A field study on using soybean waste-derived superabsorbent hydrogel to enhance growth of vegetables.

Food security is critical and has become a global concern with many of our basic food crops growing in areas with high drought risk. To improve soil water holding capacity, hydrogels are a promising solution. However, the current ones are mostly derived from petroleum products and are environmental unsustainable. In this study, the main objective is to determine if bio-based hydrogel can help in the growth of leafy vegetables while minimizing water use under field conditions. To achieve this, we developed an okara-derived hydrogel (Ok-PAA; OP) from by-products of bean curd and soybean milk production. We incorporated OP into soil and assessed the growth performance of leafy vegetables. We observed that vegetables grown with 0.2% (w/v) OP in soil with a watering frequency of 7 times per week resulted in >60 % and 35 % yield increase for the common Asian leafy vegetables, choy sum (CS) and pak choi (PC), respectively, as compared to without hydrogel supplementation. Both vegetables produced larger leaf areas (20-40 % increment) in the presence of the hydrogel as compared to those without. In addition, with OP amendment, the irrigation water use efficiency improved >60 % and 30 % for CS and PC, respectively. It is estimated that with the use of the hydrogel, a reduction in watering frequency from 21 times to 7 times per week could be achieved, and based on a per hectare estimation, this would result in 196,000 L of water saving per crop cycle. Statistical analysis and modelling further confirmed vegetables grown with 0.2 % (w/v) OP and with a watering frequency of 7 times per week showed the best growth performance and water use efficiency. Such a waste-to-resource approach offers a plant-based soil supplement for crop growers, contributes to waste valorization, and enhances the growth of plants especially under water-limited conditions.

Chemical Modification of Biomass Okara Using Poly(acrylic acid) through Free Radical Graft Polymerization.

Okara (Ok) or soybean residue is produced as a byproduct from the soybean milk and soybean curd industries world wide, most of which is disposed or burned as waste. It is important to explore the possibilities to convert okara to useful materials, because okara is a naturally renewable bioresource. Here, we report the chemical modification of okara by grafting poly(acrylic acid) (PAA) onto the backbones of okara in water medium and the characterization of the Ok-PAA graft copolymers. It was found that the received okara mainly contained insoluble contents in water. The insoluble okara component Ok(Ins) was suspended in water and activated with ammonium persulfate as an initiator, followed by grafting PAA through a free radical polymerization. After the graft polymerization, the product (Ok-PAA) was separated into precipitate and supernatant, which were dried to give Ok-PAA(pre) and Ok-PAA(sup), respectively. It was found that PAA was grafted on Ok backbones and co-precipitated with the insoluble Ok. In addition, Ok-PAA(sup) was found to be translucent as a result of the grafting of PAA. Further, the successful grafting of PAA onto okara backbones was proven by Fourier transform infrared, thermogravimetric analysis, and microscopic measurements. Ok-PAA(sup) dispersed in water formed nanoparticles with an average diameter of 420 nm, while Ok-PAA(pre) was clustered coarse particles in water. The rheological data including the storage modulus, loss modulus, and viscosity indicated that the Ok-PAA product was a viscoelastic gel-like material with potential for agricultural and environmental applications.

Expression of AoNHX1 increases salt tolerance of rice and Arabidopsis, and bHLH transcription factors regulate AtNHX1 and AtNHX6 in Arabidopsis.

