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.

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.

Infection of Early and Young Callus Tissues of Indica Rice BPT5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture （LBA4404： pSB1, pCAMBIA1301） and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6-and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants （To） revealed the presence of 1-2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.

Purification and Analysis of Abscisic Acid-Specifically-Inducible Proteins from Rice Callus

Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points （pl） of the proteins with the same molecular mass （24.5 kD） were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 （pl 6.1） protein was only detected in calli treated with ABA and seed embryos （SE）. However, the A2 （pl 6.9） protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo （or somatic embryo） formation.

Anticancer Activities of Plant Secondary Metabolites:Rice Callus Suspension Culture as a New Paradigm

Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.

Proline and Glutamine Improve in vitro Callus Induction and Subsequent Shooting in Rice

This study was conducted to evaluate the effects of proline and glutamine on in vitro callus induction and subsequent regeneration and to develop a reproducible and highly efficient plant regeneration protocol in four rice genotypes, viz. Pawana, Jaya, Indrayani and Ambemohar. Considerable variation in response to plant growth regulators and amino acid supplements used was observed in all the four genotypes. Medium supplemented with proline and glutamine was shown to be superior to medium without proline and glutamine. The best callusing from mature embryo was observed on Murashige and Skoog （MS） medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid （2,4-D）, 500 mg/L proline and 500 mg/L glutamine. Shoot induction was higher in the callus obtained from medium supplemented with 500 mg/L proline and 500 mg/L glutamine. The highest shoot regeneration frequency （83.2%） was observed on MS medium with 2.0 mg/L benzylaminopurine, 0.5 mg/L 1-naphthaleneacetic acid, 500 mg/L proline, and 500 mg/L glutamine in the callus obtained from MS medium supplemented with 2.0 mg/L 2,4-D, 500 mg/L proline and 500 mg/L glutamine. Among the four genotypes, Pawana has the highest regeneration efficiency （83.2%）, whereas the regeneration efficiency of the rest three rice genotypes was in the range of 32.0% to 72.3%. This optimized regeneration protocol can be efficiently used for Agrobacterium mediated genetic transformation in rice.

Iksan526 Rice Callus Extract Induces Dedifferentiation of Rabbit Articular Chondrocytes via ERK1/2 and PI-3K/Akt Pathways

The resveratrol-enriched transgenic rice line Iksan526(IS526),first developed by the Rural Development Administration of Korea using genetic engineering techniques,shows beneficial health effects in mitigating metabolic syndrome and obesity.However,the effects of IS526 on the differentiation of chondrocytes and the underlying mechanism have not been investigated in detail.In this study,the effects and cellular regulatory mechanisms of IS526 on rabbit articular chondrocytes were examined.Following IS526 callus extract treatment,the expression levels of differentiation-related proteins were detected via western blotting,Alcian blue staining and immune-luorescence staining.IS526 decreased the type Ⅱ collagen and proteoglycan levels in dose-and time-dependent manners.We further analyzed the effects of IS526 on skeleton genesis in zebrafish larvae using Alcian blue staining,which showed a reduction in cartilage formation along with increased production of matrix metalloproteinase(MMP)-13.IS526 also increased the phosphorylation of ERK1/2 and p38 kinase but inhibited the phosphorylation of Akt.Pharmacological inhibition of MMP-13 blocked the IS526-induced decrease in type Ⅱ collagen levels.Inhibition of p38 kinase or PI-3K/Akt with SB203580 and LY294002 enhanced the suppression of type Ⅱ collagen,but the blockage of ERK-1/2 by PD98059 rescued IS526-induced dedifferentiation.These results suggested that IS526 regulates type Ⅱ collagen and MMP-13 expression via the ERK1/2 and PI-3K/Akt pathways in rabbit articular chondrocytes.

