Zygotic Seedlings Research Articles

Development of an Optimal Set of High-Resolution Melting Markers for the Identification of Nucellar and Zygotic Seedlings in Yuzu (Citrus junos Sieb. Ex Tanaka)

Development of an Optimal Set of High-Resolution Melting Markers for the Identification of Nucellar and Zygotic Seedlings in Yuzu (Citrus junos Sieb. Ex Tanaka)

Polyembryony and morpho-genetic characterisation of zygotic seedlings through microsatellite markers in ten polyembryonic citrus rootstocks

Morpho-genetic characterisation of zygotic and nucellar seedlings of 10 citrus rootstocks was carried out to know the degree of polyembryony. 'Volkameriana' rootstock had maximum fruit and seed weight. Polyembryony, number of embryos per seed and clutch size was highest in 'Katazamir'. However, the seeds of 'Schaoub' took minimum days to germinate while 'Cleopatra' took maximum days. Significant positive correlation was observed between percent polyembryony and average number of embryos per seed, seed weight and number of seeds per fruit. Twenty SSR primers were utilised for detection of zygotic seedling from all rootstocks. None of the individual primers used in this study were able to consistently identify zygotic seedlings in all rootstocks tested. The maximum (26.7%) zygotic seedlings were identified in 'Katazamir' and minimum (5.0%) in 'X-639' rootstocks. There was not a significant correlation between seedling height and zygotic and nucellar seedlings, identified with SSR markers. Therefore, seedling height cannot be taken as a criteria to identify the nucellar and zygotic seedlings.

Leaf Volatiles as a Biomarker for Identification of Nucellar and Zygotic Seedlings in Polyembryonic Mango (Mangifera Indica L.) Genotypes

Leaf Volatiles as a Biomarker for Identification of Nucellar and Zygotic Seedlings in Polyembryonic Mango (Mangifera Indica L.) Genotypes

Increased Zygote-Derived Plantlet Formation through In Vitro Rescue of Immature Embryos of Highly Apomictic Opuntia ficus-indica (Cactaceae).

O. ficus-indica (prickly pear cactus) is an important forage and food source in arid and semiarid ecosystems and is the most important cactus species in cultivation globally. The high degree of apomixis in the species is a hindrance in plant breeding programs where genetic segregation is sought for the selection of superior genotypes. To understand if in ovulo embryo rescue could increase the proportion of zygotic seedlings, we compared the mature seed-derived seedlings with those regenerated from in vitro embryo rescue at 20, 25, 30, 35, and 40 post-anthesis days (PADs) in four Italian cultivars. The seedlings were classified as apomictic or zygotic based on molecular marker analysis using inter-sequence single repeat (ISSR) primers. Multiple embryos were recovered from all the cultured immature ovules, and plantlets were regenerated and acclimatized to the field post hardening, with success rates ranging from 62% ('Senza spine') to 83% ('Gialla'). The level of polyembryony differed among cultivars and recovery dates, with the highest being 'Rossa', producing 4.8 embryos/ovule at 35 PADs, and 'Gialla', the lowest, with 2.7 at 40 PADs. The maximum number of embryos observed within a single ovule was 14 in 'Trunzara bianca'. ISSR analysis revealed that ovule culture at 35 PADs produced the highest percentage of zygotic seedlings in all the cultivars, from 51% ('Rossa') to 98% ('Gialla'), with a high genotype effect as well. Mature seeds produced much fewer seedlings per seed, ranging from 1.2 in 'Trunzara bianca' to 2.0 in 'Rossa' and a lower percentage of zygotic seedlings (from 14% in 'Rossa' to 63% in 'Gialla'). Our research opens a pathway to increase the availability of zygotic seedlings in O. ficus-indica breeding programs through in ovulo embryo culture.

