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.

Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

The purpose of this study was to compare cell growth characteristics,ciliated cell differentiation,and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures.Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods.Epithelial cell growth characteristics were observed by inverted phase contrast microscopy.Ciliated cell differentiation was detected byβ-tubulin IV and ZO-1 immunocytochemistry.Basal and ATP-stimulated ciliary beat frequency(CBF)was measured using a high-speed digital microscopic imaging system.Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition,with both types of cultures comprising ciliated and non-ciliated epithelial cells.Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures.In both culture systems,the highest ciliated cell density appeared at 7th–10th culture day and declined with time,with the lifespan of ciliated cells ranging from 14 to 21 days.Overall,10%of the cells in explant cultures and 20%of the cells in the dissociated tissue cultures were ciliated.These two cultures demonstrated similar ciliary beat frequency values at baseline(7.78±1.99 Hz and 7.91±2.52 Hz,respectively)and reacted equivalently following stimulation with 100μM ATP.The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells,which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

Tissue Culture of Calla Lily (Zantedeschia spreng.): An Updated Review on the Present Scenario and Future Prospects

The calla lily(Zantedeschia spreng.)is a bulbousflower native to the tropical regions of Africa.Calla lily has gained significant popularity in the international market owing to its intricate morphology and prolonged flowering duration.Despite such advantages,for two sub-groups of calla lily,known as group Zantedeschia and group Aestivae,there are challenges in terms of hybrid production due to the‘plastome-genome incompatibility’there-between.Tissue culture is a fundamental biotechnological tool used in gene editing research,with a focus on disease resistance andflower color in calla lily breeding programs.The present review provides a brief background on the history and development of the calla lily,as well as a comprehensive and critical summary of calla lily tissue culture research.The regeneration pathways for both group Zantedeschia and group Aestivae can be divided into de novo organogenesis and somatic embryogenesis.Both groups are capable of obtaining replants through such means.However,only some species in group Aestivae have been reported to be successful in the somatic embryogenesis pathway.In the present review,special attention was paid to the influence of explant types,plant growth regulators,and culture conditions on both de novo organogenesis and somatic embryogenesis in calla lily tissue culture.Ultimately,future research prospects were determined based on integrated analysis of recent progress in calla lily tissue culture research.

Evaluation of Different Substrates Compositions for Acclimatization of Tissue Culture Taro Plantlets in a Propagator

Taro is cultivated in most Regions of Cameroon and it is affected by taro leaf blight disease since 2010 which has decreased its production. Lack of disease-free planting materials has been a main problem to farmers. This study was carried out at International Institute of Tropical Agriculture (IITA) Yaounde and Institute of Agricultural Research for Development (IRAD) Bambui to assess different substrates for acclimatization of tissue culture taro plantlets in apropagator. No information is available on acclimatization of Cameroonian taro plantlets in different substrates. Taro plantlets from tissue culture were acclimatised in a propagator for six weeks under different substrates, the first substrate consisted of sterile three parts of soil and one part of river sand mixed together (3:1), the second substrate consisted of sterile two parts of soil and two parts of river sand mixed together (2:2), the third substrate consisted of sterile two parts of soil, one part of rice husk and one part of river sand mixed together (2:1:1) and the fourth substrate consisted of sterile one part of soil and three parts of river sand mixed together (1:3). After acclimatisation of the different taroplantlets (Dark green petiole with small leaves (L1), Red petiole with small leaves (L2), Light green petiole with large leaves (L3) and Light green petiole with small leaves (L4) in these four substrates, it was observed that the best growth rate of plant was recorded on substrate sand + soil (1:3). The other substrates showed moderate growth of plants. Substrate sand + soil (1:3) can be recommended for acclimatization of Cameroonian taro plantlets.

Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

The Comparison in Tissue Culture Ability of Mature Embryo in Different Cultivars of Rice

