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In vitro xylem induction system is a basic tool in physiological, biochemical, and molecular studies of secondary cell wall formation, lignin biosynthesis and deposition associated with tracheary element formation. Eucalyptus bosistoana is a Class 1 durable hardwood tree species, selected by the New Zealand Dryland Forest Initiative for good quality wood and high adaptability to the NZ growing conditions. Xylogenic E. bosistoana callus culture was established and up to 40% of the callus cells were xylem-like cells (XLCs) which may have differentiated from small, cytoplasmically dense or compact dividing, and exhibited increased lignin contents during culture. The eucalyptus XLCs showed diverse sizes, patterns of secondary cell wall thickenings similar to the xylem cells in the young shoots and organized development including cell–cell connections of the XLCs to form xylem strands. This is the first report of the organized development of XLCs in E. bosistoana callus culture.

Pollen of butterfly pea (Clitoria ternatea L.) could germinate in vitro with emergence of 1, 2 and 3 tubes from individual pollen grain on germination medium (modified after Mercado et al. Sci Hortic 57:273–281, 1994). The highest percentage of pollen germination was observed on medium supplemented with 20% sucrose while addition of osmotica such as polyethylene glycol or mannitol did not have positive effect on butterfly pea pollen germination. Starch utilization, through conversion to simpler sugars including sucrose, may be the cause for pollen tube elongation. The optimum pH for maximum germination (42%) was recorded to be 5.7. Addition of various plant growth regulators including brassin-like substance and GA3 singly into medium supplemented with 20% sucrose at pH 5.7 did not result in germination percentage higher than that on the medium supplemented with only 20% sucrose. The germination percentage was increased to 71% and the pollen tube length was the greatest when butterfly pea pollen was cultured on medium supplemented with 20% sucrose, 0.25 mg L− 1 brassin-like substance and 1 mg L− 1 GA3 at pH 5.7. This suggests that brassin-like substance and GA3 may play an important synergistic role on pollen germination of butterfly pea in vitro.

Exposure to ultraviolet B radiation (UV-B) stress can have serious effects on the growth and development of plants. Germin-like proteins (GLPs) may be involved in different abiotic and biotic stress responses in different plants, but little is known about the role of GLPs in UV-B stress response and acclimation in plants. In the present study, knockout of GLP 8–14 (OsGLP1) using the CRISPR/Cas9 system resulted in mutant rice (Oryza sativa L.) plants (herein called glp1) that exhibited UV-B-dependent formation of lesion mimic in leaves. Moreover, glp1 grown under solar radiation (including UV-B) showed decreased plant height and increased leaf angle, but we observed no significant differences in phenotypes between wild-type (WT) plants and glp1 grown under artificial light lacking UV-B. Fv/Fm, Y (II) and the expression of many genes, based on RNA-seq analysis, related to photosynthesis were also only reduced in glp1, but not in WT, after transfer from a growth cabinet illuminated with artificial white light lacking UV-B to growth under natural sunlight. The genes-associated with flavonoid metabolism as well as UV resistance locus 8 (OsUVR8), phytochrome interacting factor-like 15-like (OsPIF3), pyridoxal 5′-phosphate synthase subunit PDX1.2 (OsPDX1.2), deoxyribodipyrimidine photolyase (OsPHR), and deoxyribodipyrimidine photolyase family protein-like (OsPHRL) exhibited lower expression levels, while higher expression levels of mitogen-activated protein kinase 5-like (OsMPK3), mitogen-activated protein kinase 13-like (OsMPK13), and transcription factor MYB4-like (OsMYB4) were observed in glp1 than in WT after transfer from a growth cabinet illuminated with artificial white light to growth under natural sunlight. Therefore, mutations in OsGLP1 resulted in rice plants more sensitive to UV-B and reduced expression of some genes for UV-B protection, suggesting that OsGLP1 is involved in acclimation to UV-B radiation.

Most commercial plant tissue culture laboratories still use conventional micropropagation systems, requiring air-tight vessels and gelled medium supplemented with sugar. There is, however, a more efficient and economic system available, called “photoautotrophic micropropagation system” (PAM). Application of sugar-free medium and ventilated vessels are the key features of this system, which significantly improves plant physiological processes including photosynthesis and transpiration. The genetic basis for this has become more clear with a recent transcriptomic analysis of potato plantlets cultured on sugar-free medium showing the upregulation of more than 2000 genes, many of which are associated with photosynthesis efficiency and leaf cell development. The cultured plants from PAM exhibit improved vigour and health enabling a faster and smoother acclimatisation stage. Moreover, PAM is amenable to automation and scaling up by using large culture vessels/rooms. Here, we reviewed the recent studies on PAM and the issues/barriers to its commercialisation have also been discussed. Optimal culture conditions as well as different types of culture vessels are also explained.

