Immature Flower Buds Research Articles

Identification and evaluation of internal control genes for gene expression studies by real-time quantitative PCR normalization in different tissues of Tuberose (Polianthes tuberosa)

Quantitative real time PCR has become the most popular method to study the gene expression due to its accuracy. However the expression level of the target gene may be misunderstood due to the unstable expression of the reference genes under different experimental conditions. Therefore, it is a prerequisite to identify and validate the reference genes in each species for gene expression analysis. Polianthes tuberosa, a very popular commercial flowering crop globally, lacks information on any such reference genes. In this study, we describe the first systematic evaluation of four conventional candidate reference genes; 18S ribosomal RNA (18S rRNA), Ribulose bisphosphate (RuBP), Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), Actin and four novel genes; Coatomer subunit delta (CSD), Peptidyl-prolyl isomerase (PPI), Serine/threonine-protein phosphatase, (STPP) and ATP synthase E-subunit (ATP SE) in tuberose. The transcript abundance of these genes was analyzed in eleven different tissues like young leaf, leaf sheath, root, immature flower bud, mature flower bud, open flower, stamen, ovary, stigma, petals and flower tube. Three RT-qPCR statistical analysis methods, BestKeeper, NormFinder and geNorm were used to evaluate the stability of gene expression that indicated expression of PPI and CSD to be most stable across all the tested tissues. The stability of these two genes was also confirmed across four popular commercial varieties and under biotic and abiotic stresses. Utility of PPI in tuberose as a reference gene is a pioneer report in plants whereas, usefulness of CSD as a stable reference gene has been demonstrated for the first time in a crop besides the model plant, Arabidiopsis.

First report of Alternaria dianthicola causing flower blight on carnation in China

A severe disease of carnation flowers was observed in Kunming, China in November 2009. Initial symptoms on flowers and immature flower buds were characterized by water‐soaked areas. Spots with purple margins could be seen on petals of some varieties. An Alternaria sp. was isolated from 100% of symptomatic tissues sampled. The isolates showed high virulence when inoculated onto tissue culture plantlets and on healthy carnation flowers. Results of sequence analysis of 5.8S rDNA‐ITS of isolates showed 99% sequence similarity with Alternaria dianthicola in GenBank. To the best of the authors' knowledge, this was the first sighting of blight of flowers and immature flower buds of carnation caused by Alternaria dianthicola in China.

Somatic embryogenesis and plant regeneration from cell suspension cultures of Rajeli (AAB), an endangered banana cultivar

Rajeli (AAB), a commercially valuable Indian banana cultivar, is presently under the threat of extinction due to various diseases, warranting development of the strategies for its conservation and incorporation of disease resistance. This elite genotype was successfully regenerated in vitro via establishment of cell suspensions followed by somatic embryogenesis. The immature male flower buds were cultured on gelled Murashige and Skoog medium supplemented with 4 mg/l 2,4-D, 1 mg/l each of IAA and NAA, D-Biotin, 100 mg/l Malt Extract, 100 mg/l L-Glutamine and 30 g/l sucrose. Embryogenic calli with translucent somatic embryos were observed after 4 months of male flower bud culturing. Fine embryogenic cell suspensions were established in the liquid medium of same composition but with 45 g/l sucrose. Of the various sugars tested, maltose in the maintenance medium yielded better growth of plated cells as compared to sucrose, fructose and dextrose. Embryos differentiated at a high frequency and mature somatic embryos developed into plantlets on MS medium supplemented with 0.5 mg/l BAP. Approximately 40 % of the torpedo stage somatic embryos germinated and developed into complete plants. The regenerated plants exhibited normal phenotype during acclimatization in the greenhouse. The technique can be employed for conservation of this endangered cultivar and its improvement via mutagenesis and genetic transformation.

