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.

Determination of Optimal Hormone and Mineral Salts Levels in Tissue Culture Media for Callus Induction and Growth of Industrial Hemp (<i>Cannabis sativa</i>L.)

Historically, industrial hemp (Cannabis sativa L.) has been a valuable source of metabolites and compounds, such as cannabidiols. There is a need for large amounts of plant tissue to be grown under controlled environments, and plant tissue culture is one unique way to yield this tissue. The purposes of this study were to determine: 1) the optimal concentrations (μM)/ratios of auxin:cytokinin in media and;2) the optimal mineral salts formulation for callus induction and callus growth in select hemp cultivars. To find the optimal concentration/ratios, 16 different combinations of auxin:cytokinin and three different mineral salts formulations were evaluated. The three mineral salts formulations tested were MS salts, MB5D1K and an MTSU formulation. The top performing hormone formulations were determined to be equal concentrations (1:1, 2:2, 3:3 μM) of auxin and cytokinin. The top performing media hormone formulations for callus induction were determined to be 2:1, 2:2, 2:3, and 3:2 μM (auxin:cytokinin). The optimal mineral salts formulation was determined to be MD5D1K. Therefore, the overall optimal media formulation for hemp callus production would be MB5D1K salts with the concentration/ratio of 2:2 μM (auxin:cytokinin).

Experiment on Tissue Culture Technique of Saposhnikovia divaricata(Turcz.) Schischk

The study aimed to optimize the induction and differentiation medium by exploreing different tissue culture of Saposhnikovia divaricata （Turcz.） Schischk. In tissue culture with the root, stem segments, young leaf, cotyledonary node and axillary bud of Saposhnikovia divaricata （Turcz.） Schischk as explants, a lot of plantleles were obtained and the corresponding plant regeneration-system was established. The results showed that when use MS＋1.0 mg·L^-1 6-BA＋0.2 mg·L^-1 NAA as callus induction medium, the cotyledonary node had the highest bourgeon rate, and its callus was better than any others; MS＋2 mg·L^-1 6-BA＋0.4 mg·L^-1 NAA was the best adventitious buds induction medium, and the best adventitious buds induced condition was 3% sucrose as carbon source, illumination for 12-14 h·d^-1 and pH 5.8, The best rootage medium was 1/2 MS＋0.5 mg·L^-1 NAA.

Effects of CeCl_3 and LaCl_3 on callus and root induction and the physical response of tobacco tissue culture

La3＋ and Ce3＋ have positive effects on plant growth and production. Although it is well known that rare earth elements promote cell growth. The biological effects of La^（3＋） and Ce^（3＋） on callus, shoot and root induction in tobacco are still unclear. The relationships among callus induction, rooting, enzyme activities and stomatal characteristics in tobacco are unknown. The objectives of this study were to identify the relationships between the induction of calluses, shoots, roots, stomata and enzyme activities. The induction percentages of calluses, buds, roots were recorded at 5,10,15, 20 and 25 days after La^（3＋） and Ce^（3＋） treatments. Peroxidase isoenzyme activity was determined by electrophoresis. The characteristics of the stomata were observed under an optical microscope. Our results show that low concentrations of Ce^（3＋）（〈15 mg/L） result in increases in the induction percentages of calluses,buds and roots, but La^（3＋）（〉5 mg/L） inhibits the induction of calluses, buds and roots. There are more peroxidase isoenzyme bands in Ce^（3＋） treatments than in La^（3＋） treatments. This is consistent with the induction percentages of calluses,buds and roots in Ce^（3＋） and La^（3＋） treatments. High enzyme activities may promote the induction of calluses, buds and roots. The stomata area and stomata number of leaves are significantly different between La^（3＋） treatments and Ce^（3＋） treatments. La^（3＋） improves the stomata area and number. Based on these results, we speculate that La^（3＋） may promote the development of the photosynthetic system. Ce^（3＋）may promote tobacco growth and rooting by improving enzyme activities.

