Nucellar Seedlings Research Articles

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.

Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers

A series of crosses designed for introgression of mandarin (Citrus reticulata Blanco) and pummelo (C. maxima Merr.) germplasm, to develop an alternative rootstock to sour orange (C. aurantium L.), were carried out. It is necessary to identify those hybrids that yield nucellar seedlings for rootstock propagation. Rootstocks can be developed through traditional plant breeding methods; however, the ability to screen and select for economically important traits (such as production of true nucellar seedlings) in an efficient fashion is limited by the difficulties of screening techniques based on whole plant performance. To address these problems, we have used randomly amplified polymorphic DNA (RAPD) and fluorescently labeled expressed sequence tag simple sequence repeat (EST-SSR) molecular markers. A total of 204 individual seedlings obtained from 34 hybrid parental plants were successfully characterized using five RAPD primers. Ten hybrid parents and their progenies, found to be genetically similar among themselves, were selected for more scrutiny using eight EST-SSR primer pairs. The degrees of genetic similarity (nucellars) among progeny seedlings were determined and compared with that of their parents. The mean genetic similarity varied from 67–99% among the selected rootstock candidates screened. The genetic similarity relationship identified using RAPD and EST-SSR molecular markers was highly concordant (p = 0.001). Two elite rootstock candidates (B6R5T56; B6R11T129) that seem to be ideal for future mandarin and pummelo derived rootstock breeding programs have been identified. Our results indicate that either RAPD or EST-SSR analyses could be equally successful in identifying true nucellars among the progenies obtained from introgression crosses of mandarin and pummelo, thus improving the accuracy of early selection in a citrus rootstock breeding program.

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.

Regeneration of the Vascular Bundle at the Graft Interface in Auto- and Heterografts with Juvenile Nucellar Seedlings of Satsuma Mandarin, Yuzu and Trifoliate Orange

The histology of the graft union by splice grafting epicotyls in auto- and heterografts of 4-week-old nucellar seedlings of satsuma mandarin (Citrus unshiu Marc.), yuzu (C. junos Sieb. ex Tanaka), plus common trifoliate orange (Poncirus trifoliata Raf.) and its crooked spine type variety, Hiryu (Flying Dragon) (P. trifoliata var. monstrosa (T. Ito) Swingle) was studied under light microscope. Callus formation at the graft interface that started immediately after grafting was especially active adjacent to the severed vascular bundles. Regeneration of the vascular cambium occurred within 1 week after grafting in all graft combinations that led to the formation of a new vascular bundle. The new vascular bundle was formed with exterior winding by obstruction of the callus mass proliferating at the graft interface; the winding tended to be deeper in heterografts with satsuma than that in autograft of satsuma. However, the winding tended to revert to a straight configuration by the subsequent formation of newer vascular bundles. No obvious morphological differences of cells were observed at the graft interfaces in the auto- and heterografts during the process of callus formation and vascular bundle generation during the 6 weeks after grafting. Callus formation at the graft interface as a response to wounding and vascular bundle regeneration may be spatially competitive processes, and the anatomical and physiological mismatches between scion and stock cause the delay of vascular bundle formation, resulting in the winding.

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.

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.

Maravilha: uma nova seleção de tangerina 'Sunki'

Apesar das variações somáticas identificadas em pomares comerciais e em coleções de variedades serem, geralmente, desfavoráveis, apresentando baixa produtividade de frutos, morfologia foliar atípica ou frutos anormais, inúmeras mutações espontâneas de inquestionável valor têm sido identificadas, haja vista que a grande maioria das variedades cítricas comerciais, copas e porta-enxertos surgiu como decorrência de algum tipo de mutação natural. O presente trabalho diz respeito à exploração dessa importante via de obtenção de novos clones e variedades, fazendo parte de ações do Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura. Refere-se a uma nova seleção de tangerina 'Sunki', denominada 'Maravilha', identificada dentro de um grupo de seedlings nucelares da seleção 'da Flórida'. Foram realizadas comparações da 'Sunki Maravilha' com outras três seleções dessa tangerina, 'Comum', 'da Flórida' e 'Tropical', compreendendo os caracteres: número médio de sementes por fruto, número médio de embriões por semente, intervalo de variação do número de embriões por semente, porcentagem de poliembrionia e tamanho de embrião. Comparações foram efetuadas, também, com outros importantes porta-enxertos comerciais, limões 'Cravo' e 'Volkameriano' e tangerina 'Cleópatra', relativas a caracteres relacionados à germinação de sementes e ao vigor de seedlings (altura e número de folhas verdadeiras). Os resultados obtidos permitem indicar a seleção 'Sunki Maravilha' como alternativa de uso em programas de diversificação de porta-enxertos, nas condições em que esta tangerina apresenta boa adaptação, principalmente em função de seu relativamente elevado número médio de sementes por fruto (7,7), previsível uniformidade de seedlings, esta decorrente da elevada porcentagem de poliembrionia (100%), boa germinação de sementes e vigor de seedlings, além de provável resistência à gomose de Phytophthora spp.

