In Vitro Organogenesis from Root Explants of Passiflora miniata Mast, an Amazonian Species with Ornamental Potential

ABSTRACT The present study reports a shoot organogenesis-based system for in vitro regeneration of Passiflora miniata, an Amazonia passion fruit species. Root segments were cultured in Murashige and Skoog (MS) medium supplemented with different concentrations (range 2-9 µM) of 6-benzyladenine (BA); thidiazuron (TDZ) or kinetin (KIN). Plant growth regulators were not added to the control treatment. Root explants have showed a high regenerative potential. After 30 days of in vitro culture, the root explants showed several shoots formed direct and indirectly. TDZ provided the best response in the differentiation adventitious shoots, mainly in the presence of 6.8 µM. The cytokinins BA and KIN responded producing a reduced number of shoots. After 120 days, rooted regenerated plants were transferred to a greenhouse for acclimatization. This regeneration system opens new perspectives for micropropagation and conservation of this wild Amazonic passion fruit species.

Regeneración in vitro de plantas de piña ( Ananas comosus ) ecotipo amazónico Gobernadora / In vitro regeneration of Amazonian pineapple ( Ananas comosus ) plants ecotype Gobernadora

Resumen En Venezuela existen cultivares y ecotipos de piña (A. comosus) de importancia local, entre ellos los amazónicos, cultivados principalmente por los aborígenes Piaroa. Ellos siembran los propágulos lo cual restringe la disponibilidad de material para el cultivo a gran escala. Se abordó la limitación recurriendo al cultivo de tejidos vegetales para la propagación in vitro de plantas de piña, ecotipo amazónico Gobernadora, mediante embriogénesis somática (ES) y organogénesis adventicia (OA). El material vegetal empleado correspondió a secciones basales e intermedias de hojas. Sólo las secciones de base foliar (SBF) fueron morfogénicamente inducidas. El mayor número de vitroplantas (1,58 plantas/explante) se obtuvo del callo embriogénico inducido en medio MS con Picloram 10 mg.L-1 + Tidiazuron 2 mg.L-1, transferido a MS sin hormonas. En el proceso organogénico, se obtuvo el mayor número de plantas/explante (5) por vía directa en MS con ácido naftalenoacético 5 mg.L-1 + bencilaminopurina 0,25 mg.L-1, transferido a MS. Siendo este último el mejor sistema de cultivo in vitro por su productividad y por ser una ruta que minimiza la variación somaclonal.

Abstract There are a number of pineapple (Ananas comosus) cultivars and ecotypes of local commercial importance in Venezuela, among them the Amazonian ones, cultivated mainly by the aboriginal Piaroa, are of relevance. They sow the propagules, which restricts the availability of material for large-scale cultivation. This limitation was approached by plant tissue culture for in vitro propagation of Amazonian pineapple plants, Gobernadora ecotype, through somatic embryogenesis (ES) and adventitious organogenesis (OA). Basal and intermediate sections of leaves were tested. Only the leaf base sections (FBS) were morphogenically induced. The highest number of vitroplants (1.58 plants / explant) was obtained from the embryogenic callus induced in MS medium with Picloram 10 mg.L-1 + Thidiazuron 2 mg.L-1, transferred to MS medium without hormones. In the organogenic process, the highest number of plants / explants (5) was obtained directly in MS with naphthaleneacetic acid 5 mg.L-1 + benzylaminopurine 0.25 mg.L-1, transferred to MS. The latter being the best in vitro culture system due to its productivity and for being a method that minimizes somaclonal variation.

