas o produto final da expressão gênica, o processo apomítico pode ser investigado através do estudo da expressão protéica. A técnica da eletroforese bidimensional de proteínas foi empregada nesta pesquisa, objetivando a comparação de perfis protéicos de plântulas apomíticas e zigóticas de citros obtidas a partir de cruzamento entre Citrus sinensis L. Osb. cv. Pêra e Poncirus trifoliata cv. Rubidoux. Observou-se, nestes perfis bidimensionais, a ocorrência de três classes de proteínas: a primeira presente em ambos os perfis, a segunda exclusiva do perfil apomítico e a terceira exclusiva do perfil zigótico. Dentro da classe de proteínas compartilhada por ambos os perfis, observaram-se diferenças no nível de expressão entre zigóticos e apomíticos. A classe protéica exclusiva do perfil zigótico, provavelmente, está relacionada à expressão de genes herdados do parental masculino. Já dentre as proteínas exclusivas do perfil apomítico, podem estar proteínas relacionadas ao processo apomítico. O peso molecular e o ponto isoelétrico destas proteínas de expressão diferencial foram estimados e constituem, então, um banco de dados para futuros trabalhos de purificação e seqüenciamento das mesmas.

Use of Molecular Markers in the Management of Citrus Germplasm Resources

New potential citrus germplasm accessions may be received as seed rather than budwood, thereby reducing phytosanitary risks. However, trueness-to-type may be an issue with seed materials because many varieties produce both apomictic (nucellar) and sexual (zygotic) embryos and most citrus is fairly heterozygous. To identify nucellar seedlings of polyembryonic types and to retain these as representing the type, we screened 1340 seedlings from 88 seed sources for markers amplified with two inter-simple sequence repeat (ISSR) primers. Sixteen seed sources produced no seedlings classified as being of nucellar origin. Among the remaining seed sources, seedlings classed as nucellar were identified for potential addition to the collection. In 37 accessions, both nucellar and zygotic seedlings were detected, and in some cases both types were retained. Inclusion of established accessions of the same cultivar group in the analysis allowed an initial assessment of similarity to existing accessions. This technique improved the efficiency of acquiring new germplasm of polyembryonic types by seed. The method identifies those seed sources that produce few or no nucellar seedlings, but it is not useful for determining which seedlings of monoembryonic types should be retained in collections.

Field assessment of Douglas-fir somatic and zygotic seedlings with respect to gas exchange, water relations, and frost hardiness

Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco) zygotic seedlings and seedlings produced through somatic embryogenesis were grown in a field test on southern Vancouver Island, British Columbia. The two stock types were compared in a number of adaptive attributes evaluated during two growing seasons (1999 and 2000). The attributes were related to gas exchange, water relations, phenology, and fall frost hardiness. In both years, somatic and zygotic seedlings had similar rates of net photosynthesis in optimum and reduced light conditions, similar mesophyll conductance, and dark respiration rates. Small differences (P [Formula: see text] 0.05) were found for water relation attributes measured on irrigated plants: intrinsic water use efficiency (WUEi), transpiration (E), stomatal conductance (gwv) and midday xylem water potential (Ψ). Somatic seedlings had marginally higher E and gwv and lower Ψ in 2000 and lower WUEi in 1999 and 2000. Zygotic seedlings had higher WUEi because of their lower gwv while maintaining the same rates of net photosynthesis as somatic seedlings. There were no differences between somatic and zygotic seedlings in frost hardiness throughout the fall. Zygotic and somatic seedlings had similar timing of spring bud break pointing to a comparable level of spring frost hardiness in both stock types. Findings from this study indicate that somatic and zygotic seedlings had a similar pattern of physiological performance throughout 2 years of assessment.

