Plant Biotechnology Vol.33 No.3


Original Papers

Different environmental conditions at initiation of radiata pine somatic embryogenesis determine the protein profile of somatic embryos

García-Mendiguren O, Montalbán IA, Correia S, Canhoto J, Moncaleán P. . . . . . . 143 [PDF]

Somatic embryogenesis is an important biotechnological tool in breeding and conservation programmes for woody species. It exists an increasing interest in the improvement of somatic embryogenesis induction in commercial species such as Pinus radiata. One approach to obtain markers of successful somatic embryogenesis can be the study of protein profiling in a descriptive study. In order to accomplish this objective, 2-D and MALDI-TOF/TOF-MS analysis were used in the identification and quantification of proteins from Pinus radiata D. Don somatic embryos derived from 4 cell lines that were generated under different environmental conditions at initiation stage. Based on the Swiss-Prot database, among the 139 proteins detected, 25 of them appeared only in those cell lines with low initiation percentage at the initial stage of somatic embryogenesis. Only two proteins were detected differentially in cell lines with high initiation rates. Approximately 60% of the proteins identified were related to carbohydrate metabolic process and defence response. This study gives new insights about the proteomics in forest trees, as well as provides clues on the underlying causes of the mechanisms governing Pinus radiata somatic embryogenesis process, and can serve as a tool for facing the improvement ofthe breeding programmes.

In vitro propagation and chemical profiling of Herminium lanceum (Thunb. ex Sw.) Vuijk, a medicinally important orchid, for therapeutically important phenolic acids

Deepak K. Singh, Shashi B. Babbar. . . . . . . 153 [PDF]

The paper describes an efficient and reproducible protocol for in vitro propagation of Herminium lanceum, a medicinal orchid, natural polulations of which are adversely affected because of its indiscriminate collections and other anthropogenic activities. The germination of seeds at different stages of embryo development was compared on four media: Mitra’s (M), M +0.1% peptone (M+P), 1/2 Murashige and Skoog’s+0.1% peptone (MS+P) and Knudson C+0.1% peptone (KC+P). The seeds that contained multicellular oval embryos and cultured on M+P exhibited the highest percentage (82.94%) of germination. Protocorm-like bodies (PLBs), developed from the seeds, could be multiplied by repeated sub-cultures on M+P (hereafter referred to as basal medium, BM). PLBs sub-cultured on BM, supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KN) or 1-phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea (TDZ) differentiated shoots. The maximum number of shoots per culture developed on BM+4 µM TDZ. Shoot elongation was the best on BM+0.1% AC+1 µM GA3. Among various media tested for rooting of the shoots, the best rhizogenic response was obtained on BM+4 µM TDZ+0.1 µM IBA. About 82% of in vitro regenerated plantlets survived on transfer to the potting mixture of sand : vermiculite (1 : 1). High performance liquid chromatographic (HPLC) analyses revealed the presence of medicinally important phenolic acids in leaves and tubers of both in vitro and in vivo plants. The developed protocol could be used for the large-scale production of propagules required for the cultivation of this plant as a regular source of herbal, and also for eco-restoration of its native habitats.

Enhanced cuticle accumulation by employing MIXTA-like transcription factors

Yoshimi Oshima, Nobutaka Mitsuda. . . . . . . 161 [PDF]

The cuticle covers almost the entire aerial surface of terrestrial plants, and provides protection from abiotic and biotic stresses. Cuticles basically consist of wax and cutin, and are produced with variable structures and thicknesses depending on the plant and organ. The application of plant cuticles to improve stress tolerance and wax production requires the deposition of the cuticle at specific times to avoid undesirable side effects. We previously showed that the MYB106 and MYB16 MIXTA-like transcription factors regulate cuticle formation. However, MYB106 over-expression results in severe dwarfism. In this study, we identified genes downstream of these MYB transcription factors and used their promoters to express MYB106 and MYB16 fused to the strong transcriptional activation domain, VP16. Comparisons of plant growth and cuticle morphology revealed that MYB106 and MYB16 preferentially produced cuticles that are typically observed in petals and leaves, respectively. Additionally, the CYP77A6 and CYP86A4 promoters effectively induced cuticle accumulation in leaves and petals, respectively, without inhibiting plant growth. Our strategy may be useful for increasing or altering cuticles in agronomically important plants.