REGENERATION OF BANANA (Musa spp. AAA Group) cv. WILLIAMS via SECONDARY SOMATIC EMBRYOGENESIS AND CELL SUSPENSION FROM IMMATURE MALE FLOWER BUDS

Development of embryogenic cultures having high regeneration efficiency from important, commercial varieties of banana is a prerequisite for genetic manipulation and for in vitro propagation. In the present study, we have studied the induction of somatic embryogenesis from young immature male inflorescences of the banana cultivar Williams (Musa spp. AAA group) via secondary somatic embryogenesis and cell suspension. Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l alfa naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline (MM1). Secondary somatic embryogenesis (SE2) was developed when primary embryos were subculture on SK4 medium (MS medium supplemented with 10 ml of coconut milk). Embryos differentiated when sub-cultured on SK8 medium (MS medium supplemented with 5 mg/l BA and the embryos germinated when subculture on MS free hormone medium. Suspension cultures were initiated from SE2 embryogenic tissues from when placed in liquid medium supplemented with 2,4-D (1mg/l), biotin (1 mg/l), L-glutamate (100 mg/l), malt extract (100 mg/l), and sucrose (45 g/l), pH of the medium was adjusted to 5.3. The packed cell volume (PCV) of the suspension increased 2-5 fold with each monthly cycle. The somatic embryos were developed when suspension culture were aspirated on MS medium supplemented with biotin (1 mg/l), malt extract (100 mg/l), Glutamine (100mg/l), NAA (1mg/l), Kinetin (0.5 mg/l) Zeatin (0.2 mg/l), sucrose (45 g/l), and phytagel (2.6 g/l) (SK13). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l 6-benzylaminopurine (BA) for development of mature somatic embryos, which were isolated and cultured on hormone-free MS medium for germination and development into plantlets. Approximately 55% of the somatic embryos germinated and developed into plantlets. Somatic embryogenesis via SE2 and cell suspension might be an excellent technique for mass propagation, developing a breeding strategy and genetic transformation of banana.

The fluid from the immature flower bud of Spathodea campanulata reduces intraocular pressure in dogs

The fluid from the immature flower bud of Spathodea campanulata is used in Jamaican folklore medicine for the management of glaucoma. This study investigated the effect of the fluid on the intraocu...

Characterization of Expressed Sequence Tags from Flower Buds of Alpine Lilium formosanum using a Subtractive cDNA Library

Formosan lily (Lilium formosanum), a species endemic in Taiwan, is characterized by showy and fragrant flowers. To understand the gene expression at its reproductive phase, we constructed a suppression subtractive cDNA library of immature flower buds, from which 1,324 expressed sequence tags (ESTs) were randomly selected and sequenced. These EST sequences were clustered into 974 nonredundant sequences. Based on BLAST searching, functions of 376 sequences (39%) were determined, and 80 sequences showed high similarities to genes encoding hypothetical proteins without known functions. Another 518 sequences did not show significant homology to any known sequences and were therefore classified as novel sequences. Further analyses of the 376 ESTs sequences revealed high abundance of stress-related and flower-development genes. The highly expressing stress-related transcripts include 39 with high similarities to lipid transfer proteins, five to ascorbate peroxidases, and five to heat shock proteins 70. Using real-time quantitative RT-PCR analysis, we further revealed the expression of these three genes in the immature flower buds and in the pistils or stamens of the blooming flower of Formosan lily collected from alpine regions. These results suggest that the flower of L. formosanum possesses a significantly elevated level of stress genes in response to alpine environment and the ESTs analyzed here represent a valuable resource for studying a resistance mechanism of the reproductive organs of Formosan lily.

Cloning and Characterization of MADS-box Gene in Oil Palm

Oil palm has emerged as one of the most important source of oils and fats. The mechanism of floral organs development in this plant is still at its infancy. We describe here the cloning and characterization of a MADS-box gene in oil palm (Elaeis guineensis Jacq.) named EMADS1. It belongs to the AGAMOUS-like2 family of MADS-box gene which plays critical role in flower development as defined by the ABCDE model. EMADS1 was ubiquitously expressed in the immature male and female flower buds and its expression pattern was similar to EgAGL2 and EgMADS8 of oil palm. The EMADS1 transcript also accumulated in embryos of developing seeds. These results suggested that EMADS1 is likely to function at the initial stages of flowering in determination of the inflorescence and the identity of the flower whorls and also embryo development in seeds.

