Tissue Culture and Exercising Seedlings in Scrophularia ningpoensis / 中国实验方剂学杂志

Objective:Stems,petioles,stem sections with axillary and leaves of Scrophularia ningpoensis were taken as the material in vitro to screen out the suitable plantlet regeneration system and optimal exercising seedling conditions. Method:Different explants,hormones and concentrations on the induction and proliferation of cluster bud were studied by L16(45) orthogonal test. One factor analysis of variance (ANOVA) was made on the induction of adventitious buds rooted with different concentrations of hormones. At the same time,different substrates,watering cycles and transition modes were selected to optimize key technologies of exercising seedlings of S. ningpoensis. Result:Stem sections with axillary was the best explant,which was followed by stems,leaves and petioles. The suitable media for the induction of adventitious buds was MS+6-BA 0.5 mg·L-1+NAA 0.2 mg·L-1,with the induction rate of 100.0% and the proliferation multiple of 9.84.The suitable media for root induction was 1/2 MS+IBA 0.2 mg·L-1,with the rooting rate of 100.0% and the number of roots of 39.45.For matrix,they were transplanted with nutrient soil,vermiculite and perlite (5:2:1) as the media,to keep proper matching of fertility,permeability and water retention. The container seedlings can grow well,and the survival rate was more than 95% when they were watered every 2 days,the acclimatization of plantlets took 20 days indoor and 10 days in shaded greenhouses. Conclusion:The clonal propagation system of S. ningpoensis was established to provide an effective way for the efficient,rapid and steady plantlet regeneration,the breeding of high-quality seedlings and the suitable exercising seedling conditions of S. ningpoensis.

A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 µl of Murashige and Skoog’s medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 106 cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

Establishment of an in vitro plantlet regeneration protocol for unique varieties of brinjal (Solanum melongena L.) var. Mattu Gulla and Perampalli Gulla.

Brinjal (Solanum melongena L.) var. Mattu Gulla (MG) and var. Perampalli Gulla (PG) are unique varieties with distinct flavour cultivated in Udupi, Karnataka State, and are exposed to several biotic and abiotic stresses. An efficient and reproducible in vitro regeneration method is required to expedite the manipulation of these brinjal varieties to cope up with stress by tissue culture and gene transfer methods. The present study, reports a rapid and efficient in vitro regeneration protocol for these two varieties. The in vitro growth response was studied on Murashige and Skoog (MS) medium supplemented with 2, 4-D, BAP and IAA, and the plantlets were regenerated efficiently from callus cultures of leaf, cotyledon and hypocotyl explants. Among the three explants, the hypocotyl explants were found to have better callus induction and multiple shoot regeneration. High frequency of shoot initiation was achieved from hypocotyl derived calluses in MS media with 2.0 mg/L BAP and 0.5 mg/L IAA in MG and PG. Efficient and rapid shoot proliferation, and elongation were noted in MS medium with 1.0 mg/L BAP and 0.3 mg/L GA3. The in vitro regenerated shoots produced healthy roots when they were cultured on MS medium supplemented with 0.5 mg/L IBA. A significant difference was observed in percentage of callus induction, number of shoots per callus, shoot elongation and number of hardened plantlets of MG and PG. MG showed maximum response in all stages of culture than PG. Hardening of plantlets in tissue culture was achieved in three weeks. The hardened plantlets were grown in pots for further acclimatization in green house and finally transplanted to experimental garden where they developed into flowering plants and produced mature fruits with viable seeds.