KEY MESSAGE: Expression of AoNHX1 from the mangrove Avicennia increases salt tolerance of rice and Arabidopsis, and specific bHLH transcription factors regulate AtNHX1 and AtNHX6 in Arabidopsis to mediate the salinity response. Improving crop plants to better tolerate soil salinity is a challenging task. Mangrove trees such as Avicennia officinalis have special adaptations to thrive in high salt conditions, which include subcellular compartmentalization of ions facilitated by specialized ion transporters. We identified and characterized two genes encoding Na+/H+ exchangers AoNHX1 and AoNHX6 from Avicennia. AoNHX1 was present in the tonoplast, while, AoNHX6 was localized to the ER and Golgi. Both NHXs were induced by NaCl treatment, with AoNHX1 showing high expression levels in the leaves and AoNHX6 in the seedling roots. Yeast deletion mutants (ena1-5Δ nha1Δ nhx1Δ and ena1-5Δ nha1Δ vnx1Δ) complemented with AoNHX1 and AoNHX6 showed increased tolerance to both NaCl and KCl. Expression of AoNHX1 and AoNHX6 in the corresponding Arabidopsis mutants conferred enhanced NaCl tolerance. The underlying molecular regulatory mechanism was investigated using AtNHX1 and AtNHX6 in Arabidopsis. We identified two basic helix-loop-helix (bHLH) transcription factors AtMYC2 and AtbHLH122 as the ABA-mediated upstream regulators of AtNHX1 and AtNHX6 by chromatin immunoprecipitation. Furthermore, expression of AtNHX1 and AtNHX6 transcripts was reduced in the atmyc2 and atbhlh122 mutants. Lastly, transgenic rice seedlings harboring pUBI::AoNHX1 showed enhanced salt tolerance, suggesting that this gene can be exploited for developing salt-tolerant crops.

Transcriptomics analysis of salt stress tolerance in the roots of the mangrove Avicennia officinalis.

Salinity affects growth and development of plants, but mangroves exhibit exceptional salt tolerance. With direct exposure to salinity, mangrove roots possess specific adaptations to tolerate salt stress. Therefore, studying the early effects of salt on mangrove roots can help us better understand the tolerance mechanisms. Using two-month-old greenhouse-grown seedlings of the mangrove tree Avicennia officinalis subjected to NaCl treatment, we profiled gene expression changes in the roots by RNA-sequencing. Of the 6547 genes that were differentially regulated in response to salt treatment, 1404 and 5213 genes were significantly up- and down-regulated, respectively. By comparative genomics, 93 key salt tolerance-related genes were identified of which 47 were up-regulated. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in ethylene and auxin signaling were up-regulated while those involved in ABA signaling were down-regulated. These results imply that ABA-independent signaling pathways also play a major role in salt tolerance of A. officinalis. Further, ethylene response factors (ERFs) were abundantly expressed upon salt treatment and the Arabidopsis mutant aterf115, a homolog of AoERF114 is characterized. Overall, our results would help in understanding the possible molecular mechanism underlying salt tolerance in plants.

Data in support of the proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis.

The data provides information in support of the research article, Proteomics 2014, 14, 2545-2557 [1]. Raw data is available from the ProteomeXchange Consortium via the PRIDE partnerRepository [2] with the dataset identifier PXD000837. Plasma membrane and tonoplast proteins from the leaves of Avicennia officinalis were identified using gel electrophoresis (one and two dimensional) combined with LC-MS analysis. Based on GO annotation, identified proteins were predicted to be involved in various biological processes.

Proteomic Characterisation of the Salt Gland-Enriched Tissues of the Mangrove Tree Species Avicennia officinalis.

Plant salt glands are nature's desalination devices that harbour potentially useful information pertaining to salt and water transport during secretion. As part of the program toward deciphering secretion mechanisms in salt glands, we used shotgun proteomics to compare the protein profiles of salt gland-enriched (isolated epidermal peels) and salt gland-deprived (mesophyll) tissues of the mangrove species Avicennia officinalis. The purpose of the work is to identify proteins that are present in the salt gland-enriched tissues. An average of 2189 and 977 proteins were identified from the epidermal peel and mesophyll tissues, respectively. Among these, 2188 proteins were identified in salt gland-enriched tissues and a total of 1032 selected proteins were categorized by Gene Ontology (GO) analysis. This paper reports for the first time the proteomic analysis of salt gland-enriched tissues of a mangrove tree species. Candidate proteins that may play a role in the desalination process of the mangrove salt glands and their potential localization were identified. Information obtained from this study paves the way for future proteomic research aiming at elucidating the molecular mechanism underlying secretion in plant salt glands. The data have been deposited to the ProteomeXchange with identifier PXD000771.

