Effect of Different Disinfection Methods on Calluses of Amorphophallus albus P.Y.Liu&J.F.Chen

This study aimed to examine the effect of different disinfection methods on the callus of Amorphophallus albus in the tissue culture and rapid propagation system.The calluses induced from buds of A.albus were disinfected with benzalkonium bromide,mercury bichloride,sodium hypochlorite,and ethanol,respectively,for different time periods.The growth of calluses was compared between different treatments and observed in detail by dissection.The results showed that the disinfection with 0.1% mercury bichloride for 5 min and 75% ethanol for 5 s had mild effect on the callus,with the lowest browning rates of 16.32% and 15.46% and the lowest vitrification rates of 3.24% and 9.72%,respectively,after 20 d.The disinfection with benzalkonium bromide and sodium hypochlorite had strong effect on the callus cells,with the lowest browning rate of 50.69% and 25.12% and the lowest vitrification rates of 28.47% and 28.43%,respectively.The cytological observation result showed that the calluses disinfected with ethanol and mercury bichloride showcased compact structures,while those disinfected with sodium hypochlorite and benzalkonium bromide exhibited loose structures.To sum up,it is recommended that 75% ethanol or 0.1% mercury bichloride can be used for disinfection in the tissue culture and rapid propagation of A.albus,while the use of benzalkonium bromide and sodium hypochlorite should be avoided as much as possible.

The Effect of Exogenous Phytohormones on Resistance of Wheat Calluses to <i>Tilletia caries</i>(D.C.) Tul. &C. Tul.

The influence of exogenic hormones (indole-3-acetic acid (IAA), abscisic acid (ABA) and kinetin) on defense reaction of wheat (Triticum aestivum L.) calli to the bunt agent Tilletia caries (D.C.) Tul. & C. Tul. was studied. ABA and kinetin induced the oxalate oxidase activity, increased the Н2О2 level, decreased germination of fungi teliospores and induced on calli the occurrence of dense sites non-infected by pathogen. On the contrary, IAA led to the decrease of oxalate oxidase activity, loosening of calli and increase germination of bunt agent teliospores and growth of fungi mycelium, besides stimulated rhizoids formation of wheat calli. Probably, the accumulation of Н2О2 in wheat calli under the influence of kinetin and ABA connected with activity of oxalate oxidase is one of the factors increasing defense reaction of wheat to bunt agent.

A Prospective Single-Arm Investigation Assessing Performance of a Multifunctional Foot Cream in Subjects with Tinea Pedis Interdigitalis and Heel Cracks, Calluses and/or Dry Feet

This prospective, open-label, single-arm investigation evaluated the performance of a multifunctional foot cream used twice daily for 8 weeks in the combined treatment of mild/moderate tinea pedis interdigitalis (TPI) and heel cracks, calluses and/or dry feet in 46 adult subjects. All subjects showed reductions in the severity of symptoms of both TPI (53.7% reduction) and heel cracks, calluses and/or dry feet (51.6% reduction), beginning already after 2 weeks of treatment, with reduction continuation and improvement after 4 and 8 weeks of treatment. Negative mycological cultures were obtained post-treatment in 85.7% of subjects, while 35.7% of subjects also obtained mycological cure (negative mycological culture + negative KOH test). Post-treatment questionnaires showed improved quality of life, good treatment tolerability, easy use, and that 69% of subjects would recommend the product. The results reveal that the foot cream is safe and effective in the combined treatment of TPI and heel cracks, calluses and/or dry feet.

Ethanol Metabolism in Calluses of Several Selected Plant Species on Two Typical Plant-Growth-Regulator Balanced Media

For investigation on the characteristics of ethanol metabolism in tissues of different plant species, calluses from eight selected plant species were cultured on medium supplemented with ethanol in tightly sealed culture flasks. Changes of the ethanol level were detected by gas chromatography. During the culture period, the calluses of tobacco, potato and petunia were, able to catabolize exogenous ethanol, resulting in the prominent decline of the ethanol level in the medium. The calluses of melon and peanut were also able to catabolize thanol but with lower efficiency. The other three calluses of carrot, soybean and rice did not catabolize ethanol but instead produced small to large amount of ethanol, resulting in the increase of the ethanol level in the media. It was also found that changing the balance between auxin and cytokinin could influence only the ethanol metabolism efficiency but could not change the metabolism patterns on ethanol of the cultured calluses. It can be concluded that, ethanol metabolism pattern of calluses in cultures is an innate physiological characteristic of the respective plant species.