Two Ways to Induce Gynostemma pentaphyllum Plantlets from Tissue Culture

[Objectives]This study was conducted to investigate rapid propagation systems of Gynostemma pentaphyllum.[Methods]Ten different media were tested to select the optimal media for inducing callus proliferation,bud differentiation and rooting by using tissue culture technology,with G.pentaphyllum stem segments and leaves as explants.The stem segments of G.pentaphyllum were used as explants to directly induce rooting and germination,and appropriate media were selected.[Results]The optimum callus induction medium for G.pentaphyllum stem segments was MS+6-BA 1.0 mg/L+NAA 0.4 mg/L;the optimum rooting medium for stem callus was MS+NAA 0.2 mg/L;and the optimum germination medium for stem segments was MS+6-BA 1.0 mg/L.The optimum callus induction medium for G.pentaphyllum leaves was MS+6-BA 0.5 mg/L+NAA 0.2 mg/L;and the optimum rooting medium for leaves was MS+NAA 0.4 mg/L.The optimum rooting medium for G.pentaphyllum stem segments was MS+IAA 1.0 mg/L,with which the rooting rate was 100%,the average root length was about 3 cm,and the average number of sprouts per explant was 1.48,so it is the optimal condition.[Conclusions]This study provides a new method for in-vitro cultivation of G.pentaphyllum and has far-reaching significance for improving human health.

Validating field regeneration capacity for selected accessions of Gossypium hirsutum using callus induction and regeneration capacity

Background Gossypium hirsutum undergoes rapid clonal propagation to regenerate a mature plant through tissue culture.However,the correlation between cotton leaf regeneration,callus induction,and regeneration ability was still obscure.In this research,cotton leaf regeneration level for 21 accessions in the field(new leaves)was observed after the first harvest,and a comparison between field regeneration level and callus induction with its regeneration capacity(new shoots and roots)for the same 21 accessions was carried out.Agronomic traits,including plant height,leaf area,fresh leaf weight,dry leaf weight,the number of flowers and bolls,and biochemical(proline content)and physiological(chlorophyll and carotenoid content)traits during the flowering stage of 21 upland cotton accessions,were investigated.Result A significant correlation between physiological parameters and callus induction was discovered.Callus induction and regeneration capacity of roots and shoots for hypocotyl,cotyledons,and shoot tip tissues were used to validate field leaf regeneration level after the first harvest.CCRI 24 showed significant leaf regeneration in the field and callus induction capacity through callus induction and regeneration.Conclusion We found a substantial relationship between field regeneration capability and callus induction with its regeneration capacity for the hypocotyl,cotyledons,and shoot tip.The results showed that ZS061,Lumian 378,Jimian 863,and ZS065 have the highest moisture retention capacity,while CCRI 24,Liaoyang Duomaomian,and Beizhe Gongshemian have the lowest moisture retention capacity.CCRI 24 has the highest leaf regeneration capacity in the field,while Beizhe Gongshemian has the lowest leaf regeneration capacity.All our result provides a clue for checking the regeneration capacity through leaf regeneration level in the field.

Ecophysiological aspects of in vitro biotechnological studies using somatic embryogenesis of callus tissue toward protecting forest ecosystems

This review on current biotechnological methods in forestry for in vitro tissue cultures to define the effect of stress conditions on trees,concentrates on somatic embryogenesis.Callus tissue,the key product of somatic embryogenesis,grows over a tree wound under ex vitro conditions.Callus tissue can be used in research in areas such as pathogenic susceptibility at the embryonic level,effect of heavy metals,influence of low temperatures(cryopreservation),production of secondary metabolites and transformation of plants.Callus of arborescent plants can be induced in vitro by fungal elicitors to produce secondary metabolites for pharmaceutical and cosmetic industries and are strongly repellant to herbivores and can thus act to protect forests.Analyses of dual cultures demonstrated that callus tissue exposed to a pathogenic fungus responds by synthesizing low-molecular-mass proteins belonging to an immune protein class.Cryopreservation of embryonic callus tissue also has broad applications,e.g.,for valuable plant genotypes in gene banks.Without strategies to protect forests against stressfactors,forest ecosystems will degrade to the detriment of all life,including humans.In vitro biotechnological research using callus tissue contributes to progress in forestry and the disciplines of ecology,physiology,phytopathology,culture and selection of plants.

