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.

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.

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.

The nitrate-responsive transcription factor Md NLP7 regulates callus formation by modulating auxin response

Under appropriate culture conditions,plant cells can regenerate new organs or even whole plants.De novo organ regeneration is an excellent biological system,which usually requires additional growth regulators,including auxin and cytokinin.Nitrate is an essential nutrient element for plant vegetative and reproductive development.It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants.In this study,we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants.MdNLP7 mainly participated in the regulation of callus formation,starting with pericycle cell division,and mainly affected auxin distribution and accumulation in the regulation process.Moreover,MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage.The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants.Moreover,the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

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.

Insecticidal Activities of Extracts from Brucea javanica (L.) Merr. Callus

[Objective] The research aimed to lay a foundation for the screening of cell lines producing secondary metabolites of Brucea javanica(L.)Merr.[Method] The insecticidal activities of the extracts from branch and 3 different types of calluses of Brucea javanica(L.)Merr.was detected through methods of leaf disc and potted seedlings against the diamond back moth.[Result] Extracts from four kinds of Brucea javanica(L.)Merr.tissues assumed both the activities of antifeedant and oviposition deterrency against the diamond back moth.Antifeedant effect of extracts was in turn the callus C< callus B< callus A< branches.Oviposition deterrency activity of the extracts was in turn the callus A> branch > callus B>callus C.The insecticidal activities of callus A and B were higher than that of the callus C.[Conclusion] The results show that insecticidal activity of callus and its growth rate is inversely proportional.

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.

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.

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.

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.

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.

Optimization of Inducing Conditions for Loose Callus of Pinellia ternata

[Objective] The research aimed to establish the cell suspension culture system of Pinellia ternate.[Method] The petiole of Pinellia ternate was as the explant.The orthogonal test was used to study the influences of four plant growth substances（2,4-D,NAA,picloram and KT）and their mixture ratios on the formation of loose callus.[Result] The induction effect of 2,4-D and picloram on the petiole callus of Pinellia ternate was the most significant.Then,the second ones were KT and NAA.The optimal medium which induced Pinellia ternate petiole to form the loose callus was MS＋0.5 mg/L of 2,4-D＋1.0 mg/L of NAA＋1.0 mg/L of picloram＋1.5 mg/L of KT.[Conclusion] The research laid the foundation for extracting the active ingredient from the cell suspension of Pinellia ternate and producing the artificial seed.

Sugarcane Callus Culture and Cytological Characteristics

In order to obtain sugarcane embryogenic calli for transgenosis, the effects of different soaking time of explants, medium, culture conditions and sampling time on sugarcane callus culture were investigated. The results showed that soaking ex- plants with MS liquid medium for 20 min could significantly reduce the browning rate; MS＋3.0 mg/L 2.4-D was appropriate for induction and proliferation of embryo- genic calli; dark condition was suitable for the culture of sugarcane calli; calli differ- entiated from materials collected in August exhibited low browning rate, high callus induction rate and high embryogenic callus induction rate. Calli with different pheno- types were stained, prepared into slides and observed under a microscope to ana- lyze the cytological characteristics. The resultsshowed that milky-white granular em- bryogenic calli had closely arranged cells with large nucleus and exhibited strong differentiation capacity, which were appropriate receptor materials for genetic trans- formation of sugarcane.

A Preliminary Study on Callus Induction and Proliferation of Cylocarya paliurus

[Objective] To carry out preliminary study on callus induction and prolifer- ation of Cylocarya pafiurus. [Method] The leaf and stem segments of Cylocarya paliurus as explants were cultured on MS, WPM and improved DKW3 mediums, added 6-BA and IBA with different concentration ratios to explore the optimum medium for callus induction and proliferation. [Result] It was found that MS ＋ 4.0 mg/L 6-BA ＋ 2.0 mg/L IBA was the optimum medium for inducing stem callus of Cylocarya pali- urus, MS ＋ 2.0 mg/L 6-BA ＋ 0.5 mg/L IBA was the optimum medium for inducing leaf explants callus of Cylocarya paliurus, and MS ＋ 1.0 mg/L 6-BA ＋ 1.0 mg/L IBA was the optimum medium for callus proliferation. 200 mg/L Vitamin C was best to inhibit Cylocarya paliurus callus from browning with the browning rate of only 5.71%. [Conclusion] This study provided some theoretical basis for the establishment of tis- sue culture and rapid propagation system and the development of molecular breed- ing study of Cylocarya paliurus.