Establecimiento in vitro de dos especies nativas de Costa Rica: Terminalia amazonia (Amarillón) y Vochysia allenii (Botarrama Blanco) / In vitro establishment of two species native to Costa Rica: Terminalia amazonia (Amarillón) and Vochysia allenii (Botarrama Blanco)

El objetivo de esta investigación fue lograr el establecimiento in vitro de la especie Terminalia amazonia y Vochysia allenii debido a la dificultad de propagarlas sexual y asexualmente con técnicas convencionales. Se logró establecer segmentos nodales de ambas especies en condiciones in vitro empleando el HgCl2 0,1 % con un tiempo de exposición de 5 minutos. El mejor medio de cultivo para nudos fue el WPM 100 % de sales para T.amazonia y para V. allenii fue el WPM 50 % de sales. Después de 28 días de cultivo se obtuvo un 42 % de nudos establecidos para T. amazonia y 10% para V. allenii. En ambas especies se evaluó el efecto sobre la brotación de cinco concentraciones de 6-bencilaminopurina (6-BAP) (0,0; 2.22, 4.44, 6.66, 8.88 μM L-1) y cinco de tidiazuron (TDZ) (0,0; 0.22, 0.45, 0.68, 0.90 μM L-1). Se obtuvó en promedio un brote por explante en los cinco tratamientos de BAP y TDZ utilizados.

The objective of this research was to achieve the in vitro establishment of the species Terminalia amazonia and Vochysia allenii due to the difficulty propagate sexually and asexually with conventional techniques. It was possible to establish the nodal segments of both species in in vitro conditions using 0.1 % HgCl2 with an exposure time of 5 minutes. The best nodal culture medium was 100 % WPM salts in T. amazonia and V. allenii was WPM 50% salts. After 28 days of culture 42 % of nodal segments to T. amazonia and V. allenii 10% was obtained. In both species, the effect on sprouting of five concentrations of 6-benzylaminopurine (6-BAP) (0.0, 2.22, 4.44, 6.66, 8.88 μM L-1) and five thidiazuron (TDZ) (0.0, 0.22, 0.45, 0.68, 0.90 μM L-1) was evaluated. Outbreak was scored on average per explant in the five treatments of BAP and TDZ used.

The Effect of Colchicine on Adventitious Shoot Regeneration from Cultured Leaf Explants of Petunia hybrida.

Aims: The effect of various concentrations of colchicine on adventitious shoot regeneration of two Petunia hybrid cultivars was studied. Study Design: All collected data were analyzed using the SAS software. One-way analysis of variance of the treatments was conducted for each experiment followed by means separation using the least significant difference (LSD) at 5% probability level. Place and Duration of the Study: The study was conducted during January 2012 to December 2012 at the Faculty of Agriculture, An-Najah National University, Palestine. Methodology: In vitro leaf explants of both ‘Daddy Blue’ and ‘Dreams White’ cultivars were used. Before incubation on the shoot regeneration medium, the leaves were cut transversely into two pieces and were soaked for three hours in different concentrations of colchicine solution (0.025, 0.05 and 0.1% w/v), colchicines application was done at zero incubation time and after 1, 2 or 3 weeks of culture incubation. Wet control was used by soaking explants in sterile distilled water. Results: The ability to form shoots was highly reduced with colchicine application. Significant reduction in the regeneration frequency was observed with the two cultivars. Shoot regeneration percentage was reduced from 78.3% and 90.0 % without colchicine to 13.3% and 5.2% when colchicine was added after two weeks of incubation at 0.025% in Daddy Blue’ and ‘Dreams White’, respectively. The colchicine treatments at different intervals gave significantly lower regeneration percentages than the control in both cultivars. No regeneration was observed when colchicine was added after three weeks of incubation, and at 0.1% colchicine level at all incubation treatments. A similar trend of average number of shoots produced was observed. Conclusion: This study showed a high reduction in regeneration percentage due to colchicine application with slight difference in response between the two tested cultivars; therefore, other evaluation methods are needed in the future.

An efficient method in breaking of dormancy from Bunium persicum (Boiss) Fedtsch seeds:a valuable herb of Middle East and Central Asia

