Contributions of Chinese Botanists to Plant Tissue Culture in the 20th entury

This paper looks back to the development of plant tissue culture in China in the last century. Since 1934, tissue culture studies in China has kept up with the international development in the fields. Progress has been made by Chinese in nearly every branches of tissue culture, including in vitro organogenesis, shoot tip culture, anther culture, ovary culture, endosperm culture, protoplast culture as well as mass cell culture. On the basis of reviewing the articles written by Chinese on plant tissue culture, the internationally recognized contributions are specially mentioned. The applications of plant tissue culture to agriculture and industry in China are also introduced.

Plant Tissue Culture: A Recent Progress and Potential Applications

Plant tissue culture systems have enormous potential in fundamental re- search and for commercial applications such as horticultural industry. The process of tissue culture is companied with a series of changes in respect to morphology, physiology, biochemistry, molecule and epigenetics. The changes at molecule levels mainly include genetic variation, DNA sequence variation, chromosomal variation and epigenetic regulation （DNA methylation, histone modification, chromatin remodeling, small RNA regulation）. These changes are believed to facilitate explant adaptation to culture conditions and to help subsequent morphogenesis processes. Nowadays, it has played a crucial part in commercial applications and in basic research into cell biology, genetics and biochemistry, etc. In present review, we shed light on the fun- damental of plant tissue culture, culture medium preparation, explant selection, mechanism of action of various hormones, the three major problems （explant pollu- tion, browning, plantlets vitrification） and the prevention measures in tissue culture, and elaborated on in vitro propagation of plants, virus-free seedling cultivation, cry- opreservation, artificial seeds and molecule levels changes during in vitro culture further.

Pollution and Control Measures in Plant Tissue Culture in Orchid Plant

Contamination is a phenomenon that often occurs in the operation of plant tissue culture,and it is also one of the three major problems in plant tissue culture.Compared with browning and vitrification,contamination is more likely to occur,which brings great harm to scientific research and production practice.Its appearance will greatly affect the normal growth and differentiation of tissue culture materials,and will reduce the yield of cultivated plants to a certain extent.Therefore,we cannot underestimate any step in the tissue culture operation.This study summarized the reasons for its occurrence and how to formulate prevention and control measures based on recent research combined with the actual situation.

Coscinium fenestratum: Callus and Suspension Cell Culture of the Endangered Medicinal Plant Using Vermicompost Extract and Coelomic Fluid as Plant Tissue Culture Media

In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.

Lignin Biosynthesis Studies in Plant Tissue Cultures

Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given challenges to scientists studying its structure or biosynthesis. In plants lignified tissues are distributed between other, non-lignified tissues, Characterization of native lignin in the cell wall has been difficult due to the highly cross-linked nature of the wall components. Model systems, like plant tissue cultures with tracheary element differentiation or extracellular lignin formation, have provided useful information related to lignin structure and several aspects of lignin formation. For example, many enzyme activities in the phenylpropanoid pathway have been first identified in tissue cultures. This review focuses on studies where the use of plant tissue cultures has been advantageous in structural and biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for lignin biosynthesis.

Production of Active Compounds in Medicinal Plants:From Plant Tissue Culture to Biosynthesis

Over past decades plant tissue culture has emerged as an alternative of whole plant cultivation in the production of valuable secondary metabolites.Adventitious roots culture of Panax ginseng and Echinacea purpure has reached the scale of 1-10 kL.Some molecular biological techniques,such as transgenic technology and genetic stability are increasingly used in the studies on plant tissue cultures.The studies on elicitors have deepened into the induction mechanism,including signal molecules,functional genes,and so on.More and more biological elicitors,such as A.niger and yeast are used to increase the active compounds in plant tissue cultures.We also discussed the application of synthetic biology in the studies on biosynthesis of artemisinin,paclitaxel,and tanshinon.The studies on active ingredients biosynthesis of medicinal plants provide unprecedented possibilities to achieve mass production of active ingredients.Plant tissue cultures can not only produce active ingredients but also as experimental materials for biosynthesis.In order to improve the contents of active compounds in medicinal plants,following aspects could be carried out gene interference or gene silencing,gene overexpression,combination with chemical synthesis,application of elicitors,and site-directed mutagenesis of the key enzymes.