There are over hundred species in the genus Erythrina which are of practical importance in multiple ways. The requirements for Erythrina plantlet regeneration in vitro were rarely studied. Surface-sterilized seeds of purple coral tree (Erythrina fusca Lour.) exhibited a high germination rate (80%) in vitro. The cotyledonary nodes from the one-month-old purple coral tree seedlings grown in vitro were used as explants. About 70% of the cotyledonary nodal explants exhibited shoot regeneration when the explants were cultured vertically with the cut ends inserted into basal Murashige and Skoog (MS) medium compared to only 10% of those placed lying horizontally on the surface of the medium. Supplementation of the basal MS medium with 1 mg L−1 of BAP, 1 mg L−1 kinetin and 1 mg L−1 TDZ resulted in the greatest shoot elongation and the highest number of shoots developed from the cotyledonary nodal explants. The regenerated shoots were able to root (100%) on basal MS medium while supplementation with 2 mg L−1 of either NAA or IBA was inhibitory to root elongation. This is the first study on plantlet regeneration of purple coral tree which seems to have more complex requirements for shoot regeneration in vitro than those reported for the other three Erythrina species. Since only 30% of the rooted in vitro shoots survived during acclimatization following a previously published protocol for acclimatization of another plant, it is recommended that future studies are to investigate the requirements of higher plantlet survival for the rooted in vitro shoots of purple coral tree.

Seed priming with water (hydropriming or HP) has been shown to be beneficial for seed germination and plant growth. However, there is little information on the effects of seed priming with amino acids and casein hydrolysate (CH) compared with HP, particularly in relation to early post-germinative seedling growth under salinity stress. In this study, Italian ryegrass seeds (Lolium multiflorum L.) were primed with 1 mM of each of the 20 protein amino acids and CH (200 mg l−1) before they were germinated in 0, 60 and 90 mM NaCl in Petri dishes for 4 d in darkness. Germination percentage (GP), radicle length (RL) and peroxidase (POD) activity in the root of 4-d-old Italian ryegrass seedlings were investigated. Generally, when the seeds were germinated in 0, 60 and 90 mM NaCl, there was no significant difference in GP of seeds among various priming treatments, except that a higher GP was observed in seeds of HP treatment compared with the non-primed seeds when incubated in 60 mM NaCl. When incubated in 60 and 90 mM NaCl, seedlings from seeds primed with L-methionine or CH exhibited greater RL (greater protection against salinity stress) and higher root POD activity than those from non-primed and hydro-primed seeds. Under salinity stress, there were higher levels of malondialdehyde (MDA) in the root of 4-d-old Italian ryegrass seedlings, a marker of oxidative stress, but seed priming with CH was effective in reducing the salinity-triggered increase in MDA content. These results suggest that priming with L-methionine or CH would be better than HP for the protection of seedling root growth under salinity stress and might be associated with enhanced antioxidative defence against salinity-induced oxidative stress.

Eucalyptus bosistoana is an important durable hardwood species selected by New Zealand Dryland Forest Initiative (NZDFI) for their adaptability to diverse environments and good quality wood. There is no report on in vitro methods for callus induction and plantlet micropropagation of this species, although these methods could assist further genetic improvement of plants. In this study, different combinations of α-naphthalene acetic acid (NAA) and benzyl adenine (BA) at concentrations ranging from 0.5 to 2 mg L−1 were investigated for callus induction, shoot regeneration, shoot elongation and root formation. High frequency of callus induction was obtained when cotyledon explants were cultured on MS (Murashige and Skoog medium 1962) with 1.0 mg L−1 NAA and 1.5 mg L−1 BA. For shoot regeneration from callus, MS medium with 0.5 mg L−1 NAA and 1.5 mg L−1 BA was an effective medium. Shoot elongation was observed on MS medium supplemented with 0.5 mg L−1 NAA, 1.0 mg L−1 BA and 1.0 mg L−1 GA3. High frequency of adventitious root formation in micro-cuttings of E. bosistoana was observed on half-strength MS medium with 0.5 to 1.0 mg L−1 NAA. Finally, pre-acclimatization of the micropropagated plantlets led to 100% survival under glasshouse conditions.

