PpGRAS12 acts as a positive regulator of meristem formation in Physcomitrium patens

Key message This study focused on the key regulatory function of Physcomitrium patens GRAS12 gene underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life. Abstract The miR171‐GRAS module has been identified as a key player in meristem maintenance in angiosperms. PpGRAS12 is a member of the GRAS family and a validated target for miR171 in Physcomitrium (Physcomitrella) patens. Here we show a regulatory function of miR171 at the gametophytic vegetative growth stage and targeted deletion of the PpGRAS12 gene adversely affects sporophyte production since fewer sporophytes were produced in ΔPpGRAS12 knockout lines compared to wild type moss. Furthermore, highly specific and distinct growth arrests were observed in inducible PpGRAS12 overexpression lines at the protonema stage. Prominent phenotypic aberrations including the formation of multiple apical meristems at the gametophytic vegetative stage in response to elevated PpGRAS12 transcript levels were discovered via scanning electron microscopy. The production of multiple buds in the PpGRAS12 overexpression lines similar to ΔPpCLV1a/1b disruption mutants is accompanied by an upregulation of PpCLE and downregulation of PpCLV1, PpAPB, PpNOG1, PpDEK1, PpRPK2 suggesting that PpGRAS12 acts upstream of these genes and negatively regulates the proposed pathway to specify simplex meristem formation. As CLV signaling pathway components are not present in the chlorophytic or charophytic algae and arose with the earliest land plants, we identified a key regulatory function of PpGRAS12 underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life.

Virulent Rhodococcus fascians Produce Unique Methylated Cytokinins

Some strains of Rhodococcus fascians exist only as epiphytes on the plant surface whereas others can become endophytic and cause various abnormalities including the release of multiple buds and reduced root growth. The abnormalities reflect the action of cytokinin. The strains that can become endophytic harbour a linear plasmid that carries cytokinin biosynthesis, activation and destruction genes. However, both epiphytic and endophytic forms can release cytokinin into culture, affect cytokinin metabolism within inoculated plants and enhance the expression of sugar and amino acid transporters and cell wall invertases, but only the endophytic form markedly affects the morphology of the plant. A unique methylated cytokinin, dimethylated N6-(∆2-isopentenyl)adenine (2-MeiP), operating in a high sugar environment, is the likely causative factor of the severe morphological abnormalities observed when plants are inoculated with R. fascians strains carrying the linear plasmid.

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Prolonged cyclin-dependent kinase inhibition results in septin perturbations during return to growth and mitosis

We investigated how Saccharomyces cerevisiae coordinate polarization, budding, and anaphase during a unique developmental program called return to growth (RTG) in which cells in meiosis return to mitosis upon nutrient shift. Cells reentering mitosis from prophase I deviate from the normal cell cycle by budding in G2 instead of G1. We found that cells do not maintain the bipolar budding pattern, a characteristic of diploid cells. Furthermore, strict temporal regulation of M-phase cyclin-dependent kinase (CDK; M-CDK) is important for polarity establishment and morphogenesis. Cells with premature M-CDK activity caused by loss of checkpoint kinase Swe1 failed to polarize and underwent anaphase without budding. Mutants with increased Swe1-dependent M-CDK inhibition showed additional or more penetrant phenotypes in RTG than mitosis, including elongated buds, multiple buds, spindle mispositioning, and septin perturbation. Surprisingly, the enhanced and additional phenotypes were not exclusive to RTG but also occurred with prolonged Swe1-dependent CDK inhibition in mitosis. Our analysis reveals that prolonged activation of the Swe1-dependent checkpoint can be detrimental instead of beneficial.

Multiplicação in vitro de Codonanthe devosiana

Codonanthe devosiana is a species endemic to Brazil belonging to the family Gesneriaceae. It features ornamental potential for use in hanging pots and shaded environments. The aim is to evaluate the effects of the combination of different concentrations of BAP and GA3 on the multiplication and development of in vitro explants of C. devosiana. We used MS media supplemented with concentrations of 0.0, 0.2 and 0.4 mg L-1 of BAP and 0.0, 0.25 and 0.50 mg L-1 of GA3 . The number, bud dry matter and height, callus dry matter and presence of roots were evaluated in agglomerates with multiple buds. The interaction between BAP and GA3 concentrations was verified for number of shoots, shoot dry matter and callus dry matter. For shoot height and root formation, only BAP concentrations were observed. The highest number of shoots (4.8) occurred when combined with the concentration of BAP 0.2 mg L-1 and 0.5 mg L-1 GA3 . The lowest shoot height was observed without the use of BAP, as well as the greatest rooting. It is concluded that the combination of 0.2 mg L-1 of BAP with 0.5 mg L-1 of GA3 allows for a more efficient multiplication of C. devosiana explants

First Report of Rhodococcus Isolates Causing Pistachio Bushy Top Syndrome on ‘UCB-1’ Rootstock in California and Arizona