Molecular characterization of an S-adenosylmethionine decarboxylase gene from floral organ differentiating axillary bud in calamondin (× Citrofortunella mitis)

SummaryCalamondin (× Citrofortunella mitis J. Ingram & H. E. Moore) is known for its ability to develop floral organs to flower in the adult phase during the four seasons. To provide information on molecular events during floral differentiation from axillary buds, we constructed a cDNA library from floral differentiating axillary buds of adult phase calamondin. Ninety-six cDNA clones were randomly selected from this library and sequenced. Ninety-six nonredundant ESTs were identified. Two clones encoded S-adenosylmethione decarboxylase, a key enzymes in polyamine biosynthesis. CmSAMDC 1 (Citrofortunella mitis s-adenosylmethionine decarboxylase 1) contained an 1083 bp ORF that encoded a putative SAMDC precursor of 361 amino acids. Northern blot analysis revealed that CmSAMDC 1 was expressed in axillary buds prior to floral differentiation and in axillary buds immediately after floral differentiation, in immature flower (5 mm), in fully developed flowers (120 mm long), in floral parts (petals, pistils, and stamens) of trees in the adult phase of development, but not in leaves or axillary buds of juvenile phase nucellar seedlings or leaf tissue from trees in adult phase of development. In situ hybridization revealed expression of the CmSAMDC 1 gene in the floral apex of axillary buds after differentiation, and in the phloem of axillary buds and petioles. These results suggest that SAMDC could play a role in actively differentiating and developing reproductive and vegetative organs.

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.

Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers

Citrus breeding programs normally face several biological obstacles such as apomixis and polyembryony that result in a cumbersome identification of hybrid seedlings. The main purpose of this work is to describe the combined use of visual selection based on the leaf apex morphology and SSR analysis to differentiate hybrid from nucellar seedlings derived from the cross between the ‘Murcott’ tangor [Citrus reticulata Blanco × Citrus sinensis (L.) Osb.] and ‘Pera’ sweet orange [Citrus sinensis(L.) Osb.]. A new morphological variable named leaf apex morphometric index is also described as the quantitative basis of the visual selection. The efficiency of visual selection of hybrids was tested under two growth conditions, seedlings germinated in seedbeds and in plastic tubes. Putative hybrid seedlings were also confirmed through the analysis of simple sequence repeats (SSR). The visual selection of hybrid seedlings resulted in an increase of 87.2% (p < 0.01) and 202.2% (p <0.001) in the number of correctly identified hybrids when compared to the method of random picking of seedlings in seedbeds and plastic tubes,respectively. The results indicate that the combination of visual selection and SSR analysis for the identification of hybrids derived from the cross of polyembryonic citrus cultivars will improve the accuracy of the selection,save time, and reduce the costs involved in the use of molecular markers alone in citrus breeding programs.

A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers.

In citrus breeding and genetics, it is very important to distinguish between zygotic and nucellar seedlings in order to eliminate unwanted genotypes. Usually, isozyme marker shave been employed to determine the genetic origin of young plants. In this work we propose the use of SSR markers as an alternative methodology and compare them with isozymes in this kind of screenings. Two different populations were analysed: one derives from an interspecific cross and the other from selfing. We conclude that, in most cases, microsatellites are more efficient than isozymic markers to identify the sexual origin of citrus seedlings, given their higher level of polymorphism and the scarce number of polymorphic isozymes in some populations. We describe a quick and efficient methodology for SSR analysis, including a fast DNA extraction in microcentrifuge tubes, and visualization through silver staining, which eliminates the need for a labelling step.

