In vitro multiplication of wild Manihot species with different naphthaleneacetic acid and benzylaminopurine concentrations

In vitro multiplication is an important tissue culture technique that is capable of efficiently producing seedlings at any scale. It is a propagation method based on the aseptic culture of small propagules in a suitable culture medium to enable plant regeneration. Multiplication experiments conducted in vitro to set protocols adapted to wild Manihot species have used modified mineral salts and MS vitamins as basic culture medium. Here, 25 treatments based on combinations of the regulators benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) at 0, 0.025, 0.05, 0.075, and 0.1 mg L-1 were used for in vitro multiplication of three genotypes of wild Manihot species (M. violaceae Pohl Müll. Arg., M. pseudoglaziovii Pax & Hoff., and M. flabellifolia Pohl). Plant height and the number of 1 cm minicuttings, number of roots, shoots, green leaves and senescent leaves were recorded 120 days after explant inoculation. M. violaceae Pohl. Müll. Arg. and M. flabellifolia Pohl. presented favorable results with 0.05 and 0.025 mg L-1 NAA, respectively. Culture medium lacking NAA and BAP favored the in vitro growth of M. pseudoglaziovii Pax & Hoff.

Influência de diferentes concentrações dos fitorreguladores ácido 6-benzilaminopurina e ácido naftalenoacético na propagação vegetativa de Malva sylvestris L / Effect of different BAP and NAA concentration on microprapagation of Malva (Malva sylvestris L. )

Malva sylvestris L. (família Malvaceae), conhecida como malva, é uma espécie medicinal nativa da Europa, cultivada no sul do Brasil. A espécie tem propriedade adstringente, suaviza a irritação dos tecidos e reduz inflamações, entre outras características e atributos medicinais. O estudo teve como objetivo verificar a eficiência dos hormônios ANA (ácido naftalenoacético) e BAP (ácido 6-Benzilaminopurina) na propagação vegetativa a partir do estabelecimento in vitro de segmentos nodais. Segmentos nodais obtidos de plantas matrizes mantidas em casa de vegetação foram submetidos à desinfestação e inoculados em meio MS (Murashige e Skoog) com diferentes concentrações e combinações de ANA (ácido naftalenoacético) e BAP (ácido 6-Benzilaminopurina) totalizando oito tratamentos com 60 repetições cada. Os explantes foram mantidos em sala de crescimento e, ao completar sete dias, as plântulas obtidas foram retiradas e avaliadas quanto ao número de folhas, altura total (cm) e massa fresca (g). As plântulas foram fixadas em substrato "Big bio" e transferidas para casa de vegetação com nebulização. Os dados obtidos foram submetidos à Análise de Variância, seguido pelo Teste de Tukey. As plântulas obtidas em meio MS acrescido de 2,0 mg/Lˉ¹ de BAP e de 0,5 mg/Lˉ¹ de ANA foram as que apresentaram maior média nas três variáveis avaliadas, sendo então o mais indicado para a produção de mudas. Em 10 dias foi observado o enraizamento de todas as plântulas transferidas para casa de vegetação. A aclimatização e o enraizamento ex vitro ocorrem em uma única etapa sem a necessidade da utilização de enraizadores. A técnica desenvolvida demonstra a possibilidade de produção de mudas com custos reduzidos, em larga escala, e com a garantia de fornecer mudas aptas para o cultivo em apenas 17 dias

Effect of different BAP and NAA concentrations on malva (Malva sylvestris L.) micropropagation. Malva sylvestris L. (Malvaceae family), known as mallow, is a medicinal species native toEurope, and it is grown in southern Brazil. It has astringent properties and can sooth tissue irritation and reduce inflammations among other medicinal characteristics. The study aimed to verify the efficiency of the hormones NAA (naphthalene acetic acid) and BAP (6-Benzilaminopurine acid) in propagating the species from establishing in vitro nodal segments. Nodal segments obtained from mother plants kept in greenhouses were disinfected and inoculated in MS medium with different concentrations and combinations of BAP and NAA, amounting to 8 processes with 60 repetitions each. The explants were kept in growth rooms, and after seven days the resulting seedling were removed and assessed regarding the number of leaves, total height (cm) and fresh mass (g). Subsequently, the seedling were fixed on the substrate "Big bio" and transferred to greenhouses with nebulization. The data obtained was subjected toVariance Analysis, followed by the Tukey's test. In 10 days,rooting could be observed in all the plantules transferred to the greenhouse. The plantules obtained in MS medium thatreceived 2.0mg/L-1 BAP and 0.5 mg/L-1 NAA were the ones that presented the highest average in the three variables assessed, therefore the most recommended to producing seedlings of this species. The ex vitro acclimatization and rooting occur in a single phase without the need of root promoters. The technique developed shows that it is possible to produce seedlings of this species at reduced costs, in large scale and with the guarantee to supply seedlings that can be planted in only 17 days.

Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis / 中国中药杂志

To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.

Effects of 6-benzylaminopurine and α-naphthaleneacetic acid on growth and isoflavone contents of Pueraria phaseoloides hairy roots / 生物工程学报

In order to study the effect of phytohormone on growth and isoflavones contents of Pueraria phaseoloides hairy roots, we cultured the hairy roots with different concentrations of 6-benzylaminopurine (6-BA) alone or in combination with α-naphthaleneacetic acid (NAA). Then we determined the effects of 6-BA alone or in combination with NAA on the growth and the contents of isoflavones compounds and levels of antioxidase activities of hairy roots by spectrophotometry. The results show that 6-BA inhibited the growth, and decreased biomass and total isoflavones compounds of P. phaseoloides hairy roots. Furthermore, the inhibition was increased with the concentrations of 6-BA. Compared with the controls, different concentrations of 6-BA in combination with NAA 2.0 mg/L could inhibit the growth of hairy roots and decrease the content of total isoflavone compounds, and also significantly enhanced the contents of soluble protein and levels of peroxidase (POD) activities, but decreased the activities of superoxide dismutase (SOD). DNA ladders detected by agarose gel electrophoresis can be observed after hairy roots of P. phaseoloides were cultured with 6-BA alone for 30 days, but can appear on the 20th day after culture with 6-BA in combination with NAA 2.0 mg/L. This result indicates that 6-BA or 6-BA in combination with NAA can both stimulate appearance of programmed cell death (PCD), and NAA may play a synergistic role on PCD.

Rapid in vitro plant regeneration from leaf explants of Launaea sarmentosa (Willd.) Sch. Bip. ex Kuntze

An efficient protocol for organogenesis through leaves has been established for Launaea sarmentosa (Willd.) Sch. Bip. ex Kuntze, a highly valuable medicinal plant. The leaf explants produced microshoots on MS basal medium when fortified with cytokinins and auxins. A combination of 6-benzylaminopurine (BAP) at 0.5mg/l and naphthaleneacetic acid (NAA) at 0.2mg/l resulted in the induction of high frequency microshoots in 30 days. The microshoots were successfully subcultured for shoot elongation and eventually for rooting on MS medium supplemented with indole-3-butyric acid (IBA) at 0.5mg/l. The regenerated plantlets were hardened under greenhouse conditions and transferred to garden, resulting in a 90% survival rate.

Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification / 中国中药杂志

<p><b>OBJECTIVE</b>To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.</p><p><b>METHOD</b>Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.</p><p><b>RESULT</b>Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.</p><p><b>CONCLUSION</b>Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.</p>

Induction of adventitious roots of Echinacea pallida and accumulation of caffeic acid derivatives / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of auxins 2,4-D,IAA,IBA,NAA on induction of adventitious roots as well as that of IBA concentrations on the growth of adventitious roots and the accumulation of caffeic acid derivatives, with test-tube seedling leaves Echinacea pallida as the explant, and cultivate adventitious roots in bioreactors.</p><p><b>RESULT</b>1.0 mg x L(-1) IBA was found the best for the induction of adventitious roots, with the numer of induced adventitious roots up to 22. 5 in each culture dish. Among different concentrations for suspension cultivation of IBA tested, 1.0 mg x L(-1) IBA was found the most suitable for the growth of adventitious roots and the accumulation of caffeic acid derivatives. In a 5 L balloon type bubble bioreactor, 8.98 g x L(-1) dry weight was achieved after one month, which was 2.05 times of 4.38 g x L(-1) dry weight cultivated in a triangular flask. The content of echinacoside cultivated in a bioreactor was 14.08 mg x g(-1) DW, which was 2.4 times of cultivated roots. The contents of chlorogenic acid, chicoric acid and total caffeic acid derivatives were 4.0-25.6 times of ultivated roots.</p><p><b>CONCLUSION</b>The study can provide high-quality biomedical drugs containing such caffeic acid derivatives as echinacoside for mass production of Echinacea purpurea medicines.</p>

