Induction of somatic embryogenesis in Tecomella undulata (Sm.) Seem using a pistillate explant

Desert teak (Tecomella undulata (Sm.) Seem) a multipurpose ornamental tree native to the arid and semi-arid tropics has entered the endangered plant category mainly as a result of the species' ineffective seed reproduction system. The tree usually reproduces through a few root suckers in old stands. Conventional methods of plants multiplication could not offer a viable practice for its mass multiplication. Low adventitious rooting of the cuttings has been the principal cause of failure in its vegetative propagation. Hence, the present research was planned and conducted to evaluate the feasibility of somatic embryogenesis in this species, the pathway that bypasses the need for rooting stage by developing bipolar embryos. Ovary explant was cultured in modified Murashigue & Skoog (MS) medium supplemented with different auxins and cytokinins. The results showed ?-naphthalene acetic acid (NAA) was superior in inducing embryogenic callus. NAA ranging 5.4-21.5 ?M could induce the highest embryogenic calli which exhibited developing pro-embryogenic masses (PEM) and globular somatic embryos. The calli which were induced by the use of 2,4-dichlorophenoxyacetic acid (2,4-D) were poor in quality and showed no morphogenesis potency. Individual application of Thidiazuron (TDZ) and N6-benzyladenine (BA) induced good callogenesis at low concentrations but the calli were non-embryogenic with both. The proliferation of embryogenic calli was the best in a hormone-free medium. However, the media containing 40.5 and 54 ?M NAA alone could induce somatic embryos along with callus proliferation. Low BA-contained medium (0.9-4.44 ?M) led to recurrent somatic embryogenesis. Neither BA and GA3, nor the elevating sucrose concentration could cause further development and maturation of the somatic embryos induced during previous stages (callogenesis and callus proliferation). More research is required to optimize the maturation stage. The findings of the present study can be useful for future studies in the micropropagation of this recalcitrant specieslationships in Indian mustard under heat stress and the differential remobilization efficiencies in the advanced breeding lines.

Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives

BACKGROUND: In order to produce an effective callus in Echinacea purpurea L.; determination of the explant type and growth regulators that best respond to callus induction and the optimization of the culture conditions to increase the amount of caffeic acid derivatives (CADs) in the obtained callus. CADs contents of callus cultures of E. purpurea were evaluated by establishing an effective callus induction system in vitro. RESULTS: Various medium containing different growth regulators were tested using leaf, petiole, cotyledon and root as the explants. The best callus development was achieved in MS medium with 1.0 mg l 1 2,4- D + 2.0 mg l 1 BAP in leaf, 1.0 mg l 1 NAA + 0.5 mg l 1 TDZ in petiole, 2.0 mg l 1 NAA + 1.0 mg l 1 TDZ in cotyledon and 0.5 mg l 1 NAA + 0.5 mg l 1 BAP in roots. Upon optimisation of callus growth, each type of explant was cultured for 4, 6, 8 and 10 weeks in medium for the analyses of caftaric acid, chlorogenic acid, caffeic acid and chicoric acid contents. The highest amounts of caftaric acid (4.11 mg/g) and chicoric acid (57.89 mg/g) were found from petiole explants and chlorogenic acid (8.83 mg/g) from root explants at the end of the 10-week culture time. CONCLUSIONS: As a result of the present study, the production of caffeic acid derivatives was performed by providing the optimization of E. purpurea L. callus cultures. Effective and repeatable protocols established in this study may offer help for further studies investigating the production of caffeic acid derivatives in vitro.

Rapid in vitro adventitious rooting and proliferation by leaf and nodal cultures of Momordica cymbalaria Fenzl.

An effective approach for rapid in vitro rooting and proliferation of leaf and nodal cultures of Momordicacymbalaria has been developed. To the ability of induction of rhizogenesis, both leaf and nodal explants wereused in culture on Murashige and Skoog (MS) medium. The effects of auxins such as α-naphthaleneaceticacid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) at different concentrations have beenstudied. The maximum number of roots was produced from nodal explants containing 1.5 mg/L of NAA (9.3 ±0.61), 1.0 mg/L of IBA (6.5 ± 0.41), and 1.0 mg/L of IAA (3.5 ± 0.66), and in leaf explants containing 1.0 mg/Lof NAA (5.7 ± 0.56), 1.0 mg/L of IBA (6.9 ± 0.61), and 1.5 mg/L of IAA (5.0 ± 0.73) on the half-strength MSmedium. For the root induction, NAA is the very effective auxin in node explants of M. cymbalaria. Moreover,a large amount of quercetin bioactive compound is presented in the roots, which is used in anticancer drugs, andwe have described an effective method for the in vitro rhizogenesis of the M. cymbalaria.