Objective: To develop a protocol for breaking of seed dormancy and increasing the seed germination rate of Bunium persicum. Methods:The seeds were treated with 3.1, 6.3, 12.5, 25, 50 and 100 μmol/L of benzyl aminopurine, gibberellic acid (GA3), thidiazuron (TDZ) and forchlorfenuron. Then, seeds were transferred to two different temperature conditions including room temperature (25 °C) and chilling temperature (2-5 °C). Results: The treatment of moist seeds with chilling temperature (2-5 °C) broke seed dormancy and showed maximum germination, which was 54.7%after 60 d treatment. Also, the treatment of dry seeds with chilling temperature broke seed dormancy with 9.3%germination rate after 120 d. Treatment of seeds with different level of plant growth regulators showed that under moist-room condition, there was evidence of higher and lower seed germination rate:GA3 (100 μmol/L) with 46.7%and TDZ (50 μmol/L) with 6.67%respectively. In addition, the results showed that under moist-chilling condition, TDZ (6.3 μmol/L) with 53.3%seed germination rate had higher influence on breaking seed dormancy. Treatment of seeds with combination of TDZ and GA3 under moist-chilling condition revealed higher rate of breaking of seed dormancy when 6.3 μmol/L TDZ was combined with 100 μmol/L GA3, showing 93.7%germination rate. Conclusions:The effect of plant growth regulators coupled with chilling temperature on breaking of seed dormancy could provide a large number of seedlings while the long juvenile time which is the next restricting factor of plantation still remained. Thus, the subsequent growth of seedlings to provide a large number of corms is necessary for successful plantation.

An efficient method in breaking of dormancy from Bunium persicum (Boiss) Fedtsch seeds: a valuable herb of Middle East and Central Asia

<p><b>OBJECTIVE</b>To develop a protocol for breaking of seed dormancy and increasing the seed germination rate of Bunium persicum.</p><p><b>METHODS</b>The seeds were treated with 3.1, 6.3, 12.5, 25, 50 and 100 µmol/L of benzyl aminopurine, gibberellic acid (GA3), thidiazuron (TDZ) and forchlorfenuron. Then, seeds were transferred to two different temperature conditions including room temperature (25 °C) and chilling temperature (2-5 °C).</p><p><b>RESULTS</b>The treatment of moist seeds with chilling temperature (2-5 °C) broke seed dormancy and showed maximum germination, which was 54.7% after 60 d treatment. Also, the treatment of dry seeds with chilling temperature broke seed dormancy with 9.3% germination rate after 120 d. Treatment of seeds with different level of plant growth regulators showed that under moist-room condition, there was evidence of higher and lower seed germination rate: GA3 (100 µmol/L) with 46.7% and TDZ (50 µmol/L) with 6.67% respectively. In addition, the results showed that under moist-chilling condition, TDZ (6.3 µmol/L) with 53.3% seed germination rate had higher influence on breaking seed dormancy. Treatment of seeds with combination of TDZ and GA3 under moist-chilling condition revealed higher rate of breaking of seed dormancy when 6.3 µmol/L TDZ was combined with 100 µmol/L GA3, showing 93.7% germination rate.</p><p><b>CONCLUSIONS</b>The effect of plant growth regulators coupled with chilling temperature on breaking of seed dormancy could provide a large number of seedlings while the long juvenile time which is the next restricting factor of plantation still remained. Thus, the subsequent growth of seedlings to provide a large number of corms is necessary for successful plantation.</p>

An efficient method in breaking of dormancy from Bunium persicum (Boiss) Fedtsch seeds: A valuable herb of Middle East and Central Asia

Objective: To develop a protocol for breaking of seed dormancy and increasing the seed germination rate of Bunium persicum. Methods: The seeds were treated with 3.1, 6.3, 12.5, 25, 50 and 100 μmol/L of benzyl aminopurine, gibberellic acid (GA

In Vitro Regeneration from Shoot Tip and Nodal Explants of Simarouba Glauca DC, a Promising Biodiesel Tree.

An efficient regeneration protocol was developed from shoot tip and nodal explants of Simarouba glauca DC, a promising biodiesel plant. Nodal explants appeared to have better regeneration capacity than shoot tip explants (40%) in the tested media. The highest regeneration frequency (90%) and shoot number (7.00 ± 1.00 shoots per explants) were obtained in nodal explants in Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine (BAP) 4.43 μM and α-naphthalene acetic acid (NAA) 5.36 μM.Induced shoot buds were multiplied and elongated on the MS medium supplemented with BAP (4.44 μM), NAA (5.36 μM) and TDZ (Thidiazuron) 2.27 μM with 9.66±0.33 (mean length 5.35±0.32 cm) and 9.00±0.57 (mean length 4.51±0.15cm) shoots using nodal segments and shoot tip explants, respectively. Halfstrength woody plant medium (WPM) containing 2.46μM indole-3-butyric acid (IBA) produced the maximum number of roots (6.00±1.15). The rooted plantlets were hardened on MS basal liquid medium and subsequently in polycups containing sterile soil and vermiculite (1:1) and successfully established in pots.

