Zygotic Seedlings Research Articles

Identification of 'Ubá' mango tree zygotic and nucellar seedlings using ISSR markers

Polyembryonic seeds are characterized by the development of over one embryo in the same seed, which can be zygotic and nucellar. The objective of this work was to identify the genetic origin, whether zygotic or nucellar, of seedlings of polyembryonic seeds of 'Ubá' mango tree using ISSR markers, and relating them with the vigor of the seedlings. Thus, mangos were harvested in Visconde do Rio Branco (accession 102) and Ubá (accessions 112, 138, 152 and 159), whose seeds were germinated in plastic trays filled with washed sand. Fifty days after sowing, seedlings from five seeds of each one of the accessions 102, 112, 138, 159 and from 10 seeds of the accession 152, were analyzed. These sseedlings were characterized and evaluated for plant height, stem circumference and mass of fresh aerial part and the most vigorous seedling was the one displaying at least two of these traits higher than the other seedlings from seed. Leaves were collected for genomic DNA extraction, which was amplified using seven ISSR primers previously selected based on the amplification profile and considering the number and resolution of fragments. Zygotic seedlings were found in 18 seeds, which were the most vigorous in six seeds. The results evidenced the existence of genetic variability in orchards using seedlings grown from seeds, because the farmer usually uses the most vigorous ones, assuming that this is of nucellar origin. These results also indicate that the most vigorous seedling are not always nucellar, inasmuch as of 20% of the total seeds evaluated, the zygotic seedling was the most vigorous.

Production of Colchicine-induced Autotetraploids in Pummelo (Citrus grandis Osbeck) through Indirect Organogenesis

Colchicine-induced stable autotetraploid plants were recovered through indirect organogenesis from stem sections of in vitro-grown zygotic seedlings of pink/red-fleshed pummelo (Citrus grandis L. Osbeck) selections cybrid Hirado Buntan (C-HBP) (pink), 5-1-99-3 (dark pink), and HBJL-5 (red), all derived from Hirado Buntan pink pummelo. Multiple shoot induction was achieved through indirect organogenesis from the callus produced from the cut ends of the treated explants. Ploidy levels of regenerated plantlets were determined through flow cytometry at a stage when recovered shoots had one to two expanded leaves. Recovered tetraploids proved to be stable after 2 years in the field. As expected, higher colchicine concentrations and treatment durations decreased the survival rates of the regenerated plantlets. Colchicine concentrations of 0.05% and 0.1% produced the most tetraploids; of the 19 total tetraploids produced, 10 were produced from the treatment with a colchicine concentration of 0.1% and six were produced from treatment with a concentration of 0.05%. After flowering, these stable pink/red-fleshed tetraploid plants generated should be useful as breeding parents in our grapefruit/pummelo improvement program, especially if any show canker tolerance or reduced furanocoumarins. Use of monoembryonic tetraploids in interploid citrus crosses eliminates the need for embryo rescue to recover seedless triploid progeny; this research expands our pool of available high-quality monoembryonic tetraploid parents.

Identification of Zygotic and Nucellar Individuals Produced from Several Citrus Crosses Using SSRs Markers

Turkey is an important citrus-producing country. However, new cultivars are needed to sustain citrus production and ensure its competitiveness against other crops. There are currently several citrus breeding programs that aim to help overcome the lack of local commercial varieties and to contribute to Turkey’s competitive capacity in the citrus market. In this study, we report the utilization of molecular markers in one such breeding program. Simple sequence repeat (SSR) markers were employed to eliminate nucellar individuals from a hybrid population produced by crossing. The crosses included ‘Fremont’ and ‘Robinson’ mandarins as the female parents and ‘Midknight Valencia’, ‘Rhode Red Valencia’, and ‘Valencia Late’ oranges and ‘Rio Red’ grapefruit cultivars as the male parents. Seedlings with the same banding patterns as the female parent were identified as nucellar seedlings by 11 SSR primers. Primers AG14 and TAA03 were found to be more effective at identifying zygotic individuals than other primers. ‘Fremont’ and ‘Robinson’ mandarins produced 36.91% and 31.09% of nucellar seedlings, respectively. As a pollen parent, ‘Rio Red’ grapefruit had a higher ratio of zygotic seedlings compared to ‘Midknight Valencia’, and can be recommended in breeding programs. Comparative analysis of different citrus fruits in the breeding programs allowed us to design an efficient hybridization scheme for this study.