In vitro culture of human corneal epithelial stem cells using modified explant culture / 中国组织工程研究

BACKGROUND: Corneal epithelial stem cells, also known as limbal stem cells, are distributed in the basal layer of limbal epithelium. It is extremely difficult to deal with limbal stem cell deficiency or dysfunction that is caused by severe thermal burn, chemical burn, and chronic inflammation of ocular surface. At present, in vitro culture of corneal epithelial stem cells using tissue engineering technology followed by clinical transplantation is a new and effective therapeutic direction. OBJECTIVE: To explore the feasibility of serum-free culture of human corneal epithelial stem cells in vitro using modified explant culture method. METHODS: The remaining donor corneal tissues after keratoplasty (less than 8 mm in diameter) were obtained from Henan Eye Bank, and the outer and middle limbus were dissected under surgical microscope. Two culture methods were used to culture human corneal epithelial stem cells. In the conventional explant culture group, the limbal tissues were adhered to the dish with the epithelium being upward, then Keratinocyte-serum free medium (K-SFM) was added into dishes, followed by incubation at 37 °C in a 5% CO2 incubator. In the modified explant culture group, limbal tissues were dissected to immerse in the K-SFM culture medium and incubated at 37 °C in the 5% CO2 incubator for 12 hours. The limbal tissues were then adhered to the dish with the epithelium being downward. The day whenever the cells from the limbal tissues adhered to the dish was marked as the 1st day of culture, and changes in cell morphology and growth were recorded by phase contrast microscopy every day. Immunofluorescent staining was used to detect the expression of K3 and p63 in primary cells on the 5th, 10th and 14th day of the modified explant culture. RESULTS AND CONCLUSION: The mean early stage of growth in the modified explant culture group was shorter than that of the conventional explant culture group (P < 0.05), and the mean growth rate of the modified explant culture group was higher than that of the conventional explant culture group (P < 0.05). In the modified explant culture group, cells had a good growth state, and many cells with small size gathered together on the 10th day of culture. On the 14th day, cell clones were formed, and the cells in the clone showed uniform morphology. On the 5th day, K3 highly expressed, while p63 lowly expressed in primary cells. On the 10th day, both of K3 and p63 had an increased expression. On the 14th day, there was no significant increase in the K3 expression, but the expression of p63 increased significantly. In the in vitro serum-free culture condition, the modified explant culture could significantly promote the growth of corneal epithelial stem cells, and expand corneal epithelial stem cells in vitro, which could provide sufficient seed cells for enriching corneal epithelial stem cells and constructing human limbal multilayered epithelial sheets.

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.

A new method for isolation and culture of fibroblast-like synovial cells from adjuvant arthritis rabbits / 第二军医大学学报

[Abstract] Objective To establish a new, simple method for isolation and culture of fibroblast-like synoviocytes (FLSs) from adjuvant arthritis (AA) rabbits using suspended explant culture method. Methods Sixteen healthy New Zealand white rabbits, weighing 2.0-2.5 kg, were randomly divided into two groups: the normal control group (n＝8) and the model group (n＝8). Rabbit models of AA were prepared by injection with complete Freund’s adjuvant at multiple sites and time points. The joint synovial layers were obtained. The FLSs of rabbits with AA were cultured by suspended explant culture method and explant culture method. The growth and morphological characteristics of cells were observed, and the cell viability was measured by CCK-8 assay. The expression of vimentin was detected by immunocytochemistry. Results The tarsometatarsal joints and toes of rabbits in the model group were swollen more obviously than those in the normal control group. After the toes were dissected, obvious microvascular proliferation and inflammation were observed in the model group. The results of H-E staining showed that synoviocytes in the normal control group were arranged in a regular manner like beads, while those in the model group were arranged in a disordered manner, suggesting that the AA model was successfully prepared. The operating durations required for suspended explant culture method and explant culture method were about 25 min and 1 h, respectively. The morphology and viability were similar for the cells obtained by the two methods. The positive rates of vimentin in the suspended explant culture method and explant culture method group were 98.01% and 98.35%, respectively. Conclusion Isolation and culture of FLSs by suspended explant culture method has been successfully established, and the method is simple and needs a short time.