Accumulation of main ginsenoside components in somatic embryogenesis and plantlet regeneration of Panax notoginseng / 中草药

Objective: To investigate the accumulation of total saponins and ginsenosides (Rg1, Re, and Rb1) during somatic embryogenesis and plantlet regeneration in Panax notoginseng. Methods: The dynamic accumulation of the total saponins and three components of ginsenosides (Rg1, Re, and Rb1) was analyzed by visible spectrophotometry and high-performance liquid chromatography methods, respectively. Results: The content of total saponins increased from the minimum at embryogenic callus (2.47%) to the maximum at plantlets (7.79%) during somatic embryogenesis development. Because the different specific growth rate of different materials, total saponins accumulation efficiency sort order: plantlets > earlier embryos > later embryos > embryogenic callus. We also found that the total content of the three ginsenosides (Rg1, Re, and Rb1) reached the maximum (7.05 mg/g) in the later embryos, and the minimum (2.78 mg/g) was in the embryogenic callus. The contents of ginsenosides Rg1 and Rb1 reached their maximum (2.24 and 4.03 mg/g) respectively in the earlier and later embryos, and the maximum (1.21 mg/g) of ginsenoside Re content was in embryogenic callus. The accumulation (1.947 4 mg/g) of the three ginsenosides minimum occurred in plantlets, and the maximum (5.022 5 mg/g) reached in earlier embryos. Conclusion: Both total saponin contents and accumulation efficiency reach the maximum in plantlets. Three ginsenoside contents and accumulation reach the maximum in later and earlier embryos, respectively.

Regeneration of plantlets from mature embryo calli of Western Ghats land race cultivar of rice, Oryza sativa L.

The Malnad region located in the Western Ghats of Karnataka is known for the cultivation of indigenous rain fed land race cultivar of rice. The present study was to investigate the callogenic and caulogenic potentialities of the two indigenous rice cultivar namely Karimundaga and Kanadatumba using dehusked mature embryo explants. For callus and shoot bud differentiation, the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D (1–3 mg/L), IAA (1–2 mg/L), Kn (1–4 mg/L) and BAP (1–4 mg/L). The morphogenic potentialities of the two rice cultivar differed in texture of callus. In both the cultivar callogenic frequency was optimized at 1 mg/L 2,4-D concentration, it was 94% in Karimundaga and 58% in Kanadatumba. Supplementation of IAA either alone (1–2 mg/L) or in combination with Kn or BAP at 1 to 4 mg/L concentration of each induces shoot bud differentiation from the calli. In the cultivar Karimundaga caulogenic frequency was highest (10.60±2.55) at 1.0 mg/L IAA and 4.0 mg/L BAP concentration. While in the cultivar Kanadatumba highest number of shoot buds (7.90±2.69) was differentiated at 1.0 mg/L IAA and 4.0 mg/L Kn concentration. The calli derived regenerants were successfully acclimatized in the greenhouse and agro-morphological variations were evaluated. The growth characteristics and yield related parameters exhibited by in vitro plants were lower than the in vivo plants.

Blade differentiation and plantlet regeneration of Gynostemma pentaphyllum / 中草药

Objective To construct a stably-performing and high-efficiency regeneration system of Gynostemma pentaphyllum and lay a foundation for the gene transformation of it.Methods The blades of five-leaf G.pentaphyllum were used as the explants and cultured in MS media with different portions of hormone to induce fascicled-bud,root and plantlet regeneration.Results The medium suitable for inducing the blade differentiation of G.pentaphyllum was MS+6-BA 1.0 mg/L and for the frequency of blade differentiation could reach 40%.The cultural medium MS+6-BA 1.0 mg/L was suitable for the sub-multiplication of fascicled-bud and the medium 1/2 MS for root inducement and the plantlet regeneration.Conclusion A stably-performing acceptor system of the direct differentiation for Agrobacterium tumefaciens mediated gene transformation of G.pentaphyllum blades is constructed.

Tissue culture of stem segment and plantlet regeneration of Dendrobium nobile / 中草药

Object To speed up the artificial propagation of the costly medicinal plant Dendrobium nobile Lindl. Methods The tender stems with lateral buds were tried as the explants and cultured on various culture media with different portions of hormon. Results The best medium for bud induction was the MS basic medium with addition of 6 BA (0.5 mg/L), NAA (0.2 mg/L), while MS with the addition of 6 BA (3.0 mg/L) NAA (0.5 mg/L), was suitable for propagation, MS with addition of IBA (0.3 mg/L), NAA (0.1 mg/L) was better for root growing, the striking growth rate was up to 95%. The young sprout was cultured fine on the base material mixed with coco crumbs, volcanic rocks and brick fragments (1∶1∶ 1) and covered by dried pteridophyte. Conclusion To provide the effective ways to reserve the resources of D. nobile and last its utlization.