In order to study the regeneration technology of mature embryos in different rice varieties,nine japonica,nine indica and eleven hybrid rice varieties of two line or three line or superiority combinations were selected as explants to study the callus induction,differentiation and regeneration rates on different media.The higher callus induction （61.7-89.2%） was observed in japonica rice,when cytokinin was added at lower concentration （0.3 mg L-1 6-BA） in M8 basal medium,supplemented with 30 g L-1 sucrose,8 g L-1 agar and 2 mg L-1 2,4-D.Further,the addition of two cytokinins （2 mg L-1 6-BA,0.5 mg L-1 KT） and 1 mg L-1 NAA in the M8 basal supplemented medium resulted in 9.1-100% of the callus induction in indica rice.The percent callus induction in hybrid rice varieties was 40-86.3% when addition of 1 mg L-1 6-BA and 1 mg L-1 KT was added,and the cytokinins was required by the japonica and indica rice varieties in the M8 basal supplemented medium.It was observed that when the 0.5 mg L-1 2,4-D and 1 mg L-1 6-BA were added in japonica rice,and 0.2 mg L-1 2,4-D and 0.5 mg L-1 6-BA were added in indica and hybrid rice in the MS different media,the regeneration rates were 9.2-59.5%,3.6-87.5% and 17.2-43.2% for japonica,indica and hybrid rice,respectively.Thus,the regeneration technology with higher output is established in the mature embryos of similar rice varieties.

Primary Establishment of the Tissue Culture Technique and Regeneration System for Ornamental Lupinns polyphyllus

The purpose of this paper is to develop a system for tissue culture and rapid propagation of two ornamental lupins, Minaretie and Russell Prize. In view of screening out the better explant regeneration and suitable culture medium, through adding hormone 6-BA, NAA and 2, 4-D into MS and B5 basic culture medium, a series of experiments were carried out with the shoot tips, leaves, leaf petioles and stems from the asepsis seedling. The results showed that the shoot tips had favorableness on the rapidly propagation; MS＋6-BA 0.5 mg. L-1 for first generation, the induction rate of Minaretie and Russell Prize was 90.5% and 95.86% respectivdy; Minaretie had the highest propagation index （6.35） on MS＋6-BA 0.5 mg.L^-1＋NAA 0 mg-L^-1＋GA 30.8 mg. L^1＋AC 2 g. L^-1, but Russell Prize had the highest propagation index （7.24） on MS＋6-BA 0.5 mg.L^-1＋NAA 0.15 mg.L^-1＋GA3 1.0 mg.L^-1＋AC 0.5 g.L^-1; 1/2 MS＋NAA 0.25 mg.L^-1 was the best rooting medium. The ratios of getting roots of Minaretie and Russell Prize were 94.78% and 96.32%, respectively.

Contamination and browning in tissue culture of Platanus occidentalis L.

Twigs of 2-3-year-old Platanus occidentalis L. were used as experimental material to find the causes for the contamination and browning in the initial stages of tissue cultures. To compare the degree of browning of explants picked off from different growing seasons, the experimental material was excised from trees on each of the first ten days in January, March, May and July, 2006. The results indicated that the contamination and browning rates of the material cut off in January （14.2% and 30.6%, respectively） and March were somewhat lower than those in July. The pretreatment of soaking the explants in different anti-oxidants and absorbents at the same time could diminish some side effects. The pretreatment of using 10 g·L^-1 vitamin C reduced the contamination and brown- ing rate effectively. An orthogonal experiment showed that the optimal factor and level arrangement is 0.5 mg·L^-1BA, 2.0 g·L^-1 active carbon and 1.5 g·L^-1 PVP which resulted in a browning rate of only 16.5%. In general, sampling period, physical properties and pretreatment of explants are the main factors responsible for the contamination and browning of material in the initial stages of P. occidentalis tissue cultures.

Effect of Rare Earth on Physiological-Chemical Characteristics and Its Growth of Aloe in Tissue Culture

In adopting tissue culture, effects of rare earth on Physiological and chemical characteristics, proliferation and rootage in aloe were studied. The results indicate that suitable concentration treatment of rare earth increases content of reducing sugar and protein, enhances activity intensity of superoxide dismutase (SOD), peroxidase (POD) isozymes and stripes of POD isozymes in the leaves. It also increases rate of proliferation and rootage respectively. The appropriate concentration of rare earth is 20～40 mg·L -1 in proliferation and rootage of aloe.

Effect of Coconut Milk and Rare Earth on Proliferation, Rootage and Isozymes of Dendrobium in Tissue Culture

In tissue culture, effects of coconut milk, rare earth and medium on proliferation, rootage and isozymes of Dendrobium were studied. The result indicated that coconut milk and RE could promote proliferation and rootage, with their combined treatment the most effective. In proliferation culture, coconut milk enhanced peroxidase (POD) and esterase(EST) activity and types, RE decreased POD, but increased EST activity and types and combined treatment did not show the same influence on POD and EST too. In rootage culture, adding coconut milk decreased POD activity, but improved EST activity in 1/2MS medium and declined EST activity, expression type in WPM medium. when using RE, POD and EST activity both reduced, types of the former decreased, types of the latter changed slightly with the basic medium. And combined treatment would decreased POD activity and types.