Effect on different concentrations of exogenous hormones on baicalin in Scutellaria baicalensis callus / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the content of baicalin in Scutellaria baicalensis callus induced by different doncentrations of exogenous hormones.</p><p><b>METHOD</b>HPLC system was adopted to determine baicalin in S. baicalensis callus. Chromatographic conditions: ODS column was adopted, with methanol-water-phosphate (47: 53: 0.2) as the mobile phase. The flow velocity was 1 mL x min(-1), the detective wavelength was 280 nm, and the temperature of column was room temperature.</p><p><b>RESULT</b>S. baicalensis callus induced by 6-BA 1.0 mg x L(-1) + NAA 0.5 mg x L(-1) showed the highest baicalin content, up to 49.78 mg x g(-1).</p><p><b>CONCLUSION</b>The experiment is such a simple, rapid and stable method for determining the baicalin content that it can be used for determining the baicalin content in S. baicalensis callus.</p>

Study on optimization of induction system of test-tube tuberous roots from leaves of Rehmannia glutinosa / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of sucrose and plant growth substances of different concentrations on the induction of test-tube tuberous roots of Rehmannia glutinosa, in order to establish an efficient system for the induction of test-tube tuberous roots from leaves of R. glutinosa.</p><p><b>METHOD</b>Leaves from test-tube seedlings of 85-5 R. glutinosa were used as explants. After rooting induction, they were transferred to medium with orthogonal design for inducing test-tube tuberous roots of R. glutinosa.</p><p><b>RESULT AND CONCLUSION</b>NAA played a significant role in induction of test-tube tuberous roots of R. glutinosa, followed by sucrose and 6-BA. With leaves from test-tube seedlings as the explants, the optimal medium for inducing test-tube tuberous roots of R. glutinosa was MS + BA 3.0 mg x L(-1) + NAA 0.1 mg x L(-1) + sucrose 7%. The study provides an efficient induction system for studies on artificial seeds and secondary metabolism with test-tube tuberous roots of R. glutinosa.</p>

Efeito dos ácidos naftaleno acético e indolilbutírico no enraizamento de estacas de jambolão [Syzygium cumini (L. ) Skeels] / Effect of naphthaleneacetic acid and indolebutyric acid on rooting of jambul [Syzygium cumini (L. ) Skeels] cuttings

O jambolão propaga-se normalmente por sementes o que acarreta variabilidade nas plantas descendentes e um problema quando o objetivo é a formação de pomar comercial. O desenvolvimento de protocolo de propagação vegetativa por meio da estaquia possibilitaria a reprodução de todas as características da planta matriz, uniformidade nas populações e facilidade de propagação. O presente trabalho teve por objetivo avaliar o efeito dos ácidos naftaleno acético (ANA) e indolilbutírico (AIB) no enraizamento de estacas de jambolão. Estacas da região mediana dos ramos foram confeccionadas com 12 cm de comprimento, cortadas em bisel na base e reto acima da última gema axilar, mantendo-se um par de folhas reduzidas à metade. As bases das estacas foram imersas por 10 segundos em soluções aquosas contendo ANA ou AIB nas concentrações de 0, 500, 1.000 e 1.500 mg L-1. Para o plantio foram utilizadas bandejas plásticas contendo areia de granulometria média. As estacas foram mantidas em casa-de-vegetação com nebulização intermitente e após 120 dias do plantio, foram avaliadas as variáveis: porcentagem de estacas enraizadas, com calos, vivas (não enraizadas e sem calos) e mortas, comprimento das três maiores raízes (cm) e número de raízes formadas por estaca. Os melhores resultados de enraizamento foram verificados com 1.000 mg L-1 para ambos os fitorreguladores testados. A porcentagem de enraizamento foi ligeiramente superior com a utilização de ANA quando comparada ao AIB.

Jambul usually propagates by seeds, which causes variability in the descendant plants and represents a problem in the formation of commercial orchards. The development of a protocol for vegetative propagation by cuttings would enable the reproduction of all features of the Mother plant, uniformity in populations and easy propagation. The aim of this work was to evaluate the effect of naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) on rooting of jambul cuttings. Twelve-cm-long cuttings from the median region of branches were prepared through bevel cut in the base and right cut above the last axillary bud, keeping one pair of halved leaves. Cutting bases were immersed for 10s in aqueous solutions containing NAA or IBA at 0, 500, 1000 and 1500 mg L-1 concentrations. Plastic trays containing medium sand were used in the planting. The cuttings were kept in a greenhouse under intermittent nebulization and, at 120 days after planting, the following variables were evaluated: percentage of rooted, with calluses, alive (not-rooted and without calluses) and dead cuttings; length of the three largest roots (cm); and number of roots per cutting. The best rooting was observed by using 1000 mg L-1 of both tested plant growth regulators. Rooting percentage was slightly higher under NAA relative to IBA.