Advances in D. <i>melanoxylon</i>Investigations towards Tissue Culture: Problems and Limitations

The first attempt on D. melanoxylon tissue culture was conducted from 2010 to 2013 at a high level of expectations. A total of 500 seeds were sterilized at different concentration of reagents and inoculated at different strength of the Murashige and Skoog medium for germination to obtain disease free explants for callus induction trials. A total of 400 nodal segments obtained from germinated seeds were sterilized at different concentration of reagents and inoculated at different hormonal combinations to induce callus formation for seedling multiplication. Results from this tissue culture attempt set a foundation for tissue culture success in D. melanoxylon on the future research. Only 19.8% of seeds inoculated in half strength of Murashige and Skoog medium germinated within 7 days while only 6.8% of seeds inoculated in full strength of Murashige and Skoog medium germinated within 6 days. This germination was at sterilization of 20 minutes in 35% ethanol and 20 minutes in 2.6% sodium hypochlorite. A total of 1% of inoculated D. melanoxylon seedling fragments in Murashige and Skoog media supplemented with hormone combination at 2.0 mg/l BAP + 0.5 mg/l NAA developed callus after16 days from the inoculation day. The final weight of the callus at the last record was 0.62 g. In this induction ex-plants were surface sterilized in 35% ethanol for 20 minutes and 2.6% sodium hypochlorite solution for 20 minutes. The color of callus was green and friable in nature. Other hormonal combinations in this case did not induce callus production. These results suggested that the problems which affect seed germination in the natural environment are also reflected on germination in the Murashige and Skoog medium and in callus induction. Vulnerability to fungal attack is a limitation for successful callus induction and germination in the culture room. More research under improved sterile conditions is needed to improve callus percentage for seedling multiplication.

Preliminary Study on Callus Differentiation of Hermaphrodite Papaya

With calluses of hermaphrodite papaya as the tested material and MS as the basic medium, the effects of different plant growth regulators and their combinations on adventitious bud induction of calluses and rooting induction of sterile buds were investigated. The results showed that 6-BA and TDZ all showed certain induction effect on callus differentiation of papaya; the induction effect of 6-BA was better than that of TDZ, and the optimum concentration of 6-BA was 0.05 mg/L. GA3 could promote the induction effect of 6-BA for on callus differentiation. The optimum medium combination for inducing the callus differentiation of papaya was MS ＋ 6- BA 0.5 mg/L ＋ GA3 1.0 mg/L. Compared to NAA, IBA was more suitable for inducing the rooting of adventitious buds. The optimum rooting-induction medium combination was MS ＋ IBA 0.3 mg/L.

Optimization of the Callus Induction System of Chenopodium quinoa Willd

Callus induction effects of nine varieties of Chenopodium quinoa Willd. were compared by taking stem segments and cotyledons of C. quinoa as the ex- plants. At the same time, callus JnductJon of stem segments was optimized, as well as the callus proliferation system. Research results showed that the optimal explant for callus induction was stem segment. The average callus induction rate of nine varieties reached 90% in culture medium MS ＋ 0.5 mg/L 2, 4-D. In the callus opti- mization test, treatment VI （MS ＋ 0.5 mg/L 2, 4-D ＋ 0.5 mg/L KT ＋ 0.5 mg/L NAA） and treatment II （MS ＋ 0.5 mg/L 2, 4-D） had close induction rate, but the callus morphology was greatly different. The latter had loose, glossy and yellowish white calluses. Therefore, culture medium MS ＋ 0.5 mg/L 2, 4-D was the optimal for callus induction. And using 2, 4-D together with KT and NAA could significantly increase the proliferation rate of calluses.

