Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were simultaneously investigated during the early phases of alfalfa somatic embryogenesis. These included the period of induction and subculture of callus, and 3- and 7-day suspension cultures for the induction of somatic embryogenesis. The polyamines contained in the embryogenic callus were found to include putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was much less than that of Put and Spd. There was a dramatic increase in MACC after induction of embryogenesis, and ACC levels were lower in somatic embryos than in embryogenic callus. Induction of embryogenesis for 3 days increased the levels of ACC and polyamines to a maximum level, and these then reduced as the embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during the induction. This indicated that both high levels of ACC and polyamines might be a prerequisite for early differentiation during the induction of the embryogenesis. Thus, there appears not to be competition between polyamine biosynthesis and ethylene biosynthesis at least during the induction of somatic embryogenesis, because both the polyamines and ACC were simultaneously increased during the induction period. Conversion of ACC into MACC and the maintenance of a relatively high level of polyamines, especially Spd, appear to be important for further development of the embryos. When aminooxylvinylglycine (AOA) was added at the initiation of the callus subculture, it had no significant effect on the callus growth, the ethylene production and ACC level of the callus. However, AOA increased the numbers of the embryos accompanying an increase in Spd level and S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could be associated with Spd increase rather than with the effect of ethylene biosynthesis.

A banana somatic embryogenesis receptor-like kinase (SERK) gene, designated as MaSERK1, was isolated from Musa acuminata cv. Mas (AA). It encoded a protein of 628 amino acids with above 82% identities to reported SERKs of coconut, rice, maize, Arabidopsis, carrot, and Medicago truncatula. MaSERK1 was expressed weakly in male flower clusters, but not in male flower-derived nonembryogenic calli, but it was highly expressed in male flower-derived embryogenic calli and embryogenic cell suspensions (ECS). During subculture of ECS, MaSERK1 expression and frequency of somatic embryogenesis were influenced by the duration of subculture, wherein expression decreased within 0 to 6 days of subculture, increased to highest levels at 12 days following subculture, and dropped thereafter. The frequency of somatic embryogenesis of ECS positively correlated with MaSERK1 transcript levels. Moreover, MaSERK1 expression in leaves of Musa paradisiaca L. cv. Dongguan Dajiao (ABB), known to be resistant to Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), was induced by exogenous salicylic acid (SA) or inoculation with FOC race 4. However, MaSERK1 expression levels in leaves of Mucus spp. cv. Pisang awak (ABB), known to be susceptible to FOC race 4, did not change following either treatment. These results suggested thatMaSERK1 not only could serve as a molecular marker for banana somatic embryogenesis, but also played a role in disease resistance response in banana.

The nonexpresser of pathogenesis-related gene 1 (NPR1) plays a pivotal role in systemic acquired resistance in plants. In this study, a novel full-length NPR1-like gene, designated MdNPR1 (accession number FJ357442), was isolated by RACE-PCR from cv. Dongguan Dajiao (Musa spp. ABB), a local banana cultivar known to be resistant to Fusarium oxysporum fsp. cubense (FOC) race 4. Sequence alignment showed that MdNPR1 contained an ankyrin repeat domain and a broad complex, tramtrack, and brica-brac (BTB) domain. Semiquantitative reverse transcription polymerase chain reaction revealed that MdNPR1 could be constitutively expressed at low levels in both of the FOC race 4-susceptible cultivar Fenjiao (Musa spp. ABB) and the resistant cultivar Dongguan Dajiao. However, MdNPR1 could be induced by exogenous application of salicylic acid in cv. Dongguan Dajiao, but not in cv. Fenjiao. Moreover, the accumulated level of MdNPR1 transcripts in cv. Dongguan Dajiao was higher than that in cv. Fenjiao when plants were treated with FOC race 4 inoculation. Our results implied that MdNPR1 might represent as a promising candidate for engineering resistant to broad-spectrum pathogen in banana.

Preconditioning `Mesa 659' lettuce (Lactuca sativa L.) seeds with the moist solid carrier Micro-Cel E at 15C for 20 hours removed thermoinhibition and allowed the seeds to germinate at 35C. Participation of 1-aminocyclopropane-1-carboxylic acid (ACC) and its use in the removal of thermoinhibition were indicated from the following: a) the ACC level in the preconditioned seeds peaked before germination at 35C; b) addition of aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthesis, reduced the ACC level and inhibited germination at 35C; c) response of preconditioned seeds to ethylene at 35C preceded that of ACC; d) exogenous ACC, (2-chloroethyl) phosphonic acid (ETH, ethephon), or ethylene reversed the inhibitory effect of AVG and restored the capability of the seed to germinate at 35C; e) Co+2, an inhibitor of the ACC-to-ethylene step, partially inhibited germination of preconditioned seeds at 35C, and the effect was reversed by ETH. The major free polyamines in dry seed were spermidine (Spd) and putrescine (Put). An increase in polyamine level occurred following transfer of conditioned seeds to 35C. Of the various polyamine biosynthesis inhibitors, difluoromethylarginine and cyclohexylammonium sulfate inhibited an increase in Spd and Put levels only after germination, whereas difluoromethyorinthine had no effect on polyamine biosynthesis. None of these inhibitors affected the relief of thermoinhibition. Thus, the increase in polyamine level during germination at 35C does not appear to be associated with the alleviation of thermoinhibition.

Yellowish embryogenic callus, which had been induced from the petioles of alfalfa (Medicago sativa L. cv. Jinnan) and maintained on kinetin (KN)-containing B5h medium, turned non-embryogenic with the differentiation of tracheary elements when KN in the medium was replaced by thidiazuron (TDZ). To investigate the molecular mechanisms underlying the TDZ-induced morphological competence transition, we cloned the differentially expressed sequence tags from TDZ-treated alfalfa callus using suppression subtractive hybridization. The gene expression patterns induced by TDZ were found to change throughout the period of TDZ treatment. TDZ induced the expression of stress-related genes, including trehalose-6-phosphate phosphatase (TPP) gene, 1-aminocyclopropane-1-carboxylate synthase (ACS) gene and proline dehydrogenase gene, in both early days (day 2) and late days (day 28) of treatment, which are suggestive of the stress responses from the TDZ-treated callus. The TDZ-induced expression of TPP, which tends to lower the level of trehalose-6-phosphate, and ACS, the gene involved in ethylene production, appears to be the major cause for the termination of somatic embryogenesis.