WPM Medium Research Articles

THE INFLUENCE OF SOURCE TREE ON SOMATIC EMBRYOGENESIS FROM EASTERN BLACK WALNUT (JUGLANS NIGRA) IMMATURE COTYLEDONS

The influences of stage of fruit development, media treatments and source tree (genotype) on somatic embryogenesis in Juglans nigra L. were studied over two years. Beginning at 12 weeks and continuing until 15 weeks postanthesis, 1 cm 2 explants from cotyledons of immature seeds were excised from eastern black walnut fruit during 1999 and 2000. Fruits from different trees were tested each year because of the alternate bearing habit of this species. Explants were placed on WPM, DKW, or LP medium with 1 g L -1 casein hydrolysate, 30 g L -1 sucrose, 5.0 μM thidiazuron (TDZ), 0.1 μM 2,4-D, and 7 g L -1 Sigma agar. The explants were under 16-hour photoperiod and 25 °C for four weeks. Explants were then transferred to basal media with 1 g L -1 casein hydrolysate and no plant growth regulators and incubated in darkness. Each year, the experimental design was a completely randomized design with 20 replications per treatment with factorial combinations of media salts, source tree and number of weeks post anthesis. During 1999, there was a significant interaction between genotype and media with LP being a consistently good medium for all three genotypes, with a range of 1.0 to 1.9 somatic embryos per explant and 24 to 30 percent of the explants producing somatic embryos depending on genotype. During 2000, there was a significant interaction between genotype and medium, and between genotype and number of weeks postanthesis. Explants from the best source tree produced 2.8 somatic embryos per explant plus 34 to 38% of the explants were embryogenic when DKW or LP salts were used. Medium had little effect on explants from one of the other two genotypes; the third genotype produced the most embryos when LP medium was used. Explants tended to be the most embryogenic 12 weeks postanthesis with 0.8 to 4.2 embryos per explant depending on source tree.

SUCROSE, CULTURE MEDIA, AND THEIR INTERACTIONS DURING IN VITRO PROLIFERATION OF 'ROXO DE VALINHOS'(FICUS CARICA L.)

Micropropagation is just one of many tissue culture procedures in use. The applications of this technology have multiplied rapidly in recent years. This technique produces plants that are more likely to maintain genotypic and phenotypic fidelity to the original clone. It is used for essentially all the fruit plants that are micropropagated commercially at the present time. Sucrose, media culture and their interactive effects on bud proliferation rate of Ficus carica L. cv. 'Roxo de Valinhos' were studied. The sucrose concentrations (0, 15, 30 and 45 g.L-1 each were combined factorially with MS, B5, Knudson, White and WPM media culture, were tested for optimal proliferation response. Small axillary buds were removed from these branches and culture on solid medium. Those treatments rested in growth room at 27±1 °C temperature with 16 hs photoperiod in 35 µmol.m-2.s-1 light intensity. After 60 days, we made the following assumptions in our analysis: number, length and fresh matter weight of the shoots, length and fresh weight of the roots. The best results for the aerial part were obtained at WPM medium and 15g.L-1 sucrose. There wasn't root system development.

In vitro culture of Spondias mombin L. nodal segments

Spondias mombin L. shoot cultures were initiated from nodal explants taken from plants propagated by seeds. Explants coming from 4-6 months old plants, previously disinfected, were cultivated on WPM medium supplemented with a wide range of concentrations of BAP (0.0, 0.22, 0.44, 2.22 and 4.44 muM) and NAA (0.0, 0.27 and 2.70 muM). After four weeks, the responses obtained were axillary shoot and root formation. The first response were preferentially induced with the medium containing only BAP, regardless of the BAP concentration. The addition of NAA on medium reduced significantly axillary shoot formation and induced rhizogenesis. Roots were formed on nodal explant basis, preferentially on medium supplemented with 4.44 muM NAA. The medium supplemented with BAP reduced significantly root formation.

Transgenic silver birch (Betula pendula) expressing sugarbeet chitinase 4 shows enhanced resistance to Pyrenopeziza betulicola

A sugar beet chitinase gene driven by the (4×) CaMV 35S promoter was introduced into silver birch (Betula pendula) through Agrobacterium-mediated transformation. Transgenic shoots were regenerated and grown on WPM medium supplemented with 150 mg/ml kanamycin. From a total of 220 explants, 52 transgenics were obtained and 13 transgenic lines were randomly taken for molecular analysis to confirm the presence of the introduced sugar beet chitinase 4 cDNA by polymerase chain reaction and Southern hybridisation. All 13 transgenic lines were confirmed to contain the gene and further characterised. Northern blot analysis of total RNA indicated that the transgenic lines differed with respect to the steady-state levels of chitinase mRNA. Transgenic lines with high levels of mRNA of chitinase 4 cDNA consistently showed higher levels of resistance to Pyrenopeziza betulicola than transgenics with intermediate or low mRNA levels or a non-transgenic control plant. This report demonstrates that the constitutive expression of this gene in transgenic birch lines increased the resistance of birch against the leaf spot fungus P. betulicola.