Plant Tissue Culture and Biosynthesis Provide a Fast Way to Produce Active Constituents of Traditional Chinese Medicines

The plant kingdom has provided literally thousands of natural products with widely diverse chemical structures and a vast array of biological activities.Many of them have seen subsequent application in discovery of new druids and the pharmaceutical industry and clinical therapeutic application.

Rapid Propagation of Pteris vittata L.via Tissue Culture

[Objective] The study had developed a means of rapid propagation Pteris vittata L.by tissue culture. The species was a perennial fern belonging to the genus Pteris. [Metbed] The leaf bud of P. vittata collected in field conditions as explantsand the 1/2 MS ＋ 3% sucrose ＋ 0.7% agar as the basic medium were used to screen the medium formula of the phytohormone ratio for callus induction and subculture of P. vittata. [Result] The best medium formula for each step was list below： 1/2 MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 0.1 mg/L KT ＋ 0.5 mg/L 2, 4-D for in- ducing the callus from explants; 1/2MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 1.0 mg/L KT ＋ 0.01 mg/L 2,4-D for inducing the GGB from callus and the seedlings from GGB. In addition, 1/2 MS ＋ 3% sucrose ＋ 0.7% agar ＋ 0.5 g/L PVP ＋ 0.5 mg/L 2,4-D for the subculture could make the continued proliferation of callus. [Cen- clusioa] This study makes an applicable procedure by the direct use of field materi- als, for propagating P. vittata in a simplified and rapid mode.

Fourier transform infrared spectral features of plant biomass components during cotton organ development and their biological implications

Background:The majority of attenuated total reflection Fourier transform infrared(ATR FT-IR)investigations of cotton are focused on the fiber tissue for biological mechanisms and understanding of fiber development and maturity,but rarely on other cotton biomass comp on ents.This work examined in detail the ATR FT-IR spectral features of various cott on tissues/organs at reproductive and maturation stages,an a lyzed and discussed their biological implications.Results:The ATR FT-IR spectra of these tissues/organs were an a lyzed and compared with the focus on the lower wavenumber fingerprinting range.Six outstanding FT-IR bands at 1730,1620,1525,1235,1050 and 895 cm^(-1) represented the major C=O stretching,protein Amide I,Amide II,the O-H/N-H deformation,the total C-O-C stretching and the β-glycosidic linkage in celluloses,respectively,and impacted differently between these organs with the two growth stages.Furthermore,the band intensity at 1620,1525,1235,and 1050 cm^(-1) were exclusively and significantly correlated to the levels of protein(Amide I bond),protein(Amide II bond),cellulose,and hemicellulose,respectively,whereas the band at 1730 cm^(-1) was negatively correlated with ash content.Conclusions:The resulting observations indicated the capability of ATR FT-IR spectroscopy for monitoring changes,transportation,and accumulation of the major chemical components in these tissues over the cotton growth period.In other words,this spectral technology could be an effective tool for physiological,biochemical,and morphological research related to cotton biology and development.

Sterilization of Seed <i>Carthamus tinctorius</i>(Safflower) Plant and Investigation of the Effectiveness of the Sterilizants

Plant tissue culture studies are one of the pretreatments carried out to increase crop yield by preventing germination in plant seeds. In this research, repeated plant tissue culture studies were conducted with sterilizers specific to safflower seed, which will increase production efficiency but do not cause genetic polymorphism and corrosion in endosperm with 3N chromosomes. Corrosives were used by dilution, and this did not damage the 3N chromosome endosperm, targeting the protein walls of microorganisms on the seed surface without eroding the seed surface, thereby providing biological sterilization. Besides, because it does not contain heavy metals, it did not cause polymorphism, that is, a mutation in the genetic sequence of the seed. Moreover, the environment and the equipment were sterilized with 2 - 3 repetitions, sterilizer treatment, planting, and germination operations were performed in a sterile environment this, in turn, allowed an isolated assessment of the yield of solution G.

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.