Plantlet regeneration from long-term callus cultures of Citrus acida Roxb. and the uniformity of regenerated plants

Callus cultures were initiated from epicotyl explants of in vitro grown nucellar seedlings of Citrus acida. Best callus induction occurred on MS medium containing BAP and 2,4-D and when explants were incubated in the dark. High levels of MS vitamins enhanced callus initiation and growth. These calli could be maintained in culture for upto two years and plantlets regenerated from them on transfer to light. Greening of callus and regeneration of shoot-buds from callus occurred on transfer to MS media with BAP. These shoots grew further in media with both, BAP and GA 3, Rooting of shoots occurred on media with NAA or IBA and the rooted plantlets could be transferred to soil. Regeneration of shoots and roots decreased with increase in age of callus. Cytological study of the regenerants revealed no change in chromosome number. Thus, genetically uniform plantlets could be regenerated continuously from established callus upto two years old, thereby helping in multiplication and conservation of the species.

Genetic analysis of apomixis in Citrus and Poncirus by molecular markers

Propagation of citrus rootstocks depends upon the production of clonal plants from nucellar seedlings. This makes apomixis one of the host important traits in breeding programs for citrus rootstocks. The genetic control of apomixis was studied in a 50-tree progeny derived from the cross C. volkameriana×P. trifoliata using 69 molecular markers and bulked segregant analysis. The proportion of nucellar seedlings was estimated by isoenzymatic analysis of 25 seedlings per tree for 2 consecutive years. The type of embryony (polyembryonic versus monoembryonic seeds) was also determined for fruit-yielding trees. Separate genetic maps for each parental species were developed. The integration and comparison of these maps could be accomplished using common multiallelic segregant loci. Differences in gene synteny between the two species-specific genetic maps were shown. Important distortions in the segregation of markers at several genomic regions, some of them also involving differences in the C-methylation pattern, have been observed, especially for the pollen parent. Analysis of quantitative trait loci (QTLs) revealed the presence of six genomic positions (two in P. trifoliata and four in C. volkameriana) contributing individually up to 24% of the total variation for apomixis. Within the same species, QTLs with positive and negative allele effects were present, even in the same linkage group. One of the markers associated to apomixis (Apo2) is also associated to embryony type. Therefore, the genetic control of apomictic reproduction found in citrus (nucellar embryony) is quite complex compared to what has been reported for gametophytic apomixis. Molecular markers linked to QTLs governing apomixis will be useful to assist selection of future apomictic rootstocks for citrus varieties.

Transgenic grapefruit plants obtained by Agrobacterium tumefaciens-mediated transformation

The economically important citrus crop plant, grapefruit, was genetically transformed, which has not been previously reported. Seedling epicotyl tissue was infected with Agrobacterium tumefaciens carrying a T-DNA vector plasmid containing genes for the scorable marker, β-glucuronidase (GUS) and the selectable marker NPTII. Shoots that regenerated from epicotyl segments from nucellar seedlings in the presence of kanamycin were analyzed. Histochemical staining verified expression of the GUS reporter gene in 43.5% of the regenerated shoots examined; however, the majority were chimeric for GUS expression while 11.9% stained solid blue. Transgenic plants were recovered from regenerated shoots by direct rooting using a simple protocol. The presence of the transgene was confirmed in 25 regenerated grapefruit plants by histochemical staining, PCR and Southern hybridization.

In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis

AbstractEmbryo development in vivo has been studied in four Citrus aurantium L. polyembryonic genotypes. Seeds were collected 65, 85, 105, 125 and 220 days after pollination (DAP). None of the immature seeds harvested 65 and 85 DAP contained visible embryos. A single embryo at a more advanced developmental stage was observed in the central position at the micropylar apex of the embryo sac in about 74% of seeds harvested at 105 DAP, while at 125 and 220 DAP the majority of seeds had two or more embryos at the same developmental stage crowded together. Restriction fragment length polymorphism (RFLP) analysis of lowand high‐copy‐number nuclear DNA was used to distinguish zygotic from nucellar seedlings. Analysis of plantlets derived from in vitro culture of the bigger embryos, located in the central position at the micropylar apex of the embryo sac of seeds harvested at 105 DAP, established that the frequencies of zygotic embryos ranged from 82 to 88%. Media for immature embryo germination in vitro were based on the nutrients and vitamins of Murashige and Skoog (MS) and Murashige and Tucker (MT) media supplemented with various concentrations of sucrose and growth regulators. A total of 76% of globular stage embryos (&lt;0.3mm) germinated on MT medium containing 150 mM sucrose and 14.4 μM gibberellic acid. Heart stage embryos (0.3‐0.8 mm) germinated at 95% on MT medium supplemented with 150 mM sucrose and 2.9 μM gibberellic acid. The addition of 500 mg/l malt extract to MS medium increased the germination of early cotyledon stage (0.8‐2.0mm) embryos to 98%. The optimum sucrose concentration for embryo rescue was 150 mM for the three embryo developmental stages. The ability to form plants in vitro strongly increased with increasing embryo developmental stage.