In this study, different concentrations of 6-benzyladenine (BA) on in vitro shoot and inflorescence inductions of P. lanceolata were investigated. The in vivo and in vitro floral characteristics of this plant were also compared. Nodal explants of P. lanceolata were cultured vertically with the cut ends inserted into semi-solid Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1, 2, 4, and 8 mg L–1 BA. The results showed that the explants formed the highest numbers of shoots even when cultured in MS basal medium without any addition of BA, while the shoots formed in the explants cultured in MS medium supplemented with 1 mg L–1 BA were the longest. No inflorescence was found in the shoots cultured in MS medium supplemented with 8 mg L–1 BA, while the highest percentage of inflorescence induction was found in the shoots cultured in the medium supplemented with 0.5 mg L–1 BA. The apperances of in vivo and in vitro flowers of P. lanceolata were the same in many aspects except that the number of flower/inflorescence formed was different. In addition, water accumulation was observed only inside the in vitro flowers. Water deposit in the long tubular structure of P. lanceolata flower could cause anther injury, suggesting that flowers developed in vitro may not always produce pollen.

Pseudomonas syringae pv. actinidiae (Psa) causes the bacterial canker disease on kiwifruit vines. The disease outbreak has been reported in several countries worldwide, including New Zealand. Here,...

Immature sponge gourd fruit is consumed as a vegetable with a limited shelf life. Although cold storage is a simple and powerful tool for maintaining postharvest fruit quality, storage at a low temperature may not be appropriate for vegetables as some chilling injury (CI) of the immature sponge gourd fruit may occur. Therefore, this research aimed to elucidate the relationship between CI, oxidative stress, and the antioxidative defense mechanisms in the exocarp and mesocarp of immature sponge gourd fruit. After storage at 5°C for 6 days, visual CI symptoms, including browning and surface pitting, were found in the peel (exocarp) but not in the mesocarp. There were, however, more dead cells (stained by Evans blue) in the mesocarp of the fruit stored at 5°C. There was a more considerable increase in the electrolyte leakage rate in both fruit tissues held at 5°C than 25°C. The CI was correlated with malondialdehyde (MDA) levels in the tissues. The MDA of fruit exocarp at 5°C was 1.6 fold higher than that at 25°C on day 6, while the lipoxygenase (LOX) activity in mesocarp was 50% higher in fruit stored at a lower temperature. The action of ascorbate peroxidase (APX) was high in the exocarp of the fruit stored at 5°C, but there appeared to be a continuous depletion of the co-substrate or ascorbic acid. In conclusion, the CI in the exocarp was mainly associated with a high level of reactive oxygen species (ROS). In contrast, the CI in the mesocarp appeared to be primarily associated with increased lipid peroxidation by the elevated LOX activity under cold stress compared to storage at 25°C.

There is a lot of market for squalene (C30H50) for industrial and therapeutic applications such as oil, biofuel, antioxidant, antimicrobial and anticancer agents. Squalene was used to be derived from liver oil of sharks and whales, but a sustainable alternative source such as plants is needed. An ayurvedic plant, Nilgirianthus ciliatus, was found to contain a limited amount of squalene. This study hypothesizes that squalene content in callus culture of N. ciliatus could produce an increased level of squalene. For efficient callus induction, various parameters such as explants, plant growth regulators, light and dark conditions were optimized. Leaf produced the highest frequency of callus induction (89.5%) with 2.3 fold enhanced squalene production on MS medium containing 4.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.4 mg l−1 benzyladenine. The wild and in vitro tissues were evaluated for metabolic profiles using GC–MS and antioxidant properties. The leaf callus exhibited increased level of total phenolic, flavonoid and squalene contents which confers the highest level of antioxidant (IC50 = 88.8 μg ml−1) activity compared to wild explants. The GC–MS profiles of wild and callus tissues showed twelve compounds, in which squalene was 9.3% in wild and 21.4% in callus. Yield of squalene was found to be 70.9 μg g−1 (dry weight) DW and 146.3 μg g-1 DW for wild and callus tissues, respectively. Furthermore, FT-IR analysis also confirmed the presence and increased level of squalene in callus compared to that of wild. This callus could be an ideal source for mass squalene production without depleting natural sources and simultaneously fulfilling the pharmacological demands.