Obtaining citrus hybrids by in vitro culture of embryos from mature seeds and early identification of hybrid seedlings by allele-specific PCR

Two trifoliate oranges (Poncirus trifoliata var. monstrosa and P. trifoliata cv ‘Xiaoganzhi’) were used separately as pollinators to cross with Shantou-Suanju (Citrus sunki Hort. ex. Tan.) for breeding new citrus rootstocks. Embryos from the seeds of mature fruits were rescued and individually cultured in vitro. On average, 9.0–10.0 embryos per seed were harvested from the two crosses. The zygotic seedlings were only derived from large- to medium-sized embryos. The small embryos were not able to germinate or develop into seedlings. The numbers of seedlings per seed produced from the two crosses were similar, ranging from 1 to 11 and 1 to 14, at average of 4.17 and 4.90, respectively, with in vitro culture in this study. A dominant trifoliate leaf trait from P. trifoliata was used as a morphological marker to discriminate hybrid from nucellar seedlings. Of 43 and 14 zygotic seedlings were obtained from 98 and 65 seeds of the two crosses, respectively. In addition to the use of morphological marker, single nucleotide polymorphism based allele specific PCR (AS-PCR) was also successfully employed as a handy tool to confirm and identify zygotic seedlings.

Precocious flowering of Citrus seedlings and its use for determination of cultivars generating male sterile progenies

Precocious flowering in several-month-old to one-year-old seedlings is known in many Citrus cultivars with very long juvenile phase of around ten years. The Citrus accessions generating precocious flowering seedlings are interesting materials for rapid genetic analysis. Precocious flowering was observed in the zygotic seedlings obtained from the most pummelo and pummelo-relative accessions used, and many of yuzu, mandarin and their relative accessions with the range between less than 1% and about 20%. The rates indicated that some pummelo and orange cultivars have high ability to generate precocious-flowering seedlings. Male sterility in the seedlings showed stability in the flowers of first, second, and third years after seed germination. Male sterile seedlings did not segregate in the progenies of pummelo and pummelo-relative accessions used as pistillate parents, whereas they segregated in those of accessions related to ‘Yuzu’ (C. junos) and mandarin, indicating pummelo cytoplasm is fertile for male fertility, whereas ‘Yuzu’ and mandarin cytoplasm was sterile. The segregation of sterility in the progenies also indicated that most Citrus accessions are heterozygous for male fertility restoration genes.

Techniques to Differentiate Zygotic and Nucellar Seedlings in Polyembryonic Fruit Crops

Different methods are in use for differentiating zygotic and nucellar seedlings in these polyembryonicfruit crops which include morphological methods, isozymes, RAPD, AFLP, TLC, ISSR, SSR etc. Polyembryonic varieties produce seeds which originate zygotic and nucellar plantlets. Zygotic plantlets are sexual whereas nucellar plantlets come from the maternal tissues which are assexual plantlets. Therefore, nucellar plantlets are preferred for grafting, because they maintain the same genetic background of the rootstock mother-plant whereas zygotic plantlets preferred for breeding programmes because they do not maintain same genetic background of the mother plant. Identifying the sexual embryo in each hybrid seed, in order to preserve only the nucellar ones, would help to maintain the genetic characteristics of the rootstock and also to progress fruit breeding by avoiding the nucellar ones to advance generations.

Morphology of Sterile Anthers and Inheritance of Cytoplasmic-genetic Male Sterility in Zygotic Seedlings of Polyembryonic Acid Citrus

Morphology of Sterile Anthers and Inheritance of Cytoplasmic-genetic Male Sterility in Zygotic Seedlings of Polyembryonic Acid Citrus

Identification of zygotic and nucellar seedlings in polyembryonic mango cultivars

The objective of this work was to evaluate the occurrence of polyembryony in the mango cultivars Manila and Ataulfo, and to determine whether seedlings cultured in vitro are zygotic or nucelar. Percentage of polyembryony was calculated and the number of embryos in 100 seeds of each cultivar was recorded. 'Manila' exhibited 97% polyembryony with 3.4 embryos per seed, while 'Ataulfo' had 95% polyembryony with 3.2 embryos per seed. Later, 20 seeds of each cultivar were established in vitro, and it was analyzed those in which all embryos germinated (12 seeds from 'Manila' and 7 from 'Ataulfo'). DNA was extracted from seedling leaf tissue, and its origin was identified with 14 RAPD primers. The polymorphic markers recognized the seedlings of sexual origin in seven of nine 'Manila' polyembryonic seeds, and in four of seven 'Ataulfo' ones. Also, in polyembryonic seeds not all zygotic seedlings were produced by small embryos located at the micropyle.

Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

RAPD와 SRAP 방법을 이용한 '성전온주'(C. unshiu Marc.)와 '병감'(C. reticulate Blanco) 교잡실생 식별

감귤 '성전온주'(C. unshiu Marc)와 '병감'(C. reticulate Blanco)을 교배하여 얻은 다배성종자에서 교잡실생을 생육초기에 효과적으로 식별할 수 있는 방법 얻고자 PCR 기법에 바탕을 둔 RAPD와 SRAP 방법을 수행하였다. UBC (9, 27, 229, 230, 254) 프라이머와 SRAP (F4/R27, F7/R14, F12/R10, F44/R62) 프라이머 조합들을 사용하여 55개의 교배종자에서 얻은 실생들을 조사한 결과 37개의 종자에서 교잡실생을 식별할 수 있었다. F7/R14프라이머 조합에서는 45.5% (25/55)의 교잡실생을 식별할 수 있었고, UBC27 프라이머에서는 50.9% (28/55)의 식별효율을 보였다. 성전온주와 병감의 교배종자에서 UBC27 프라이머와 F7/R14 프라이머조합을 동시에 적용하였을 때에는 33개(60%, 33/55)의 종자에서 교잡실생을 식별할 수 있었다. 따라서 RAPD와 SRAP를 이용하였을 때 다배성 종자에서 교잡실생을 생육초기에 효율적으로 식별할 수 있었다. The purpose of this study was to evaluate the methods of identifying zygotic seedlings of crosses between 'Morita unshiu' (Citrus. unshiu Marc.) and 'Ponkan' (C. reticulata Blanco). In order to investigate the frequency and position of zygotic seedlings and to determine the efficiency of zygotic seedling identification, random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were performed using UBC (9, 27, 229, 230, and 254) primers and F4/R27, F7/R14, F12/R10, and F44/R62 primer sets, respectively. A total of 37 putative zygotic seedlings out of 55 individuals were selected by RAPD and SRAP. The F7/R14 primer pair showed a screening ability of 45.5% (25/55), whereas the primer UBC27 revealed the highest efficiency of zygotic seedling identification (50.9%, 28/55). When both UBC27 and F7/R14 were properly used for selection of hybridized seedlings of 'Morita unshiu' (C. unshiu Marc.) and 'Ponkan' (C. reticulata Blanco), screening efficiency was increased to 60% (33/55) for putative zygotic seedlings. Thus, it is possible to select putative hybrid zygotic seedlings in an accurate and effective manner by RAPD and SRAP.

Performance and Genetic Parameters of Somatic and Zygotic Progenies of Coastal Douglas-fir at 71/2-Years across Washington and Oregon, USA

Abstract Five genetic tests involving 37 somatic clones of coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) were planted March 2000 in Weyerhaeuser plantations across western Washington and Oregon States, USA. Four of the tests are in Longview, Twin Harbors and Vail regions of Washington, and one test is in Springfield, Oregon. Each test is based on single-tree plots with 12 randomized complete-blocks. The 37 coastal Douglas-fir clones were propagated by somatic embryogenesis from four full-sib families. Zygotic seedlings from two of these full-sib families were planted across all five tests to allow comparison between somatic and zygotic trees of the same pedigree. Results are reported for survival, stem height, diameter at breast-height (DBH), volume and stem sinuosity at 71/2- years. On average the total population of 37 somatic clones had less stem sinuosity, but grew more slowly than zygotic trees across the five tests studied. However, the best 20% of somatic clones for growth produced 25% greater stem volume at 71/2-years than the zygotic fullsib families. Height had a clonal heritability of 0.61 ± 0.09, DBH 0.64 ± 0.06, volume 0.58 ± 0.08 and stem sinuosity 0.26 ± 0.06. The clonal genetic correlation between height and DBH at 71/2-years was 0.98 ± 0.01, while stem sinuosity was adversely genetically correlated with growth. Clonal performance for growth and stem sinuosity was stable across tests with overall between-test correlations of 0.96 to 0.98. This clonal stability resulted in little variance due to clone x test interactions.

