Efficient propagation with in vitro seed germination of Vanda falcata / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Vanda falcata (Thunb.) Beer (Orchidaceae), a famous native orchid of China, Japan, and Korea, is known as one of the most beautiful and charming orchid species in the world (Ohwi, 1965; Lawler, 1984; Arditti, 2008). V. falcata is widely cultivated and delights the world with its compact plant shape, elegant white blooms, and sweet coconut-like scent. However, vegetative propagation by division has limited the development of V. falcata because of its inefficiency (Mitsukuri et al., 2009a, 2009b).

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R. Br. ex Sm

BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and antidiabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

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.

Callus induction and establishment of plant regeneration system of Saposhnikovia divaricate / 中草药

Objective: To establish the regeneration system of Saposhnikovia divaricate (Turcz.) Schisck. Methods: The effects of different explants, temperature, medium, and illumination was studied on the induction of S. divaricate. Response surface test design was used to optimize the hormone ratio of callus induction and proliferation. Results: The leaves of S. divaricate were used as the experimental materials, the optimal medium for the callus induction was MS + 6-BA 0.90 mg/L + 2,4-D 1.05 mg/L + KT 0.96 mg/L, with temperature of 25 ℃ and light of 12 h/d. The optimal medium for callus proliferation was MS + 6-BA 0.56 mg/L + 2,4-D 0.48 mg/L + KT 0.40 mg/L, and the subculture cycle was 35 d. The optimal medium for adventitious shoots was MS + NAA 0.6 mg/L + 6-BA 1.0 mg/L. The optimal medium for root growing was MS + NAA 0.4 mg/L + 6-BA 0.1 mg/L. Conclusion: S. divaricate callus was successfully induced and a stable and efficient plant regeneration system was established.

Tissue culture of callus and establishment of regeneration system of Tussilago farfara petiole / 中国中药杂志

Young petiole of Tussilago farfara was used as the material to investigate the plant growth regulators which could influence in vitro culture and plant regeneration and to establish rapid propagation technique. The ideal sterilization method was that young petiole of T. farfara was sterilized with 75% ethanol for 30 s, and then transferred to saturated bleaching power supernatant for 15 min. The suitable medium for callus induction was MS+6-BA 3.0 mg•L⁻¹+2,4-D 2.0 mg•L⁻¹ with 96.2% induction rate. The seedlings had better differentiation with 91% differentiation rate and 8.26 buds on the medium containing MS+ZT 2.0 mg•L⁻¹+NAA 0.3 mg•L⁻¹. The preferred enrichment medium of adventitious bud was MS+KT 1.0 mg•L⁻¹+IBA 0.3 mg•L⁻¹ with 11.81 enrichment times and 4.9 cm seedling height. The rooting medium included 1/2MS+IBA 0.2 mg•L⁻¹ with the average number of rooting was 5.86 and the rooting rate was above 95.22%. The container seedlings can grow well and the survival rate was more than 90% when they were transplanted on the medium added with river sand and organic fertilizer with the ratio of 3∶1. The field experiments indicated that significant differences in increment and yield of pollen grains among the tissue-culture, cultivation and wild type of T. farfara under the same cultivation conditions. The cultivated plants were relatively high on the increment and yield of pollen grains. The active ingredient content of the tissue culture and the wild materials was basically the same.

Phytochemical and Morphological Evidences for Shikonin Production by Plant Cell Cultures of Onosma sericeum Willd

ABSTRACT Shoot regeneration, callus growth, and biosynthesis of shikonin in callus cultures of Onosma sericeum were examined. Plant tissue culture was used as an alternative method for increasing the production of shikonin, a secondary metabolite. The isolated cultures were subjected to abiotic factors such as light, plant growth regulators, and nutritional factors. Identification was carried out by High- Performance Liquid Chromatography (HPLC) after 10th subculture. Nodal explants were incubated in Murashige and Skoog (MS) medium along with different combination of growth hormones. Shoot regeneration from calli were achieved on MS basal medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l Naphthalene acetic acid (NAA) under light cycle. Shikonin was formed in dark culture. Calli grown on MS (ammonium ion-free) medium supplemented with 3 mg/l BAP and 0.5 mg/l NAA contained the maximum shikonin level (15.26 µg/mg DW). Minimum shikonin content (9.85 µg/mg DW) was observed in calli cultured on MS (ammonium ion-free) medium supplemented with 3 mg/l BAP and 0.5 mg/l indole-3-acetic acid (IAA). In establishing cell culture, the ammonium ion, and light cycle inhibited shikonin formation. This is the first report on the establishment of isolated cultures of O. sericeum for shikonin production and callus growth.