Ecological stoichiometry and biomass response of Agropyron michnoi Roshev.under simulated N deposition in a sandy grassland,China

Sandy grassland in northern China is a fragile ecosystem with poor soil fertility.Exploring how plant species regulate growth and nutrient absorption under the background of nitrogen(N)deposition is crucial for the management of the sandy grassland ecosystem.We carried out a field experiment with six N levels in the Hulunbuir Sandy Land of China from 2014 to 2016 and explored the Agropyron michnoi Roshev.responses of both aboveground and belowground biomasses and carbon(C),N and phosphorus(P)concentrations in the plant tissues and soil.With increasing N addition,both aboveground and belowground biomasses and C,N and P concentrations in the plant tissues increased and exhibited a single-peak curve.C:N and C:P ratios of the plant tissues first decreased but then increased,while the trend for N:P ratio was opposite.The peak values of aboveground biomass,belowground biomass and C concentration in the plant tissues occurred at the level of 20 g N/(m2•a),while those of N and P concentrations in the plant tissues occurred at the level of 15 g N/(m2•a).The maximum growth percentages of aboveground and belowground biomasses were 324.2%and 75.9%,respectively,and the root to shoot ratio(RSR)decreased with the addition of N.N and P concentrations in the plant tissues were ranked in the order of leaves>roots>stems,while C concentration was ranked as roots>leaves>stems.The increase in N concentration in the plant tissues was the largest(from 34%to 162%),followed by the increase in P(from 10%to 33%)and C(from 8%to 24%)concentrations.The aboveground biomass was positively and linearly correlated with leaf C,N and P,and soil C and N concentrations,while the belowground biomass was positively and linearly correlated with leaf N and soil C concentrations.These results showed that the accumulation of N and P in the leaves caused the increase in the aboveground biomass,while the accumulation of leaf N resulted in the increase in the belowground biomass.N deposition can alter the allocation of C,N and P stoichiometry in the plant tissues and has a high potential for increasing plant biomass,which is conducive to the restoration of sandy grassland.

The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme

Objective: To determine the effects of different strength of Murashige and Skoog(MS)media(full,1/2and1/4) in solid and liquid media on in vitro growth of Typhonium flagelliforme(T. flagelliforme), whereby an optimum media composition can be provided for mass propagation of T. flagelliforme.Methods: Rhizome bud of T. flagelliforme was obtained from the axenic in vitro established T. flagelliforme plantlets in Plant Tissue Culture Laboratory, Universiti Teknologi MARA, Shah Alam. Rhizome bud was used as explant and cultured onto shoot proliferation medium under different strength of MS media(full,1/2,1/4) in solid and liquid culture media.Results: After 6 weeks of culture, the number of shoot, number of leaf, number of root,height of shoot, fresh weight, dry weight and chlorophyll content of T. flagelliforme were analyzed. A comparison was made between liquid and solid culture media. The results revealed that the liquid culture media were more effective for all the growth parameters(shoot height, shoot number, leaf number, root number, fresh weight, dry weight, chlorophyll a and chlorophyll b content) compared to solid culture media. Apart from that,this study revealed the positive relationship between strength of MS media and type of culture media(solid and liquid media) to the growth of T. flagelliforme. Growth of T. flagelliforme was improved when MS strength was increased in liquid media. In contrast, growth of T. flagelliforme was improved when MS strength was decreased in solid media.Conclusions: Through this study, an optimum media composition for mass propagation of T. flagelliforme had been established by observing effects of MS media strength and type of culture media(solid and liquid media) on the growth of T. flagelliforme.

In Vitro-Propagation of Agave tequilana Weber cv.azul in a Temporary Immersion System