Inducing Adventitious Buds from Tomato Callus and Their Rooting

[ Objective] The aim was to establish efficient regeneration system of tomato so as to study the genetic transformation of chloroplast in tomato. [ Meth- od] The tomato seeds were sterilized and cultured into plantlets. Then, the leaves were cut from plantlets and placed in the MS with 3.0 mg/L 6-BA ＋ 0.3 mg/L IAA to induce callus. Finally, the effect of different hormones and concentrations on induction of adventitious buds from tomato callus and rooting was compared. [Result] The best medium for the induction of differentiation of adventitious buds from callus was： MS ＋ 2.0 mg/L 6-BA ＋ 0.3 mg/L sugar. The best medium for rooting was： 1/2MS ＋ 1.0 mg/L IAA. [ Conclusion] Appropriate sdection of hormone concentrations is the key to establish efficient regeneration system for tomato.

Study on Callus Induction and Plant Regeneration of Physalis alkekengi

[ Objective] This study aimed to investigate optimal conditions for callus induction and plant regeneration of Physalis alkekengi. [ Method ] P. alkek- eng/was employed as the experimental material and different concentrations of 6-BA and c^-NAA were added to MS medium to prepare the differentiation medium, to investigate the effect of different concentrations of plant hormones on callus differentiation of P. alkekengi. [ Result ] Under low or high concentrations of 6-BA, ratio of the total number of adventitious buds/explants and differentiation rate were reduced with the increasing concentration of ct-NAA ; when the concentrations of 6-BA and ct-NAA were respectively 1.0 and 0.2 mg/L, ratio of the total number of adventitious buds/explants and differentiation rate reached the maximum. [ Conclusion] When the concentrations of α-NAA were maintained unchanged, ratio of the total number of adventitious buds/explants reached the maximum under the moderate concentrations of 6-BA, while the differentiation rate showed irregular variations. In MS medium with supplement of 1.0 mg/L 6-BA and 0.2 mg/L α-NAA, ratio of the total number of adventitious buds/explants and differentiation rate reached the peak. This study provided reference for callus induction of P. alkekengi.

激素及pH对大蒜愈伤组织诱导研究

探讨大蒜愈伤组织诱导及增殖分化的影响因素,建立大蒜愈伤组织诱导、增殖及器官分化的培养体系。结果表明,适合大蒜愈伤组织诱导及增殖的培养基激素浓度及pH为2 mg/L NAA,2 mg/L 6-BA,1 mg/L 2,4-D和pH 5.8;苍山蒜愈伤组织出芽分化的最佳培养基激素浓度及pH为1 mg/L NAA,3 mg/L 6-BA,pH 5.8;苏联蒜愈伤组织出芽分化的最佳培养基激素浓度及pH为2 mg/L NAA,2 mg/L 6-BA,pH 6.1;苍山蒜和苏联蒜愈伤组织生根分化的最佳培养基激素浓度及pH均为1 mg/L NAA,pH 5.8,表明高浓度的2,4-D有利于大蒜愈伤组织的诱导,但并不利于其分化。

6 Kinds of Isozymes after Long-term Subculture of Emmenopterys henryi Oliv.

[Objective] The aim was to study the changes of zymography in 6 kinds of isozymes after long-term subculture of Emmenopterys henryi Oliv.. [Method] Non-denaturing polyacrylamide gel electrophoresis was used to analyze isozyme patterns such as esterase (EST),acid phosphatase (ACP),ATP enzyme (ATPase),amylase (AMY),superoxide dismutase (SOD) and peroxidase (POD) in long-term subculture callus of Emmenopterys henryi Oliv. [Result] The research showed that there were differences among the 6 kinds of isozymes in embryogenic callus and non-embryogenic callus of Emmenopterys henryi Oliv.,and both levels could be taken as the basis for identification,the EST,ACP,and POD of non-embryogenic callus were significantly higher than embryogenic callus. The browning of non-embryogenic callus was non-level in the AMY,SOD and POD isozymes when was compared with normal non-embryogenic callus,while the EST,ACP and ATPase isozymes decreased; When the browning of embryogenic callus was contrasted with normal embryogenic callus,EST isozyme increased and the other 5 kinds of enzymes decreased. [Conclusion] The study provided theoretical basis for research morphological difference and browning of long-term tissue culture of Emmenopterys henryi Oliv..