Proteome profile of salt gland-rich epidermis extracted from a salt-tolerant tree species.

Preparation of proteins from salt-gland-rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland-rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland-rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D-LC-MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol-based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.

Identification of salt gland-associated genes and characterization of a dehydrin from the salt secretor mangrove Avicennia officinalis.

BACKGROUND: Salt stress is a major challenge for growth and development of plants. The mangrove tree Avicennia officinalis has evolved salt tolerance mechanisms such as salt secretion through specialized glands on its leaves. Although a number of structural studies on salt glands have been done, the molecular mechanism of salt secretion is not clearly understood. Also, studies to identify salt gland-specific genes in mangroves have been scarce. RESULTS: By subtractive hybridization (SH) of cDNA from salt gland-rich cell layers (tester) with mesophyll tissues as the driver, several Expressed Sequence Tags (ESTs) were identified. The major classes of ESTs identified include those known to be involved in regulating metabolic processes (37%), stress response (17%), transcription (17%), signal transduction (17%) and transport functions (12%). A visual interactive map generated based on predicted functional gene interactions of the identified ESTs suggested altered activities of hydrolase, transmembrane transport and kinases. Quantitative Real-Time PCR (qRT-PCR) was carried out to validate the expression specificity of the ESTs identified by SH. A Dehydrin gene was chosen for further experimental analysis, because it is significantly highly expressed in salt gland cells, and dehydrins are known to be involved in stress remediation in other plants. Full-length Avicennia officinalis Dehydrin1 (AoDHN1) cDNA was obtained by Rapid Amplification of cDNA Ends. Phylogenetic analysis and further characterization of this gene suggested that AoDHN1 belongs to group II Late Embryogenesis Abundant proteins. qRT-PCR analysis of Avicennia showed up-regulation of AoDHN1 in response to salt and drought treatments. Furthermore, some functional insights were obtained by growing E. coli cells expressing AoDHN1. Growth of E. coli cells expressing AoDHN1 was significantly higher than that of the control cells without AoDHN1 under salinity and drought stresses, suggesting that the mangrove dehydrin protein helps to mitigate the abiotic stresses. CONCLUSIONS: Thirty-four ESTs were identified to be enriched in salt gland-rich tissues of A. officinalis leaves. qRT-PCR analysis showed that 10 of these were specifically enriched in the salt gland-rich tissues. Our data suggest that one of the selected genes, namely, AoDHN1 plays an important role to mitigate salt and drought stress responses.

Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis.

In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 (http://proteomecentral.proteomexchange.org/dataset/PXD000837).

Role of root hydrophobic barriers in salt exclusion of a mangrove plant Avicennia officinalis.

Salt exclusion at the roots and salt secretion in the leaves were examined in a mangrove, Avicennia officinalis. The non-secretor mangrove Bruguiera cylindrica was used for comparative study of hydrophobic barrier formation in the roots. Bypass flow was reduced when seedlings were previously treated with high salt concentration. A biseriate exodermis was detected in the salt-treated roots, along with an enhanced deposition of hydrophobic barriers in the endodermis. These barriers reduced Na(+) loading into the xylem, accounting for a 90-95% salt exclusion in A. officinalis. Prominent barriers were found in the roots of B. cylindrica even in the absence of salt treatment. A cytochrome P450 gene that may regulate suberin biosynthesis was up-regulated within hours of salt treatment in A. officinalis roots and leaves, corresponding with increased suberin deposition. X-ray microanalysis showed preferential deposition of Na(+) and Cl(-) in the root cortex compared with the stele, suggesting that the endodermis is the primary site of salt exclusion. Enhanced salt secretion and increased suberin deposition surrounding the salt glands were seen in the leaves with salt treatment. Overall, these data show that the deposition of apoplastic barriers increases resistance to bypass flow leading to efficient salt exclusion at the roots in mangroves.

Dynamic secretion changes in the salt glands of the mangrove tree species Avicennia officinalis in response to a changing saline environment.