Comparison of leaf volatile aroma constituents and phenolic acid profiles of the seedling originated polyembryonic mango (Mangifera indica L.) genotypes

In mango, leaf and fruit volatile aroma profiles are variety specific which can be used as fingerprint of a variety. Such biochemical markers can also discriminate the nucellar and zygotic seedlings in polyembryonic mango varieties. In order to validate the applicability of volatile as well as phenolic acid profiles as biomarkers, the open pollinated seedlings of three polyembryonic varieties of mango were compared with their mother trees. Leaf volatile and phenol acid profiling were done using Gas Chromatography/Mass Spectrometry (GCMS) and Liquid Chromatography/Mass Spectrometry (LCMS) methods respectively. The sesquiterpene hydrocarbons were the most abundant in all the genotypes studied. Monoterpenoids were the major compounds in cultivars Vellaikolumban and Olour, while the sesquiterpenoids were the major compounds in cv. Turpentine. While terpinolene was the major monoterpenoid compound in Vellaikolumban and limonene in cv. Olour, the sesquiterpene á-gurjunene was the major compound in cv. Turpentine. Volatile profiling showed clear differences between the varieties but was similar within a variety. Among the 15 phenolic acids quantified in the leaves, P-coumaric acid, gallic acid, and ferulic acids were predominant whereas, vanillic acid, syringic acid, gentisic acid, benzoic acid, and sinapic acids were low in quantity. Phenolic acid profile did not show significant diversity among the varieties and therefore cannot be used for identification of varieties. The volatile profiling can be used for the identification and differentiation of polyembryonic mango genotypes.

Stipagrostis pennata (Trin.) De Winter Artificial Seed Production and Seedlings Multiplication in Temporary Immersion Bioreactors

This study was conducted to develop the protocol for artificial seed production of Stipagrostis pennata (Trin.) De Winter via somatic embryo encapsulation as well as test a temporary bioreactor system for germination and seedling growth. Embryogenic calli were encapsulated using sodium alginate and calcium chloride and then sowed in the Murashige and Skoog (MS) germination medium in in vitro cultures. The experiments were conducted as a factorial based on a completely randomized design with three replications. The treatments include three concentrations of sodium alginate (1.5%, 2.5%, and 3.5%), two ion exchange times (20 and 30 min), and two artificial seed germination media (hormone-free MS and MS supplemented with zeatin riboside and L-proline). Germination percentage and number of days needed until the beginning of germination were studied. The highest percentage of artificial seed germination was obtained when 2.5% sodium alginate was used for 30 min (ion exchange time) and when the seeds were placed on the MS germination medium supplemented with zeatin riboside and L-proline. The results of the analysis of variance in the temporary immersion bioreactor system showed that the main effects observed on the seedling growth were associated with different growth hormones in culture media and the number of feeding cycles. Experimental results also indicated that the total protein analyses of zygotic seedlings and seedlings originating from the synthetic seeds showed no statistically significant differences between these samples.

Screening of Mango Landraces for Polyembryony and Confirmation of Seedling Origin using Microsatellite Markers

Background: The introduction of polyembryonic rootstocks in the area of propagation is of great importance since they produce one zygotic and several nucellar plantlets. Proper identification of sexual embryo from each hybrid seed is necessary in order to preserve only the nucellar seedlings, which would help to maintain the rootstock’s genetic characteristics as well as to overcome the major constraints in the area of fruit breeding especially in hybridization programme by eliminating the nucellar ones to advanced generations. Contrasting reports exists regarding the vigour of zygotic seedlings of polyembryonic mango genotypes. It is necessary to identify/ distinguish the zygotic seedling from the nucellar population at an early stage, for which, microsatellite analysis could be a reliable tool. Methods: The experiment was laid out in completely randomized design (CRD) with 20 treatments replicated thrice. The twenty local mango landraces from Thiruvananthapuram (Kerala) were screened for polyembryony and were geo-referenced. Germination studies were conducted. Microsatellite analysis of all the plantlets from two varieties which exhibited the highest polyembryony were done using SSR primers and their banding patterns were compared with those of their respective mother plants. Result: Out of twenty mango varieties screened, seventeen were polyembryonic. Kappa Manga recorded the highest germination, germination index and seedling vigour index-I. Kotookonam Varikka recorded the highest polyembryony and followed by Kochu Kilichundan. Microsatellite analysis revealed that all the seedlings obtained from the respective stones of Kotookonam Varikka and Kochu Kilichundan had identical SSR profile to the mother plant, which indicated nucellar origin of seedlings having similar genetic composition to the mother plant.

Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing.

Huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), is the primary biotic stress causing significant economic damage to the global citrus industry. Among the abiotic stresses, salinity affects citrus production worldwide, especially in arid and coastal regions. In this study, we evaluated open-pollinated seedlings of the S10 (a diploid rootstock produced from a cross between two siblings of the Hirado Buntan Pink pummelo (Citrus maxima (Burm.) Merr.) with the Shekwasha mandarin (Citrus reticulata Blanco)) for their ability to tolerate HLB and salinity stresses. In a greenhouse study, ‘Valencia’ sweet orange (either HLB-positive or negative) was grafted onto six clonally propagated lines generated from the screened seedlings in the greenhouse and the trees were irrigated with 150 mM NaCl after eight months of successful grafting and detection of CaLas in the leaf petioles. Cleopatra mandarin was used as a salt-tolerant and HLB-sensitive rootstock control. CaLas infection was monitored using a quantitative polymerase chain reaction before and after NaCl treatments. Following three months of NaCl treatment, ‘Valencia’ leaves on the S10 rootstock seedlings recorded lower levels of chlorophyll content compared to Cleopatra under similar conditions. Malondialdehyde content was higher in HLB-infected ‘Valencia’ grafted onto Cleopatra than in the S10 lines. Several plant defense-related genes were significantly upregulated in the S10 lines. Antioxidant and Na+ co-transporter genes were differentially regulated in these lines. Based on our results, selected S10 lines have potential as salt-tolerant rootstocks of ‘Valencia’ sweet orange under endemic HLB conditions. However, it is necessary to propagate selected lines through tissue culture or cuttings because of the high percentage of zygotic seedlings derived from S10.

English

Identification of zygotic and nucellar seedlings of Harumanis mango by morphological approach is impossible or hard to be performed. Therefore, this research were to identify zygotic or nucellar seedlings from Harumanis mango polyembryonic seeds through simple sequence repeat (SSR) molecular markers in relation with germination sequence and vigour of seedlings for source of true-to-type cutting material. The results showed that there were 3 seedlings per seed on average. The result from the molecular analysis of 136 seedlings (45 seeds) showed four zygotic seedlings in seeds 1, 15, 29 and 43 representing 8.9% of seeds evaluated. Most of the zygotic seedlings were found towards the end of germination sequence except for seed number 15 and the rest were considered as nucellar seedlings. Based on the fitted logistic regression, the predicted sequence to obtain 90% nucellar seedlings is 5.47. This means that, the germination sequence of less than 6 has 90% chance of getting nucellar seedlings compared to zygotic seedlings. Morphological characters such as number of leaves, stem diameter and leaf area could also be used as references with germination sequence. The results showed that there were significant (p<0.01) relationships between germination sequence and all the growth variables. All growth variables were negatively correlated with germination sequence. This suggested that in order to have 90% chances of getting nucellar seedling (germination sequence below 6), the seedling needs to exhibit several morphology characters; big stem girth, tall plant, high leaf number and large leaf area. Therefore, choosing vigour seedling will increase chances of getting nucellar seedlings, which can be used as cutting source for true-to-type planting material or for breeding purposes.   Key words: Harumanis, molecular, morphology, nucellar, SSR markers, zygotic.