Freqüência de híbridos em cruzamentos controlados de citros: cultivo de sementes versus cultivo in vitro de embriões

Os objetivos deste trabalho foram estudar a freqüência de híbridos oriundos de cruzamentos controlados em citros, comparando a germinação de sementes em canteiros com o cultivo in vitro de embriões, e analisar o efeito da remoção dos tegumentos da semente na germinação. Como parentais femininos foram utilizadas as tangerineiras 'Clementina Palazelli' e 'Sunki', os limoeiros 'Cravo Santa Bárbara' e 'Volkameriano Catânia 2' e as laranjeiras Azedas 'Comum' e 'Double Calice'. Os parentais masculinos compreenderam Poncirus trifoliata e híbridos desta espécie. No cultivo das sementes em canteiros foi usado o delineamento experimental inteiramente casualizado, considerando-se os tratamentos de semente: íntegra; sem o tegumento externo (testa); e sem os tegumentos externo e interno (tégmen). O cultivo in vitro de embriões foi empregado como controle. Avaliou-se: velocidade de germinação das sementes, porcentagem de germinação e de poliembrionia, tamanho e número de embriões por semente, e porcentagem de híbridos. A remoção dos tegumentos das sementes acelerou o início de germinação de todas as variedades; afetou favorável e significativamente a porcentagem de germinação somente em tangerineira 'Sunki' e em limoeiro 'Volkameriano'. A porcentagem de indivíduos zigóticos resgatados foi maior no cultivo in vitro de embriões. A vantagem de uso desta técnica é diretamente proporcional ao grau de poliembrionia dos parentais femininos empregados nos cruzamentos.

Long-term effects of rootstocks on growth and fruit yielding patterns of ‘Alphonso’ mango ( Mangiferaindica L.)

Results of a 21-year study on the performance of ‘Alphonso’ mango grafted on eight rootstocks established the influence of the rootstocks on the growth and fruit yielding patterns of ‘Alphonso’ scion. Nucellar seedlings of ‘Muvandan’ ‘Bappakai’ and ‘Olour’ were vigorous rootstocks in decreasing order of vigour, while those of ‘Vellaikulamban’ imparted dwarfing, in comparison with the scion grafted on its own open-pollinated zygotic seedlings. The cumulative fruit yield per tree was higher on the vigorous rootstocks, particularly ‘Muvandan’ and ‘Olour’; the latter rootstock also resulting in early higher yields of the scion. However, the fruit yields per unit canopy volume and per unit land area were higher with the dwarfing ‘Vellaikulamban’ rootstock. Based on the analysis of variance and game theory analysis of growth and yield data of several years, nucellar seedlings of ‘Vellaikulamban’ and ‘Olour’ were suggested as potential rootstocks for intensive cultivation of ‘Alphonso’ scion. Correlation studies indicated trunk cross sectional area to be a good indicator of vigour in mango and cumulative fruit yield during the initial 16 years was highly correlated with that during the subsequent years. The bienniality parameters worked out with the yield data over a number of years suggested ‘Alphonso’ to be of irregular bearing nature which was controlled more by exogenous factors than endogenous ones and slightly influenced by the rootstocks.

Clonal variation in morphology, growth, physiology, anatomy and ultrastructure of container-grown white spruce somatic plants

We assessed clonal variation in morphological variables, mineral nutrition, root growth capacity, net photosynthesis, tannin distribution, and cuticle and epicuticular wax features within four families of white spruce (Picea glauca (Moench) Voss). Seeds were collected from four families obtained through controlled crosses among selected genotypes. For each family, plants were produced either from seeds (zygotic) or by somatic embryogenesis (clones). Each family was therefore represented by its zygotic seedlings and three clones. Within a family and under similar growth conditions, several clones differed significantly from the zygotic seedlings in height, root-collar diameter, needle dry mass, branch density, shoot dry mass, root dry mass, and length of needles. Branch density (number of first-order branches per cm height) of zygotic seedlings and clones varied from 0.8 to 1.4 branches cm(-1) and from 0.6 to 1.3 branches cm(-1), respectively. Mean needle length of zygotic seedlings and clones ranged from 11 to 14 mm and from 11 to 17 mm, respectively. For many variables (height, dry mass of new roots, needle dry mass and branch density), differences among clones were significantly greater than differences among zygotic seedlings within a family. Tannins were more abundant in needles of clones than in needles of zygotic seedlings. In some clones, tannins occurred as a ribbon along the central vacuole, whereas in others they appeared as aggregates dispersed in the vacuole. Within a family, N, P and K showed considerable variations in their use efficiency. Interclonal variations were observed in root growth potential and net photosynthesis. Variations in growth and physiology reflect genetically determined differences among clones within a family.

