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.

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.

Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

AIM： To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells （ADSCs） generated by enzyme （EN） and explant （EX） culture methods.METHODS： We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescent （IMF） staining.RESULTS： Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION： Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method.

Tissue Culture and Rapid Propagation of Spathiphyllum kochii Engl. et Krause

[Objectives] This study was conducted on tissue-culture rapid propagation techniques of Spathiphyllum kochii Engl. et Krause. [Methods] With lateral buds of S. kochii as explants, the effects of such four basic media as MS, B5, Nitsch and Wpm and the ratio of two hormones(1, 2, 3 mg/L 6-BA and 0.1, 0.3, 0.5 mg/L NAA) on bud proliferation of S. kochii were studied by the complete test method, and the effect of the ratio of the two hormones(0.25, 0.50, 1.00 mg/L NAA and 1.0, 1.5 mg/L IBA) on rooting of S. kochii as well as the effect of substrate ratio(perlite and peat soil at 1∶9, 2∶8, 3∶7, 4∶6 and 5∶5) on its transplanting survival rate were also studied. [Results] The best basic medium for the rapid propagation of S. kochii was MS. The best hormone ratio suitable for bud proliferation was 2 mg/L6-BA+0.1 mg/L NAA, and the average number of buds proliferated at 45 d was 3.04. The average bud height was 2.05 cm; the most suitable medium for rooting was 1/2 MS+1 mg/L IBA+0.25 mg/L NAA, and its rooting rate was 100%; and the best transplanting substrate was 5∶5 perlite and peat. The soil ratio had a survival rate of 94%. [Conclusions] This study provides a theoretical basis for the improvement of the transplanting survival rate of test-tube S. kochii plantlets.

Optimization of Rooting Medium for Tissue Culture Seedlings of Dendrobium officinale by Response Surface Methodology

[Objectives] To increase the survival rate of tissue culture seedlings of Dendrobium officinale,and optimize the conditions of rooting medium by the response surface methodology( RSM). [Methods]The effects of 6-BA concentration,NAA concentration,potato amount and the amount of mashed banana on the growth of seedlings were determined by single factor experiment and were analyzed by Box-Behnken design and response surface methodology. [Results]The optimal culture conditions: rooting medium 1/2 MS + 6-BA 0. 24 mg/L + mashed banana 87. 63 g/L + potato 89. 30 g/L + NAA 0. 52 mg/L + sucrose 20. 0 g/L + activated carbon 4. 0 g/L + agar 7. 0 g/L,p H 5. 8,and light intensity 2 000 Lx. [Conclusions]The model established by response surface methodology has a good predictability and could be used to optimize the conditions of tissue culture and rooting medium of D. officinale.

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.

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.

Wound Healing: From Epidermis Culture to Tissue Engineering

The skin is the largest organ of the individual, being the interface between the body and the microenvironment. In severely burned patients and other diseases, the physiological processes of wound healing are not sufficient to complete the closure of their wounds. The in vitro culture of autologous epidermis, which has represented the beginning of Tissue Engineering, is a valuable tool for the treatment of these patients. Keratinocytes can be cultured and stratified in vitro, and an entire epidermal sheet can be obtained. The epidermis cells can be amplified in the laboratory from a skin sample to obtain a surface equivalent to that required for each patient. This technology was first used clinically in 1981 and in Argentina since 1991. Wound repair is a complex process that involves dermal and epidermal cells, extracellular matrix, soluble factors and the sum of interactions between them, providing physical, biological and chemical keys capable of guiding cell function. Seeking to improve the results obtained with cultured epidermis, tissue engineering was directed towards the development of substitutes that not only involve epidermis but also the dermal component. The tissue engineered skin and its therapeutic applications reported in this review demonstrate the feasibility and effectiveness of these approaches. It represents a clear benefit in wound healing. Now, focus must be directed on the development of new scaffolds, developed by different technologies, such as polymer science, or nanotechnology, able to be used as templates to direct the growth of cells, in an attempt to better regenerate the lost skin.

INFLUENCE OF ZINC，MANGANESE AND SELENIUM ONSUPEROXIDE DISMUTASE ACTIVITY IN LUNGCANCER TISSUE AND CELL IN CULTURE

In this experiment,the cancer tissues and cells,Which were derived from Lewis lung cancer and A549 lung Cancer cell line,were respectively divided into four groups and zinc, manganese and selenium were respectively added to the medium for 24 hours. The superoxide dismutase activity in the tissues and the cells was estimated. It was found that the SOD activity was enhanced by zinc and manganese and the effect of zinc on SOD activity was superior to that of manganese. We supposed that the enhance of the SOD activity was relative to the activation of the SOD apoenzymes. This experimental result indicated that the inhibitory effect of zinc and manganese on carcinogenesis was achieved by SOD and the elements might be considered a SOD activator.

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）.

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.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

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.

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.

A Phenotyping Protocol for Detailed Evaluation of Apple Root Resistance Responses Utilizing Tissue Culture Micropropagated Apple Plants

In the post-genomics era, reliable phenotypes are considered the bottleneck for unraveling the genetic control over the biology of interest. Phenotyping resistance response of roots to infection by soilborne pathogen is more challenging compared to that of plant aerial parts. In additional to the hidden nature and small stature of fine roots where infection occurs, extra obstacles exist for rosaceae tree crops such as apple. Due to self-incompatible reproduction and high-level heterozygosity of apple genome, genetically identical apple plants cannot be produced through apple seed germination. Here we report an established phenotyping protocol which includes a streamlined tissue culture procedure for micropropagation of uniform apple plants, standardized inoculation procedure using Pythium ultimum, and multilayered evaluating methods on apple root resistance traits. Because of the implementation of tissue culture based micropropagation procedure, constant availability of the uniform plants with defined genetic background, equivalent age and non-contaminated roots overcame a longstanding barrier of systematic and detailed phenotypic characterization of apple root resistance traits. Repeated infection assays by root-dipping inoculation demonstrated the reproducible and wide-range plant survival rates, from single-digit to over 90% survived plants for a given genotype. Genotype-specific values due to P. ultimum inoculation on shoot and root biomass reduction, maximum root lengths, leaf number and cumulative leaf areas were quantified between mock-inoculated and P. ultimum infected plants. Use of a glass-box container offered enhanced accessibility and minimized invasiveness for continuous and non-disruptive observation on the necrosis progression patterns along inoculated roots. With the assistance of a dissecting microscope, the genotype-specific resistance responses along the infected apple roots were captured and analyzed in detail. This reported phenotyping protocol represents a major development and should be easily adopted for other rosacea tree fruit crops with minor modifications.