Tratamentos pré-germinativos em sementes de <i>Didymopanax morototoni</i> (Aubl.) Dcne. et Planch.

Didymopanax morototoni (Aubl.) Dcne. et Planch, (caixeta), é uma espécie nativa com vasta distribuição natural, rápido crescimento, grande porte e ainda pela sua rusticidade, é considerada uma espécie pioneira de potencial florestal para o Brasil. Porém, as suas sementes apresentam baixa germinabilidade. O objetivo deste trabalho foi avaliar o efeito de tratamentos pré-germinativos que pudessem maximizar o processo de germinação desta espécie. Foram testados os seguintes tratamentos: pré- -embebição em H20 por 8 h; escarificação química (HCl e H2SO4 a 70%); escarificação mecânica (por picotes no tegumento e com lixamento); lavagem com água destilada e lavagem em água + álcool (1:1) por 15, 30 e 45 min. As sementes foram colocadas para germinar em diferentes substratos. Realizou-se ainda teste de germinação in vitro. Sementes foram inoculadas em meio WPM, solidificado com ágar na presença de cinetina e ácido giberélico nas concentrações (0; 0,1; 0,5; 1,0 mg/l), e caseína hidrolisada na concentração 250 mg/l. A incubação foi a 25°C 1°C em câmara de germinação sob fotoperíodo de 16 horas luz, numa intensidade de 14,3 μE.m-².s-¹ ou no escuro. A germinação foi favorecida pelos pré-tratamentos de lavagens com água e água + álcool, sendo o tempo mais eficaz o de 45 min. A adição de cinetina e ácido giberélico provocou um aumento na germinabilidade de 40 a 70 %. Estes resultados confirmam a dormência tegumentar parcial e indicam a presença provável de substancias inibidoras nos tegumentos, removidas pelas lavagens.

Regeneration of Lonicera tatarica plants via adventitious organogenesis from cultured stem explants

We describe an efficient process for the regeneration of Lonicera tatarica plants from cultured stem sections. Induction of multiple shoots was achieved directly from cultured stem cuttings. The highest regeneration rate was achieved on Gamborg's B5 medium supplemented with 4% sucrose and 0.8% Difco bacto-agar in the absence of hormones. Differentiated shoots were elongated for 5–7 days on induction medium supplemented with 0.5 mg/l GA3. Shoot induction and elongation experiments were carried out using original stem explants from either 2-, 6-, or 18-month-old donor plants. The age of the donor plant had no noticeable effect on either process. However, rooting of elongated shoots occurred only with shoots derived from 2-month-old donor plants. Rooting efficiency and proliferation were highest on half-strength WPM medium supplemented with 2 µM indole-3-butyric acid and 0.6% Keylis agar. The plants regenerated from stem explants were morphologically normal, and levels of loganin and secologanin were comparable to those detected in plants grown from seed and maintained through vegetative propagation.

Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian

About 70% of the shoots developed from nodal explants ofGentiana triflora flowered in vitroondouble strength WPM medium containing 3% (w/v) sucrose, 0.5mg/l BA after 12 weeks of culture in a growth room at 22°Cwith continuous illumination (PPFD = 60μmolm−2 s−1). The influences oninvitro shoot development and flowering of several factors includingthe position of the explant, requirements for sucrose, cytokinin orGA3, variations of pH and photosynthetic photon flux density (PPFD)were investigated. In vitro flowering but not shootdevelopment of G. triflora decreased notably withincreaseddistance from the apex of the shoot, indicating the presence of a “floralgradient” in the micropropagated shoots. Conversely, as little as 0.01mg l−1 GA3 in the medium promotedshootdevelopment but even up to 0.2 mg l−1GA3 did not induce in vitro flowering.Even though BA could substitute GA3 for a high level of shootdevelopment, it also promoted a high level of in vitroflowering at the PPFD of 60 μmolm−2 s−1. Sucrose was required for shootdevelopment and flowering in vitro and higher levels ofPPFD could not compensate effectively for the omission of the sugar from themedium. In general, the effects of different concentrations of BA in the mediumor variations of pH on shoot development and flowering invitro were found to be influenced by PPFD. A novel observation isthat precocious flowering of micropropagated gentian shoots did not occur ifthey were first cultured for 5 weeks in the dark before transfer to the lightcondition.