IN VITRO INDUCTION OF AUTOTETRAPLOID ACTINIDIA PLANTS AND THEIR FIELD EVALUATION FOR CROP IMPROVEMENT

IN VITRO INDUCTION OF AUTOTETRAPLOID ACTINIDIA PLANTS AND THEIR FIELD EVALUATION FOR CROP IMPROVEMENT

Sequential Florivory/Saproflorivory of Anaxagorea crassipetala (Annonaceae) by Diathoneura tessellata (Drosophilidae)

Abstract Diathoneura tessellata Duda, 1925 (Diptera: Drosophilidae) uniquely and effectively uses the fleshy tepals of Anaxagorea crassipetala (Annonaceae), a small, understory tree of the primary lowland rain forest of Costa Rica, as a larval substrate and pupation site. This study is the first to document 1) the brood substrate for larvae of this species and the 2) use of flowers in the Annonaceae as a drosophilid larval substrate. Oviposition into the tough, immature flower buds is made possible by an enlarged oviscape. This relationship is unique in that these flowers support two sequential cohorts of larvae, one cohort mining the living tepals of immature and mature flowers and the second cohort consuming the fallen, postanthesis tepals. We refer to this phenomenon as sequential florivory/saproflorivory. The first cohort consists of fewer, larger larvae, whereas the second cohort consists of more numerous, smaller larvae. Both cohorts exhibit a female skewed sex ratio.

Natural History Studies for the Preliminary Evaluation of <I>Larinus filiformis</I> (Coleoptera: Curculionidae) as a Prospective Biological Control Agent of Yellow Starthistle

We studied the life history, geographic distribution, behavior, and ecology of Larinus filiformis Petri (Coleoptera: Curculionidae) in its native range to determine whether it is worthy of further evaluation as a classical biological control agent of yellow starthistle, Centaurea solstitialis (Asteraceae: Cardueae). Larinus filiformis occurs in Armenia, Azerbaijan, Turkey, and Bulgaria and has been reared only from C. solstitialis. At field sites in central and eastern Turkey, adults were well synchronized with the plant, being active from mid-May to late July and ovipositing in capitula (flowerheads) of C. solstitialis from mid-June to mid-July. Larvae destroy all the seeds in a capitulum. The insect is univoltine in Turkey, and adults hibernate from mid-September to mid-May. In the spring, before adults begin ovipositing, they feed on the immature flower buds of C. solstitialis, causing them to die. The weevil destroyed 25-75% of capitula at natural field sites, depending on the sample date. Preliminary host specificity experiments on adult feeding indicate that the weevil seems to be restricted to a relatively small number of plants within the Cardueae. Approximately 57% of larvae or pupae collected late in the summer were parasitized by hymenopterans [Bracon urinator, B. tshitsherini (Braconidae) and Exeristes roborator (Ichneumonidae), Aprostocetus sp. (Eulophidae), and unidentified species of Eurytomidae and Ormyridae]. This weevil may be a better choice than the other capitula insects already established in the United States, particularly in colder parts of the plant's range.

Plant Regeneration and Floral Bud Formation from Intact Floral Parts of African Violet (Saintpaulia ionantha H. Wendl.) Cultured in vitro

Intact immature flower buds of African violet (Saintpaulia ionantha H. Wendl.) were used as explant sources for in vitro studies. The effect of exogenous hormones, NAA and BAP on the indirect organogenesis of this species was observed. Callus was formed on the cut end (base) of pedicels of floral buds where they were in contact with the medium. When maintained on the same medium, callus was differentiated into adventitious shoots after 10 weeks in culture. MS media supplemented with 2.0 mg L(-1) NAA and 1.0 mg L(-1) BAP gave the highest number of sterile or vegetative floral buds from the surface of callus of the explants, but these buds failed to develop further. The floral buds were expanded as abnormal flowers. The floral structures were smaller in size compared to intact flowers. Petals (corolla) were white to purple in colour but did not form any reproductive organs, i.e., stamens or pistils. All sterile or vegetative floral buds and abnormal flowers survived for 3 months in culture but failed to reach anthesis.