Effects of Cultivar and Explant Sources on Callus Induction and Plant Regeneration in Rice （Oryza sativa L.）

In vitro callus induction and plant regeneration potentiality were studied from mature embryo of three Indian rice （Oryza sativa L.） groups at Field Crops Department, Agricultural Faculty, Ondokuz May？s University, Samsun, Turkey. The study was done by using callus induction MS medium having different concentration of four. The present research was conducted according to the design of randomized blocks trial. A total of 696 calluses, 193 plants and 917 seeds were obtained from Indica group; 2,110 calluses, 103 plants and 235 seeds were obtained from Japonica group; 1,243 calluses and 13 plants were obtained from Javanica group. With regard to number of calluses obtained from each explant source, 52 calluses were obtained from whole-plant explants, 1,668 calluses from root explants, 629 calluses from shoot explants, 649 calluses from the 1st node explants, 240 calluses from the 2nd node explants, 269 calluses from the 1st internode explants and 12 calluses from the 2nd internode explants. With regard to number of plants obtained from each explant source, 27 plants were obtained from whole-plant explants, 195 plants from shoot explants, 43 plants from the 1st node explants, 40 plants from the 2nd node explants and four plants from the 1st internode explants. With regard to number of seeds, 823 seeds were obtained from shoot explants and 329 seeds were obtained from the 2nd node explants. Germination rate of harvested seeds was over 90%. The establishment of this regeneration system is essential for the development of a genetic transformation system for commercial rice cultivars.

S-诱抗素对杂交稻产量及品质的影响

为探寻提高杂交稻产量和品质的栽培调控措施,对甬优4949、玮两优8612和乔两优17开展早、晚稻栽培试验,研究S-诱抗素对其产量及品质的影响。结果表明:S-诱抗素提高了各品种齐穗期SPAD值、光合参数及齐穗、成熟期干物质生产能力。各品种S-诱抗素处理的有效穗数、每穗粒数、结实率及千粒重等产量构成因子均有提高,增产7.8%~11.1%,乔两优17的增产效果更显著。S-诱抗素提高了各品种加工品质中的糙米率、整精米率及蒸煮品质中的碱消值、胶稠度,同时降低了外观品质中的垩白度、垩白粒率及蒸煮品质中的直链淀粉含量。早稻季各品种S-诱抗素处理的整精米率、垩白度、垩白粒率及碱消值与对照相比达到显著差异,S-诱抗素对早稻品质的改善效果优于晚稻。综合结果表明,施用S-诱抗素能够有效提高杂交稻的产量和品质。

In vitro callus induction and plantlet regeneration of Achyranthes aspera L.,a high value medicinal plant

Objective:To study callus induction from different explants(internode,leaf,root)and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.Methods:Sterilized explants were prepared by uning 0.1%HgCl_2 and 0.5%Bavistin and callus was obtained when cultured onto Murashige Skoog's(MS)medium by using different concentrations and combination of 2,4-D.NAA.BAP,IAA,IBA with 3%sucrose and 0.8%agar.Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.Results:Sterilization treatment of 0.1%HgCl_2.for 2-3 min and Bavistin 0.5%for 10-12 min showed the highest percentage of asepsis and survival rate.Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf.Highest shootlets number(4.83±0.l7)and length(3.8±0.16)cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L.Concerted efforts of BAP 10 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number(6.77±0.94).In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations.Experimentally,3.0 mg/L IBA was enabled to induce maximum rootlets number(10.0±9.82)on full strength MS medium.Afterwards,regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized.The survived plantlets showed 66.67%survival frequency without any morphological abnormality.Conclusions:The results demonstrated that different explants were good source of callus induction,morphology analysis as well as indirect plantlets regeneration.

Effects of variations in culture media and hormonal treatments upon callus induction potential in endosperm explant of Barringtonia racemosa L.