Estaquia de Ginkgo biloba L. utilizando três substratos / Ginkgo biloba L. cutting using three substrates

Ginkgo biloba é arbórea, decídua, cuja folhagem se torna amarelada no outono antes da queda das folhas, o que a torna valorizada em jardinagem. A estaquia é um método de propagação vegetativa baseado na capacidade das células em retomarem o processo de divisão celular, formando raízes em estacas destacadas de ramos provenientes de plantas matrizes. O presente trabalho teve como objetivos verificar a influência de diferentes substratos, assim como, a aplicação da auxina sintética o ácido indol butírico (AIB) no enraizamento de estacas de Ginkgo biloba. No inverno de 2005, ramos foram coletados e transportados até o Laboratório de Macropropagação, onde foram confeccionadas estacas sem folhas, com 10-12 cm de comprimento. Os tratamentos com regulador vegetal (T) foram T1- 0 mg L-1 AIB em solução; T2- 4000 mg L-1 AIB em solução; T3- 8000 mg L-1 AIB em solução; T4- 0 mg kg-1 AIB em talco; T5- 4000 mg kg-1 AIB em talco e T6- 8000 mg kg-1 AIB em talco. Para cada tratamento foram utilizados três diferentes substratos (S), S1- areia, S2- fibra de casca de coco (coxim) e S3- casca de arroz carbonizada. Após 120 dias da instalação, foram avaliadas as porcentagens de estacas enraizadas, vivas, com calos e mortas; o número de raízes por estaca e o comprimento das três maiores raízes por estaca. Os melhores resultados no enraizamento foram obtidos com estacas tratadas com 4000 e 8000 mg kg-1 AIB em talco, utilizando o coxim como substrato (45,00 e 46,25 por cento de enraizamento, respectivamente).

Ginkgo biloba is an arboreal and deciduous species, the foliage of which becomes yellowish in the autumn, before leaf drop, increasing its value for gardening. Cutting is a method of vegetative propagation based on the capacity of cells to recover the cell division process, originating roots in cuttings detached from branches of stock plants. This study aimed to verify the influence of different substrates, as well as the application of the synthetic auxin indole-3-butyric acid (IBA) in Ginkgo biloba cutting rooting. In the winter of 2005, branches were collected and sent to the Macropropagation Lab, where cuttings of 10-12cm length were made without leaves. The treatments with plant growth regulator (T) were T1- 0 mg L-1 IBA solution, T2- 4000 mg L-1 IBA solution, T3- 8000 mg L-1 IBA solution, T4- 0 mg kg-1 IBA in talc, T5- 4000 mg kg-1 IBA in talc, T6- 8000 mg kg-1 IBA in talc. Each treatment was planted in three substrates (S), S1- sand, S2- coir and S3- carbonized rice hull. After 120 days, the percentages of cuttings that were rooted, alive, with callus and dead were evaluated, besides the number of roots per cutting and the length of the three highest roots per cutting. The best results regarding rooting were obtained for cuttings treated with 4000 and 8000 mg kg-1 IBA in talc, by using coir as substrate (45.00 and 46.25 percent rooting, respectively).

Application of SPSS orthogonal design in tissue culture of Anoectochilus roxburghii / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of the different constitutions of plant hormone on the development of Anoectochilus roxburghii.</p><p><b>METHOD</b>A. roxburghii were harvested after having been cultured for 60 days. An orthogonal design was used to study the effect of NAA and 6-BA on the leaf number, eustipe number, lateral branch number of the stem tip and stem section, and the height of the stem tips. All of the data were processed by SPSS.</p><p><b>RESULT AND CONCLUSION</b>It is reported for the first time that NAA could make different development of A. roxburghii at low concentration ( < 1 mg L(-1)) and high concentration ( > 1 mg L(-1)). The optimum constitution of MS medium was NAA 0.5 mg L(-1) + 6-BA 1 mg L(-1) for the growth of the stem tip of A. roxburghii, and NAA 1 mg L(-1) + 6-BA 2 mg L(-1) for the differentiation of bud and the formation of lateral branch of the stem section. The different concentrations of NAA and 6-BA had different effects on the growth and differentiation of the stem tip and the stem section of A. roxburghii.</p>

Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypocotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.