Callus production, somatic embryogenesis and plant regeneration of Lycium barbarum root explants

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm−3 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm−3 lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.

Parentais femininos monoembriônicos na obtenção de porta-enxertos híbridos de citros

Visando a evitar os efeitos da poliembrionia, os quais dificultam a distinção entre indivíduos híbridos e aqueles de origem materna (nucelares). O Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura Tropical selecionou híbridos monoembriônicos, com potencial de geração de novos porta-enxertos, e utilizou-os como parentais femininos em polinizações abertas. Esses parentais foram: trifoliados HTR-001; HTR-002; HTR-005; HTR-037; HTR-073 e HTR-135, além de LCR (limoeiro 'Cravo' Citrus limonia) x LRF (limoeiro 'Rugoso da Flórida' C. jambhiri) - 005. As progênies de meios-irmãos obtidas compreenderam, respectivamente, 364; 55; 12; 58; 51; 551 e 59 seedlings (plantas oriundas de sementes) zigóticos. Os híbridos dentro de progênies foram extremamente variáveis quanto à altura da planta e diâmetro do caule, também apresentando diferenças na presença de ramificações, formato, cor e textura do limbo foliar e tamanho de espinhos. A progênie mais vigorosa relacionou-se ao parental HTR-073, dando-se o contrário com aquela relativa ao híbrido LCR x LRF-005. Este trabalho indica que, em citros, a utilização de parentais femininos monoembrônicos em cruzamentos abertos constitui método simples e fácil de obtenção de híbridos.

IDENTIFICATION OF ZYGOTIC AND NUCELLAR SEEDLINGS FROM CITRUS ROOTSTOCK CANDIDATES USING RAPD

The preliminary approach in citrus breeding is to eliminate undesirable genotypes by distinguishing between zygotic and nucellar seedlings. The identification of true nucellar seedlings with the desired genotype is a daunting process as many citrus varieties produce both nucellar and zygotic embryos. We have carried out a series of crosses aimed at introgression of mandarin (Citrus reticulata) and pummelo (C. grandis) germplasm to develop alternative rootstocks to sour orange (C. (C. grandis), and it is necessary to identify those hybrids that yield nucellar seeds for rootstock propagation. Randomly Amplified Polymorphic DNA (RAPD) marker characterization was used to select hybrids that produce primarily nucellar seedlings. Five random primers were screened to classify various citrus seedlings from a total of 276 individual progeny obtained from 46 hybrid parental plants. The degrees of genetic similarity (nucellar) among progeny seedlings for each primer and among all primers were determined and compared with that of the parents. The usefulness and efficiency of RAPD-PCR method as a quick screening technique for citrus hybrids are discussed.

Genetic analysis of early field growth of loblolly pine clones and seedlings from the same full-sib families

Field trials established with clones and seedlings from the same families provide an opportunity for comparing full-sib family performance across propagule types. More than 1200 different clones together with over 14 000 zygotic seedlings from the same 61 full-sib families of loblolly pine (Pinus taeda L.) were tested on multiple sites across Florida and Georgia. The genetic variance associated with several early growth traits partitioned differently depending on propagule type. Most of the genetic variance associated with growth in the clonal population was additive, while the estimate of dominance in the seedling population was greater than estimates of dominance in the clonal population, based on single-site analyses. Apparently, a lack of randomization of the seedlings prior to field establishment caused full-sib families to appear more different, inflating estimates of dominance genetic variance. Parental and full-sib family ranks were stable regardless of propagule type as indicated by type B genetic correlations. In the clonal population, little genotype × environment interaction was observed across sites at the parental, family, and clonal levels for all traits. The high genetic correlations between propagule types provide further assurance that selections made through traditional tree-improvement activities for recurrent selection for general combining ability in seedling trials can also be used successfully for breeding families to test in a clonal forestry program.