Ginsenosides in American Ginseng: Comparison of in vitro Derived and Field-Grown Plant Tissues

ABSTRACT Ginsenoside content and profiles were evaluated in plant tissues of field and in vitro grown American ginseng (Panax quinquefolius L.). The six major ginsenosides, Rg1, Rc, Rb1, RC, Rb2 and Rd were present in all materials tested. The total ginsenoside content of two-year-old roots was 31 mg/g dry matter, with Rb1 and Re as the main ginsenosides. Although in vitro grown materials such as callus and somatic embryo-derived plantlets contained relatively lower total ginsenosides, somatic embryo-derived plantlets had two-fold higher level of ginsenosides than the embryogenic callus. The two-month-old, zygotic embryo-derived, in vitro grown seedlings contained 24.4 mg/g total ginsenosides. In addition, higher Re, Rb2 and Rd were observed in zygotic embryo-derived seedlings than in the two-year-old roots of field grown plants, indicating a potential for producing high-quality ginsenoside products under controlled culture conditions.

Vigor híbrido em tangerina 'Sunki'

Estimou-se a freqüência de plantas zigóticas em cruzamentos controlados de tangerina 'Sunki' com Poncirus trifoliata e híbridos desta espécie, comparando-se seu vigor com o das de origem nucelar. A taxa média de vingamento de frutos foi de 40%, indicando uma boa compatibilidade dos parentais nos cruzamentos. A freqüência observada de plantas zigóticas foi de 48%, devendo estar subestimada em razão do emprego de híbridos de Poncirus nas polinizações. Estimativas dos caracteres comprimento do epicótilo e número de folhas verdadeiras mostraram que 91% dos híbridos identificados podem ser semelhantes, ou mesmo superiores, aos nucelares em relação ao vigor de planta.

Factors affecting shoot regeneration from zygotic embryo and seedling explants of Cycas revoluta thunb

Adventitious shoot induction from zygotic embryo and seedling explants of Cycas revoluta was studied, with reference to both nitrogen formulation of the medium and light. The presence of nitrate as a sole source of nitrogen in Klimaszewska and Keller (KK) medium did not promote shoot induction; on the other hand, shoot induction was promoted by the use of ammonium-containing media, i.e., Schenk and Hildebrandt (SH) and Murashige and Skoog (MS). In the case of zygotic embryos, the percentage of responding explants and the number of regenerated shoots were significantly higher on SH medium than on MS medium. With seedling explants, shoot induction was not affected by the use of SH medium versus MS medium. Photoperiod also influenced bud formation and development. The absence of light during shoot induction stimulated callus formation and very few differentiated shoots. Shoot induction from zygotic embryos was positively affected by the application of 4- and 8-wk photoperiod exposures, the combination SH medium/8-wk photoperiod exposure giving the best results in terms of shoot induction and development. Rooting of shoots was improved by their culture in medium containing NAA.

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.).

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1-50 µM). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 µM and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2-3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem.

Identification of zygotic and nucellar tangerine seedlings (Citrus spp.) using RAPD

The randomly amplified polymorphic DNA (RAPD) technique was used to distinguish nucellar and zygotic seedlings resulting from crosses between the ‘Montenegrina’ (Citrus deliciosa Tenore) and‘ King’ (C. nobilis Loureiro) tangerines. The aim of the present study was to develop tangerine varieties with a reduced number of seeds and organoleptic characteristics similar to the ‘Montenegrina’ tangerine. Embryos were isolated from seeds, cultivated in vitro, and acclimated in a greenhouse. Four random primers were used to identify 54 plants of sexual origin from a total of 202 individuals. The degree of polymorphism of each primer was reflected in the number of zygotic plants obtained per primer. Cluster analysis of parents and progeny separated the individuals into distinct groups with a maximum genetic dissimilarity of 20%.