Plant regeneration of Fraxinus angustifolia by in vitro shoot organogenesis

The in vitro regeneration of Fraxinus angustifolia through bud/shoot organogenesis on embryonic explants was investigated. Embryo axes and cotyledons from mature seeds underwent adventitious regeneration after exposure to growth regulators, the embryo axes showing a greater regenerative potential than cotyledons. While the MS medium proved effective for the induction and initiation phases, it was inadequate for bud development, and was replaced by DKW medium. A constant supply of benzyladenine (BA) appeared essential for all phases. The gelling agents used had an important role in morphogenesis: Duchefa Gelrite positively affected the number of new buds per explant. The rooting of developed shoots was easily achieved on auxin-free WPM medium and most of the rooted plantlets “hardened” after a period of acclimation in a mist greenhouse.

MICROPROPAGATION OF A SELECTED ROOTSTOCK OF ACER PALMATUM

Explants taken from axillary shoots -forced or spontaneous- of a 4-year-old selected rootstock of Acer palmatum (cultivar 'Osakii') were established in vitro on WPM medium. Subsequently, new in vitro formed shoots were subcultured and rooted. Cytokinin TDZ has proved to be useful for establishment and multiplication at a concentration of 0,01mgL-1. Adition of 0,lmgL-1 IAA conjunctly with TDZ had no effect. Two agar brands were assayed during the multiplication stage (Difco Bacto Agar and Panreac's Agar Bacteriologico Tipo Europeo) and the best results were obtained with DBA. In vitro rooting has been achieved on WPM with adition of 1,0 mgL-1 IBA.

Protocolo de micropropagação da goiabeira serrana (Acca sellowiana (Berg) Burret)

Visando ao desenvolvimento de um novo protocolo para a micropropagação da goiabeira serrana (Acca sellowiana (Berg) Burret) foram estabelecidos experimentos com segmentos nodais e microestacas. As citocininas 6-Benzilaminopurina (BAP), Cinetina (Kin) e 2-Isopenteniladenina (2-iP) foram adicionadas ao meio de cultura Woody Plant Midium-WPM, visando à proliferação de brotações múltiplas dos genótipos 53B-7, 101, 529 e 152-12 x 458. As microestacas obtidas in vitro foram submetidas a diferentes períodos de indução, em 20 miM de ácido indolbutírico (AIB) e, posteriormente, transferidas para meio de cultura isento desse fitorregulador para a indução de raízes ou, alternativamente, submetidas a diferentes concentrações e tempos de exposição em AIB e transferidas para substrato. As citocininas empregadas não promoveram aumento na taxa de proliferação de brotos em relação à testemunha. O meio de cultura basal WPM, adicionado de Kin (5 miM), proporcionou maior altura média dos brotos. Segmentos nodais do acesso 101 cultivados em meio de cultura WPM, isento de fitorreguladores, apresentaram maiores taxas médias de proliferação. Pulsos de seis dias com AIB (20 miM) induziram uma maior taxa de enraizamento (68,9%), um maior número médio de raízes (1,3 raízes) e raízes com maior comprimento médio (5,6miM). Microestacas enraizadas ex vitro, pela imersão em AIB (100 miM) por 60 minutos, resultaram em maior altura das plantas (45,3mm), número de raízes secundárias (11,3 raízes), massa fresca (1069mg) e seca das raízes (282mg).

Field and in vitro methods of propagation of the African locust bean tree (Parkia biglobosa (Jacq.) (Benth.)

SummaryStem cutting in non-mist propagators, air layering and in vitro propagation were investigated as means of vegetative propagation of Parkia biglobosa. The results of the non-mist propagation experiment show that it is possible to root single-node stem cuttings from juvenile P. biglobosa plants in non-mist propagators. The highest percentage of rooting (35%) was achieved by the application of 1000 g m±3 IBA and NAA. Propagation of single-node stem cuttings from mature trees was, however, not a success. The results from the air layering experiment indicate that it is also possible to propagate P. biglobosa by air layering. The application of hormone (200 g m±3 IBA and NAA) produced the highest percentage of rooting of air layers (16%). The results of the in vitro propagation experiment reveal that the method offers an alternative opportunity for rapid multiplication of the tree. MS medium supplemented with 6.mg.l±1 IAA was the best for shoot initiation (81.7%) whereas 4±5 mg l±1 IAA gave the highest percentage of explants with roots (8.3%). Root promotion did not correspond well with IBA in both MS and WPM media. Transplanted in vitro rooted shoots were well acclimatized in the greenhouse and all plantlets exhibited some mature characteristics including compound leaves and multiple roots.

