Eng-Chong Pua

Effect of AgNO3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes.

The presence of 1–10 μM aminoethoxyvinylglycine (AVG) or 5–30 μM AgNO3 markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 μM), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO3 and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO3 was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 μM.

Ethylene inhibitors enhanced de novo shoot regeneration from cotyledons of Brassica campestris ssp. chinensis (Chinese cabbage) in vitro

Abstract A tissue culture system for obtaining high frequency shoot regeneration of Brassica campestris ssp. chinensis cv. Speedy and 2B-21-64 was optimized via judicious selection of explants and manipulation of culture medium with respect to hormonal combination and addition of AgNO 3 , Ag 2 SO 4 , aminoethyoxyvinylglycine (AVG), aminooxyacetic acid (AOA) or 2,4-dinitrophenol (DNP). Three-day-old cotyledons of both cultivars grown on Murashige and Skoogs medium supplemented with 4.4–17.6 μM benzyladenine and 2.7–5.4 μM naphthaleneacetic acid (NAA) in the presence of 7.5–60 μM AgNO 3 or Ag 2 SO 4 or medium containing 8.8 μM BA, 5.4 μM NAA and 0.1–10 μM AVG formed shoots at 70–85% frequency after 3–4 weeks, whereas explants grown in the absence of Ag + or AVG or in the presence of AOA or DNP were poorly regenerative (20–30%). Shoot tips originating from regenerants and seedlings rooted equally well in hormone-free medium or medium containing 0.05–5 μM indolebutyric acid. All rooted shoots were successfully acclimatized. The acclimatized plants were phenotypically indistinguishable from those derived from seeds.

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.

Expression of glutathione-S-transferase and its role in plant growth and development in vivo and shoot morphogenesis in vitro.

The enzymes glutathione-S-transferases (GSTs, E.C.2.5.1.18) have been associated with detoxification of xenobiotics, limiting oxidative damage and other stress responses in plants. In this study, we report the isolation of a mustard gene, BjGSTF2, homologous to the phi class GSTs and changes in plant growth in vivo and shoot regeneration in vitro were related to GST expression. GST transcripts accumulated differentially in mustard organs, where transcript was most abundant in root. Tissues incubated at high temperature or in the presence of exogenous H2O2, HgCl2, 1-aminocyclopropane-1-carboxylate, salicylic acid and paraquat upregulated GST expression, whereas spermidine was inhibitory. To investigate the in vivo function of GST, transgenic Arabidopsis thalianaplants expressing sense (GST-S6), antisense (GST-A4) and double-stranded BjGSTF2(GST-DS1) RNAs were generated. GST-S6 was shown to flower two days earlier and was relatively more tolerant to HgCl2 and paraquat, whereas GST-DS1 with least stress tolerance flowered one week later compared to WT and GST-A4. In shoot regeneration response, tissues originated from GST-S6 were highly regenerative, whereas no shoot regeneration was observed in GST-DS1 tissues after 30 days of culture. Results of this study provide the evidence showing that GST plays a role in plant growth and development in vivo and shoot regeneration in vitro.

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

SummaryThe role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 μM aminoethoxyvinylglycine (AVG) or AgNO3] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N6-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO3 in combination with AVG. Culture medium solidified with agarose in the presence of AgNO3 but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 μM AgNO3 or AVG. Explants with high regenerability grown in the presence of AgNO3 or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.