In Vitro Propagation of Chamomilla Recutita From Capitulum Inflorescence: A Medicinal Plant With Multiple Therapeutic Applications

A protocol has been developed for induction of somatic embryogenesis from whole inflorescence explants of Chamomilla recutita L. (chamomile). Chamomile is a well-known medicinal plant from the Asteraceae family often referred to as the “star among medicinal species.” Nowadays, it is a highly favoured medicinal plant in folk and traditional medicine. Its multitherapeutic, cosmetic and nutritional values have been established through the years of traditional and scientific use and research. Chamomile has an established domestic (Indian) and international market, which is increasing day by day. Among the various major constituents, α-bisabolol and chamazulene have been reported to be more useful than others. Chamazulene occurs in the capitula of the flowers in minute quantities and has been demonstrated to exert antiinflammatory activity in-vivo. Moreover, chamomile is a seasonal 4-5 months winter crop in India but is extensively required in various medicinal applications. Therefore, to increase the overall yield of this plant, its in-vitro propagation is needed. In the present study, somatic embryos were developed from capitulum explants after 2-4 weeks of culture on MS medium supplemented with 26.8 µM NAA and 11.5 µM Kin. The somatic embryos were further subcultured in-vitro, where new plantlets regenerated from embryos. It is concluded that in-vitro propagation is possible in case of chamomile and can be used to increase the overall yield of chamazulene present in the capitula of flowers as well as augment the overall yield of this important plant, which is conventionally propagated by seeds.

Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Stevia rebaudiana.

In order to develop a high-efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum, incubation time, plant material type and age were studied. Protoplasts were successfully isolated from leaves of fourweek- old in vitro grown plants using an enzyme mixture comprising of 2% (w/v) Cellulase Onozuka R-10, 1.5% (w/v) Macerozyme Onozuka R-10, 0.2% (w/v) Driselase and 0.1%(w/v) Pectolyase Y- 23 in 0.5 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES ) at pH of 5.8. Approximately 8.4±0.40x106 protoplasts g-1fresh weight with 98.8±1.39% viability was obtained after incubating in enzyme solution for 4 hours in dark. Viable protoplasts were collected by centrifugation in the presence of 16% sucrose solution. Protoplasts at density of 5x105 mL-1were cultured on modified KM8P medium supplemented with 0.2 mg L-1 2,4-dicholorophenoxyacetic acid (2,4-D), 1 mg L-1 α-naphthalene acetic acid (NAA), 0.5 mg L-1 zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead or thin layer liquid culture technique. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days and microcolonies were formed within 4 weeks. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Compared to liquid culture, agarose bead culture improved division frequency almost 1.5 times effectively and showing a plating efficiency of 13% and 9.1% respectively with survival rate of 23.5% to 14.8%. Upon transfer to Murashige and Skoog’s medium (MS) with 1 mg L-1BA, alone or in combination with NAA or 2, 4-D at 0.1 mg L-1, protoplast-derived calli produced complete plantlets through somatic embryogenesis in 8-weeks. The regenerated plants survived in soil and all were normal with respect to morphology and growth characters. This protocol might lead to the improvement of the Stevia through somatic hybridization, somaclonal variation and genetic engineering by using protoplast based regeneration System.

In vitro Callus Induction and Plant Regeneration of Celosia argentea- An Important Medicinal Plant

Celosia argentea (Var.) cristata (Amaranthaceae) is a widely cultivated ornamental plant, which has antibacterial, astringent, haemostatic, hypertensive, ophthalmic, and parasitic significance. This study describes a protocol for in vitro callus induction and plant regeneration from leaf and stem explants of C. argentea using Murashige and Skoog (MS) medium. Callus culture was initiated and established from seedling, leaf, and stem explants. Explants were cultured on MS medium supplemented with auxin alone (0.5 mg/L Naphthaleneacetic acid (NAA), 2, 4-Dicholorophenoxyacetic acid (2, 4-D). Green and red compact callus (98%) were induced using MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L Benzyladenine (BA). Two different concentrations (1.0 mg/L BA + 0.5 mg/L NAA and 1.0 mg/L BA + 1.0 mg/L NAA) successfully induced plant regeneration with multiple shoots (1.5 and 0.9 shoots per explants, respectively). Successful shoots were transferred to rooting medium supplemented with 1.0 mg/L Indole-3-acetic acid (IAA) (80%) at 35th day. Acclimatization was done, which resulted in 90% of the plantlets surviving in garden soil. This protocol could be used to micropropagate C. argentea for conservation, commercial natural product production. The high frequency of callus indicated potential of C. argentea for secondary metabolite production (celosin) in pharmaceutical industry.