Organogenesis of Abelmoschus moschatus Medik. Using Aseptic Seedling Explants

Aim: Abelmoschus moschatus have been extensively used in traditional medicine as well as in perfume industries. The primary goal of the present research was to develop an efficient plant regeneration protocol of Abelmoschus moschatus from aseptic seedling explants such as cotyledon, internode, leaf and root. Methodology: The seeds of Abelmoschus moschatus were surface sterilized with 0.1% mercuric chloride and 70% ethanol were cultured on ½ MS basal media for developing aseptic seedlings Aseptic seedling explants were cultured on different concentrations of auxins for callus induction. Later callus was transferred on to different concentrations of cytokinins for shoot regeneration and for in vitro, rooting different concentrations of auxins were used. Finally, such in vitro developed plantlets were acclimatized. Results: Half strength MS medium with 1% sucrose was used for raising aseptic seedlings. Maximum of 92% response of callus induction was obtained from leaf explants on MS medium + 2 mg/L 2, 4-dichlorophenoxyacetic acid. An average of 2.4 shoots per callus were observed on MS + 2 mg/L benzyl-6-aminopurine from leaf explant. The regenerated shoots were best rooted on 1/2 MS + 0.5 mg/L indole-3-butyric acid. The regenerated plantlets were established with 70% survival. Conclusion: An efficient plant regeneration protocol of Abelmoschus moschatus was developed.

Concentration of ms medium and cutting of seeds on in vitro establishment of baruzeiro (Dipteryx alata Vog. ) / Concentração do meio ms e corte da semente no estabelecimento in vitro do baruzeiro (Dipteryx alata Vog. )

Savannah is the second biome in biodiversity in Brazil, presenting great vegetation endemism. Baruzeiro (Dipteryx alata Vog.), native from this biome, is an economically important species, with an incipient market due to the lack of commercial plantations. This highlights the need to develop and provide the basis for the domestication of this species. Thus, this study evaluated different concentrations of MS medium, using baruzeiro intact or cut seeds for in vitro establishment. Seeds from baruzeiro ripe fruits were decontaminated and were left intact or partially cut; subsequently, the seeds were inoculated into flasks with different concentrations of MS culture medium. The experimental design was completely randomized as a 5 x 2 factorial, consisting of 5 MS medium concentrations (0, 25, 50, 75 or 100%) and 2 types of seeds (intact or partially cut seeds), with three replications. Each experimental unit consisted of five flasks and 10 plants. After five months of incubation, the contamination of explants, seed germination, the number of fully developed plants and the dry masses of shoot and root of baruzeiros were evaluated. Intact seeds provided better results for all characteristics evaluated. The increased concentration of MS medium resulted in mass gain of plants; however, the use of MS medium 0% provided greater percentage of fully developed plants, the most interesting feature for baruzeiro in vitro establishment.

O Cerrado é o segundo bioma em biodiversidade do Brasil, apresentando grande endemismo vegetal. O baruzeiro (Dipteryx alata Vog.) é uma espécie economicamente importante, com mercado incipiente devido à escassez de cultivos comerciais. Isto deixa notória a necessidade de desenvolver e aperfeiçoar subsídios para a domesticação dessa espécie. Assim, neste trabalho, objetivou-se avaliar diferentes concentrações do meio MS, utilizando sementes de baruzeiro (Dipteryx alata Vog.) íntegras e com corte para o estabelecimento in vitro. Sementes retiradas de frutos maduros de baruzeiro foram descontaminadas e mantidas intactas ou receberam cortes parciais, sendo posteriormente inoculadas em frascos, com diferentes concentrações de meio de cultivo MS. O experimento foi instalado em sistema de delineamento inteiramente casualizado em esquema fatorial 5 x 2 - meio MS (0, 25, 50, 75 e 100%) x tipos de sementes (sementes íntegras e sementes com corte) com três repetições, totalizando 30 parcelas. Cada unidade experimental foi constituída de cinco frascos e 10 plantas. Após cinco meses, foram avaliados: a contaminação dos explantes, a germinação das sementes, o número de plantas totalmente desenvolvidas e as massas secas da parte aérea e da raiz dos baruzeiros. As sementes íntegras proporcionaram melhores resultados, para todas as características avaliadas. O aumento da concentração do meio MS colaborou no ganho de massa das plantas, no entanto, o uso do meio MS 0% foi o que proporcionou maior percentual de plantas formadas, a característica mais interessante para o estabelecimento in vitro do baruzeiro.