Fingerprinting trifoliate orange germ plasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers

Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource.

Growth, yield, fruit quality and leaf nutrient status of thirteen year old ‘Alphonso’ mango trees on eight rootstocks

SummaryNucellar seedlings of mango cv. Vellaikolamban were dwarfing when used as a rootstock for cv. Alphonso scions, while those of cvs Muvandan, Bappakai and Olour behaved as vigorous rootstocks, in decreasing order. There was more flowering, and less vegetative flushing when ‘Alphonso’ was grown on this dwarfing rootstock. An inverse relationship between the phenolic content of stem apical buds of the scion and tree size was observed. Differences in leaf nutrient status of ‘Alphonso’ on the various rootstocks were significant for N, K, Fe, Ca, Mn, and Cu, but not for P, Mg and Zn. Vigorous rootstocks led to higher leaf N contents and the least vigorous rootstock resulted in the lowest values of N, K, and Fe. The highest content of the last two nutrients were also recorded on the vigorous rootstocks cvs. Olour and Muvandan. Although fruit yield per tree was high on the vigorous rootstocks and low on cv. Vellaikolamban, productivity, expressed as fruit yield per unit space occupied by canopy, was higher on the latter, indicating the potential of Vellaikolamban as a dwarfing rootstock for high-density orchards. In areas where land availability is not limiting, nucellar seedlings of ‘Olour’ may be better rootstocks when planting ‘Alphonso’ at wider spacings. Zygotic seedlings of ‘Alphonso’ as well as nucellar seedlings of cvs Chandrakaran, Mylepelian and Kurukkan seemed not to hold promise as rootstocks for ‘Alphonso’. Fruit quality was not affected by any of the rootstocks tested.

Relationships of Seed Size and Shape with Polyembryony and the Zygotic or Nucellar Origin of Citrus spp. Seedlings

We examined the relationship between seed size and shape in Citrus and the number and type of seedlings produced by individual seeds for each of three citrus cultivars. Seed size and shape were related to the number of seedlings produced and the likelihood of recovering a zygotic seedling. The relationship between seed size and shape and the likelihood of recovering a zygotic seedling most often was connected with weight and thickness of a seed. This relationship might be of sufficient strength to use in some aspects of cultivar development. However, the relationship did not appear strong enough to be of practical value for application in commercial production of purely nucellar rootstock seedlings.

DNA Amplified Fingerprinting, A Useful Tool for Determination of Genetic Origin and Diversity Analysis in Citrus

We used three short repetitive nucleotide sequences [(GTG)5, (TCC)5, and (GACA)4] either as radiolabeled probes for hybridization with restricted citrus DNA or as single primers in polymerase chain reaction amplification experiments with total genomic DNA. We tested the ability of the sequences to discriminate between seedlings of zygotic or nucellar origin in the progeny of a Volkamer lemon (Citrus volkameriana Ten. & Pasq.) tree. The genetic variability within two species [Citrus sinensis (L.) Osbeck (sweet oranges) and Citrus reticulata Blanco and relatives (mandarins)] also was evaluated. DNA amplified fingerprinting with single primers was the more successful technique for discriminating between nucellar and zygotic seedlings. Although we were not able to distinguish among 10 cultivars of C. sinensis, all 10 C. reticulata cultivars tested were distinguishable. However, it still is difficult to identify the putative parents of a hybrid plant when the two parental genomes are closely related.