Study on Seed Germination Conditions and in vitro Tissue Culture of Lilium formolongi

To facilitate the rapid production of lily （Liliumformolongi）, conditions favoring the germination of dormant lily seeds and in vitro regeneration from tissue cultttre were studied, using the germinating plumule as the explants. The optimal inducing conditions for dormant lily dry seeds to germinate were low tempera- ture （ 10 ℃ ） treatment for one hour followed by incubating at 18 ℃ with light illumination. After 8 days of incubation/induction, over 90% of the seeds were ger- minated. MS ＋ 6-BA 1.0 mg,/L ＋ NAA 0.5 mg/L was the most efficient medium formula in lily callus induction. MS ＋ 6-BA 2.0 mg/L ＋ NAA 0.5 mg/L was the optimal medium for calli growth. 1/2MS ＋ NAA 1.0 mg/L was the medium gave the highest root inductive rate.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils

BACKGROUND Liver transplantation is a therapy for irreversible liver failure;however,at present,donor organs are in short supply.Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes.AIM To investigate the possibility that hepatic progenitor cells(HPCs)prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine,we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells.METHODS In vitro liver organoid tissue has been generated by accumulating collagen fibrils,fibroblasts,and HPCs on a mesh of polylactic acid fabric using a bioreactor;this was subsequently implanted into syngeneic wild-type mice.RESULTS The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy.CONCLUSION Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system.This tissue was comparable to liver lobules,and with fibroblasts embedded in the network collagen fibrils of this artificial tissue,it is useful for reconstructing the hepatic interstitial structure.

Tissue Culture Rapid Propagation and Transplantation Techniques of Anoectochilus roxburghii (Wall) Lind.

[Objectives]To screen the tissue culture rapid propagation formula suitable for each tissue culture stage of Anoectochilus roxburghii( Wall) Lind. [Methods] The stem segments of Fujian A. roxburghii were used as explant to study the tissue culture rapid propagation and transplantation techniques. The comparative experiment was carried out to study the effects of different hormone concentrations on the induction of stem segments,proliferation of cluster buds,rooting and seedling hardening of A. roxburghii,and study the effects of transplantation matrix on the transplantation of A. roxburghii. [Results]MS + 0. 5 mg/L NAA + 2 mg/L BA + 20 g/L sucrose + 6 g/L agar was suitable for induction of stem segments of A. roxburghii; MS + 0. 5 mg/L NAA + 2 mg/L BA + 1 mg/L KT + 25 g/L sucrose + 6 g/L agar was most suitable for proliferation of cluster buds of A. roxburghii; MS + 1. 0 mg/L IBA + 1. 0 mg/L NAA + 1 g/L activated carbon + 50 g/L mashed banana + 25 g/L sucrose + 6 g/L agar was most suitable for rooting and seedling hardening of A. roxburghii; using peat soil: fine sand( 3∶ 1) as transplantation matrix,the survival rate was the highest. [Conclusions] The experiment results are expected to provide references for factory production of A. roxburghii.

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.

Study on the Tissue Culture of Coleus blumei

This paper studied on the way of Coleus blumei, the leaf was chosen as explants and was inoculated on 1/2 MS medium with different combinations of 6-benzyladenine （6-BA）, α-naphthaleneacetic acid （NAA） and indole-3-butyric acid （IBA）. The optimal conditions of callus induction from explants were achieved on the medium containing 6-BA （2.0 mg· L^-1） and NAA （1.0 mg· L^-1）. Shoot tips were induced on the medium containing 6-BA （4.0 mg·L^-1） and NAA （0.5 mg·L^-1）. The same media conditions were found suitable for shoot multiplication, we multiplied shoots rooted best on 1/2 MS medium supplemented with IBA （0.1 mg·L^-1）.

Establishment of Tissue Culture Regeneration System in Bama Hemp (Cannabis sativa L.)