Pattern of Plant Regeneration from Shoot Tip Explants of Pigeonpea (Cajanus Cajan L Milllsp) var LRG-41.

An efficient direct shoot bud differentiation and multiple shoot induction from shoot tip explants of pigeon pea (Cajanus cajan L.) has been achieved. The frequency of shoot bud regeneration was influenced by the type of explants, genotype and concentrations of cytokinin. Explants viz. shoot tip isolated from 10 day old seedlings showed better explants response Explants were cultured on Murashige and skoog (MS) medium augmented with different concentrations of BAP and NAA. Among the various concentrations tested, 2.0mg/l BAP (Benzyl amino purine) and 0.1 mg/l Napthalene acetic acid (NAA) were found to be the best for maximum shoot bud differentiation. Percentage, as well as the number of shoots per explant showing differentiation of shoot buds was higher on MS media supplement with BAP and optimal BAP concentration for shoot regeneration was 2mg/l. The elongated shoots were successfully rooted on MS medium containing different concentrations of auxins. Among them indole buteric acid (IBA) at 1.0mg/l induced maximum frequency of rooting. Regenerated plants were successfully established in soil where 91% of them have been developed into morphologically normal and fertile plants. This method can thus be advantageously applied in the production of transgenic pigeon pea plants.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant / Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.

The Effect of Picloram on Somatic Embryogenesis of Different Explants of Strawberry (Fragaria ananassa Duch.).

The present investigation was conducted to study the effects of different concentrations of picloram on somatic embryogenesis induction, development and maturation of three strawberry (Kurdistan, Paros and Camarosa) cultivars. For this purpose, leaf blade, nodal, petiole, stamen and flower bud calli were cultured on MS medium supplemented with picloram at 0.25, 0.5, 1 and 2 mg/L concentrations. The concentration of growth regulator, cultivar and explant type were found critical to somatic embryogenesis induction, development and maturation. Results obtained from the studies revealed that all explants with the exception of petiole and stamen incubated on medium formed embryonic calli. 2 mg/L picloram yielded the highest percentage of embryonic calli and number of globularstage embryos and 1 mg/L picloram yielded the highest number of cotyledonary-stage embryos in all types of explants. The leaf explant calli and Paros cultivar were the most responsive to produce somatic embryogenesis induction, development and maturation.

Plant regeneration from inflorescence cultures of Gynandropsis pentaphylla DC.

An efficient protocol for shoot regeneration from immature inflorescence of Gynandropsis pentaphylla DC (Capparidaceae) is described. Healthy inflorescences were cultured on MS medium supplemented with BAP, Kn, zeatin, TDZ (0.5 - 5.0 mg/l) alone and in combination with IAA, NAA, IBA (0.5 - 2.0 mg/l). BAP (3.0 mg/l) with IBA (1.0 mg/l) as well as Kn (1.0 mg/l) with TDZ (0.1 mg/l) were found to be effective in inducing callus and in production of shoots and roots. The present investigation also describes the histological studies depicting various stages that occurred during the development of embryoids and shoot buds. The species is so potential that the flower buds on the medium induces callus from each part of the flower bud. All the in vitro raised shoots were transferred to MS rooting liquid medium supplemented with 0.5 - 1.0 mg/l IAA, NAA and IBA. Well rooted plantlets were transferred to polycups containing soil : vermiculite (1:1) for hardening. Finally the hardened plantlets were transferred to field conditions for maximum survivability. The flower is mixoploid and hence the regenerated roots were squashed in acetoorcein to know the ploidy level of the regenerated shoots. All the plants regenerated were shown to be diploid.

Direct and indirect method of plant regeneration from root explants of Caesalpinia bonduc (L.) Roxb.— A threatened medicinal plant of Western Ghats.