In Mexico,there is a need to produce large quantities of plantlets for the establishment and replanting of blue(cv.azul)agave production areas.Most of these plots are within the origin denomination area(DOT,Spanish acronym)of the distilled product of this plant,known as tequila.The objective of this study was to develop an in vitropropagation protocol for Agave tequilana Weber cv.azul using segmented stems in both:solid and liquid media.A disinfection and in vitro technique were developed to obtain shoots,through plantlets collected in commercial plots,which attained 100%surface-disinfection and budding rate.At the multiplication stage,the effects of 6-Benzylaminopurine(BA)(0.0,4.4 and 13.2μM)and kinetin(0.0,9.4,18.8 and 37.6μM)were evaluated on lateralshoot production of segmented sagittal stems.These were cultivated on Murashige&Skoog(MS)medium,with the addition of 3.0%sucrose and 8 g L−1 agar.It was observed that BA and kinetin increased the number of shoots per explant,obtaining up to 18 and 26,respectively.Furthermore,it was found that just the sagittal segmentation of explants increased axillary budding.On the other hand,segmented-stem bases were grown in MS liquid medium with 3.0%sucrose,inside a RITA
system,programmed by a 5 min immersion step with a frequency of every 4 h.The effect of Indole−3-Acetic acid(IAA)(0.57,2.9,5.7μM)was evaluated,while maintaining a concentration of BA(13.2μM).It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant.These results offer a new methodology to increase the efficiency of A.tequilana Weber cv.azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.

Thin Cell Layer (TCL) Culture System for Herbal Biomass Production and Genetic Transformation of Bacopa monnieri L. Wettst.

Bacopa monnieri (L.) Wettst. (Scrophulariaceae) is a highly sought after medicinal plant with therapeutic properties as cognition enhancer as well as for other brain and body functions. Research was conducted to optimize a thin cell layer explant based micropropagation system to assist mass propagation. Thin cell layers (TCL) derived from leaf and internode segments were used as explants. Murashige and Skoog medium was used to formulate shoot induction, elongation, and rooting media. Shoot induction media were prepared by supplementing three concentrations (0.1, 1.0, and 10.0 μM) of four cytokinins 6-benzylaminopurine, 2-isopentenyl-adenine, 6-3-Hydroxybenzylaminopurine, and thidiazuron to study adventitious shoot bud induction response. An optimum shoot bud induction response was observed on MS medium supplemented with 10.0 μM 6-benzylaminopurine for both leaf and stem transverse thin cell layer (tTCL) explants. The average number of shoot buds from leaf tTCL explants was 59, whereas, on an average, 33 shoot buds were regenerated from internode tTCL explants. Elongation of adventitious shoot buds was achieved best in a liquid medium using Liquid Lab Rocker® system. Elongated shoots recorded 100% rooting in MS medium supplemented with 5 μM indole butyric acid. Bacopa micropropagation employing tTCL explants for initial shoot bud induction and using LLR® boxes in subsequent elongation step can achieve cost effective way to regenerate high volume of plantlets and biomass required for herbal industry. Leaf and stem tTCL explants both were suitable for Agrobacterium tumefaciens (EHA105) mediated genetic transformation. Successful transformation was scored within three days of co-cultivation with Agrobacterium suspension on the basis of Enhanced Green Fluorescent Protein (EGFP) expression as an early and non-destructible screening device. Transformation frequencies of 83% and 76% were accomplished for leaf and stem tTCL explants, respectively. Greenhouse grown Bacopa plants were analyzed as fresh and dry methanolic extracts for total polyphenol content (811.93 ± 7.98 and 814 ± 17.64 GAE mg g-1) and the Trolox Equivalent Antioxidant Capacity values were 1918.25 ± 173.12 and 3163.14 ± 403.25 μmol/g, respectively.

Evaluation of differential antitermitic activities of Lantana camara oven-dried tissues against Reticulitermes virginicus （Isoptera： Rhinotermitidae）

Chemical-treated soil or physical barriers have been the most commonly used approach for termite management. We hypothesized that a barrier of soil incorporated with oven-dried Lantana camara L. tissues could prevent termite infestation. We first examined the antitermitic effects of the dried tissues from two cultivars （＇Mozelle＇ and ＇New Gold＇） on the subterranean termite, Reticulitermes virginicus （Banks） （Isoptera： Rhinotermitidae）. Results show that all of the tissues of Mozelle had greater antitermic activity than corresponding tissues of New Gold, and leaves had greater termiticidal effects than flowers and stems. When termites were exposed to the test materials in a no-choice bioassay, the 24-day test resulted in a significant reduction of survival （52.5%-88.6%）, running speed （18.2%-37.3%）, live weight （21.8%-53.5%） and body water content （33.2%-56.2%） compared to the control. The consumption of leaves and flowers was exiguous. When used as 25% tissue mulch-barrier, the oven-dried lantana tissues decreased termite tunneling and wood consumption and increased termite mortality. The decreased survival, vigor, and low consumption indicate a toxic and anti-feeding property of the materials tested. The results therefore support our hypothesis that the dried lantana tissues possess antitermitic activities.