360 ORIGIN, INHERITANCE, AND EFFECTS OF A DWARFING GENE FROM THE CITRUS ROOTSTOCK PONCIRUS TRIFOLIATA `FLYING DRAGQN'

The `Flying Dragon' cultivar of Poncirus trifoliata L. Raf. is a strongly dwarfing rootstock for Citrus cultivars, reducing canopy volume of 9 year-old `Valencia' orange trees to 1/3 that of trees on standard rootstocks Open-pollinated seed of `Flying Dragon' was screened with isozyme markers to distinguish zygotic from nucellar (apomictic) seedlings. All zygotics had genotypes consistent with an origin by self-pollination. Zygotic seedlings were budded with `Valencia' orange scion and planted in the field. Of 46 progeny evaluated as rootstocks, 35 produced small trees similar to those on nucellar `Flying Dragon' and 11 produced large trees. This ratio is consistent with the 3:1 segregation expected for a single dominant gene. The dwarfing gene was closely linked, or pleiotropic with a gene causing curved thorns and stems. Several RAPD markers close to the dwarfing gene were identified with bulked segregant analysis. `Flying Dragon' apparently originated as a mutation because it had au identical genotype to non-dwarfing strains of trifoliate orange at all 38 isozyme and RFLP markers tested

Eliminating Zygotic Seedlings in `Turpentine' Mango Rootstock Populations by Visual Roguing

The ability to eliminate zygotic seedlings from the polyembryonic mango (Mangifera indica L.) rootstock `Turpentine' by visual roguing was investigated. Four selected populations, A) randomly selected plants, B) plants selected as off-types, C) seedlings that were of `Turpentine' phenotype, and D) seeds where a single seedling emerged, were examined using electrophoretic analysis and five enzyme systems. Significant differences (χ2 = 39.63, P< 0.001) were found among the four categories, with 28% of the random, 66% of the off-type, 10% of the true-to-type, and 54% of the monoembryonic seedlings being zygotic. These data indicate that visual selection for trueness-to-type and roguing for off-types is useful in reducing the frequency of zygotic seedlings among `Turpentine' rootstock plants.

カラタチにおける極わい性の遺伝および極わい性個体のGA<SUB>3</SUB>に対する反応

The inheritance of two types of dwarfism, namely "rosette" and "match" types originating from selfing and crossing of trifoliate orange strains, 'Hiryu' and 'Small leaf C' were investigated. One-year-old seedlings were classified into four tree forms; straight, crooked ('Hiryu'-like), rosette and match. Rosette type seedlings had extremely short internodes, less than 1 mm. Match type ones grew very little after germination. From the segregation patterns of tree form in the populations investigated, it was considered that rosette type and match type were controlled by a single recessive gene and two recessive ones, respectively. The rosette type seedlings responded to GA3 with a significant elongation of their internodes, whereas the match type seedlings did not respond to GA3treatment. The zygotic seedlings occurred at a high rate (47%) in the population from a selfing of 'Hiryu'. Rosette type seedlings in the population were divided into two types, one had straight twigs and the other one had crooked twigs as a result of GA3 treatment.

Isozyme Analysis of "Citrus" Rootstock Populations to Identify Zygotic Seedlings.

Isozyme Analysis of "Citrus" Rootstock Populations to Identify Zygotic Seedlings.

Glucosinolate Metabolism in Zygotic and Microspore-Derived Embryos of Brassica napus L.

Summary Microspore-derived embryos of Brassica napus L. cultured in vitro fail to accumulate detectable levels of glucosinolates until they are placed in a germination medium that supports true shoot and root formation. The resulting induction of glucosinolate accumulation is restricted initially to indole derivatives, but the pattern then becomes very similar to that observed in zygotic seedlings. The induction of glucosinolate accumulation in cultured microspore-derived embryos results specifically from reduction of the osmotic potential in the embryo environment. The failure of early-stage microspore-derived embryos to accumulate glucosinolates is not entirely due to a lack of biosynthetic capability, since labelled tryptophan is incorporated into indole glucosinolate derivatives in these embryos. Methionine, on the other hand, is not incorporated into alkenyl glucosinolates at this stage.