An in vitro regeneration protocol has been standardized via direct and indirect methods from excised root explants of C. bonduc, a threatened woody legume used for the treatment of contagious diseases, inflammation, leprosy, antiperiodic, febrifuge, anthelmenthic, urinary disorders, leucorrhoea, piles and to heal wounds. MS medium supplemented with 17.75 µmol BAP and 2.46 µmol IBA, induced a mean of 3.40 ± 1.07 shoots directly from the surface of excised root explant. Subsequently, the shoots rooted readily on MS half strength medium with out growth regulators. In indirect organogenesis, callogenic frequency was optimized (96.66%) at the concentration of 9.04 µmol 2, 4-D and 0.88 µmol BAP. An average, 15.30 ± 5.25 shoots were differentiated from the root callus at the concentration of 17.57 µmol BAP and 2.85 µmol IAA. Shoots regenerated through callus were rooted well on MS half strength medium with growth regulators at 2.95 µmol IBA. Rooted plantlets were transferred to the pots containing sterilized soil and were successfully hardened at greenhouse condition for three weeks then exposed to the natural environment. Survival rate was more (95%) in plantlets derived through direct organogenesis than (60%) the plantlets regenerated through root calli.

Callus induction and plant regeneration of Ulex europaeus

A callus induction and plant regeneration protocol was developed from leaf and thorn explants for the plant Ulex europaeus. Explants were incubated on 2 percent sucrose half-strength Murashige and Skoog Medium (MS) with various combinations of plant growth regulators and antioxidants. The best frequency of callus and shoot formation was obtained with 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/l x kinetin (Kin) 0.2 mg/l (DK Medium; callus induction) and zeatin (Z) 1 mg/l (DK medium; shoot induction). Both media were supplemented with ascorbic acid 200 mg/l to prevent browning and death of the explants. The regenerated shoots transferred to rooting medium (half-strength MS Medium, 2 percent sucrose) showed rapid growth and development of roots (100 percent). Rooted plantlets were successfully transferred to soil in pots containing a 3:1 mixture of soil and vermiculite.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective: To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants. Methods: Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3-4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration.Results:Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field. Conclusions: The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.

Callus induction and plant regeneration of the endemic Astragalus nezaketae in Turkey

A callus induction and plant regeneration protocol was developed from leaf and petiole explants of the endemic Astragalus nezaketae. Explants were cultured on Murashige and Skoog medium (MS) supplemented with different plant growth regulators (PGRs) [a-naphthaleneacetic acid (NAA), benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), thidiazuron (TDZ)]. The combinations and concentrations of PGRs were shown significant variations for the frequency of callus formation, appearence of callus and the potential of callus differentiation. NAA x BA have been found highly affective in callusing and plant regeneration. Other PGRs have not resulted in callus differentiation for shoot formation. The highest number of shoots (6/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. The regenerated shoots transferred to rooting medium (MS with 0.5 mg/l indole-3-butyric acid) were successfully rooted (100 percent) and showed rapid elongation. Rooted plantlets were acclimatized in pots containing 1:1 mixture of peat and perlite.

Plant Regeneration through Somatic Embryo in Herpetospermum pedunculosum,an Endangered Tibetan Medicinal Herb / 中草药·英文版

Objective An effective reproducible protocol for complete plant regeneration via somatic embryogenesis has beendeveloped for Herpetospermum pedunculosum,an endangered Tibetan medicinal herb.Methods The cotyledonexplants used in this study were excised from seedlings germinated in vitro.Callus was induced from cotyledonexplants on Murashige and Skoog's medium,supplemented with 2,4-dichlorophenoxyacetic acid(2,4-D,0.1-1.0mg/L)alone or in combination with 6-benzylaminopurine(BA,0.5,1.0,and 2.0 mg/L).Results The calli showeddifferentiation of globular embryos after three weeks of incubation on MS medium supplemented with variouscombinations of BA and NAA.Sixty-two percent of the embryogenic calli produced somatic embryos in MS basalmedium supplemented with BA(1.0 mg/L)+NAA(2.0 mg/L).The addition of KN(0.5 mg/L)to MS mediumcontaining both BA and NAA(2.0 mg/L each)significantly increased the frequency of somatic embryogenesis.Themaximum percentage of embryogenic calli formation was 83%,and globular embryos formed and germinatedsuccessfully in this medium.Then,transferring the regenerated plants from this medium to hormone-free MSmedium will further enhanced the development of the plants,and the healthy plantlets are formed successfullywithin four weeks.The plantlets were transferred to soil to acclimatize under greenhouse conditions and 75%survived.Conclusion Somatic embryogenesis protocol as reported here can play a key role in the propagation andconservation of this endangered species.