Implications of the gene for F1–ATPase β subunit (AtpB) for the grain quality of rice matured in a high-temperature environment

Hiroaki Kusano, Yohei Arisu, Jun Nakajima, Mitsuhiro Yaeshima, Kao-Chih She, Hiroaki Shimada. . . . . . . 169 [PDF]

Rice grain filling is impaired by high-temperature stress during seed development. This event results in reduced grain yield and quality because of formation of smaller and chalky grains, and often causes problems in agriculture. High-temperature stress causes disorders in primary metabolism pathways including ATP accumulation in the developing seeds. To determine the effect on the genes for ATP biosynthesis, we created the transformant rice plants in which expression of an F1–ATPase β subunit gene (AtpB) was reduced by its RNAi gene, and elevated in developing seeds by its overexpression using the GlutelinB4 promoter. The transformants, which showed a high level of ATP accumulation as well as abundant AtpB transcript in the developing seeds, showed acquired tolerance to high-temperature stress during seed development because the ratio of perfect grains without any chalky part was significantly increased. In contrast, transformants harboring the RNAi gene showed reduction of grain quality with increase of ratio of white-core and white-berry grains when they were developed in the normal growth condition. Most of these seeds never germinated, and the RNAi transgene was poorly inherited by the progenies. These results suggest that AtpB plays important roles in rice seed development and that its gene expression is strongly influenced by high-temperature stress during the grain filling stage.

Doubled Haploids generated through anther culture from an elite long duration rice hybrid, CRHR32: Method optimization and molecular characterization

Prachitara Rout, Nupur Naik, Umakanta Ngangkham, Ram Lakhan Verma, Jawahar Lal Katara, Onkar Nath Singh, Sanghamitra Samantaray. . . . . . . 177 [PDF]

An improved procedure has been developed for high frequency androgenesis in an elite long duration indica rice hybrid. The effects of cold temperature pretreatment, duration of treatment and media with different plant growth regulators on callus induction and shoot regeneration were examined for generation of doubled haploids. N6 medium supplemented with 2.0 mg l−1 2,4-D, 0.5 mg l−1 BAP and 30 g l−1 maltose was found to be most effective for callusing when compared with MS and SK1. The N6 media grown calli showed maximum green shoot regeneration frequency in MS medium supplemented with 0.5 mg l−1 NAA, 0.5 mg l−1 Kinetin, 1.5 mg l−1 BAP and 30 g l−1 sucrose after 2 week of culture. The cold temperature treatment of spike at 10°C for 2 days alongside by 8 days was found to be most suitable conditions for callusing and green shoot regeneration producing 186 green plants from indica rice hybrid, CRHR32. The ploidy status assessed on the basis of morpho-agronomic characters revealed fertile diploids at a frequency of about 81.10%; 16.10%, 2.68% and 1.08% were polyploids, haploids and mixploids respectively. Microsatellite marker analysis showed 1 : 1 ratio of the alleles of CMS and restorer lines used for development of CRHR32. Homozygosity was detected for all the marker loci in 150 DHs and only one plant was identified as heterozygote. This investigation identified the favorable media composition and condition for callus induction and green plant regeneration which would further increase the knowledge and better understanding in rice hybrids for development of DHs.

A MYB-related transcription factor affects nodule formation in Lotus japonicus

Supriadi, Mallika Duangkhet, Apiraya Thepsukhon, Rahayu Widyastuti, Dwi Andreas Santosa, Shigeyuki Tajima, Mika Nomura. . . . . . . 187 [PDF]

The MYB family is one of the largest groups of transcription factors in plants. Our previous microarray data revealed that a MYB-related gene (Ljmybr) was induced upon nodule formation in Lotus japonicus. As shown using real-time PCR, the expression of the Ljmybr in L. japonicus was up-regulated after M. loti infection. To determine the role of Ljmybr in the nodulation process, we created transgenic hairy roots in L. japonicus and then performed Ljmybr overexpression (MYBOX) or RNAi-mediated suppression (MYBRNAi). The results of these experiments indicated that overexpressing Ljmybr accelerated L. japonicus nodulation and increased acetylene reduction activity (ARA) in the nodules. Suppressing Ljmybr (MYBRNAi) delayed nodulation and decreased ARA compared to control suppression (GUSRNAi). Seven days post infection, the expression levels of Nin, Enod40-1 and Enod40-2 were higher in MYBOX-treated nodules and lower in MYBRNAi-treated nodules. We propose that the Ljmybr transcription factor plays a role in regulating nitrogen fixation in L. japonicus.