Determination of Emodin and Chrysophanol Contents in Callus of Cassia tora L. Leaf

[ Objective] Through inductive culture, emodin and chrysophanol contents in cassia seed were increased. [ Method ] MS culture medium was used to bourgeon seedlings of cassia seed and to induce cotyledon callus of cassia seed while HPLC method was adopted to determine emodin and ehrysophanol eontents.[ Result] Emedin and chrysophanol contents in cotyledon callus were 0. 099% and 0. 312%, respectively, while they were 0.029% and 0. 190% respectively in cassia seed. [ Conclusion] The method of inducing cotyledon callus was helpful for increasing emodin coment in cassia seed.

Effects of Four Culture Media on Callus Induction Rate of Wheat Anther

Effects of four culture media including MS, N6, C17 and K on wheat anther callus induction in vitro culture were studied. The results showed that the callus in- duction rate of four kinds of culture medium was in the order of K〉C17〉N6〉MS.

Callus Induction of Alfalfa and Evaluation of Callus Tolerance to Acid and Aluminum

Medicago is an important grass in the legume family,which is suitable to be grown in neutral or alkalescent soils,and hence,can be widely spread in south of China with low pH value.It is one of major objectives to cultivate acid-aluminum tolerant species.In the research,6 high-quality species with high-yielding potential were taken as materials to make evaluation on acid-aluminum tolerance by cell-culture technique,and the results showed that tolerance capacity from high to low was GT13R＞Muge 701=Muge 702＞Acrora＞AC-3＞Sheshou No.2=Medicago sativa.

Callus Induction and Cell Suspension Culture of Eggplant(Solanum melongena L.)

[Objective] The aim of this article is to establish a cell suspension culture system for eggplant. [Method] Using orthogonal design, appropriate media were screened for callus induction, subculture and cell suspension culture. [Result] The appropriate medium for callus induction was MS supplemented with 0.2 mg/L 6-BA and 0.2 mg/L NAA; the appropriate subculture medium was MS supplemented with0.2 mg/L 6-BA, 0.2 mg/L NAA, and 0.1 mg/L KT; the suitable medium for cell suspension culture was liquid MS medium supplemented with 0.4 mg/L NAA and 0.2mg/L. Cell growth curve was similar to ＂S＂ shape in the above suspension medium.The cell growth included four phases, the initial phase（1-3 d）, the logarithmic phase（3-7 d）, the steady phase（7-8 d）, and the decline phase（8-11 d）. With the increasing culture time, the number of suspension cells increased, and it reached the maximum value at the 7thd, about 3.8 ×105cells/ml. Then the number of cells began to decline rapidly. The cell vigor was the highest at the initial phase. Suspension cells grew best in the liquid MS medium supplemented with NAA（0.4 mg/L）and 6-BA（0.2 mg/L）. The mitotic index reached the maximum, about 4.1% in the above medium, which suggested that this medium was suitable for cell suspension culture of eggplant. [Conclusion] Cell suspension culture system of eggplant provides a significant method for eggplant biotechnology. Genetic transformation and mutants screening can be carried out with this system.

A Genetic Transformation System for Rosa multiflora Thunb. var. cathayensis Rehd. et Wils through Callus Induction

An efficient genetic transformation system is a preparation for Rosa multi-flora Thunb. var. cathayensis Rehd. et Wils to diversify its flower color through ge-netic engineering. We firstly optimized the explants and culture conditions on callus induction, hormone concentrations and dark period of culture time on bud differentia-tions in particular, with sterilized seedlings to establish the regeneration system of R. multiflora. It showed that callus induction frequency reached 100% after the ex-plants being cultured in dark for 21 d when MS was chosen to be the initial culture medium. The bud differentiation rate was 48% after cal i being cultured under dark for 8 d on MS medium supplemented with TDZ （1.5 mg/L） and NAA （0.05 mg/L）. The cal i was used as the explants that were infected with Agrobacterium tumefa-ciens harboring a DFR-RNAi construct. The transformation rate reached as high as 50%. The establishment of a highly efficient rose gene transformation system out-lined in this report is prerequisite for genetic improvement in rose flower colors.