Frequent Occurrence of Precocious Flowering in Zygotic Seedlings Derived from Crosses with a Monoembryonic and Male Sterile Acid Citrus Hybrid between Yuzu (C. junos Sieb. ex Tanaka) and Hanayu (C.hanaju Hort.exShirai)

Frequent Occurrence of Precocious Flowering in Zygotic Seedlings Derived from Crosses with a Monoembryonic and Male Sterile Acid Citrus Hybrid between Yuzu (C. junos Sieb. ex Tanaka) and Hanayu (C.hanaju Hort.exShirai)

The type of explant plays a determining role in the micropropagation of Ensete ventricosum

Three to five shoots per shoot tip were produced when in vitro-grown zygotic seedlings of enset genotype Oniya were used as donor plants and the shoot tips split longitudinally through the apex into two and cultured on a basal Murashige and Skoog (MS) medium supplemented with 11 μM BA and 6 μM IAA. Only one shoot per shoot tip of greenhouse-grown suckers was regenerated from about 60% of the explants on a similar medium, while the remainder died, presumably due to blackening and necrosis of the tissues. Increasing the BA concentration from 11 μM to 22 μM or to 44 μM did not increase to the number of shoots from the halved shoot tips. On MS medium supplemented with 11 μM BA and 6 μM IAA or 5 μM NAA, the intact shoot tip gave rise to a single shoot that originated from the apical bud. From the intact shoot tips that were grown in vitro on medium with 44 μM, 89 μM, 178 μM, 266 μM, 355 μM and 444 μM BA, 72% gave rise to a single shoot/bud from the apical bud without induction of any lateral buds, while the rest produced one or two lateral buds that were mainly hyperhydric. Therefore, wounding the apical dome by splitting appears necessary to release lateral buds from apical dominance of the tip of the monopodial corm of enset during micropropagation. The greater response in production of multiple shoots by zygotic seedlings cultured in vitro could be due to the absence of blackening and the juvenile nature of the explants.

Diversidade genética de porta-enxertos cítricos baseada em marcadores moleculares RAPD

Este trabalho teve como objetivo caracterizar a diversidade genética, através do marcador molecular RAPD, dos porta-enxertos da Coleção de Citros da Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul (EEA/UFRGS) e acessos de porta-enxertos cítricos coletados em viveiristas da Região do Vale do Rio Caí do estado do Rio Grande do Sul. Para tanto, coletaram-se folhas de nove porta-enxertos cítricos da EEA/UFRGS e de dez acessos de trifoliata (Poncirus trifoliata) de viveiristas. Com o uso de nove seqüências inicializadoras, foi possível separar os porta-enxertos cítricos em dois grupos principais, um formado pelo limoeiro ‘Cravo’ e outro pelo trifoliata e seus híbridos, apresentando alta dissimilaridade genética entre os grupos. Marcadores moleculares RAPD foram eficientes para caracterizar variedades de porta-enxertos de citros e para separar o porta-enxerto P. trifoliata de seus híbridos podendo serem utilizados para caracterização de plantas matrizes, análise de variabilidade genética entre genitores em programas de melhoramento genético de porta-enxertos e para identificar a origem sexual ou nucelar de mudas de trifoliata em viveiros comerciais.

Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD

The objectives of this work were to evaluate the frequency of polyembryony, and to identify zygotic and nucellar seedlings of Citrus volkameriana using RAPD. Twenty-five polyembryonic and eight monoembryonic seeds were cultivated in vitrofor six months. DNA from seedlings was extracted and used in combination with five RAPD primers to identify zygotic or nucellar origin of the seedlings. Environmental conditions of the year affected significantly (P<0.05) the morphological characteristics of fruitsand the number ofembryos per seed. Polyembryonic seeds ranged from 30.9%, 44.8% to 54.4% over three years. Morphological characteristic was not correlated with polyembryony. In vitro culture enable all embryos of each seed to grow, favoring the percentage of seedlings identified as zygotic. In polyembryonic and monoembryonic seeds, 25.9% and 87.5% of the seedlings, respectively, were sexually originated. In polyembryonic seeds, not all zygotic seedlings were produced by small embryos located at the micropyle.

Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets

Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μM α-naphthaleneacetic acid (NAA) and 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2–100%, while somatic embryos were produced from these calluses at a frequency of 25–87.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μM gibberellic acid and 5 μM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.