The specialized salt glands on the epidermis of halophytic plants secrete excess salts from tissues by a mechanism that is poorly understood. We examined the salt glands as putative salt and water bi-regulatory units that can respond swiftly to altering environmental cues. The tropical mangrove tree species (Avicennia officinalis) is able to grow under fluctuating salinities (0.7-50.0 dS m(-1)) at intertidal zones, and its salt glands offer an excellent platform to investigate their dynamic responses under rapidly changing salinities. Utilizing a novel epidermal peel system, secretion profiles of hundreds of individual salt glands examined revealed that these glands could secrete when exposed to varying salinities. Notably, rhythmic fluctuations observed in secretion rates were reversibly inhibited by water channel (aquaporin) blocker, and two aquaporin genes (PIP and TIP) preferentially expressed in the salt gland cells were rapidly induced in response to increasing salt concentration. We propose that aquaporins are involved and contribute to the re-absorption of water during salt removal in Avicennia officinalis salt glands. This constitutes an adaptive feature that contributes to salt balance of trees growing in saline environments where freshwater availability is limited.

A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies.

BACKGROUND: Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy. RESULTS: Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®). CONCLUSIONS: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels.

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.

Early in vitro flowering and seed production in culture in Dendrobium Chao Praya Smile (Orchidaceae).

Plantlets of Dendrobium Chao Praya Smile maintained in vitro were induced to flower, which produced viable seeds within about 11 months. A two-layer (Gelrite-solidified layer topped with a layer of liquid medium of the same volume and composition) culture system containing benzyladenine (BA) at 11.1 muM induced the highest percent of flowering (45%) in plantlets within 6 months from germination. The percentage of inflorescence induction was increased to 72% by pre-selecting morphologically normal seedlings prior to two-layer culture. Plantlets in culture produced both complete (developmentally normal but smaller than flowers of field grown plants) and incomplete flowers. Pollen and female reproductive organs of in vitro-developed complete flowers were morphologically and anatomically similar to flowers of field grown plants. In addition, 65% of the pollen grains derived from in vitro-developed flower were tetrad suggesting that regular meiosis occurred during microsporogenesis. The percentage of germination of pollen grains derived from in vitro-developed flowers and flowers of field grown plants, incubated on modified Knops' medium for 8 days, were 18.2 and 52.8%, respectively. Despite a lower percentage of germination of the pollen grains derived from in vitro-developed flowers, flowers induced in culture could be self-pollinated and developed seedpods with viable seeds. Nearly 90% of these seeds developed into protocorms on germination in vitro. These seedlings were grown in culture and induced to flower in vitro again using the same procedure.

High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae).

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.

Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein.

Complementation of movement and coat proteins of the orchid-infecting potexvirus Cymbidium mosaic virus (CymMV) and tobamovirus Odontoglossum ringspot virus (ORSV) was investigated. Nicotiana benthamiana, which is susceptible to both CymMV and ORSV, was used as a model system. Four transgenic lines, each harbouring one of the movement protein (MP) or coat protein (CP) genes of CymMV or ORSV, were constructed. The MP of CymMV consists of three overlapping open reading frames, together called the triple-gene block (TGB). CymMV and ORSV mutants, each carrying an inactivated MP or CP, were generated from the respective biologically active full-length cDNA clones. Complementation was studied by infecting transgenic plants with in vitro transcripts generated from these mutants. The cell-to-cell movement of a movement-deficient CymMV was restored in transgenic plants carrying the ORSV MP transgene. Similarly, CymMV TGB1 transgenic plants were able to rescue the cell-to-cell movement of a movement-deficient ORSV mutant. ORSV CP transgenic plants supported systemic movement of a CymMV CP-deficient mutant. However, in these plants, neither encapsidation of CymMV RNA with ORSV CP nor CymMV CP expression was detected. Long-distance movement of an ORSV CP-deficient mutant was not supported by CymMV CP. The complementation of MPs and CPs of CymMV and ORSV facilitates movement of these viruses in plants, except for long-distance movement of ORSV RNA by CymMV CP.

Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development.

BACKGROUND: Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development. RESULTS: The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C (C, DNA content of the unreplicated haploid chromosome complement) peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C. CONCLUSIONS: Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed.

Induced mutations in cassava using somatic embryos and the identification of mutant plants with altered starch yield and composition.

A cyclic somatic embryogenic system was used to induce mutations in cassava variety PRC 60a in vitro. Globular-stage somatic embryos were selected as suitable experimental materials, and 50 Gy of gamma-rays was determined to be the optimal dose for inducing mutations. During subsequent field trials, more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, we used this approach to induce genetic variability for obtaining novel cassava cultivars. Among the different mutant lines obtained, lines S14 and S15 showed large morphological variations. In 10-month-old S14 and S15 mutant lines, storage root yield was reduced 17-fold and 60-fold, respectively, compared to wild-type plants, while the storage roots of S15 mutant plants also exhibited an almost 50% decrease in starch content and a significant reduction (30%) in amylose content. These two features were observed throughout the different developmental stages of the storage roots in S15 plants.

Endopolyploidy in Vanda Miss Joaquim (Orchidaceae).

• Occurrence of endopolyploidy in somatic tissues of the hybrid orchid Vanda Miss Joaquim (Vanda hookeriana × Vanda teres) was investigated with respect to tissue type and developmental stage. Effects of naphthaleneacetic acid (NAA) and gibberellic acid (GA3 ) on endopolyploidy during embryo development were also studied. • For the study of endopolyploidy, flow cytometric analysis was employed to determine nuclear DNA content of cells of somatic tissues. • Multiploid cells were observed in leaves, roots and column, but not in shoot apex, stem, perianth and pedicel. Furthermore, differential distribution of multiploid cells was found among different parts of leaves and roots. The degree of endopolyploidy in embryos increased with development. NAA was shown to induce endoreduplication in germinating embryos to a much larger extent than GA3 . • The pattern of endopolyploidy was characteristic of tissue type and developmental stage. The implications of endopolyploidy during differentiation and development, as well as the relevance of endopolyploidy to somaclonal variation, are discussed.

Differential expression and characterization of three metallothionein-like genes in Cavendish banana (Musa acuminata).

Metallothioneins (MTs) are cysteine-rich polypeptides that are involved in metal detoxification and homeostasis in both prokaryotes and eukaryotes. In this study, we report the isolation and characterization of three members (MT2A, MT2B and MT3) of the MT-like gene family from ripening banana fruit and their differential expression in various banana organs and during fruit development and ripening. All members of the MT-like gene encode small cysteine-rich polypeptides of 65-79 amino acid residues. MT2A shared a high sequence similarity (54-77%) with several type-2 MTs in plants, while MT3 was highly homologous (51-61%) with type-3 MTs. The three members expressed differentially in various organs but transcripts were generally more abundant in reproductive than vegetative organs. During fruit development, the MT2A transcript was barely detectable in ovary but increased to a high level in young fruit at 20 days after shooting (DAS) and declined gradually thereafter as fruit developed. In contrast, both MT2B and MT3 expressed poorly in young fruits (20-60 DAS) and transcripts were detected only in fruits at later stages of development. As ripening progressed, expression of MT2A decreased but that of MT3 increased. Expression of MT members during ripening appeared to be differentially regulated by ethylene, whose levels were low in FG and TY fruit but surged climacteristically in MG and declined sharply as ripening advanced further. Exogenous application of ethylene at 5 ppm or higher concentrations down-regulated MT2A expression and the inhibitory effect of ethylene could be partially suppressed by the presence of norbornadiene, an inhibitor of ethylene action. Ethylene had no effect on transcript accumulation of MT2B and MT3. However, MT3 expression was greatly enhanced in response to metals such as CdSO4, CuSO4 and ZnSO4. These results suggest that increased MT3 expression may be associated with excess metal ions present in ripening fruit tissues. This study also provided evidence, for the first time, that ethylene and metals play a regulatory role in expression of MT-like genes in banana.