The Pivotal Roles of the Plant S-Adenosylmethionine Decarboxylase 5′ Untranslated Leader Sequence in Regulation of Gene Expression at the Transcriptional and Posttranscriptional Levels

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key rate-limiting enzyme located in the polyamine biosynthesis pathway. When compared with other organisms, the plant SAMDC genes possess some distinct features because they are devoid of introns in the main open reading frame (ORF) but have an intron(s) in their 5′ untranslated leader sequences, in which two overlapping tiny and small upstream ORFs (uORFs) are present. Our results show that the presence of the 5′ leader sequence plays important roles in transcriptional and posttranscriptional regulation of SAMDC expression. This sequence may help to keep the transcript of its downstream cistron at a relatively low level and function together with its own promoter in response to external stimuli or internal changes of spermidine and spermine to initiate and regulate SAMDC expression. Under stress and high spermidine or spermine conditions, the tiny uORF shows the same function as its overlapping small uORF, which is involved in translational repression and feedback controlled by polyamines. The presence of introns is necessary for the SAMDC up-regulation process when the internal spermidine level is low. Our results suggest that plants have evolved one network to adjust SAMDC activity through their 5′ leader sequences, through which transcriptional regulation is combined with an extensive posttranscriptional control circuit.

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

SummaryThe promotive effect of ethylene inhibitors (Els), i.e. AgNO3 and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO3 at 30 μM did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 μM AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.

Enhanced de novo shoot morphogenesis in vitro by expression of antisense 1-aminocyclopropane-1-carboxylate oxidase gene in transgenic mustard plants

The role of ethylene in shoot morphogenesis in vitro was investigated via the production of transgenic mustard (Brassica juncea (L.) Czern & Coss cv. Indian Mustard) plants with impaired ethylene biosynthesis using the antisense-RNA approach. Mustard plants transformed with antisense MEFE5 encoding 1-aminocyclo propane-1-carboxylate (ACC) oxidase, which catalyses the conversion of ACC to ethylene in mustard, were characterized with respect to enzyme activity, ethylene production and the capacity for shoot morphogenesis in vitro. Most transgenic plants exhibited a decrease in the amount of endogenous 1.4-kb transcript and ethylene production, which was concomitant with a marked increase of shoot regenerability of cultured tissues. Tissues of four individual highly regenerative transgenic plants (T0, T2, T4 and T8) consistently showed lower relative activity of endogenous ACC oxidase and ethylene production than control tissues during a three-week culture period. The highly regenerative phenotype of transgenic tissues reverted to the poorly regenerative control phenotype in the presence of 50 μM 2-chloroethylphosphonic acid. The inverse relationship between shoot regenerability and ethylene production possessed by individual transgenic plants was also manifest in R1 progeny. This study presents strong evidence that ethylene plays a key regulatory role in the de novo shoot formation of mustard in culture, and also indicates a possible general role of ethylene in plant morphogenesis in vitro.

Expression of a UDPglucose pyrophosphorylase cDNA during fruit ripening of banana (Musa acuminata)

We report the isolation of a banana cDNA, designated MWUGPA, encoding uridine diphosphoryl (UDP)-glucose pyrophosphorylase (UGPase, EC.2.7.7.9) that catalyses the reversible conversion between glucose 1-phosphate and UDPglucose in plants and animals. Furthermore, UGPase expression in fruit during ripening and in response to exogenous ethylene and sugars was also investigated. MWUGPA encodes a polypeptide of 467 amino acid residues and shares a high degree of sequence similarity (85–90%) with other plant UGPase homologs. In northern blot analysis, a 1.7-kb UGPase transcript was detected in both the vegetative and reproductive organs, but the former was considerably less abundant than the latter. In fruit, the level of accumulated transcripts was higher in pulp than peel at all ripening stages. Transcript abundance in both fruit tissues was relatively constant during ripen-ing, but pulp transcripts surged in the ‘more green than yellow’ category fruit when ethylene also increased. Further analysis revealed that UGPase expression in fruit was ethylene-inducible, but the response was tissue-specific, as evidenced by the promoting effect of exogenous ethylene on accumulation of UGPase transcripts in pulp but not peel. Exogenous application of sucrose and fructose also increased UGPase transcript abundance in leaf and fruit tissues, especially pulp, whereas exogenous glucose had little or no effect. The results of this study indicate that ethy-lene and soluble sugars may play a regulatory role in UGPase expression during ripening in banana fruit.