Ultraviolet-C (UV-C) radiation is well-known for its germicidal effect but its mutagenic potential in aiding new plant variety development has not been explored extensively. Persian violet (Exacum affine Balf.f. ex Regel) is a well-known decorative plant in Thailand. In this research, the effects of 0–4 h of UV-C irradiation on shoot proliferation and in vitro flowering of Persian violet plantlets derived from non-encapsulated and encapsulated microshoots were investigated. The number of multiple shoots was found to decrease as UV-C exposure time increased. Shoot-tip decapitation increased floral number in plantlets derived from both non-capsulated and encapsulated microshoots. The plantlets developed from non-encapsulated microshoots exposed to 4 h of UV-C irradiation exhibited tiny flower buds within 3 days from shoot-tip decapitation of the plantlets. However, the time for flower initiation was 35 days in control plantlets developed from non-encapsulated microshoots without irradiation. Another interesting novel finding is that 4 h of UV-C irradiation of the non-encapsulated microshoots resulted in the highest number of flowers (6.80 flowers) in the plantlets after 10 weeks compared to all the other treatments including the control that had only 3.20 flowers. This finding of Persian violet flowers with 7 petals has never been reported before. However, this UV-C treatment of the non-encapsulated microshoots diminished viability and in vitro germinability of pollen more than those in the treatment of encapsulated microshoots. Thus, UV-C irradiation of Persian violet microshoots affected multiple shoot proliferation and in vitro flowering. Encapsulation of the microshoots could counteract many of these effects of UV-C irradiation.

The effect of low inorganic nitrogen (N) supply on potato plantlets developed from nodal explants cultured in vitro on modified Murashige and Skoog media supplemented with 3% sucrose was investigated. Lowering the inorganic nitrogen supply from 60 mM to 1.88 mM, while keeping the ratio of ammonium and nitrate at 0.52, did not result in a significant decrease in the fresh and dry weights of the plantlets, the number of new leaves formed and shoot length. Moreover, the plantlets cultured on the medium supplemented with 3.75 and 7.5 mM inorganic N exhibited greater biomass and shoot lengths than those on the medium with 60 mM inorganic N. Lowering the inorganic N supply in the media resulted in increased chlorosis with accompanying decreases in the chlorophyll and carotenoid contents of the leaves, but faster development of lateral roots and increased phenolic content in the roots.

Urea, the most commonly used nitrogen fertiliser in New Zealand, can be quickly lost from the system via ammonia volatilisation or nitrate leaching following hydrolysis of urea by urease producing soil microorganisms (UPSMs). This study investigated UPSMs involved in urea degradation for upcoming research to reduce soil urease activity. Soils from under dairy pasture management across New Zealand, with a pasture species component of ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) and aged between 9 months to 60 years old, were collected, and UPSMs were isolated and identified using both polymerase chain reaction (PCR)-based molecular and conventional methods. The fungal genera belonged to diverse taxonomical groups including the phylum Ascomycota: class: Dothideomycetes, Eurotiomycetes, Leotiomycetes and Sordariomycetes, the phylum Basidiomycota: class: Tremellomycetes and the phylum Zygomycota: order: Mucorales, all of which have a role in urea degradation in soil. Pasture soil-resident urease producing bacteria belonged to the Gammaproteobacteria and Betaproteobacteria. Cupriavidus sp. and Mucor hiemalis showed strong urease activity when cultured on urease medium. This is the first report on the urease activity of the pasture soil inhabitants Pochonia bulbillosa, Mariannaea elegans and Gliomastix sp. This study was part of a larger study underway to investigate control of UPSMs in soil to improve the efficiency of urea utilisation.

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H2O2) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.

The response of the microshoot explants (1 mm long) from Persian violet ( Exacum affine Balf. f. ex Regel) to various in vitro manipulations was investigated. The best multiple shoot induction (about 100 shoots per explant) was observed after the microshoot explants were cultured on semi-solid Murashige and Skoog (MS) medium supplemented with 2.0 mg l −1 BAP for 6 weeks. Transfer of the new shoots (2 cm long) to MS medium containing 2.0 mg l −1 IBA for 6 weeks resulted in the best rooting response (18 roots per shoot). Subsequently, 82% of the rooted shoots (plantlets) were successfully hardened after ex-flasking and grown in soil. The shoots induced from microshoot explants were also found to form flowers under in vitro conditions on MS medium without addition of any plant growth regulator. Cutting off the shoot tip led to the development of more branches and in vitro flowers. A comparative study of the in vitro flowers and those formed in plants grown in soil ( in vivo flowers) showed that the floral structures were mostly similar, except that the ovary and style of the in vitro flowers were slightly smaller than the in vivo ones. The viability of the pollen grains from in vitro flowers seemed to be higher than the pollen of in vivo flowers, but the highest pollen germination response (about 33%, in darkness at room temperature for 24 h) was exhibited by the pollen grains from both sources on an in vitro germination medium containing 5% sucrose was not different.