In vitro Propagation and Plantlet Regeneration from Doritaenopsis Purple Gem ‘Ching Hua’ Flower Explants

The effects of four types of explants removed from 10-cm flower stalks of Doritaenopsis Purple Gem ‘Ching Hua’ (immature apical flower buds, immature lateral flower buds, flower stem nodes, and flower pedicel sections) and combinations of two plant growth regulators [naphthalene acetic acid (NAA) and thidiazuron (TDZ)] on direct in-vitro shoot induction and multiplication were studied. Immature apical flower buds were the only explants that showed induction and multiplication of shoots in vitro. NAA at 5.4 and 10.7 μm combined with either 4.5 or 9.1 μm TDZ provided the fastest and greatest percentages of shoot induction (27% to 40%) and the greatest numbers of shoot multiplication (111–160 shoots per explant). In vitro–induced shoots were rooted on medium containing 5.4 μm NAA and developed into plantlets with normal vegetative and reproductive morphology. Regenerated plantlets were acclimatized, showing 100% survival and establishment in greenhouse. Plantlets were grown to maturity and showed normal flower morphology. No floral off-types were observed. The high rates of shoot multiplication obtained offer a means for mass clonal propagation of this and possibly other related Doritaenopsis hybrids.

Polyamines are involved in the gynogenesis process in onion

Haploidization in onion (Allium cepa L.) using immature flower buds simulates zygotic embryogenesis with no fecundation. In order to know the involvement of polyamines (PAs) in this process, we determined the concentration of endogenous PAs in flower buds and experimented the addition of various combinations of PA molecules in the medium. At the inoculation stage, high levels of free and conjugated spermidine and low putrescine + hydroxyputrescine/spermidine + spermine ratio characterized the highest responsive varieties. During in vitro culture, high levels of putrescine and its derivatives characterized the lowest responsive varieties, whereas high levels of spermidine and spermine characterized responsive varieties. The putrescine + hydroxyputrescine + homospermidine/spermidine + spermine ratio remained low in responsive varieties. The addition of spermidine or spermine (2 × 10−3 M) to the culture medium improved significantly the embryo production. Our results suggest that the arginine decarboxylase pathway is involved in PA biosynthesis during the in vitro culture of flower buds. Our study showed that specific ratios of PAs are required for successful gynogenesis in onion.

A vegetative line of asparagus (Asparagus officinalis) with a homeotic change in flower development is correlated with a functional deficiency in class-B MADS-box genes

SummaryWe describe a vegetative line of asparagus (Asparagus officinalis) that bears flowers with highly altered floral organs. Flower buds in a female line (designated ‘GS#2’) of asparagus ‘Gold Schatz’ develop small tepals that are green in colour. Stamens are not found, but floral organs resembling carpels differentiate at positions where stamens would form. As these features appeared to indicate a homeotic mutant caused by loss of B-function in the ABC genetic model, expression analysis of class-B MADS-box genes was performed. Northern blot analysis revealed that appreciable amounts of transcripts for GLO- and DEF-like gene products were present in immature male and female flower buds of wild-type asparagus, but were lacking in those of ‘GS#2’. Southern blot analysis of genomic DNA showed that there was no difference between ‘GS#2’ and wild-type ‘Gold Schatz’ when probed for DEF- and GLOlike genes.. These results demonstrate that a failure of expression of class-B genes was correlated with aberrant morphology in the floral organs of ‘GS#2’. The stamens of flowers in wild-type female asparagus ceased to grow; however, the floral organs of ‘GS#2’ continued to develop without showing abortion.Thus, the line ‘GS#2’ may provide novel material for further examination of the expression of floral organ identity genes in dioecious plants that have been implicated in the sex-specific abortion of floral organs.

Transient accumulation of .ALPHA.-ketol linolenic acid (KODA) in immature flower buds of some ornamental plants

9-Hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (KODA) induces flowers in Lemna paucicostata after reacting with catecholamine (Yokoyama et al. 2000; Yamaguchi et al. 2001), and changes in endogenous KODA levels during the flower-inductive phase in short-day-induced cotyledons are closely related to flower induction (Suzuki et al. 2003). Here, we examined the change of KODA level after the flower induction period. KODA showed a transient increase in immature flower buds in all the plants we examined, i.e., Pharbitis nil, Dianthus caryophyllus L., Dendranthema grandiflorum Kitam. and Eustoma russellianum Griseb. No such increase of KODA was seen in foliar buds of P. nil. These phenomena indicate that KODA may be involved in flower formation, as well as flower induction.