Functional analysis of the promoter of a rice 18 kDa oleosin gene

Yuko Ishibashi, Hideki Takanashi, Kaoru T. Yoshida. . . . . . . 195 [PDF]

The 18 kDa oleosin (Ole18) is a seed-specific protein expressed specifically in embryos and the aleurone layers of rice (Oryza sativa L.) seeds. Sequence analysis revealed that the promoter of the gene Ole18 contains many cis-acting elements, including seed-specific elements, ABA-responsive elements, and drought-responsive elements, mainly in the region between −400 and −100. To elucidate the regulatory mechanism of Ole18 gene expression, the 1249-bp Ole18 promoter and its internal deletion-derivatives were fused to the GUS reporter gene and introduced into rice. Histochemical analysis and fluorometric quantitative analysis in transgenic rice showed that GUS activity varied depending on the deletion-derivative construct. GUS activity decreased as the deletion region increased (from −378 to −187, to −180, to −169, and to −130), suggesting the importance of known cis-acting motifs such as ABA-responsive elements and drought-responsive elements, and a novel motif within these regions. In addition, either the region between −378 and −179 or that between −179 and −130 was sufficient to induce high expression in the embryos and aleurone layers. This suggests the importance of the common cis-acting elements in the two regions: the drought-responsive element DRE2 and the ABA-responsive element ABI4-binding motifs.


Analysis of TTG1 and CPC-like MYB genes during Arabidopsis epidermal cell differentiation

Rumi Tominaga-Wada, Takuji Wada. . . . . . . 201 [PDF]

The development of Arabidopsis thaliana epidermal cells includes the differentiation of trichomes and root hairs. The TRANSPARENT TESTA GLABRA 1 (TTG1) gene encodes a WD40 protein that induces trichome differentiation and reduces root hair formation in Arabidopsis. The CAPRICE (CPC) gene family includes CPC, ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), and CPC LIKE MYB3 (CPL3), which encode R3-type MYB transcription factors that inhibit trichome differentiation and promote root hair formation. CPC expression is positively regulated by a transcriptional complex that includes TTG1. To determine whether ETC1, ETC2, and CPL3 are also regulated by the TTG1 complex, we examined the functional relationship between TTG1 and CPC-like MYB genes. Double mutant analysis showed that the ttg1 mutant is epistatic to the cpc, etc1, etc2, and cpl3 mutants in trichome cell fate determination but not in root hair development. In roots, the cpc mutant is epistatic to the ttg1 mutant in root epidermal cell fate determination. Promoter-GUS analysis indicated that TTG1 is necessary for the expression of ETC1 and CPL3, but not for ETC2 expression. These results indicate that TTG1 had a stronger effect on trichome formation than CPC-like MYBs. By contrast, CPC had a stronger effect on root hair formation than TTG1. Our results suggest that ETC1 and CPL3 are also regulated by the TTG1 complex as is the case for CPC; however, ETC2 is not regulated by this complex. We concluded that ETC2 does not have a role in trichome and root hair formation.

Cytokinin Response Factor 2 positively regulates salicylic acid-mediated plant immunity in Arabidopsis thaliana

Tackmin Kwon. . . . . . . 207 [PDF]

The phytohormone cytokinin affects various growth and developmental processes and plant immune responses to various pathogens. However, understanding of the molecular mechanism by which cytokinin regulates plant immunity is at the early stage compared to that of growth and development. Therefore, the role of Arabidopsis thaliana CYTOKININ RESPONSE FACTOR2 (AtCRF2) in plant immunity were investigated. AtCRF2 is a plant specific APETALA2 (AP2)/ethylene-responsive factor (ERF) type transcription factor that is transiently upregulated by cytokinin in a type-B Arabidopsis response regulator (ARR) dependent manner. Arabidopsis transgenic plants overexpressing AtCRF2 showed an accelerated leaf senescence phenotype, but extreme AtCRF2 overexpression was lethal at an early stage of growth with lesion-mimic phenotypes in leaves. Moreover, growth of the compatible hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, was strongly compromised in transgenic than in wild-type plants. Furthermore, there was a strong correlation between the expressions of AtCRF2 and pathogenesis related (PR) genes. However, the phenotypes of transgenic plants overexpressing AtCRF2 were completely suppressed by expressing bacterial salicylic hydroxylase, NahG. These results suggest that AtCRF2 may enhance salicylic acid (SA) biosynthesis, leading to autoimmune responses and leaf senescence.