Establishment of plantlet regeneration system of Rubus idaeus / 中草药

Object\ To establish a plantlet regeneration system of Rubus idaeus L for the purpose to obtain a large number of high quality seedling in a short time Methods\ Stem apex and part of the stem were used as the explant and the optimal culture media and conditions were selected by orthogonal design Results\ An optimum culture medium for the induction of callus, adventitious bud and root was obtained which can be carried out in the laboratory with comparative ease and good repeatability Conclusion\ A basic medium + BA 0 2 mg/L+NAA 1 0～1 5 mg/L was most suitable for the induction of callus; a medium + BA 1 mg/L+NAA 0 1～0 2 mg/L+GA 3 6～8 mg/L+CH 300 mg/L was good for the induction of bud; a medium +BA 1 mg/L+NAA 0 1 mg/L+GA 3 2 mg/L was suitable for propagation of the bud; and the basic medium+IBA 0 1 mg/L+NAA 0 5 mg/L was good for the induction of root

Somatic embryogenesis and plantlet regeneration of Aconitum coreanum / 中草药

Object To develop new resources of medicinal plan t of Aconitum coreanum (L?vl.) Rapaics by the method of plant cel l biotechnology. Methods The segments of stems, leaves, embr yonal axises, radicles, and cotyledon of A. coreanum were used a s the explants. The MS medium supplied with different auxin and cytokinin was e xamined. Results The calli were induced from explants on the MS medium supplied with 2, 4-D 1 mg/L and 6-BA 0.5 mg/L in the period of 30 d. And the calli were differentiated into green conus or globe embryo after se veral subcultures period on medium MS supplied with 2, 4-D 0.2 mg/L and 6-BA 0.5 mg/L. The adventitious buds differentiated could be developed from the so matic embryo callus into roots on medium MS supplied with IBA 0.5 mg/L, and the n the intact plantlets were obtained. Conclusion The plantle ts of A. coreanum can be obtained by the techniques of somatic embryogenesis and it is indicated that this may be a new way in developing new r esource and protecting of A. coreanum.

Tissue culture of shoot-tip and plantlet regeneration of Camptotheca acumunata / 中草药

Object To explore the artificial propagation of the medicinal plant, Camptotheca acuminata Decne. Methods The shoot-tip was tested as the explants and cultured on culture media with different portions of hormone. Results The best medium for bud induction was the B5 basic medium with additions of 6-BA (0.2 mg/L), IBA (0.05 mg/L), and AS (afenine sulfate, 10 mg/L). While B5 with the additions of IBA (0.5 mg/L), KT (0.1 mg/L), and AS (10 mg/L) was suitable for rooting. The seedling was cultivated and grew well on the base material mixed with perlite-soil (3∶7). The survival rate of transplant was up to 96%. Conclusion The above mentioned method provides a new effective way to exploit this plant resources.

Establishment of plantlet regeneration system for Aconitum carmichaeli / 中草药

Object To establish an effective plantlet regeneration system of Aconitum carmichaeli Debx. for the purpose to obtain a large number of high quality seedling in a short time. Methods Leaves in vitro were tried as the explants and cultivated in different media with the various portion of hormones. Results The medium of MS+BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.2 mg/L was the most suitable one for the induction of shoots; the medium of MS+BA 1.5 mg/L+NAA 0.1 mg/L was beneficial to the propagation of shoots; the medium of MS+BA 0.5 mg/L+NAA 0.1 mg/L was good for the elongation of shoots; and the medium of 1/2 MS without any hormone was suitable for the induction of roots. Conclusion Rapid propagation of A. carmichaeli could be achieved by tissue culture and this will lead to the possibility for its seedling in the industrial production.