SSR marker based differentiation of zygotic and nucellar seedlings in mango (Mangifera indica)

Non-availability of standard clonal rootstocks in mango (Mangifera indica L.) is considered as an important hurdle, which has significant impact on orchard productivity. To obtain grafted mango plants, it is important to use polyembryonic rootstocks since they produce a zygotic and several nucellar plantlets from their seed, which are trueto- the type and also uniform. It is therefore imperative to device some reliable approach to ascertain the nucellar origin of seedlings to be used as rootstocks and thus culling out the variable zygotic seedlings in a polyembryonic genotypes for their use as uniform clonal rootstock. Differentiation of zygotic and nucellar seedlings using SSR markers in mango rootstock genotypes was undertaken during 2012-2015. Of the 42 SSRs used, 9 primer pairs (LMMA1, LMMA2, LMMA8, LMMA15, ESTD1, ESTD2, ESTD6, ESTD9 and ESTD10) were found to be informative, while 33 primer pairs were monomorphic. These nine primer pairs were used for differentiating zygotic and nucellar seedlings. In Olour rootstock, LMMA1, LMMA2, LMMA8, ESTD6 and ESTD10 primer pairs were informative and ascertained the zygotic and nucellar origin of seedlings. In Kurukkan rootstock, ESTD1, ESTD2, ESTD6 and ESTD9 primer pairs differentiated zygotic from nucellar seedlings. In 13-1 rootstock, LMMA8, LMMA15 and ESTD9 discriminated nucellars from zygotic seedlings. It is concluded that SSR markers were useful in differentiating the zygotic and nucellar seedlings in polyembryonic mango rootstocks and can be used in combinations to ascertain the origin of seedlings in polyembryonic mango rootstocks.

Identification of true to type and open pollinated progenies of polyembryonic Mangifera indica cv. Harumanis using microsatellite markers

The study was conducted on a polyembryonic mango, Harumanis which contains more than one embryos including one zygotic andsingle or more number of nucellar one. In this paper, we used microsatellite markers to identify whether the seedlings of Harumanis were zygotic or nucellar. A total of 95 progenies of Harumanis were evaluated using 13 polymorphic microsatellite markers. The genetic profiling revealed that a total of 14 Harumanis progenies were zygotic or open pollinated seedling as their genetic profile was different with Harumanis at least at one loci. Nevertheless, 76 Harumanis progenies were true to type or nucellar seedlings as their genetic profile was similar to Harumanis. The remaining five progenies could not be identified with the call rates of the genotypic data. Identifying true to type or nucellar seedling is useful for nursery growers to determine the true to type Harumanis progenies from the seed. Meanwhile, the open pollinated seedling or zygotic seedlings are preferred by breeders as they are considered as a new variety which increases the mango genetic variability.

Ex vitro recovery of rough lemon (Citrus jambhiri) hybrids and identification with SSR markers

Rough lemon (Citrus jambhiri Lush) is one of the leading citrus rootstock around the world besides sour orange. In the present studies, rough lemon was crossed with X 639 and Swingle citrumelo at Department of Fruit Science, PAU Ludhiana during 2015-16. Cent per cent seed germination was recorded for both the crosses under in vitro conditions but, the germination rates with growing media in plug trays were significantly lower (91.3 and 89.2% for RL× X and RL × SC, respectively). Higher proportion of multi-foliate seedlings (26.89%) was recovered by in vitro seed culture over the ex vitro seed germination (5.20%). All the multi-foliate seedlings were confirmed by SSR analysis using two primers (F 29 and F 87). The SSR analysis showed that only 85% multifoliate seedlings were zygotic while, the rest were not confirmed to be zygotic. The SSR analysis of the 50 unifoliate F1 seedlings each from both the crosses with 5 SSR primers differentiated seedlings into two groups, viz. zygotic and nucellar seedlings. In RL × X cross, the most efficient SSR marker was F29, which identified 30% F1 unifoliate seedlings to be zygotic followed by CCSME31, which identified 26% unifoliate seedlings as zygotic seedlings while the remaining seedlings were nucellar. In the cross RL × SC, the primer F29 was most efficient as it identified 28% zygotic seedlings and 72% nucellar seedlings whereas, the primer F87 identified 24 and 76% zygotic and nucellar seedlings, respectively.