爆裂玉米自交系愈伤组织诱导和再生体系研究

为了优化爆裂玉米自交系N10和N04的胚性愈伤组织诱导和再生体系,以爆裂玉米自交系N10和N04的幼胚为外植体,研究影响幼胚愈伤组织诱导和再生的重要因素。结果表明,幼胚大小为1.0~1.3 mm时,胚性愈伤组织诱导形成较好。自交系N10和N04的适宜愈伤组织诱导培养基分别为MS和MB培养基,AgNO_(3)浓度分别为13μmol/L和36μmol/L,2,4-D浓度分别为2 g/L和0.4 g/L,L-脯氨酸浓度分别为2.76 g/L和5.0 g/L。6-BA浓度为0.2 mg/L时,自交系N10和N04愈伤组织分化较好;NAA浓度为0.2 mg/L时,幼苗生长状况较好。

Preliminary Study on the Anther Culture in Lycium barbarum L.

To investigate the culture technique in anther of Chinese wolfberry,we optimized the culture medium(including hormone combination)and culture conditions.The results showed that calluses were induced from all the six tested Chinese wolfberry materials,but the induction rate of callus varied toward the materials with different genotypes.When the experimental materials were cultured on medium appended with 2,4-D 1.0 mg/L and KT 1.0 mg/L under dark,the callus induction rate reached 20.0 % in this study,and this hormone combination should be the optimum for anther culture of Chinese wolfberry.With MS appended with 6-BA 0.5 mg/L and NAA 0.1 mg/L as differentiation medium and that appended with NAA 0.1 mg/L,the plants could be yielded in 20 days.

黄花菜组织培养常见问题及建议

黄花菜兼具食用价值、观赏价值和药用价值,现今越来越受到人们的广泛关注。长期以来,黄花菜新优品种缺乏、繁殖手段单一,限制了产业健康快速发展。组织培养拥有繁殖系数大、周期较短、不受季节约束、苗整齐度高以及节省育苗用地等优点,因此,利用组培技术快速扩繁对黄花菜的市场开发、应用和推广具有重要意义。旨在为应对黄花菜的组培中出现的各类问题、提高黄花菜组培苗成活率提供一定的参考,为黄花菜遗传转化以及新优品种工厂化育苗提供理论依据,以黄花菜为研究对象,阐述了在组织培养过程中存在的各项内在影响因素,包括植物材料的基因型、选取的外植体类型;以及外在影响因素,包括外植体取材时期、基本培养基类型、植物生长调节剂以及培养条件。结果发现,黄花菜的组织培养体系仍不成熟,存在着外植体及其采集标准的不确定、愈伤组织诱导困难、最优培养基配方不统一、再生植株移栽成活率低等问题。为优化这样的问题,仍需摸索一套适合黄花菜的再生体系,并加强对组培苗的移栽后管理。此外,对组织培养过程中存在的污染、褐化及玻璃化问题作出归纳总结,并对黄花菜组织培养未来的发展趋势进行了展望。