Objective: To induce callus from the medicinally valuable species, Barringtonia racemosa L.(B. racemosa) whereby the formation of callus is essential for micropropagation studies and in vitro plant secondary metabolites production.Methods: The callus induction potential in B. racemosa was assessed from endosperm explant cultured on different culture media and plant hormonal treatments. Lloyd and Mc Cown's woody plant medium and Murashige and Skoog's medium were used in the study as culture media. On the other hand, various concentrations and combinations of2,4-dichlorophenoxyacetic acid(1.0–2.0 mg/L) and kinetin(0.5–2.5 mg/L) had been incorporated in the culture media to exert the effects of auxin and cytokinin on callus induction.Results: From the present study, it was found that the profuse [(1.681 ± 0.770) g fresh weight,(0.239 ± 0.239) g dry weight] and friable callus formation was optimally produced with desirable morphology and considerable percentage of callus induction(56.70%) in endosperm explants cultured on 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 1.5 mg/L kinetin in Murashige and Skoog's medium.Conclusions: A reliable protocol for inducing callus formation of profuse and friable morphology in endosperm explant of B. racemosa had therefore been successfully established.

A simple and efficient seed-based approach to induce callus production from B73 maize genotype

The wild type maize genotype, B73, is not amenable for callus production and an efficient protocol for B73 maize callus induction has never been reported up-to-date. Scientific efforts in producing B73 maize callus using all known callus inducible media have been unsatisfactory. Here we developed and described an efficient protocol for callus induction from B73 maize seedlings. The protocol is based on well known callus inducible media CM4C where we have sequentially subtracted some chemical compounds and added some new compounds mediating cell proliferations. This newly described protocol was able to induce callus production in a wide range of crop species including rice and soybean. We found that cell proliferation factors, NAA (auxin analog) and 2,4 D (auxin influx carrier) were not only very crucial but required for positive B73 maize callus induction. The absence of one or the other will lead to the failure of B73 maize callus production. The well known CM4C callus induction composition lacks NAA. Our findings will advance genetic studies of maize mutants generated from B73 genotype background.

过表达野生大豆耐盐碱基因GsGSTU13提高了水稻苗期耐盐碱性

谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)是一类高度保守的功能酶,在植物对非生物胁迫应答过程中发挥重要作用。本研究通过生物信息学分析发现,野生大豆GsGSTU13与水稻GSTU17蛋白序列相似性最高,均含有高度保守的结构域。为探究GsGSTU13基因对水稻耐盐碱性的影响,通过遗传转化、PCR、RT-PCR和GST活性检测,获得了2个GsGSTU13过表达转基因水稻纯合株系。苗期耐盐碱性分析显示,200 mmol L^(-1) NaHCO_(3)处理下,GsGSTU13转基因株系的活性氧积累显著低于野生型,而存活率、相对含水量、超氧化物歧化酶、过氧化物酶、过氧化氢酶和GST活性均显著高于野生型。综上,在水稻中过表达GsGSTU13基因增强了活性氧清除能力,提高了转基因水稻耐盐碱性,为耐盐碱水稻新种质创制提供了参考。