[Objectives]This study was conducted to establish a tissue culture regeneration system in Bama hemp(Cannabis sativa L.).[Methods]Using hemp seeds as explants,a regeneration system was established through explant sterilization,callus induction,callus differentiation,and rooting culture.[Results]The results showed that the best sterilization effect was achieved when sterilizing with 75%ethanol for 30 s,followed by 0.1%HgCl 2 solution for 9 min,with a contamination rate as low as 11.4%.In presence of 3 mg/L 2,4-D and 0.1 mg/L6-BA,the callus induction effect from hemp seeds was better.The formula for better differentiation of callus was MS+2.0 mg/L 6-BA+0.2 mg/L NAA.IBA had a promoting effect on the rooting of hemp aseptic plantlets.The highest rooting rate reached 80%when MS+0.3 mg/L IBA were used.[Conclusions]This study established a hemp seed regeneration system to provide technical support for the conservation and breeding of hemp germplasm resources.

Tissue Culture and Plant Regeneration of Chicory

[ Objecllve] To establish a high-frequency regeneration system of chicory （ Cichodum intybus L. ） using leaf segments of aseptic seed- lings. E Method] Calluses and adventitious buds of chicory were induced by inoculating explants on MS medium supplemented with 6-BA （6-benayl aminopurine） and NAP, （naphthylacetic acid） at different final concentrations. [ Result] When lower part of leaves derived from 20-day-old seedlings was used as explant and inoculated on MS medium containing 2.0 rng/L 6-BA, 0.5 mg/L NAA and 40 g/L sucrose, the frequency of adventitious bud formation was 90.0%. When the regenerated shoots were cultured in 1/2 MS medium containing 0.1 mg/L NAA, the frequency of root forma- tion was 88.3%. All rooted plants transplanted in pots could survive and grew well without abnormal shape. [ Conclusion] Better differentiation of adventitious buds can be achieved by inoculating the lower part of leaves derived from 20-day-old seedlings on MS medium containing 2.0 mg/L 6- BA, O. 5 mg/L NAP, and 40 g/L sucrose. The 1/2 MS medium containing O. 1 mg/L NAP, is most suitable for rooting.

Study on Surface Sterilization Method for Tissue Culture of Haloxylon ammodendron(C.A.Mey.) Bunge Seeds

[ Objective ] The aim was to select appropriate sterilization methods and explants for tissue culture of Haloxylon ammodendron （ C. A. Mey. ）. [Method] H. ammodendron （C. A. Mey. ） seeds were used as experimental materials to investigate the sterilization results of different sterilization treatments and the effect of different seed sizes on the survival rate of aseptic seedlings. [ Result] Sterilization of H. ammodendron （ C.A. Mey. ） seeds achieved the best result by using 75% alcohol for 15 min and 0.1% mercuric chloride for 8 min. Seeds with diameter φ 〉 2.0 mm were used as explants and achieved relatively high surviv- al rate of aseptic seedlings, which reached above 65%. [ Conclusion] This study established a surface sterilization method for tissue culture of H. ammodendron （ C. A. Mey. ） Bunge seeds.

Interaction Effect of Auxin and Cytokinin on in Vitro Shoot Regeneration and Rooting of Endangered Medicinal Plant Valeriana jatamansi Jones through Tissue Culture

Micropropagation of Valeriana jatamansi Jones by using small segments of rhizome on full strength MS medium having various concentrations and combinations of auxin Naphthaleneacetic acid (NAA) and cytokinin Benzylaminopurine (BAP) was conducted. The highest mean shoot length (3.71 cm) was achieved when media was fortified with BAP 2 mg/L in combination with NAA 1 mg/L. The highest mean leaf number i.e. 6.00 was observed when BAP was used alone at 2 mg/L. Average root length (0.77 cm) was recorded when BAP 1.5 mg/L along with NAA 0.5 mg/L was used. Maximum mean root numbers 2.57 were obtained when BAP and NAA were used at equal concentrations i.e. 1.5 mg/L. Observed data demonstrated that BAP up to 1 mg/L, 1.5 mg/L and 2 mg/L promotes shoot length, leaf number and leaf growth when used along with NAA at 0 mg/L, 0.5 mg/L and 1 mg/L. However lower quantities of both NAA (0 mg/L, 0.5 mg/L) and BAP (1 mg/L and 1.5 mg/L) produced significantly higher root length of Valeriana jatamansi Jones but the higher concentrations of plant growth hormones BAP (2 mg/L, 3 mg/L) and NAA (1 mg/L, 1.5 mg/L) were found unfavorable for increase in root length but the root number increases at higher concentration of NAA (1 mg/L and 1.5 mg/L).