Direct regeneration from wild pear (Pyrus syriaca) leaf explant

The effects of growth regulator, light, expiant, sugar, agar, pH and leaf age on adventitious shoot formation were tested using leaf expiants of endemic wild pear ( Pyrus syriaca ). Shoots regenerated from whole leaf and leaf sections when either adaxial or abaxial leaf surfaces were in contact with the media. Expiants placed in darkness for two or three weeks yielded regeneration frequencies of 76% and 11.8 regenerants/leaf were obtained on thiadiazuron (TDZ at 2.0 |liM) supplemented medium. When zeatin and benzyleadenine (BA) were substituted as the cytokinin source, frequencies of regeneration and shoot productivity were lower than when TDZ was used. Increasing sucrose concentration to 0. 15 M resulted in increased regeneration compared to the use of fructose and glucose. Maximum regeneration percentage (88%) and number of regenerants/leaf (13.2) were obtained on medium containing 0.15 M sucrose and 2.0 |liM TDZ. MS and WPM medium produced higher regeneration frequencies and number of regenerants/leaf than did TK, LP, NN, or B5 medium. Medium with 7 to 8 g/1 agar and myo-inositol at 50 to 100 mg/1 were optimal for regeneration. Leaves of intermediate age possessed better regeneration ability than did young and old leaves. Cells from callus and suspension cultures did not exhibit regeneration in any of the media that supported organogenesis from leaves. Regenerants were successfully micropropagated on MS medium containing 6.6 juM BA and 0.5 jliM naphthaleneacetic acid (NAA). Microshoots were rooted on medium containing 17.1 |liM indoleacetic acid (IAA) and rooted microshoots had a 95% survival rate under ex vitro acclimatization. Plantlets produced developed in the greenhouse and did not show phenotypic variation.

The role of rejuvenated and adult forms of English oak (Quercus robur) in in vitro cultures

In vitro plant material of clones (Q. robur) NL 100 A (adult) and NL 100 R (rejuvenated) received from Germany (A. Meier-Dinkel, 1995) were used in these experiments. WPM medium was used for the multiplication phase. Plantlets were subcultured monthly. Differences in quality and colour of the adult and rejuvenated cultures induced us to follow and compare the changes of mineral- and chlorophyll content and dry weight during the propagation phase. Mineral and chlorophyll content as well as dry weight were measured weekly on three samples during the subculture period.
 In the case of propagation rates we stated, they were similar around the year, but both clones had a high peak in April. Examining the cation-content, we detected that, the plantlets had a highest quantity of several elements during the 2nd and 3rd week of subculture. The iron content was the highest in the 1st week and after that it decreased continuously. It is supposed, that the content of iron is not enough in the media. The chlorophyll content of the rejuvenated clone was higher than that of the adult one.
 In the rooting experiments it was stated that, after one-week cold treatment the rooting ability was the best.

Micropropagação de caixeta, <i>Didymopanax morototoni </i>(Aubl.) Dcne. et Planch.

O presente trabalho objetivou estabelecer um protocolo básico para a micropropagação da caixeta Didymopanax morototoni (Aubl.) Dcne. et Planch. Segmentos nodais foram desinfestados em hipoclorito de sódio 10 % por diferentes periodos de exposição, e inoculados em dois meios básicos: WPM (Lloyd & McCown, 1981) e MS (Murashige & Skoog, 1962) suplementados com BAP, TDZ, ANA, GA3 , AIB e carvão ativado em diferentes combinações e concentrações para cada estádio de desenvolvimento. A desinfestação dos segmentos nodais com hipoclorito de sódio por 10 minutos proporcionou os melhores resultados para o estabelecimento asséptico das culturas. A maior taxa de regeneração e alongamento de brotações foi obtida com o meio WPM contendo 1,0 mg.l-1 de BAP. Contudo após três subcultivos neste mesmo meio observou-se redução na taxa de multiplicação. O enraizamento das brotações foi obtido no meio WPM suplementado com 1,0 mg.l-1 de AIB. O processo de aclimatização possibilitou a sobrevivência das plântulas micropropagadas às condições ex vitro.