‘UCB-1’ (Pistacia atlantica × Pistacia integerrima) rootstock is a hybrid cultivar widely used by the U.S. pistachio industry. In the last three years, a large number of micropropagated UCB-1 pistachio rootstocks planted in California and Arizona orchards exhibited shortened internodes, stunted growth, swollen lateral buds, bushy/bunchy growth, stem galls with multiple buds, and twisted roots with minimal lateral branching. Field T-budding success in affected orchards was reduced to approximately 30% with unusual bark cracking often observed around the bud-union. The percentage of abnormal rootstocks within affected orchards varied from 10 to 90%. We have termed the cumulative symptoms “pistachio bushy top syndrome” (PBTS) to describe these affected trees. Two isolates, both containing virulence factors from the phytopathogen Rhodococcus fascians, were identified on symptomatic trees in field and nursery samples. Micropropagated UCB-1 trees inoculated with the Rhodococcus isolates exhibited stunted growth, shortened internode length, swollen lateral buds, sylleptic branching, and differences in root morphology, compared with control UCB-1 trees. To our knowledge, this is the first report of Rhodococcus isolates, related to Rhodococcus fascians, causing disease on a commercial tree crop and the results presented indicate that this organism is responsible at least in part for PBTS in California and Arizona.

МОНГОЛ ОРНЫ ЭНДЕМИК УРГАМАЛ МОНГОЛ ДОГАР-CARYOPTERIS MONGOLICA BGE.-ИЙГ IN VITRO НӨХЦӨЛД ҮРЖҮҮЛСЭН ДҮНГЭЭС

Caryopteris mongolica Bge. буюу Монгол Догарын үрийг ариутгаад гиббереллиний хүчил(ГХ) 0.5мг/л, 1мг/л, 2мг/л тус тус агуулсан болон дан МС(хяналт) тэжээлт орчин дээр соёолуулсан. ГХ 2мг/л агуулсан хувилбар нь хяналттай харьцуулахад 20 хувиар үрийн соёололтыг нэмэгдүүлсэн. 28 хоногтой цухуйцаас хажуугийн нахиа бүхий ишний үе болон үрийн талын хэсгийг эксплант болгон сонгон авч Бензиламинопурин(БАП) 1мг/л, БАП 2мг/л ба Индол-3-цууны хүчил(ИЦХ) 0.3 мг/л, БАП 3мг/л концентрациар тус тус агуулсан, мөн Кинетин(КИН) 1мг/л, КИН 2мг/л ба α-нафталин цууны хүчил(НЦХ) 0.3мг/л, КИН 3мг/л ба НЦХ 0.4мг/л харьцаагаар тус тус агуулсан МС үндсэн тэжээлт орчинд өсгөвөрлөсөн. 21 хоногийн дараа найлзуур ургаж, нахиа олширсон байсан учир хэмжилт авч, 28 хоногийн дараа субкультур хийсэн. Нахиа үүсгэхэд хамгийн тохиромжтой орчны хувилбараар үрийн талын эксплант дээр 3 мг/л БАП, харин нахиа бүхий ишний үеийн эксплант дээр КИН=3мг/л, НЦХ=0.4мг/л өсөлтийн бодис агуулсан МС тэжээлт орчин байв. Үүссэн найлзууруудаа ямар нэг өсөлтийн бодис агуулаагүй ½ МС тэжээлт орчинд өсгөвөрлөхөд 7-10 хоногийн дараа үндэс үүсч эхэлж байсан бөгөөд 21-28 хоногт 95% нь үндэслэж байв.IN VITRO PROPAGATION THE MONGOLIAN ENDEMIC PLANT CARYOPTERIS MONGOLICA BGE.Caryopteris mongolica Bge. is rare and endemic plant of Mongolia, which used in traditional medicine of various diseases. We have initiated in vitro propagation the Caryopteris mongolica Bge. from seeds. Seeds were surface sterilized by immersing in 70% ethanol for 90 sec, 10% hydro peroxide for 10 minutes and 5% sodium hypochlorite for 20 minutes. Finally, rinsed with sterile distilled water for 4 times. Sterilized seeds were germinated on MS medium supplemented with 0.5, 1, 2 mg/l gibberillic acid, respectively. Nodal segment and cotyledon explants from 4 weeks old seedling were cultured on the MS basal medium supplemented with benzylaminopurine(BAP) 1 mg/l, BAP 2 mg/l and Indole acetic acid(IAA) 0.3 mg/l, BAP 3 mg/l, Kinetin(KIN) 1 mg/l, KIN 2 mg/l and Naphthalene acetic acid(NAA) 0.3 mg/l, KIN 3 mg/l and NAA 0.4 mg/l concentrations, respectively. After 3 weeks, measured of shoot height and bud number and 4 weeks later subcultured in a same medium. High frequency multiple buds were formed on MS medium supplemented with BAP 3mg/l, from cotyledon explants, but KIN 3 mg/l and NAA 0.4 mg/l was best version of nodal segment explants. All shoots were cultured in the ½ MS basal without any growth regulators for induction of root. After 7-10 days, initiation of root form and 21-28 days later 95% of cultured shoots were rooted.Key words: Caryopteris mongolica Bge., MS basal medium, Gibberillic acid, Benzylaminopurine, Kinetin, Indole-3-acetic acid, α-Naphthalene acetic acid, explants.DOI: http://dx.doi.org/10.5564/pmas.v52i2.356 Proceedings of the Mongolian Academy of Sciences Vol.52(2) 2012 p.23-28