Isolation and identification of nucleus pulposus mesenchymal stem cells from tissues removed by percutaneous endoscopic lumbar discectomy / 中国病理生理杂志

AIM:To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells (hNP-MSCs) in vitro and to identify their biological characteristics.METHODS:The explant culture method was employed to isolate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy (PELD).The isolated cells were passaged for purification and cultured in vitro followed by morphological observation.The cell proliferation ability was detected by CCK-8 assay.Growth curves of the cells were drawn and surface antigens were detected by flow cytometry.The cells at the 3rd~6th passages were induced for adipogenic, osteogenic and chondrogenic differentiation, and examined by oil red O staining, alizarin red staining and Alcian blue staining.RESULTS:The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD.The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45.The proliferative capacity was consistent with the growth characteristics of MSCs and multilineage differentiation potential was identified.CONCLUSION:A novel method to efficiently isolate and culture hNP-MSCs,PELD combined with explant culture method,was established, which would promote the study of regenerative medicine based on hNP-MSCs.

An experimental study of isolation of SD rat adipose-derived stem cells using suspended explant culture method / 中国免疫学杂志

Objective:To isolate SD rat adipose-derived stem cells(ASCs)by suspended explant culture method.Methods: The healthy rat inguinal fat pads were obtained.The SD rat ASCs were isolated by suspended explant culture method and explant adherent culture method.The growth status and morphology were observed.The growth curve and cell surface markers CD29,CD44,CD45 of the 3rd passage cells were analyzed respectively by CCK-8,immunocytochemistry;the 3rd passage cells were induced individually by adipogenic differentiation medium and osteogenic differentiation medium,and cells were examined by oil red O staining and alizarin red staining.Results: The SD rat ASCs obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curves were typical of S type.Immunocytochemistry analysis revealed that the third passage of SD rat ASCs were positive for CD29,CD44,but were negative for CD45.SD rat ASCs were positive for oil red O staining at 14 days after adipogenic induction,and positive for alizarin red staining at 14 days after osteogenic induction.Conclusion: Isolation of SD rat ASCs by suspended explant culture method is successfully established.The method is simple.It provides a new method for the isolation of SD rat ASCs in vitro.

Model construction of rat coronary artery smooth muscle cell endoplas-mic reticulum stress induced by thapsigargin / 中国病理生理杂志

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.

Comparison of explant culture methods for human corneal epithelial cell with free-serum / 眼科新进展

Objective To compare and analyze the time of cell eruption formation and the rate of cell eruption of human corneal epithelial cells cultured by different explant culture methods under serum-free conditions.Methods Explant tissues were randomly divided into A,B,C and D groups.In A group,the cornea epithelial cells were put side up after stripping corneal endothelium and scraping the corneal epithelium;in B group,the corneal epithelium was put side down after stripping corneal endothelium and scraping the corneal epithelium;in C group,the corneal epithelium was put side up after only stripping corneal endothelium;and in D group,the corneal epithelium was put side down after only stripping corneal endothelium.The morphological changes of the cells in the 4 groups were observed by phase-contrast microscope,and both aforementioned variable were recorded.Results The average time of cell eruption formation and the rate of cell eruption in A,B,C and D group was (7.00 ± 3.00) d and 47.50％ (19/40),(7.55 ±2.58)d and 45.83％ (11/24),(8.43 ±3.32)d and 63.64％ (14/22),(7.49 ± 2.20) d and 72.22％ (39/54),respectively.The chi-square test showed that there was significant difference in the cell eruption rate between A and D group as well as B and D group (all P ＜ 0.05),but there was no significant difference between the other groups (all P ＞ O.05).Conclusion The time of cell eruption formation in the four different explant culture methods was about one week in free-serum conditions.And D group has higher cell eruption rate,and pure plus stable cultured cells when compared with the other three groups.