Micropropagation and shoot regeneration of Cistus creticus ssp. Creticus.

Cistus creticus L. ssp. creticus is a medicinal aromatic shrub native in Crete (Greece). The protocol described in this paper provides optimal levels of growth regulators required to obtain high regeneration rates of Cistus in vitro. Micropropagation has been achieved through rapid proliferation of shoot-tips on Murashige and Skoog (MS) basal medium supplemented with 0.2 mg l-1 6-benzylaminopurine (BAP). After four weeks shoots were transferred to MS medium without growth regulators for further development and rooting. The highest percentage of regenerated shoots was obtained with 0.1 mg l-1 TDZ and 0.1 mg l-1 NAA after 4 weeks. Elongation and rooting was readily achieved when multiple shoots more than 1 cm in length were singled out and cultured on the MS medium without growth regulators. The plantlets were successfully adapted and grew vigorously in greenhouse conditions. This is the first report of shoot regeneration in the genus Cistus. The regeneration protocol developed in this study provides a basis for further investigation of the medicinally active constituents of this elite medicinal plant.

Aumento da produção de pereira asiática pelo uso de fitorreguladores / Fruit production increase in Asian pear trees by use of plant growth regulators

A baixa frutificação de pereiras nas condições do Sul do Brasil é um dos limitantes à produção da cultura no país, podendo o problema ser minimizado pelo uso de fitorreguladores. Objetivou-se avaliar a efetividade de diferentes fitorreguladores e a combinação dessas substâncias no aumento da frutificação de pereira asiática 'Shinseiki. A aplicação dos fitorreguladores foi realizada quando as pereiras atingiram o estádio de plena floração. O delineamento experimental foi em blocos casualizados, com quatro repetições de uma planta, sendo avaliados os seguintes tratamentos: 1. testemunha (sem aplicação); 2. thidiazuron (TDZ) 20mg L-1; 3. ácido giberélico (AG) 20mg L-1; 4. proexadione cálcio (PCa) 600mg L-1; 5. PCa 600mg L-1 + TDZ 20mg L-1; 6. PCa 600mg L-1 + AG 20mg L-1; e 7. AG 20mg L-1 + TDZ 20mg L-1. Os fitorreguladores thidiazuron e ácido giberélico, e a combinação dessas substâncias, ambos na concentração de 20mg L-1, quando aplicados na floração, determinam aumento significativo da frutificação e da produção de peras 'Shinseiki'. A utilização do proexadione cálcio na floração, tanto isoladamente quanto associado ao thidiazuron e ao ácido giberélico, não repercutiu no aumento da frutificação.

The low fruit set in the Southern Brazil conditions is one of the limiting factors to pear production in the country. The use of plant growth regulators may minimize this problem. This study was carried out aiming to evaluate the effectiveness of different growth regulators and the combination of these substances on fruit set increase of 'Shinseiki Asian pears. The application of growth regulators was performed when the pears have reached the full bloom stage. The experimental design was randomized blocks with four replications of one tree, with the following treatments: 1. control (no application); 2. thidiazuron (TDZ) 20mg L-1; 3. gibberellic acid (GA) 20mg L-1; 4. prohexadione calcium (PCa) 600mg L-1; 5. PCa 600mg L-1 TDZ + 20mg L-1; 6. PCa 600mg L-1 + AG 20mg L-1; and 7. AG 20mg L-1 + TDZ 20mg L-1. When sprayed on full bloom stage, thidiazuron and gibberellic acid, and combinations of these substances, both at a concentration of 20mg L-1, increased significantly the fruit set and the fruit production of 'Shinseiki' pears. The use of prohexadione calcium, single or in combination with thidiazuron and gibberellic acid, did not increase the fruit production.