Production of lithospermic acid B and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium rhizogenes ATCC 15834, and the transformation was proved by direct detection of the inserted T-DNA by the polymerase chain reaction. As determined by HPLC, these hairy root cultures had the ability to produce lithospermic acid B (LAB), rosmarinic acid (RA) and other related phenolic compounds, the water-soluble active components of the plant. The effect of five different basal media, MS, MS-NH<INF>4</INF> (MS without ammonium nitrate), B5, WPM and 6,7-V on the root growth and phenolic compound production was studied. It was found that MS-NH<INF>4</INF> and 6,7-V media were superior to MS, B5 and WPM media in terms of both root growth and phenolic compound production. The time course of biomass accumulation and phenolic compound formation was also examined in the culture using MS-NH<INF>4</INF>medium. During cultivation, the content of RA in the roots was stable being approximately 0.48% of dry weight while the content of LAB fluctuated between 0.73% and 1.61% of dry weight, and decreased gradually at the stationary phase of growth. The highest production of LAB and RA was about 64 mg L−1 and 23 mg L−1, respectively.

Micropropagation of Camptotheca acuminata

Camptotheca acuminata (Chinese happy tree, Nyssaceae) is a source of the anticancer phytochemical, camptothecin. An endangered species in its native China, Camptotheca has been grown in this country on an experimental basis, but the germplasm base is extremely narrow. As a prelude to the establishment of a plant improvement effort designed to increase the efficiency of camptothecin production, in vitro studies that will enable plant regeneration and shoot proliferation from selected clones have been undertaken. Shoot proliferating cultures were established from shoot tip explants and were maintained on WPM medium containing 4 μm BA. MS medium and nodal explants proved unsatisfactory. Shoot proliferation was highest when in vitro shoot tips were cultured on 4 μm BA compared to media containing no growth regulator or the cytokinins zeatin, thidiazuron, or kinetin. In vitro—produced shoot tips were rooted by direct sticking in plastic containers filled with RediEarth mix, and were successfully adapted to the greenhouse environment.

Indução in vitro de raízes adventícias em explantes de salix (Salix humboldtiana Willdenow)(Salix humboldtiana Willdenow)

With the objective to investigate the in vitro behavior of salix (Salix humboldtiana Willdenow), leaf and nodal segments were inoculated in WPM medium supplemented with NAA and IBA. In leaf explants, the presence of these growth regulators, isolated or in combination, induced the formation of adventicious roots. Root hairs were only observed when NAA was present. In nodal explants, while the combination of 2,68 µM ANA + 2,46 µM IBA induced bud growth, the other treatments (4,92 µM IBA; 2,68µM ANA + 4,92 µM IBA; 5,37 µM ANA + 4,92 µM IBA; 5,37µM ANA) induced the formation of adventicious roots.

Somatic Embryogenesis and Regeneration of Plantlets In Common Oak (Quercus Robur L.)

ABSTRACTImmature embryos of common oak (Q. robur L.) were used as initial explants for inducing the process of somatic embryogenesis. The best embryogenic response (53.3%) was registered on MS medium supplemented with either 1.0 mg/l BAP alone or in combination with 1.0 mg/l GA3. The embryos collected during the first week of July demonstrated highest embryogenic competence that rapidly decreased and got lost by the end of the period tested. Embryogenic cultures could be easily maintained in darkness by regular transfers on hormone-free MS medium. Some mature somatic embryos successfully germinated after being transferred to WPM medium with 0.2 mg/l BAP. Such embryos showed further development and converted into plantlets.

In vitro regeneration of Norway maple (Acer platanoides L.)

Regenerants were produced from axillary buds, but not from petiole segments, greenwood cuttings and leaf discs. Petiole segments and greenwood cuttings responded by massive callus cell proliferation without adventitious shoot formation. The development of induced buds into shoots occurred on WPM medium containing kinetin. Vigorous shoots larger than 2.0 cm in length were successfully rooted in half strength WPM medium supplemented with 1.0 mg dm-3 indole-3-butyric acid.

Generation and Identification of New Viburnum Hybrids

Abstract The controlled crossing of fragrant flowered viburnums (V. carlesii and its hybrids) with the hardier, rugose foliaged V. lantana is unattainable through traditional breeding methods due to incompatibilities within the developing seed. Hybrid plants from these crosses have now been obtained using embryo rescue techniques. Embryos were removed from developing seed 17 days following pollination and placed on WPM medium to promote continued maturation. Genotypes from two crosses, V. lantana ‘Mohican’ x V. carlesii ‘Aurora’ and V. lantana ‘Mohican’ x V. x juddii, were obtained and verified using RAPD markers. These plants are presently being evaluated for ornamental characteristics and cold hardiness.