Single-cell transcriptome atlas of the leaf and root of rice seedlings

As a multicellular organism,rice flourishes relying on gene expression diversity among cells of various functions.However,cellular-resolution transcriptome features are yet to be fully recognized,let alone cell-specific transcriptional responses to environmental stimuli.In this study,we apply single-cell RNA sequencing to both shoot and root of rice seedlings growing in Kimura B nutrient solution or exposed to various abiotic stresses and characterize transcriptomes for a total of 237,431 individual cells.We identify 15 and 9 cell types in the leaf and root,respectively,and observe that common transcriptome features are often shared between leaves and roots in the same tissue layer,except for endodermis or epidermis.Abiotic stress stimuli alter gene expression largely in a cell type-specific manner,but for a given cell type,different stresses often trigger transcriptional regulation of roughly the same set of genes.Besides,we detect proportional changes in cell populations in response to abiotic stress and investigate the underlying molecular mechanisms through single-cell reconstruction of the developmental trajectory.Collectively,our study represents a benchmark-setting data resource of single-cell transcriptome atlas for rice seedlings and an illustration of exploiting such resources to drive discoveries in plant biology.

A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones

One of the hottest topics in plant hormone biology is the crosstalk mechanisms,whereby multiple classes of phytohormones interplay with each other through signaling networks.To better understand the roles of hormonal crosstalks in their complex regulatory networks,it is of high significance to investigate the spatial and temporal distributions of multiple-phytohormones simultaneously from one plant tissue sample.In this study,we develop a high-sensitivity and high-throughput method for the simultaneous quantitative analysis of 44 phytohormone compounds,covering currently known 10 major classes of phytohormones(strigolactones,brassinosteroids,gibberellins,auxin,abscisic acid,jasmonic acid,salicylic acid,cytokinins,ethylene,and polypeptide hormones[e.g.,phytosulfokine])from only 100 mg of plant sample.These compounds were grouped and purified separately with a tailored solid-phase extraction procedure based on their physicochemical properties and then analyzed by LC–MS/MS.The recoveries of our method ranged from 49.6%to 99.9%and the matrix effects from 61.8%to 102.5%,indicating that the overall sample pretreatment design resulted in good purification.The limits of quantitation(LOQs)of our method ranged from 0.06 to 1.29 pg/100 mg fresh weight and its precision was less than 13.4%,indicating high sensitivity and good reproducibility of the method.Tests of our method in different plant matrices demonstrated its wide applicability.Collectively,these advantages will make our method helpful in clarifying the crosstalk networks of phytohormones.

Effects of red and blue LEDs on in vitro growth and microtuberization of potato single-node cuttings

The objectives of this study were to investigate the effects of red and blue LEDs on in vitro growth and microtuberization of potato(Solanum tuberosum) singlenode cuttings. Explants were incubated under 6 light treatments: 100% red LEDs(R), 75% red LEDs + 25%blue LEDs(3 RB), 50% red LEDs + 50% blue LEDs(RB),25% red LEDs + 75% blue LEDs(R3 B), 100% blue LEDs(B) and white LEDs(W). Most of the growth and physiological parameters were significantly higher in3 RB than W. Enhancement of leaf area and chlorophyll concentrations were obtained in B. Leaf stomata were elliptical with the lowest density in 3 RB. However, those in W were round in shape, and those with the smallest size and the highest density were observed in R. Most of the characteristics of microtuberization were also improved in3 RB. The combined spectra of red and blue LEDs increased the number of large microtubers. The fresh weight of individual microtubers in R and W were increased, but not their number. These results suggest that, of the treatments assessed, 3 RB is optimal for the in vitro growth of potato plantlets and the combination of red and blue LEDs is beneficial for microtuberization.