Adventitious root induction and in vitro culture of Panax notoginseng / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the induction and culture of adventitious root of Panax notoginseng.</p><p><b>METHOD</b>Three ways, induction from the explants of three-year-old P. notoginseng. The explants of regenerated shoots and calluses, were used to induce adventitious roots. The effects of 2, 4-dichlorophenoxyacetic acid, indole-3-butyric acid and naphthylacetic acid on adventitious root induction were investigated respectively. The effects of four modes of separating adventitious roots from the parent tissues on culture in vitro were compared.</p><p><b>RESULT</b>Adventitious roots were successfully induced by three methods, of which the young flower bud callus was the best material for the induction of adventitious root. Indole-3-butyric acid possessed the strongest potency for induction. The liquid culture system was established by continuous culture of adventitious roots together with their parent tissues before separated.</p><p><b>CONCLUSION</b>The acquisition and culture in vitro in liquid culture system of adventitious roots of P. notoginseng lay a foundation for the next investigation.</p>

A new approach for enhanced multiplication of arbuscular mycorrhizal fungi and isolation of ITS regions from Glomus deserticola and Laccaria fraterna.

Rootlets induced from the petiole base of L. purpureus, using IAA and kinetin was used for enhanced multiplication of arbuscular mycorrhizal (AM) fungus, G. deserticula. Using conserved short arbitrary oligonucleotides, as specific primers, we amplified the ITS-region, a molecular marker for fungal identification, from the genomic DNA extracted from cultured spores of G. deserticola, and genomic DNA extracted from the mycelium of L. fraterna. The capacity of fungal colonization and subsequent spore formation of G. deserticola, compared with the natural root system was evaluated. This technology would provide a simple way to multiply AM fungi and to produce spores without microbial contamination useful for further molecular characterization.

Endophytic colonization and in planta nitrogen fixation by a diazotrophic Serratia sp. in rice.

Nitrogen fixing endophytic Serratia sp. was isolated from rice and characterized. Re-colonization ability of Serratia sp. in the rice seedlings as endophyte was studied under laboratory condition. For detecting the re-colonization potential in the rice seedlings, Serratia sp. was marked with reporter genes (egfp and Kmr) using transposon mutagenesis. The conjugants were screened for re-colonization ability and presence of nif genes using PCR. Further, the influence of flavonoids and growth hormones on the endophytic colonization and in planta nitrogen fixation of Serratia was also investigated. The flavonoids, quercetin (3 microg/ml) and diadzein (2 microg/ml) significantly increased the re-colonization ability of the endophytic Serratia, whereas the growth hormones like IAA and NAA (5 microg/ml) reduced the endophytic colonization ability of Serratia sp. Similarly, the in planta nitrogen fixation by Serratia sp. in rice was significantly increased due to flavonoids. The inoculation of endophytic diazotrophs increased the plant biomass and biochemical constituents.

Studies on the seed embryo germination and propagation of Dendrobium candidum in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To determine optimum culture conditions for the seed embryo culture and rapid propagation of Dendrobium candidum.</p><p><b>METHOD</b>Seed embryos of D. candidum were incubated in the medium containing a combination of 6-benzylaminopurine (BA) and 1-naphthaleneacetic acid (NAA), potato extract, banana extract and activated carbon in order to induce seed embryo germination, protocorm differentiation, plantlet propagation and plantlet rooting.</p><p><b>RESULT AND CONCLUSION</b>The maximum embryo germination percentage was obtained in the 1/2 MS media supplemented with 20% potato extract. The 1/2 MS medium supplemented with 1.0 mg x L(-1) BA and 0.1 mg x L(-1) NAA was very beneficial to the protocorm differentiation and propagation of D. candidum. The highest protocorm propagation index was obtained from the medium containing the activated carbon. The highest root numbers and length were observed in plants growing in 1/2 MS medium containing 0.5 mg x L(-1) NAA.</p>

Microcutting culture and morpho-physiological changes during acclimation in two Lycium chilense cytotypes

Lycium chilense, a deciduous perennial shrub, is one of the endangered native species of Patagonia due to sheep overgrazing. Chances of recolonization by seeds are scarce due to the limited density of propagules in the soil and very specific requirements for germination. The objective was to develop an in vitro propagation protocol that would help to perform reestablishment of this species in degraded areas of the Patagonian steppe. Seeds came from two provenances with different somatic number due to differences in ploidy level. Defoliated microcuttings were planted in test tubes with different growing media and taken to a growth chamber. Rooting percentage did not differ between origins, but higher values were encountered for medium without hormones. Subcultures increased significantly rooting percentage and reduced time to rooting. The leaves from micropropagated plants were thinner, did not exhibit hairs, and had poorly developed palisade parenchyma and less epicuticular waxes. In vitro leaves had lower stomatal density and their stomata were less functional when compared to acclimated leaves. A repopulation program of Lycium chilense based on microcutting culture, specialy using subcultures, is feasible.

Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats.

Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins. Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production. Berberine was synthesized and released continuously into the liquid medium. Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis. On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth. Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D. IAA and IBA were not favourable for cell growth and berberine synthesis.

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol.

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.