Primary culture of rabbit fibroblast-like synoviocytes using an improved explant culture method / 中国比较医学杂志

Objective To establish an easier and better method for primary culture of rabbit fibroblast-like synoviocytes(FLS) by modified tissue cell culture.Method Healthy rabbit joint synovial layers were cut into small pieces and siphoned off water attached on the tissue with sterile filter paper and then cultured.The growth status and morphology were observed.The growth curve of the 3rd passage cells was measured by CCK-8 assay, and the expression of vimentin was detected by immunocytochemistry.Results The FLS cultured in vitro exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curve was typical of S type, and the cells highly expressed vimentin.Conclusions Primary culture of rabbit FLS by the improved explant culture method is successfully established.The method is simple and highly efficient.It provideds a new method for isolation of FLS.

Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method

Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC’s surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.

Primary culture of murine spleen-derived mesenchymal stem cells by explant culture / 中国比较医学杂志

Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells ( MSCs) by tissue explant culture.Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues.Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks.The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope.Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS).The cells were induced to differentiate into osteoblasts and adipocytes.Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance.The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established.The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

Primary culture of rabbit fibroblast-like synoviocytes using suspended explant culture method / 中国免疫学杂志

Objective:To isolate rabbit fibroblast-like synoviocytes(FLSs) by suspended explant culture method. Methods:The healthy rabbit joint synovial layers were obtained. The cells were cultured by suspended explant culture method and compared with the explants adherent culture method. The growth status and morphology were observed. The growth curve of the 3rd passage cells was measured by CCK-8 assay,and the expression of vimentin was tested by immunocytochemistry. Results: The FLS obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand. The cell growth curve was typical of S type, and the cells highly expressed vimentin. Conclusion:Primary culture of rabbit fibroblast-like synoviocytes by suspended explant culture method were successfully established. The method was simple and highly efficient. It provided a new method for the isolation of FLS in vitro.

New light sources for in-vitro potato micropropagation / Novas fontes de luz para micropropagação in vitro de batata

This research work objective was to optimize the micropropagation of potato cultivars through the use of new light sources in the growth rooms. Treatments consisted of three potato cultivars (Asterix, Catucha and Macaca), and five light sources (blue, green and red LEDs; Growlux and white fluorescent lamps). The explants consisted of nodal segments containing one bud, isolated from plantlets grown in vitro. The experimental design was completely randomized arranged in a 3x5 factorial, with eight replications. Each experimental unity consisted of a flask with five explants. Three 28-day consecutive subcultures were carried out in MS semi-solid medium, in growth-room under controlled conditions (temperature = 25+2 ºC; photoperiod = 16 hours; light intensity = 20 µmol m-2 s-1). At the end of each subculture, the bud number per plantlet, plantlet length and internode length were evaluated. After the third subculture, the concentrations of carotenoids and a- and b-chlorophylls were also determined. Different micropropagation efficiencies were found among potato cultivars grown in vitro conditions: 'Macaca' was the most and 'Catucha' the least responsive cultivar. The growth room light sources differently affected the potato plantlet development: red and green LEDs were the most and least recommended for plantlet development, based on the results of bud number per plantlet, plantlet length, and leaflet concentrations of a- and b-chlorophylls and carotenoids.

Este trabalho objetivou otimizar a micropropagação de cultivares de batata por meio do uso de novas fontes de luz no ambiente de cultivo. As cultivares avaliadas foram Asterix, Catucha e Macaca e as fontes de luz foram LEDs azuis, LEDs verdes, LEDs vermelhos, lâmpadas Growlux e lâmpadas fluorescentes brancas. Como explantes foram utilizados segmentos nodais contendo uma gema, isolados de brotações estabelecidas in vitro. O delineamento experimental foi inteiramente ao acaso em um fatorial 3 x 5 (cultivar x fonte de luz), com oito repetições por tratamento. Cada unidade experimental foi composta por um frasco contendo cinco explantes. O experimento compreendeu três subcultivos consecutivos de 28 dias em meio semi-sólido MS a 25+2 ºC, 16 horas de fotoperíodo e intensidade luminosa de 20 µmol m-2 s-1. Ao final de cada subcultivo foram avaliados o número de gemas produzidas por explante, o comprimento das brotações e o comprimento dos entrenós. Ao final do terceiro subcultivo, determinaram-se, ainda, as concentrações de carotenoides e de clorofilas a e b. Verificou-se efeito da cultivar na eficiência do processo de propagação de batata, sendo a 'Macaca' a mais e a 'Catucha' a menos responsiva in vitro. A fonte de luz do ambiente de cultivo afetou o desenvolvimento dos explantes de batata. Os LEDs vermelhos e os verdes foram, respectivamente, os mais e os menos indicados tanto para o desenvolvimento vegetativo quanto para a formação de clorofilas a e b e de carotenoides.