Tuber cadmium (Cd) accumulation was evaluated in ten potato cultivars grown in the same field with an average soil Cd level of 0.06 mg kg−1 dry weight (DW). Analysis of tuber metal levels revealed a significant difference in mean Cd concentrations among different cultivars, which varied from 0.05 to 0.21 mg kg−1 DW. No cultivars exceeded the maximum permissible concentration (MPC) for Cd in potato. Correlation analysis showed a strong positive relationship between bioaccumulation of Cd and zinc (Zn), copper (Cu) or manganese (Mn). There was also a negative correlation between Cd and iron (Fe) accumulation which was significant only within three cultivars including Summer Delight, Russet Burbank and Agria. Summer Delight, with the lowest Cd and highest Fe content, was identified as a low Cd accumulating cultivar as well as a Fe-enriched tuber vegetable making it of significant interest. Laura, Yukon Gold and Purple Passion were identified as strong Cd accumulators. One of the implications is that growing these three potato cultivars in soils with Cd levels greater than 0.06 mg kg−1 DW might be of human health concern.

The efficient utilization, storage and buffering of iron (Fe) is necessary to prevent the development of iron deficiency or toxicity related stresses in plants. It is of practical interest to develop effective in vitro plant tissue culture schemes for generating chlorosis-tolerant and/or Fe-biofortified plants. The objectives of this study were to induce the formation of compact and friable calli from leaf explants of potato and to investigate adaptive responses to Fe nutrition using these in vitro cell culture-based systems. The severity of chlorosis of the callus cultures increased with the withdrawal of Fe supplementation from the growth medium and the culture duration. Ferric chelate reductase (FCR) activity was found to be higher in the compact compared to the friable potato callus cultures. Chlorophyll and carotenoids content in compact calli increased with an increase in Fe supply but in friable calli carotenoids content declined and the chlorophyll level was unaffected. Phenolic content in Fe-deficient compact calli was significantly higher than in friable ones but a reverse trend was observed as the culture period was extended. Peroxidase activity was reduced in both compact and friable calli cultured under Fe-deprived condition compared to those on Fe-sufficient medium. Friable and compact calli displayed differential morphological and biochemical responses to Fe deficiency. These calli also appeared to vary in their sensitivity to Fe-deficiency and the response to Fe nutrition seemed to be related to the culture duration.

Seed germination is a complex process. Upon imbibition, many factors including phytohormones (gibberellin and abscisic acid) and reactive oxygen and nitrogen species [hydrogen peroxide (H2O2) and nitric oxide (NO), respectively] are involved in a complicated web of interactions. While there are some impressive recent progresses made in our understanding of these interactions, it is also of great interest to investigate treatments that help seeds with difficulties to germinate under unfavourable conditions including abiotic stress factors such as chilling and heavy metals. In this chapter, an update and critical interpretations of some recent investigations into the relationships among H2O2, NO, catalase activity and gene expression in cold stratification, light signal and abiotic stress are provided.

In-vitro grafting involves grafting a micro-scion onto a micro-rootstock under aseptic conditions. Due to juvenility and health status of the source plants for microscion / microrootstock, in-vitro grafting possesses many advantages over traditional field / bench grafting methods. So far, in-vitro grafting has been used for producing virus-free plants as well as studying scion-rootstock interactions, long-distance signalling and virus-indexing. However, this technique has other potential applications, such as propagation of fruit trees and crop improvement, which has been neglected. This article reviews recent applied methodologies and their findings in the last 20 years, and discusses challenges and future direction of in-vitro grafting.

The size of the staminodes and ring stamen of Couroupita guianensis (cannonball tree) grown in the Nonthaburi province, Thailand as well as some characteristics of their pollen were investigated. The staminodes were clearly larger than the ring stamens. This finding is at variance with other previous studies. Viability staining showed that almost all the cannonball tree pollen from hood staminode were not viable but about 85% of the ring stamen pollen were viable. When both types of cannonball tree pollen were cultured on modified Mercado medium, hood staminode pollen did not germinate whereas the sucrose concentrations in the medium had a promotive effect on germination of ring stamen pollen. About 65% of the ring stamen pollen germinated on the medium supplemented with 20% sucrose. These pollen studies were in agreement with other similar studies on cannonball trees, suggesting that the relative sizes of the male reproductive organs of cannonball trees may not be related to fertility of their pollen.