DISTINGUISHING OF ZYGOTIC AND NUCELLAR SEEDLINGS IN CITRUS ROOTSTOCKS USING ISSR TECHNIQUE

The objective of this study is to evaluate the morpho-based method (common system) which used to distinguish between zygotic seedling and nucellar seedling of Cleopatra mandarin and Sour orange rootstocks using ISSR technique. Eight ISSR primers were used to check true-to-type of three mother-plants and nine seedlings of two citrus species, Cleopatra mandarin from Giza nursery and Sour orange from Benha nursery. Results showed that ISSR markers were a utility to identify nucellar or zygotic seedlings and check seedlings true to the type of mother plant. Three Cleopatra mandarin seedlings (33.3%) were identified as nucellar seedlings and six seedlings (66.7%) were identified as zygotic seedlings when using ISSR5 primer. While this percentages were different when using ISSR5 primer with Sour orange seedlings, which revealed one of them (11.1%) was identified as a nucellar seedling and eight seedlings (88.8%) were identified as zygotic seedlings. This suggested that the common method used until now for selection the nucellar seedlings in nursery is not perfect. Finally, that screening of seedlings for early identification of putative zygotic seedlings by ISSR analysis for random and various combinations is essential in citrus to maintain the genetic characteristics of the citrus rootstocks in orchards.

Identification of zygotic and nucellar seedlings inCitrus limon: the search for molecular markers

Identification of zygotic and nucellar seedlings in<i>Citrus limon</i>: the search for molecular markers

Identification of zygotic and nucellar seedlings inCitrus limon: the search for molecular markers

Identification of zygotic and nucellar seedlings in<i>Citrus limon</i>: the search for molecular markers

Identification of zygotic and nucellar seedlings inCitrus limon: the search for molecular markers

Identification of zygotic and nucellar seedlings in<i>Citrus limon</i>: the search for molecular markers

Molecular Verification of Putative Zygotic Seedlings in Different Intra-Specific Crosses in Mandarins (Citrus reticulata) by SSR Markers

The molecular verification of different intra-specific mandarin hybrids was explored during 2014–2015. Simple sequence repeat (SSR) markers were employed to identify putative zygotic seedlings from hybrid progeny produced from different crosses in mandarin. The most informative SSR marker in the F1 hybrids of ‘Daisy × Kinnow’ cross was CCSM170 which identified 73% of zygotic seedlings followed by CMS4, AG14, CiBE6006 and CiBE3397 which identified 38.5%, 31.5%, 27% and 24% of zygotic seedlings, respectively. Most efficient SSR marker in ‘Kinnow × Daisy’ cross was CiBE6006 which identified 22.5% zygotic seedlings followed by CCSM170, CiBE3397 and CMS4 which identified 17.5%, 12.5% and 5.5% of zygotic seedlings, respectively. In ‘W. Murcott × Kinnow’ hybridization, CMS31 marker identified 28.5% zygotic seedlings in F1 progeny followed by CiBE3298 which identified 14.5% zygotic seedlings. The SSR marker CiBE3397 identified 19.5% zygotic seedlings in ‘Kinnow × W. Murcott’ cross. The results show that the SSR markers can differentiate the zygotic seedlings of F1 progeny in different intra-specific crosses in mandarins.