PLANT REGENERATION AND DIFFERENTIATION OF ADVENTIVE BUDS FROM T-DNA-CALLUSES IN Solanum nigrum

Solanum nigrum is a herb useful for medicine and can also be used as a receptor system in gene transfer. Teratomas can be obtained in Solanum nigrum by infection of Agrobacterium tumefaciens and T-DNA plantlets differentiated from tumor tissues. The differentiation of leaves and stems of such T-DNA plantlets can be observed.

Population genomic analysis reveals key genetic variations and the driving force for embryonic callus induction capability in maize

Genetic transformation has been an effective technology for improving the agronomic traits of maize.However,it is highly reliant on the use of embryonic callus(EC)and shows a serious genotype dependence.In this study,we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline(MQ2Gpipe).Based on the induction rate of EC(REC),these inbred lines were categorized into three subpopulations.The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms.By integrating a genome-wide selective signature screen and region-based association analysis,we revealed 95.23 Mb of selective regions and 43 REC-associated variants.These variants had phenotypic variance explained values ranging between 21.46 and 49.46%.In total,103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci.These genes mainly participate in regulation of the cell cycle,regulation of cytokinesis,and other functions,among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction.Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci,implying a potential synergistic selection of REC and leaf size during modern maize breeding.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures(RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

In Vitro Propagation and Artificial Seed Production of Fritillaria cirrhosa D. Don, an Endangered Medicinal Plant

Fritillaria cirrhosa D.Don(Liliaceae)is an endangered perennial bulbous plant and its dry bulb is a valuable med-icinal material with antitussive and expectorant effects.Nevertheless,lack of resources and expensive prices make it difficult to meet clinical needs.This study presents a regeneration system aimed at overcoming the challenge of inadequate supply in F.cirrhosa,focusing on:(1)callus induction,(2)bulblets and adventitious bud induction,and(3)artificial seed production.Callus development was achieved in 84.93%on Murashige and Skoog(MS)medium fortified with 1.0 mg·L^(-1) picloram.The optimal medium for callus differentiation into regenerated bulb-lets was MS medium supplemented with 1.0 mg·L^(-1)6-benzyladenine(6-BA)and 0.2 mg·L^(-1)α-naphthaleneacetic acid(NAA).Subsequently,bulblets and adventitious buds were induced from regenerated bulblet sections cul-tured on MS medium fortified with 0.3 mg·L^(-1)6-BA+1.0 mg·L^(-1)2,4-dichlorophenoxyacetic acid(2,4-D),2.0 mg·L^(-1)6-BA+0.5 mg·L^(-1),and the induction rates were 88.17%and 84.24%,respectively.The regenerated bulblets were transplanted into a substrate of humus soil,river sand,and pearlite(1:1:1)after low-temperature treatment.The germination rate was 42.80%after culture for 30 days.Regenerated bulblets were used for encap-sulations in liquid MS medium containing 3%sucrose(w/v)+0.5 mg·L^(-1) NAA+2.0 mg·L^(-1)6-BA+3%sodium alginate(w/v)with a 10 min exposure to 2%CaCl_(2).Under non-aseptic conditions,the germination rate reached 81.67%,while the rooting rate was 20.56%after 45 days.The capsule added 1.0 g·L^(-1) carbendazim and 1.0 g·L^(-1) activated carbon was the best component of artificial seeds.This study successfully established an efficient regen-eration system for the rapid propagation of F.cirrhosa,involving in vitro bulblet regeneration and artificial seed production.This method introduces a novel approach for efficient breeding and germplasm preservation,making it suitable for large-scale industrial resource production.

Chemical profiling of bioactive compounds in the methanolic extract of wild leaf and callus of Vitex negundo using gas chromatographymass spectrometry