Regeneración natural de plantas leñosas en una plantación costarricense de Vochysia guatemalensis (Vochysiaceae) y el efecto de la fertilización con P y NPK / Natural regeneration of woody species in a Costa Rican plantation of Vochysia guatemalensis (Vochysiaceae) and the effect of P and NPK fertilization

Natural regeneration of woody species in a Costa Rican plantation of Vochysia guatemalensis (Vochysiaceae) and the effect of P and NPK fertilization. Forest plantation management strategies, including the selection of species, may have positive or negative effects over plant regeneration in the tropics. in this case, understory woody plants density and richness were studied in Tabarcia de Mora, Costa Rica, in a monoculture of Vochysia guatemalensis (ten year old plantation). Nineteen 80 m²plots, with several fertilization treatments (0-0; 0-50; 50-0, 50-50 g/plant of P, and NPK, during the first years, P placed once at the hole) in a completely randomized factorial design, were analyzed. Afterwards, the NPK fertilizer was increased from 150 to 200 g/ plant/year until the plantation was six year old. The plots, established after the coffee plantation was eliminated, had a minimum management schedule, basically the elimination of herbaceous vegetation once or twice a year during the first three years, and a tree thinning when the plantation was four year old, to increase spacing from 2x2 to 4x4 m. All woody vegetation taller than 0.5 m was tallied. A total of circa 10 000 ind/ha, distributed in 90 species, were found, mostly native of the region, some identified for forestry use, others important for the fauna. The majority of the species had low relative densities and frequencies. Sixteen percent of the plants reached heights greater than 2.5 m. Several factors seem to explain this regeneration pattern: a canopy with an intermediate openness, a low intensity forestry management, the nearness of the plantation to a mature forest fragment, and that the Vochysia plantation substituted a coffee plantation where soil conservation strategies and an annual fertilization management plan were applied. Finally, plots with only P had significantly higher species richness and abundance (χ2=15.364, gl=3, p=0.002) probably because the trees in this treatment were less developed (when compared with the others). Rev. Biol. Trop. 57 (Suppl. 1): 111-118. Epub 2009 November 30.

Las estrategias de manejo y las especies seleccionadas en plantaciones forestales pueden tener efectos positivos o negativos sobre la regeneración vegetal en los trópicos. Esta investigación estudió la abundancia y riqueza de plantas leñosas bajo el dosel de un monocultivo de Vochysia guatemalensis de diez años. Se evaluaron 19 parcelas de 80 m²en Tabarcia de Mora, con varios tratamientos de fertilización (0-0; 0-50; 50-0, 50-50 g/ planta de P, y NPK, respectivamente, este ultimó se aumentó de 150 a 200 g hasta que la plantación alcanzó los seis años), en un diseño factorial totalmente al azar. Se contaron e identificaron todas las especies leñosas con más de 0.5 m de altura, con un total de aproximadamente 10 000 ind/ha en 90 especies, siendo éstas principalmente nativas de la zona (varias maderables, otras importantes para la fauna), la mayoría con bajos índices de importancia (suma de la densidad y frecuencias relativas). Un 16% alcanzaron alturas superiores a 2.5 m. Se considera que varios factores pudieron favorecer dicha regeneración, como un dosel con una apertura inter-media, un manejo forestal de bajo impacto, la cercanía de un fragmento boscoso maduro, y el establecimiento de la plantación en sustitución de un cafetal donde se aplicaban estrategias de conservación de suelos y se fertilizaba anualmente. Finalmente, se determinó una mayor abundancia y riqueza en las parcelas con solo P (χ²=15.364, gl=3, p=0.002), probablemente porque los árboles de Vochysia tendieron a ser menos desarrollados en comparación con los otros tratamientos.

Plant regeneration of natural tetraploid Trifolium Hum pratense L

The regeneration of natural tetraploid T. pratense, originated from Erzurum-Turkey, is reported in this study. This plant has low seed setting and hard seed problems due to polyploidy. Hypocotyl, cotyledon, apical meristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing different concentrations of BAP and NAA as growth regulators. The best shoot formation has been observed on explants initiated from apical meristem placed on PC-L2 medium that includes 2 mg dm-3 BAP and 1 mg dm-3 NAA. 94.4 percent of the shoots originated from calli were rooted on PC-L2 medium with 1 mg dm-3 NAA. In vitro organogénesis has been accomplished in the natural tetraploid T. pratense regenerated plants successively transferred to the field.