Early Identification of Citrus Zygotic Seedlings Using Pollen-specific Molecular Markers

Abstract: This study was carried out to develop molecular techniques to allow the selection of zygotic seedlings in the early sta ge of the plant development. We identified 37 pollen-specific molecular markers from RAPD analysis and successfully used them for identification of the zygotic seedlings from various hybrid crosses. Three Satsuma mandarin cultivars (‘Morita unshiu’, ‘Na ngan20’ and ‘Miyagawawase’) were used as mother parents and seven cultivars (‘Ponkan’, ‘Lee’, ‘Kinokuni’, ‘Shiranuhi’, ‘Tamnaneunbo ng’,‘Shinyegam’, and ‘Sunburst’ mandarins) served as pollen parents. PCR analysis showed that 2 primers could identify zygotic h ybrid seedlings. Among them, an UBC-27 primer was used to identify the zygotic seedlings from hybrid crosses of “‘Nangan 20’ בKinokuni’” mandarin, “‘Nangan 20 × Ponkan’” mandarin and “‘Miyagawawase × Sunburst’” tangerine. In total 29 out of 40 seedlin gs (73%), 9 out of 47 seedlings (19%), and 13 out of 45 (29%) were identified as zygotic seedlings, respectively. These results can show that the pollen-specific markers selected in this study can be used effectively for early identification of zygotic se edlings from Citrus hybrid crosses.Additional key words:

English

Somaclonal variation induced in vitro during tissue culture can be a problem in clonal micropropagation of elite plants. This study investigated the extent of morphological and genetic similarity or dissimilarity between Melia volkensii in vitro plants (somatic seedlings) obtained via somatic embryogenesis and normal seedlings. Comparisons were made between in vitro plants regenerated directly from cotyledon explants, indirectly from zygotic embryos and normal seedlings of the same parent trees. Regeneration was achieved using half MS medium supplemented with 0.05 mg/l thidiazuron. Shoots were elongated in half MS with 0.1 mg/l BAP plus 0.01 mg/l IAA then rooted in half MS with 0.1 mg/l IBA and 0.1 mg/l NAA. Six morphometric and five meristic characters were used for the morphological characterization. PCR-RAPD markers were used for assessment of genetic similarity or distance. Multivariate analysis using principal coordinates, cluster analysis, analysis of similarities (Anosim) and similarity percentages analysis (SIMPER) revealed significant dissimilarities (p< 0.0001) in morphometric and meristic characters between the in vitro plants and normal seedlings. However, significant similarity (p<0.01) was observed in the RAPD-genic characters of the in vitro plants and normal seedlings. Out of six morphometric traits, taproot length, internode length and shoot height were the most important sources of dissimilarity, cumulatively accounting for 72.37% of overall morphometric dissimilarity. Number of lateral roots was the single most important source of meristic dissimilarity, with 77.02% contribution. Plants regenerated directly from cotyledons were more similar to the normal seedlings in morphological and RAPD-marker characters than those regenerated indirectly from zygotic embryos. This study paves the way for identification of trait-specific RAPD markers for further characterization through sequence-characterized amplified regions (SCARs). Key words : Morphometric, meristic, PCR-RAPD, Melia volkensii, tissue culture, somaclonal variation.

Application of simple sequence repeat markers for demarcation of Citrus reticulata nucellar and hybrid seedlings

The Polyembryony trait is a characteristic feature of the genus Citrus. This trait is regarded as a nuisance in hybridization but could be effectively utilized for Citrus propagation. The occurrence of nucellar embryony could be regarded as a replacement of costly tissue culture technique and identification of nucellar seedlings of Citrus reticulata may lead to proper management of the elite germplasms in mass scale propagation. Conventional techniques of ‘Off type’ rouging and other morphological identification techniques are not always correct. Molecular marker may be used for detection of nucellar seedlings. Simple sequence Repeat (SSR) markers were used for detection of nucellar seedlings. They are reliable markers and remain consistent under different environmental conditions. Four microsatellite primers with AG repeat CCSM13, CCSM17, CCSM18 and CCSM147 were able to demarcate the nucellar seedlings from the sexual ones. Three primers from TTA series TTA15, TTA27 and TTA33 were not able to discriminate seedlings. The four primers were able to discriminate the nucellar and zygotic seedlings developed from a local selection of an open pollinated population of Citrus reticulata of Darjeeling region of West Bengal. This experiment identified a few reliable markers useful for Citrus orchard management programme and could be applied for screening of asexual seedlings from an open population and also for controlled crossing.