Cytokinin preconditioning enhances multiple shoot regeneration in Pongamia pinnata (L) Pierre - a potential, non-edible tree seed oil source for biodiesel

An efficient, highly reproducible protocol for multiple shoot induction and plant regeneration of Pongamia pinnata has been successfully developed using cotyledonary node explants. This study also demonstrates that preconditioning of explant stimulates production of multiple shoots from cotyledonary nodes of P. pinnata. The highest direct shoot regeneration (90 percent) with an average of 18.4 +/- 3.1 shoots/explant were obtained when cotyledonary node explants were excised from seedlings germinated on Murashige and Skoog (MS) media supplemented with benzyladenine (BA) 1 mg l-1, and subsequently cultured on MS media with 1 mgl-1 thidiazuron (TDZ). Scanning electron microscope observations of cotyledonary node (CN) explants excised from pre-conditioned and normal seedlings, revealed larger buds with rapid development in BA-preconditioned CN explants. The addition of adenine sulphate significantly increased the average number of shoots per explant. The highest direct shoot regeneration (93 percent) with an average of 32.2 +/- 0.93 shoots/explant was obtained from BA-preconditioned CN when cultured on MS media supplemented with 1 mg l-1 TDZ and 200 mg l-1 adenine sulphate (ADS). Repeated shoot proliferation was observed from BA preconditioned CN explants up to 3 cycles with an average of 15 shoots/explant/cycle when cultured on MS media supplemented with 1 mg l-1 TDZ and 150 mg l-1 L-glutamine, thus producing 45 shoots/CN explant. Shoots were elongated on hormone free MS media and rooted on 1/2 MS media supplemented with 1 mg l-1 of IBA. Rooted shoots were successfully acclimatized and established in soil with 80 percent success. The highly regenerative system developed in this investigation for this important tree could be a useful tool for genetic transformation.

Efeito de thidiazuron na indução e manutenção de calos de erva-baleeira (Cordia verbenacea L) / Effects of thidiazuron on callus induction and maintenance of Cordia verbenacea L

O trabalho teve como objetivo avaliar a influência do Thidiazuron (TDZ) na formação e manutenção de calos de Cordia verbenacea. Segmentos caulinares com 3 a 4cm de comprimento provenientes de mudas cultivadas em casa de vegetação foram desinfestados em solução contendo hipoclorito de sódio comercial a 30 por cento e duas gotas de detergente por 10 minutos, sendo 5 minutos em agitação. Posteriormente, foram excisados e inoculados com 5mm de tamanho em meio sólido a 0,6 por cento de Murashige & Skoog (MURASHIGE & SKOOG, 1962) - MS, complementado com 0,22; 0,68: 2,04 e 6,l3uM de TDZ. A formação de calos ocorreu em todos os tratamentos com 10 a 15 dias de cultivo Os tratamentos que apresentaram o explante com a maior área coberta com calos, em média 75 por cento, foram 2,04 e 6,13miM de TDZ. Somente nesses tratamentos os calos aumentaram de volume e mantiveram a coloração inicial quando transferidos para o meio fresco de cultura. Os tratamentos com menores concentrações de TDZ apresentaram explantes com 50 por cento da área coberta com calos e quando transferidos para o meio fresco de cultura ocorreu a paralisação do crescimento com a posterior morte dos calos.

The influence of thidiazuron on calius formation and maintenance of Corola verbenacea was evaluated. Stem segments with 3 to 4cm length were excisedfrom greenhouse plants and surface sterilized with 30 percent commercial sodium hypochiorite for 10 minutes. Explants were inoculated with 5mm on Murashige and Skoog (MVRASHIGE & SKOOG, 1962) - MSsolid médium (0.6 percent) suppiemented with 0.22, 0.68, 2.04 and 6.13muM TDZ. Calius induction was observed 10 to 15 days after incubation. The media contaming, 2.04 and 6.13uM TDZ induced 75 percent expiam with calius. The calius when transferred to fresh culture médium exhibited active growth. The treatments containing lower concentration induced 50 percent expiam with caltus that when transferred to fresh culture médium, showed no development.