BACKGROUND The investigation of plant-based therapeutic agents in medicinal plants has revealed their presence in the extracts and provides the vision to formulate novel techniques for drug therapy.Vitex negundo(V.negundo),a perennial herb belonging to the Varbanaceae family,is extensively used in conventional medication.AIM To determine the existence of therapeutic components in leaf and callus extracts from wild V.negundo plants using gas chromatography-mass spectrometry(GCMS).METHODS In this study,we conducted GC-MS on wild plant leaf extracts and correlated the presence of constituents with those in callus extracts.Various growth regulators such as 6-benzylaminopurine(BAP),2,4-dichlorophenoxyacetic acid(2,4-D),α-naphthylacetic acid(NAA),and di-phenylurea(DPU)were added to plant leaves and in-vitro callus and grown on MS medium.RESULTS The results clearly indicated that the addition of BAP(2.0 mg/L),2,4-D(0.2 mg/mL),DPU(2.0 mg/L)and 2,4-D(0.2 mg/mL)in MS medium resulted in rapid callus development.The plant profile of Vitex extracts by GC-MS analysis showed that 24,10,and 14 bioactive constituents were detected in the methanolic extract of leaf,green callus and the methanolic extract of white loose callus,respectively.CONCLUSION Octadecadienoic acid,hexadecanoic acid and methyl ester were the major constituents in the leaf and callus methanolic extract.Octadecadienoic acid was the most common constituent in all samples.The maximum concentration of octadecadienoic acid in leaves,green callus and white loose callus was 21.93%,47.79%and 40.38%,respectively.These findings demonstrate that the concentration of octadecadienoic acid doubles in-vitro compared to in-vivo.In addition to octadecadienoic acid;butyric acid,benzene,1-methoxy-4-(1-propenyl),dospan,tridecanedialdehyde,methylcyclohexenylbutanol,chlorpyrifos,n-secondary terpene diester,anflunine and other important active compounds were also detected.All these components were only available in callus formed in-vitro.This study showed that the callus contained additional botanical characteristics compared with wild plants.Due to the presence of numerous bioactive compounds,the medical use of Vitex for various diseases has been accepted and the plant is considered an important source of therapeutics for research and development.

Study on Tissue Culture and Effective Clone Establishment of Hemistepta lyrata Bunge

[Objective] The research aimed to study the rapid propagation technology and establish effective clone of Hemistepta lyrata Bunge. [Method] With tender stem of Hemistepta lyrata Bunge as material, the conditions needed in calluses induction and differentiation, adventitious bud differentiation and radication, test tube seedling cutting and transplantation were studied. [ Result] The results showed that the optimum medium for granulated calluses induction from tender stem was MS ＋ BA 0.3 mg/L ＋2,4-D 1 -1.5 mg/L, for granulated calluses and adventitious bud differentiation was MS ＋ AgNO31.5 mg/L ＋ BA 0.4 mg/L ＋ NAA 0.1 mg/L. 1/2 MS ＋ IAA 0.6 mg/L was suitable for test tube seedling rooting and regeneration, and cinder was used as transplantation and cutting substrate. [ Conclusion]This study will provide the scientific reference for choosing the feasible medium in tissue culture of Hemistepta lyrata Bunge.

云南红豆杉愈伤组织培养及其生产紫杉醇的研究

云南红豆杉愈伤组织培养及其生产紫杉醇的研究甘烦远彭丽萍郑光植（中国科学院昆明植物研究所昆明６５０２０４）从红豆杉科红豆杉属植物如短叶红豆杉（Ｔａｘｕｓｂｒｅｖｉｆｏｌｉａ）等的树皮或枝叶提取到的紫杉醇（Ｔａｘｏｌ）是一种具有强抗癌活性的二萜烯类化合...

Establishment of In Vitro Regeneration System of the Atrichum Mosses

In vitro regeneration systems of Atrichum mosses, Atrichurn undulatum (Hedw.) P. Beauv. and A. undulatum var. minus (Hedw.) Par. were established. After one month, soft, friable and green calli were induced successfully from inoculated protonema of Atrichum mosses on MS medium containing glucose (4%) and 6-BA (0.2-2.0 mg/L). The suitable culture medium for the callus induction and regular subculture was MS medium with 1.0-2.0 mg/L 6-BA and 4% glucose. The calli of Atrichum mosses developed into protonema, when it was transferred to phytohormone-free MS medium with 4% glucose. Meanwhile, the calli developed into erect gametophytes through protonema phase on carbohydrate-free Benecke medium.

Study on Explants Sterilization and Callus Induction of Aquilegia oxysepala

[Objective] The aim was to study the explants sterilization and callus induction of Aquilegia oxysepala.[Method] the seeds of Aquilegia oxysepala were sterilized by different kinds and concentrations of disinfectants,and the pollution rate and pollution speed were investigated so as to find the best way to build sterile seedling setup.Taking the roots,stem segments and leaves of the sterile seedlings from Aquilegia oxysepala seeds as explants,the optimum explants and medium were screened by adding MS basic medium with different hormone proportions.[Result] The best germicidal treatment was as follows：explants were soaked in 75% alcohol for 30 s firstly,washed by sterile water for 5 times,then soaked in 0.2% mercuric chloride liquid for 2 min,finally washed by sterile water for 5 times again.The sterilization treatment could get the lowest pollution rate,the highest germinating capacity and the best sterile seedling.Roots were the optimum explants for the callus induction of Aquilegia oxysepala,meanwhile the optimal medium was MS＋0.6 mg/L 2,4-D＋0.5 mg/L 6-BA.[Conclusion] The research provides technical support for the large scale production of Aquilegia oxysepala and also makes a contribution to the medicinal and ornamental value of Aquilegia oxysepala.