S型控释氮肥运筹对水稻产量形成及氮素利用特征的影响

缓控释肥具有施用次数少、肥料利用率高的特点,S型热固性树脂包膜控释氮肥(S型控释氮肥)受土壤pH影响小、肥料稳定性好。本研究以S型控释氮肥为试材,分别设置了6个水稻品种的响应比较、4种氮肥施用量、5种氮肥施用时期及7种氮肥配施比例的4个盆栽试验,研究其对水稻产量形成、氮素吸收利用等的影响。结果表明:1)品种比较试验中,S型控释氮肥提高了6个水稻品种的产量,增幅在2.95%~18.57%,平均增加12.60%;产量提高的原因是穗数或每穗颖花数的增加;S型缓控释氮肥可显著提高供试水稻成熟期的氮素积累量、氮肥利用率,但氮素籽粒生产效率有所下降。2)控释肥运筹试验中,随着S型控释氮肥施用量和施用比例的增加,武运粳24号的产量呈先增加后下降的趋势,分别以每盆3g纯氮处理和控释氮肥占比80%处理产量增幅最大,作基肥时增产效应更显著;成熟期氮素积累量呈增加和先增加后下降的趋势,分别以每盆4.5 g纯氮处理和控释氮肥占比80%处理增幅最大,作基肥时增幅效应更显著;氮素籽粒生产效率和氮肥利用率呈降低趋势,氮素籽粒生产效率分别以控释氮肥占比0%和控释肥作保花肥最大,氮肥利用率分别以控释氮肥占比80%和控释肥作基肥时最大。3)控释肥对根系性状影响试验中,S型控释氮肥处理提高了武运粳24号水稻栽后30天单株不定根总长、根干重与吸收面积,提高了抽穗期单株不定根总长、根干重和根活力;单株不定根数、不定根总长、根干重、根活力的增加是产量、氮素积累量和氮肥利用率协同提高的根系基础。总之,S型控释氮肥可显著提高水稻的产量和氮肥利用率,作为基肥时最优。每盆3 g纯氮处理、控释氮肥占比80%左右时产量、成熟期氮素积累量、氮肥利用率协同增加,与其栽后30天、抽穗期根系性状优化有密切关系。

优质籼型光温敏核不育系荃时S的选育与应用

介绍了荃时S的选育过程、特征特性、配组应用及繁殖技术要点。荃时S是以银312S为母本,与配合力好的优良恢复系YR0822杂交,采用系谱法选育而成的籼型光温敏核不育系,具有育性转换起点温度较低、不育性稳定、异交率高、抗倒伏、米质优等特点,2021年通过安徽省农作物品种审定委员会组织的专家技术鉴定。该不育系与恢复系银泰香占配制的籼型两系杂交水稻品种时两优泰香具有中抗稻瘟病、米质达部标优1级、丰产等特点,并获得2022年(第四届)安徽省优质稻品种食味品质籼稻金奖,2022年通过国家农作物品种审定委员会审定。

Comparative Studies on the Changes of Microtubule Distribution and Reorganization During the Meiotic Stages of Development in Normal (IR36) and a Temperature/photoperiod Sensitive Male Sterile Line (Peiai 64S) of Rice ( Oryza sativa )

Changes in the pattern of organization of microtubules in the meiotic stages of development of pollen (i.e. from pre-meiotic interphase to more or less metaphase I) of a normal (IR36) and a temperature/photoperiod sensitive male sterile line (Peiai 64S) of rice were studied using immunofluorescence confocal microscopy. In IR36, from pre-meiotic interphase to metaphase I, the pattern of microtubule distribution in the meiocytes underwent a series of changes. Some new organizational patterns of microtubules (that have not been described before) were observed during microsporogenesis, including the existence of a broad band of perinuclear microtubules at the diakinesis stage of development. The pattern of microtubule distribution in the meiocytes of the male sterile line, Peiai 64S, was quite different front that seen in IR36. In Peiai 64S, the microtubules showed abnormal patterns of distribution from pre-meiotic interphase to metaphase I. For example the broad band of perinuclear microtubules seen at diakinesis in IR36 was much disorganized and loosened in Peiai 64S. The spindles formed were also very abnormal and different from the normal spindle. The appearance of abnormal microtubule distribution in the early stages of microsporogenesis may contribute to the malformation and ultimate abortion of pollen in Peiai 64S.