Natural variation in cadmium (Cd) uptake and accumulation has been found between different cultivars of a crop plant as well as among different plant species although Cd uptake can be influenced by environmental factors, particularly soil properties. This knowledge can be of practical importance for agricultural industry and plant breeders. For instance, in the case of crops, obviously, identification of cultivars with low Cd accumulation in their edible parts would be of particular interest. Cultivation of these cultivars even in soils with elevated Cd level would be a promising land-independent approach to minimize human dietary Cd intake, which is a food-safety concern. Low Cd crop cultivars meeting particular criteria, including acceptable yield performance, can be introduced as Cadmium-Safe Crop Cultivars (CSCCs). On the other hand, identification of high-Cd accumulating cultivars would enable the farming industry to exclude those cultivars in cropping for consumption purposes and employ them...

Multiple abiotic stress conditions are known to trigger production of reactive oxygen species such as hydrogen peroxide. Generally, the related antioxidative defense reactions including catalase capable of scavenging hydrogen peroxide are also activated. A closer look at some of the recent studies including those on exogenous application of abiotic stress-relief agents, a no catalase1 mutant and plant transformation on catalase changes in plants under abiotic stress conditions was undertaken here. Some areas worthy of further investigations to gain a better understanding of the role and regulation of catalase activity as a response of plants to abiotic stress have been identified.

The biochemical and enzymatic properties of four highly similar rice oxalate oxidase proteins (OsOxO1-4) were compared after their purification from the leaves of transgenic plants each overexpressing the respective OsOxO1-4 genes. Although alignment of their amino acid sequences has revealed divergence mainly in the signal peptides and they catalyze the same enzymic (oxalate oxidase) reaction, divergence in apparent molecular mass, Km, optimum pH, stability and responses to inhibitors and activators was uncovered by biochemical characterization of the purified OsOxO1-4 proteins. The apparent molecular mass of oligomer OsOxO1 was found to be similar to that of OsOxO3 but lower than the other two. The molecular mass of the subunit of OsOxO1 was lower than that of OsOxO3. The Km value of OsOxO3 was higher than the other three which had similar Km. OsOxO1 and OsOxO4 possessed peak activity at pH 8.5 which was close to that at the optimum pH 4.0. The activity of OsOxO2 at pH 8.5 was only 65% of that at its optimum pH 3.5, while the activity of OsOxO3 did not vary much at pH 6-9 and was also much lower than that at its optimum pH 3. OsOxO2 and OsOxO3 still maintained all their activities after being heated at 70°C for 1h while OsOxO1 and OsOxO4 lost about 30% of their activities. Pyruvate and oxaloacetic acid inhibited the activity of OsOxO3 more strongly than the other three. Interestingly, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-biphosphate related to photosynthetic assimilation of triose phosphate greatly increased the activities of OsOxO3 and OsOxO4. In addition to the differences in the biochemical properties of the four OsOxO proteins, an intriguing finding is that the purified OsOxO1-4 exhibited substrate inhibition, which is a typical of the classical Michaelis-Menten enzyme kinetics exhibited by a majority of other enzymes.

Callus culture has been used to study the cellular basis of sensitivity of plants to toxic trace elements including cadmium. Callus friability may be related to plant response to exposure to trace elements as callus friability is related to the plant cell wall which plays a role in resistance of plant cells to trace element toxicity. The aim of this study was to investigate the relationship between two types of potato callus with different friability and their sensitivity to cadmium. A high frequency (about 80%) of leaf or internodal explants of potato (Solanum tuberosum L., cv, Iwa) formed a friable, pale green callus (type-A callus) on half-strength basal Murashige and Skoog medium supplemented with 12.42 μM picloram. On medium supplemented with 4.43 μM 6-benzyladenine (BA) and 5.37 μM 1-naphthalene acetic acid (NAA), a compact, non-friable callus (type-B callus) was induced in about 80% of the expiants. Type-B callus was greener than type-A callus. Callus formation in both the leaf and internodal explants were completely inhibited on the medium used for induction of type-A or type-B callus when the respective medium was supplemented with 54 μM cadmium chloride (Cd). The type-B callus was found to be more resistant (less necrosis and higher relative growth rates) to 27, 54, and 109 μM of Cd than the type-A callus. The type-B callus also exhibited a higher level of peroxidase activity (a marker antioxidant enzyme counteracting oxidative stress) than the type-A callus when cultured on these Cd concentrations. This is the first study showing the importance of callus friability in plant cell response to Cd treatment.