Review of Food and Medicinal Uses of Capparis L. Subgenus Capparis (Capparidaceae)

Capers of commerce are immature flower buds which have been pickled either in vinegar or preserved in granular salt. Semi-mature fruits and young shoots with small leaves may also be pickled for use as a condiment. The use of capers can be traced to the prehistory. Although Capparis spinosa from the western Mediterranean is the most widely used species, the subgenus comprises 23 species and subspecies occupying large territories from the Atlantic coasts to the Pacific in the Old World. We have recorded medicinal and food uses for 19 species.

Regeneration of banana (Musa spp. AAB cv. Dwarf Brazilian) via secondary somatic embryogenesis

A method has been developed for the regeneration of the banana cultivar Dwarf Brazilian (Musa spp. AAB group). Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l α-naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline. Subsequent transfer to MS supplemented with 10% coconut water produced rapidly proliferating embryogenic callus that developed into secondary somatic embryos (SE2); these were subcultured on half-strength MS supplemented with 5 mg/l 6-benzylaminopurine (BA). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l BA for development, and mature SE2 were isolated and cultured on hormone-free MS for germination and development into plantlets. Approximately 90% of the SE2 germinated and developed into plantlets, and these were subcultured onto MS medium plus 0.1% activated charcoal, 1 mg/l BA and 1 mg/l IAA where complete plantlets developed. Morphologically normal banana plants developed from all of the regenerated plantlets, the first of which were produced within 6 months of culture initiation.

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus.

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.

Factors affecting flower infection and disease severity of lowbush blueberry by Botrytis cinerea

Infection of lowbush blueberry (Vaccinium angustifolium Ait. and V. myrtilloides Michx.) flowers by Botrytis cinerea Pers.:Fr. and subsequent disease was determined for differing stages of flower bud development, temperature, and duration of postinoculation wet periods. Conidium germination of B. cinerea at 20°C was poor on immature flower buds when the corolla was beginning to protrude from the calyx (floral stage F4) and when the corolla was half developed (F5), but better when flowers were at the pink bud prebloom stage (F6) or fully open (F7). Infection incidence followed a similar trend; there was none on the F4 flowers, slightly more on F5 flowers, and an increase to over 45 and 98% on F6 and F7 flowers, respectively. Immature green berries were resistant to infection, but nonpollinated ovaries from which corollas had abscised were susceptible to infection. At 4, 8, 12, 16, and 20°C, low levels of infection occurred on F7 flowers after 24, 13, 10, 8, or 6 h of postinoculation wetness, respectively. The infection incidence increased progressively with temperature and wetness duration. Lesions spread from the corolla to the peduncle of flower clusters over a 96-h wet period at 16, 20, and 24°C, but the disease failed to spread beyond the corolla at 4, 8, and 12°C. When flower clusters were inoculated with 105 or 106 conidia/mL at 20°C and incubated over a 96-h wet period, lesions spread from the corolla to the peduncle, but when the inoculum concentration was 103 or 104 conidia/mL, they did not extend beyond the corolla. These results could help to reduce fungicide applications when disease management programs are based on monitored weather conditions.

Partial male sterility in transgenic tobacco carrying an antisense gene for alternative oxidase under the control of a tapetum-specific promoter

The alternative oxidase of plant mitochondria is the terminal oxidase of the cyanide-insensitive respiratory pathway and is encoded by a nuclear gene. A 1 kb genomic fragment including exon 3 of the alternative oxidase was amplified by PCR from the genome of Arabidopsis thaliana. This fragment was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. The pollen viability in three transgenic plants ranged from 2% to 60%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. Immunolocalization of alternative oxidase protein in the immature flower bud section indicated that expression of alternative oxidase protein in tapetum of the transgenic plant was much lower than that of the non-transformant. The histological observation and protein gel-blot analysis showed that the development of pollen grains in the transgenic plant did not progress after the degradation of the tapetum, and the amount of alternative oxidase in pollen grains of the transgenic plant became lower than that of the non-transformant. These results suggested that the alternative oxidase activity in the tapetum has a significant effect on the pollen development.