Induction of Callus from Seedling Explants of Citrus macrophylla W. Rootstock

Citrus macrophylla is an important citrus rootstock for Mexican lemon (Citrus aurantifolia S.). This study was conducted to select explant type and to optimize cultural requirements for induction callus of C. macrophylla in vitro. The explants tested were leaf, epicotyl, cotyledon, and root segments excised under sterile conditions from 4-week-old nucellar seedlings. The various medium comprising either basal of Murashige and Skoog (MS) or Murashige and Tucker (MT) salts suplemented with various concentrations of plant growth regulators, including naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin) were used for the establishment of the explants. All cultured explants initiated callus from the cut ends after 2 weeks, when cultured on a modified MT medium suplemented with 6 mg NAA and 0.2 mg kinetin; cotyledon segments were the best explant for callus induction and development (43 mm2). Root segments were the lowest explants for callus induction.

Inheritance of Procumbent Habit from `Cipo' Sweet Orange in Crosses with `Clementine' Mandarin

`Cipo' sweet orange [Citrus sinensis (L.) Osbeck] combines typical midseason fruit characteristics with a unique procumbent growth habit. This distinctive habit may be of value in breeding smaller and more procumbent scion cultivars if the growth habit is transmitted to hybrid seedlings. Two hybrid populations were created using `Clementine' mandarin (Citrus reticulata Blanco) as the female parent and either `Cipo' sweet orange or `Pineapple' (another midseason sweet orange with a more typical upright growth habit) as the male parent. The `Clementine' × `Cipo' cross yielded many hybrids with the procumbent habit, many with the upright habit, and some that appeared intermediate. Both hybrid populations were compared with nucellar seedling populations from `Cipo' and `Pineapple' using two morphological characteristics that differentiate between the procumbent habit of `Cipo' and the upright habit of `Pineapple'. All the `Clementine' × `Pineapple' hybrids were of upright growth habit, while the `Clementine' × `Cipo' progeny segregated into two groups based on growth habit (upright and procumbent). The two measured characteristics were tightly correlated in the segregating population and are probably pleiotropic effects of the same genetic mutation. The observed population distributions were as expected if the procumbent habit in `Cipo' is controlled by a single dominant allele in the heterozygous condition.

幼苗期の葉油成分によるカンキツ珠心胚実生と交雑実生の職別

幼苗期の葉油成分によるカンキツ珠心胚実生と交雑実生の職別

Screening Trifoliate Hybrid Citrus Rootstocks for Resistance to Phytophthora parasitica by in Vitro Inoculation

Phytophthora parasitica Dast. causes several root and trunk diseases of citrus, including damping-off, root rot, foot rot, and gummosis. Phytophthora resistance is needed in Citrus rootstocks and is available in Poncirus trifoliata (L.) Raf. and some hybrids between Citrus and P. trifoliata. Field or greenhouse tests of rootstocks require large amounts of space and time. To provide a preliminary indication of rootstock resistance to P. parasitica, nucellar seedlings of P. trifoliata selections, and Citrus × P. trifoliata hybrids were tested for response to P. parasitica by in vitro inoculation. Seeds of individual selections were germinated in sterile culture and 3-week-old shoots were excised and inoculated with a cultured isolate of Phytophthora. After 1 week of incubation, response to the disease organism was measured by length of stem discoloration. Progression of Phytophthora in the stem also was measured by plating sequential 5-mm segments of the shoot and determining presence or absence of Phytophthora in individual segments. Stem discoloration length corresponded with location of Phytophthora in the stem. Relative resistance, as measured by this technique, approximated field resistance for several common rootstock cultivars. Resistant, intermediate, and susceptible selections were found in populations of Citrus × P. trifoliata rootstock hybrids using in vitro inoculation.

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.