Studies on GA_3 Spraying Dosage for eui TGMS Rice Changxuan 3S in Its Hybrid Seed Production

[Objective] Changxuan 3S was thermo-sensitive genicmale sterile（TGMS）rice selected from irradiated seeds of Peiai 64S by 350 Gy^（60）Coγ-ray.The aim of the study was to confirm GA3 spraying dosage of Changxuan 3S with eui gene in its hybrid seed production.[Method] Changxuan 3S possessing eui gene and its parent Peiai 64S were chosen as materials.Comparison studies on sensitivity to GA3 in their hybrid seed production were carried out.[Result] The suitable stage for spraying GA3 in the hybrid seed production of Changxuan 3S was at 10% of panicles headed;The optimal dosage was 90 g/hm2 with 2 split sprayings,the first spraying of 45 g/hm2 at heading of 10% panicles and the second one of 45 g/hm2 on the following day.Under the condition of spraying GA3 at the rate of 90 g/hm2,the panicle neck exsertions of Changxuan 3S was ＋1.78 cm,and exserted stigma rate and seed setting rate of Changxuan 3S were 96.87% and 36.44%,being 21.46% and 16.33% more than those of Peiai 64S,respectively.The theoretical yield of ＂Changxuan 3S/9311＂ reached 2 931.90 kg/hm2,which was increased by 1 259.40 kg/hm2 comparing with ＂Peiai 64S/9311＂.[Conclusion] Compared with Peiai 64S,Changxuan 3S is more sensitive to GA3,which results in no or little using GA3 in seed production of Changxuan 3S.Moreover,Changxuan 3S showed higher yield potential than Peiai 64S.

Isolation, Expression Characteristics and Chromosomal Locations of Three cDNA Fragments Under Salt Stress in Rice

cDNA libraries were constructed from the leaves of a rice (Oryza sativa L.) salt tolerancevariety Tesan抋i 2 growing in solutions with 150 mmol/L NaCl for 3 h or without salt stress. Three salt-responsive cDNA clones, Ts1, Ts2 and Ts3 were isolated by differential screening. Northern blottinganalysis showed that the transcription levels of Ts1 and Ts2 increased within 3 h salt stress and kept onincreasing within 24 h, while the transcription level of Ts3 reached its peak within 3 h. Sequence analysisindicated that there were no homologies between the three cDNA clones and any known gene. The threecDNA clones were mapped using a doubled haploid (DH) population derived from an indica variety ZYQ8,which was a salt tolerance parent of Tesan抋i 2, with a japonica variety JX17. Ts1, Ts2 and Ts3 werelocated on chromosomes 1, 3 and 7, respectively. It was noted that Ts1, Ts2, and Ts3 were in or near theregions of major or minor salt tolerance quantitative trait loci (QTLs), which were mapped in the same DHpopulation in a parallel study.

Breeding and Application of Indica PTGMS Line Yan 161S and Its Hybrid Yanliangyou 1618 in Rice

In 2006, the indica photoperiod- and thermo-sensitive genic male sterile （PTGMS） line Yan 161S was successful y bred by crossing the Guangzhan 63S （♀） and the medium indica breeding material Yan 229 （♂）, which was bred by the Institute of Agricultural Sciences in Coastal Region of Jiangsu Province, after years＇ and generations’ selection in different areas. It passed the identification by Jiangsu Provincial Crop Variety Appraisal Committee in 2013. Crossing the Yan 161S with Yanhui 888, a new high-yielding and high-quality two-line hybrid rice combination Yanliangyou 1618 was developed, and it passed the variety identification in 2014. This paper introduced the breeding processes and characteristics of Yan 161S and Yanliangyou 1618 and the cultivation and seed production techniques of Yanliangyou 1618.

Breeding and Application of Indica PTGMS Line Yan 220S in Rice

Yan 220S, which is derived from a natural variant plant discovered among the population of Guangzhan 63S and developed by Jiangsu Agricultural Research Institute for Coastal Regions through pedigree selection over years, is an indica photo- and thermo-sensitive genic male sterile （PTGMS） line in rice. In 2013, it was technically identified by Jiangsu Crop Variety Appraisal Committee. Its hybrid Yan-liangyou 2208 （Yan 220S/Yanhui 888）, a new two-line hybrid rice combination with high yield and good grain quality, was subsequently developed and released for commercial production by the National Crop Variety Appraisal Committee in 2013.