Iron (Fe) deficiency induces chlorosis (IDC) in plants and can result in reduced plant productivity. Therefore, development of Fe-efficient plants is of great interest. To gain a better understanding of the physiology of Fe-efficient plants, putative novel plant variants were regenerated from potato (Solanum tubersosum L. var. ‘Iwa’) callus cultures selected under Fe deficient or low Fe supply (0–5 μM Fe). Based on visual chlorosis rating (VCR), 23% of callus-derived regenerants were classified as Fe-efficient (EF) and 77% as Fe-inefficient (IFN) plant lines when they were grown under Fe deficiency conditions. Stem height was found to be highly correlated with internodal distance, leaf and root lengths in the EF plant lines grown under Fe deficiency conditions. In addition, compared to the IFN plant lines and control parental biotype, the EF plants including the lines named A1, B2, and B9, exhibited enhanced formation of lateral roots and root hairs as well as increased expression of ferritin (fer3) in the leaf and iron-regulated transporter (irt1) in the root. These morphological adaptations and changes in expression the fer3 and irt1 genes of the selected EF potato lines suggest that they are associated with resistance to low Fe supply stress.

Tagetes patula (marigold) and Petunia grandiflora (petunia) have been shown to exhibit potential in phytoremediation of environmental pollutants including heavy metals and textile dyes. To investigate their phytoremediation potential of diesel, it was necessary to evaluate diesel phytotoxicity of these two ornamental plants. Marigold and petunia seeds were incubated, for 10 and 15 days, respectively, in deionised water contaminated with 0 to 4%, v/v, diesel in Petri dishes in a growth room with continuous lighting at 25 °C. It was found that as far as seed germination was concerned, petunia was less sensitive than marigold to 4% diesel in water. In contrast, petunia exhibited poorer seedling root growth than marigold in the presence of diesel contamination. This finding of differential sensitivity of these two ornamental plants to diesel-contaminated water during germination and seedling growth has not been reported before. Therefore, the implications of phytotoxicity evaluation and comparison between different species or genotypes of plants at both seed germination and postgermination seedling growth should both be taken into consideration in screening tolerant plants for phytoremediation.

Plant science research may help to reduce the potential exposure of people to environmental arsenic pollution. In particular, arsenic phytoremediation and development of low arsenic accumulating plants are two main research foci that hold much promise in helping to tackle the arsenic pollution problem. Plant tissue culture has been useful to aid arsenic toxicity and resistance studies in the laboratory which could better inform phytoremediation studies, for example, involving the arsenic hyperaccumulating Pteris vittata (Chinese brake fern). Transfer of arsenic metabolism-related glutaredoxin genes, for example, has been shown to reduce arsenic accumulation in transgenic plants. In vitro plant cell selection may be, however, an attractive alternative to generation of transgenic plants to yield low arsenic accumulating crop plants (somaclonal variants) aiming to lower dietary arsenic intake.

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 μM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 μM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

We investigated the benefits of restoring native plants to intensive agricultural landscapes of New Zealand. As a browse crop on paddock borders, endemic species potentially could add value to more sustainable food systems. Their foliage was found to contain less nitrogen but higher concentrations of a range of trace elements and tannins than both ryegrass and border-planted willows. While native plants could reduce the need for feed supplements, we conclude that the small amount consumed would provide negligible overall nutritional benefits to stock. Nevertheless, this may provide added justification for endemic species and agroecology in landscapes with a depauperate native biodiversity.

Nilgirianthus ciliatus is of interest for its extensive applications in Ayurvedic medicine. The study focuses on the assessment of genetic diversity and phylogenetic relationships in N. ciliatus populations to aid conservation of this species at risk of extinction. Thirty five inter simple sequence repeats (ISSR) and thirty random amplified polymorphic DNA (RAPD) markers were employed in PCR yielding 298 and 99 bands, respectively. Using ISSR markers, bands of 200–3500 bp were obtained and accounted for 77.0% of polymorphism observed. The polymorphism information content (PIC) and resolving power (Rp) values were 0.59 and 20.8, respectively. RAPD analysis resulted in 99 total bands of which 89 were polymorphic (89.9%). The PIC value for RAPD was 0.61 and Rp value was 18.8. The two different cluster analyses (UPGMA and Neighbor Joining) and principal component analysis showed the genetic variation among the populations. Clustering of N. ciliatus populations from different geographical regions in distinct groups can be a good sign for clear separation that is supported by high bootstrap values. Overall, both ISSR and RAPD analyses were valuable for identifying genetic variations and phylogenetic relationships in N. ciliatus populations at the DNA level and these differences provides the basic knowledge for better conservation management policies and genetic identification of the different geographical populations of this plant.