Direct Multiple Shoot Induction and Plantlet Regeneration from Cotyledonary Explants of Sapindus emarginatus Vahl (Soapnut).

germination of S. emarginatus in vitro cotyledon explants in BAP/Kn/TDZ (1.0-3.0 mg/L) supplemented MS medium and (2) in plant treatment with BAP/Kn/TDZ (3.0 mg/ L) in combination of 1AA (0.5 mg/L) of the cotyledon explants of plants and maintained under sterile conditions. While the former method resulted in as many as (7.5±8.6 shoot buds) from the cotyledonary explants within four weeks, the latter yielded on average approximately 8 shoot buds from each treated node in eight weeks. The cytokinin treatment in plant consisted of placing sterile filter paper moistened with sterile distilled water over the node and adding different concentrations of 6- benzylaminopurine. The best results for shoot bud regeneration were obtained with cotyledons, when cultured in the presence of (0.5 mg/L) IAA in combination with (3.0 mg/L). The shoots elongated and rooted directly in vermiculite after a pulse treatment with IBA (2.5 mg/L) for 15 min. Fungus growth, a serious problem in S. emarginatus tissue culture, was controlled using a fungicide, Bavistin, along with elimination of organic nutrients from the growth medium.

Indirect Regeneration of Withania somnifera from Nodal Explants.

Withania somnifera is an important medicinal plant and used to cure many diseases. Indirect regeneration protocol for multiple shoots development was established using nodal explants of W. somnifera from 50-60 days old seedlings. The callus induction was observed from nodal explants, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn). Maximum level of callusing response (80.0%) was recorded on MS medium supplemented with a combinations of 2.0 mg/l 2,4-D and 0.2 mg/l Kn. The callus (greenish compact) was transferred into MS medium containing various concentrations (0.5–2.0mg/l) of 6-benzyl amino purine (BAP) alone and in combination (0.1–0.4mg/l) with indole-3-acetic acid (IAA) for shoot initiation and proliferation. The maximum number of shoots was initiated from callus on 1.0mg/l BAP along with 0.2 mg/l IAA and proliferation of shoots achieved by subsequent subcultures at 4 weeks interval in the same medium. The maximum of 31.4 shoots/explant were achieved in the second subculture. MS medium along with 1.0 mg/l gibberellic acid (GA3) induced maximum elongation (96.7%) of regenerated shoots and MS medium supplemented with 0.8 mg/l indole-3-butyric acid (IBA) induced maximum rooting (96.7%) from elongated shoots. After a hardening period, the plantlets were transferred to the field with 98% of survival.

Research on tissue culture and plant regeneration for roots of Ficus hirta / 中草药

Objective: To solve the shortage problem of large-scale planting the seedlings in the roots of Ficus hirta by rapid propagation in vitro tissue culture. Methods: The MS and 1/2 MS media were used as basic media, The multi factor combination of plant growth regulator (6-BA, NAA, 2, 4-D, IAA, KT, and IBA) on the seedling subculture and root culture was studied by orthogonal design. Results: The best medium for the adventitious bud induction was MS + 6-BA 1.0 mg/L + NAA 0.3 mg/L, 72 adventitious buds were obtained by differentiation with 20 d induction of explants; The best medium for cluster inducing and subculture was MS + 1.0 mg/L 6-BA + 0.3 mg/L IAA + 0.3 mg/L KT; The best rooting medium was 1/2 MS + 1.0 mg/L IBA + 0.3 mg/L NAA, and the rooting rate was 100%. Being suitably Transplanted to peat-perlite (1:1) matrix, the 30 d survival rate was 93%. Conclusion: The tissue culture for the roots of F. hirta could be used to produce test-tube seedlings for large scale planting.