槲树不同外植体愈伤组织诱导及继代培养研究

【目的】以槲树(Quercus dentata)的根与叶为外植体,以期筛选出愈伤组织诱导和继代培养的适宜条件,为槲树组培快繁体系的建立提供支撑。【方法】在WPM、MS、1/2MS三种培养基中,加入不同浓度的NAA和6-BA,以槲树幼苗根与叶为外植体诱导愈伤组织,记录、分析和比较不同条件下的诱导率。再将根诱导后的愈伤组织接种于WPM培养基中,添加不同浓度的2,4-D和6-BA进行愈伤组织的增殖,计算和比较增殖系数。【结果】叶片诱导愈伤组织的最佳培养基为MS+NAA 1.0 mg/L+6-BA 0.5 mg/L。根诱导愈伤组织的最佳的培养基为WPM+NAA 2.0 mg/L+6-BA 1.0 mg/L。经比较,以根作为外植体的诱导率高于叶片。愈伤组织增殖的最佳培养基为WPM+2,4-D 1.2 mg/L+6-BA 0.9 mg/L。【结论】筛选出了最适合槲树愈伤组织诱导和增殖的培养基配方,为槲树组培快繁体系的建立奠定了基础。

‘红颜’草莓组培快繁技术试验

以‘红颜’草莓匍匐茎的茎尖为试材,经初代培养和继代培养获得脱毒苗。结果表明:初代愈伤诱导培养最适宜的培养基配方为MS+NAA 1.0 mg·L^(-1)+6-BA 5 mg·L^(-1);最适宜愈伤增殖的培养基配方为MS+NAA 1 mg·L^(-1)+6-BA 10 mg·L^(-1)+TDZ 1 mg·L^(-1);最适宜诱导萌芽的培养基配方为MS+NAA 0.40 mg·L^(-1)+6-BA 10 mg·L^(-1)+活性炭1g·L^(-1)。在继代培养中最适宜诱导愈伤的部位为叶片,最适宜诱导萌芽的部位为茎尖处;最适宜叶片诱导出愈伤组织的培养基配方为MS+NAA 0.50 mg·L^(-1)+TDZ 20 mg·L^(-1)+活性炭1g·L^(-1);最适宜茎尖诱导萌芽的培养基配方为MS+IBA 5 mg·L^(-1)+活性炭3 g·L^(-1);最佳生根培养基配方为1/2MS+IBA 5 mg·L^(-1)+活性炭3 g·L^(-1)。

扭果花旗杆组织培养与再生体系的建立

【目的】以扭果花旗杆(Dontostemon elegans)根和下胚轴为外植体,建立扭果花旗杆组织培养体系,为后续遗传转化体系的建立提供技术基础。【方法】以MS培养基为基础培养基,利用萘乙酸(NAA)探究根和下胚轴直接诱导不定芽情况,利用2,4-二氯苯氧乙酸(2,4-D)、6-苄氨基嘌呤(6-BA)、NAA、苯基噻二唑基脲(TDZ)、吲哚丁酸(IBA)进行愈伤组织诱导及分化和不定芽生根试验,统计愈伤组织诱导及分化和不定芽生根情况,确定扭果花旗杆组培体系最佳培养基类型。【结果】(1)由根和下胚轴直接诱导不定芽的最适培养基分别为MS+0.3 mg/L NAA和MS+0.6 mg/L NAA,直接诱导不定芽的最佳外植体为根。(2)扭果花旗杆根和下胚轴都能诱导愈伤组织,其中根系诱导愈伤组织速度最快且状态最佳,其次为下胚轴。根系愈伤组织诱导的最佳培养基为MS+1.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.1 mg/L NAA,诱导率可达100.00%;下胚轴愈伤组织诱导的最佳培养基为MS+0.3 mg/L 2,4-D+1.5 mg/L 6-BA+1.5 mg/L NAA,诱导率可达100.00%。(3)根系和下胚轴的最佳愈伤组织分化培养基均为MS+0.5 mg/L 6-BA+0.3 mg/L NAA,分化率分别为100.00%和86.66%。(4)最佳生根培养基为MS+0.5 mg/L IBA,生根率可达50%以上。(5)将组培苗移至土壤后,在光照培养室培养15 d,其存活率可达83.33%。【结论】建立的扭果花旗杆组培体系可获得与实生苗形态一致的组培苗,为扭果花旗杆遗传转化体系的建立奠定了技术基础。