Optimization of Callus Induction Medium for Schisandga chinensis Baill via Uniform Design

[Objective]This study was to optimize callus induction medium for Schisandga chinensis Baill.[Method]Using callus induction rate as an indicator,uniform design was employed to optimize hormone combination at poly-factors and poly-levels for callus induction from Schisandga chinensis Baill.[Results]Optimal hormone combinations depended on different explants：optimum medium for both tender leaf and petiole was MS ＋3 mg/L 6-BA,for stem segment was MS ＋3 mg/L 6-BA ＋0.6 mg/L NAA.[Conclusion]Uniform design is a time-saving and convenient method for the optimum medium for callus and yields a higher callus induction rate.

Effects of 2,4-D and 6-BA on Callus Induction and Plantlet Regeneration from Mature Embryos of Hsien Rice

[ Objective] In order to study the effects of 2,4-D and 6-BA on callus cultivation from mature embryos of hsien rice. [ Method] 2,4-D and 6-BA were set at different concentrations in callus induction and differentiation mediums to study their effects on callus induction, seedling formation and regenerated seedlings rooting. [ Result] In the callus induction medium treated with 0.5 mg/L 2,4-D, the callus induction effects on the varieties like Jiayu 948, Yanghui 559, Yangxian 6547, Zhong＇erruanzhan, Minghui 86, Guanghui 998 and Zunxian 3 were the best; If 0.2 mg/L 6-BA was added into the callus induction medium containing the optimum level of 2,4-D, there was no obvious effect on induction rate of callus, but the differentiation and seedling of callus were inhibited; If the concentration of 6-BA was reduced appropriately in the differentiation medium, the seedling rate of callus would be not only no decreased but increased, meanwhile the quality of regenerated plants would be improved. [ Conclusion] The study results provided some references for the reasonable uses of 2,4-D and 6-BA in callus culture of hsien rice.

Agrobacterium-mediated Transformation of Kentucky Bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

Effects of Genotypes and Basic Medium on Culture of Maize Mature Embryos

[Objective]This study was to screen out suitable genotypes and basic medium for the culture of maize mature embryos.[Method]Using mature embryos of nine maize genotypes as explants,the effects of genotypes and basic medium on callus induction and subculture were investigated.[Result]The genotypes performed better in callus induction and subculture were found in turn 853-35,853-209,Dan 34 and 81162.MS medium is better than N6 medium in the callus induction from maize embryos,while N6 medium is more suitable for callus subculture.[Conclusion]Our study further improved the tissue culture system in maize with mature embryos as explants.

Callus Induction from Mature Embryos of Maize

In this study we studied the factors influencing the callus induction from mature embryos of maize inbred lines Qi 319, Zhen 58, Chang 7 -2, Lx 9801 and 81162, such as genotype, combination of plant growth regulators, and low-temperature pretreatment. The results showed that the induction rate of Qi 319 was the highest among the four genotypes tested; combination of 4.0 mg/L 2,4-D ＋ 0.5 mg/L 6-BA was suitable for inducing callus from mature embryos; three days of 4℃ pretreatment can promote the callus induction significantly. The indices optimized in the present study are helpful for establishing genetic transformation system in maize without considering seasonal variation.

The Factors of Genetic Transformation for Indica Rice Kasalath

Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.

Callus induction from leaves of different paulownia species and its plantlet regeneration

The experiment was carried out on five different species of Paulownia for callus induction from leaves. MS medium was adopted as basic medium, and from different combinations of NAA and BA the suitable media were determined for callus induction, bud differentiation, and root differentiation of five different species. MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5NAA+4BA, MS+0.3NAA+6BA, and MS+0.3NAA+8BA were suitable media of callus inductions of leaves, respectively, for Paulownia tomentosa, Paulownia australis, Paulownia fortunei, Paulownia elongata and P. tmentosa x P. fortunei, and MS+0.3NAA+12BA, MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA, and MS+0.7NAA+12BA were suitable media for bud differentiation from leaf callus respectively for above five species. The rooting media was determined as 2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, 1/2MS+0.3NAA, and 1/2MS+0.5NAA. These results provide reference data for breeding new fine va-rieties with different kinds of Paulownia protoplasts fusions.