The total oxalate contents of various vegetables, legume seeds, cereals, fruits and nuts commonly available in Southern China were determined using an improved enzymatic method. Spinach, xeric water spinach (grows on moist soil), amaranth, bamboo shoot, ginger, Chinese wolfberry, rice bean and black glutinous rice contained more than 100mg oxalate/100gFW, but leaf mustard, white radish, broccoli, cauliflower, cabbage, onion, gourd except bitter melon, tomato, pepper, chufa, kelp, mushroom, soybean sprout and cowpea contained less than 10mg oxalate/100gFW. The oxalate contents of star fruit and dragon fruit were 111.4 and 97.1mg/100gFW, respectively, and those of other fruits except strawberry and black plum were less than 20mg/100gFW. Almond, cashew, hazel, pine nut and abalone fruit contained more than 150mg total oxalate/100gFW, and the oxalate levels in Chinese torreya fruit, peanut, pistachio and walnut ranged from 54.1 to 83.1mg/100gFW. Based on the results obtained, water spinach, Chinese wolfberry, black glutinous rice, dragon fruit, rice bean, abalone fruit and Chinese torreya fruit should also be considered as high oxalate-foods. The enzymatic method used in the present study enabled us to perform a large-scale quantitative investigation of oxalate contents of different foods.

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.

Introduction – Proteinaceous inhibitors of animal trypsin occur naturally as isoforms in seeds and some are of interest as antinutritional or anti-pest agents. Objective – To establish a simplified electrophorectic, in-gel method for rapid and direct detection of trypsin isoinhibitors present in crude plant extracts that are particularly suitable for many studies including rapid evaluation of cultivars. Methodology – Azoalbumin (3%, w/v) is immobilised in 7.5% polyacrylamide gels before electrophoresis under non-denaturing conditions. Results – This improved method eliminates the need for both time-consuming and labourious staining and destaining or renaturation steps. Conclusion – Immobilised azoalbumin in polyacrylamide gels, run under non-denaturing electrophoresis conditions, can be used to assist rapid evaluation of trypsin isoinhibitors in numerous crude plant extracts. Copyright © 2010 John Wiley & Sons, Ltd.

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.

Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates.

Uptake of the non-essential element, lead (Pb), by 7-days-old Arabidopsis thaliana seedlings caused inhibition of root growth. Pb accumulated mainly in the root and only a small proportion of the total uptake was transported to the shoot. At the ultrastructural level, Pb was localized mainly in the cell walls, intercellular spaces and vacuoles, which may serve to minimize Pb toxicity to vital functions of the cytosol. Pb exposure induced the generation of reactive oxygen species (ROS) and increased the level of lipid hydroperoxide. These changes were accompanied by up-regulation of the activities of antioxidative enzymes in a dose-dependent manner. Although no major destructive changes to the cytoplasm and vital organelles were observed, it is postulated that at least part of the cost of Pb exposure to A. thaliana seedlings is reflected in reduced root growth associated with increased free radical production by cell wall-accumulated Pb.

The effects of carbohydrate supply and light on rhizosecretion during micropropagation of potato plantlets (Solanum tuberosum L., cv. ‘Iwa’) in liquid medium were investigated. Soluble protein content was higher in the spent medium for plantlets grown under light conditions than in the dark. For those plantlets grown under light conditions and on different sugar-supplemented media, they rhizosecreted the highest amount of soluble protein when grown in the presence of maltose, while they rhizosecreted the lowest amount of soluble protein when grown on medium containing glucose. Moreover, plantlets grown under light and on a medium containing sucrose were the most vigorous, and exhibited the highest levels of rhizosecreted acid phosphatase activity. However, there was no direct relationship between plantlet growth and rhizosecretion. When plantlets were grown in the dark and on medium containing maltose, a higher α-glucosidase activity was detected than those grown on medium containing sucrose. These results suggested that rhizosecretion of certain proteins from plantlets grown in vitro might not require exposure to light conditions.