Effects of Media composition on Asymbiotic Seed Culture of Vanda tessellata : An Approach to In vitro Conservation

For initiation of seed germination and protocorm growth in Vanda tessellata (Roxb.) Hook. ex G. Don different media, namely Knudson’s C basal medium, modified Knudson’s C basal medium, Murashige and Skoog basal medium, Vacin and Went basal medium and Lindeman basal medium either alone or supplemented with vitamin or 0.1% peptone (w/v) were used. Vitamin mixture was consisted of nicotinic acid, pyridoxine and thiamine HCl at 1 : 1 : 10, respectively. For the initiation of germination, all the treatments exhibited promising results without showing significant variation. But the rate of survival of the germinated seedlings was remarkably low in all the basal media. Maximum survival rate of germinated seedlings was recorded in MS + 0.1% peptone. Necrosis of protocorms was a common phenomenon in all the treatments and 100% necrosis was recorded in LM basal medium and LM + vitamin. Addition of vitamin mixture and peptone in the basal media increased the rate of survival as well as differentiation of the germinated protocorms. Maximum rooted plantlets were recorded in VW + 0.1% peptone Plant Tissue Cult. & Biotech. 30(2): 285-295, 2020 (December)

Download Full-text

Organic Additives Improves the in Vitro Growth of Native Orchid Vanda helvola Blume

Notulae Scientia Biologicae ◽

10.15835/nsb729546 ◽

2015 ◽

Vol 7 (2) ◽

pp. 192-197

Author(s):

Devina DAVID ◽

Roslina JAWAN ◽

Hartinie MARBAWI ◽

Jualang Azlan GANSAU

Keyword(s):

Seed Germination ◽

Rapid Development ◽

Basal Medium ◽

Tomato Juice ◽

Organic Additives ◽

Orchid Species ◽

Orchid Conservation ◽

Hand Pollination ◽

Basal Media

In vitro seed germination has been proven to be the most efficient technique to propagate orchid. The application of this aseptic technique has contributed to conservation of many endangered orchid species. In this study, undehisced capsules of Vanda helvola Blume were collected from Orchid Conservation Centre in Lagud Sebrang Agriculture Park, after 120 days from hand pollination and aseptically cultured on three types of basal media such as Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW). After 90 days of culture, 66.40 ± 4.14% of seeds successfully germinated on KC medium. The effect of organic additives such as tomato juice, coconut water, peptone and yeast extract at different level of concentrations in KC basal medium were also tested on seed germination and seedling development of this native orchid. After 90 days of culture, over 90% of seeds were tremendously germinated on KC medium supplemented with 10% or 15% (v/v) of tomato juice. The incorporation of peptone at 0.1% (w/v) in KC basal media promoted rapid development of protocorm to seedling. Seedlings on this treatment produced an average of three leaves and two roots after 90 days of culture and were successfully acclimatized.

Download Full-text

In vitro Seed Germination, Protocorm Formation and Plantlet Regeneration in Aerides ringens Fisher

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v29i1.41978 ◽

2019 ◽

Vol 29 (1) ◽

pp. 49-62 ◽

Cited By ~ 1

Author(s):

Ashok N Pyati

Keyword(s):

Survival Rate ◽

Seed Germination ◽

Plantlet Regeneration ◽

Ex Vitro ◽

Coconut Husk ◽

Half Strength ◽

The Media ◽

Basal Media ◽

Protocorm Formation

An attempt was made to in vitro seed germination and plantlet regeneration in Aerides ringens, a threatened, endemic and epiphytic orchid. Five basal media Knudson C (KC), Vacin and Went (VW), Burgeff’s (N3f), half strength MS, MS were examined for their effectiveness. Germination occurred in all the media. However, KC medium proved to be the best for seed germination (70.8%) and half strength MS was superior for the differentiation of protocorm like bodies (PLBs) into seedlings. The seeds were also cultured on various plant growth regulators (PGRs) to evaluate their effectiveness on seed germination and further differentiation. Among the PGRs tested, 0.57 μM IAA (IAA 89.3%), 4.64 μM Kn (88.6%) and 2.21 μM BAP (90.7%) and 0.57 μM IAA + 0.55 μM BAP (96.5%) were stimulated the germination and further differentiation of PLBs into seedlings. The concentrations of 2.21 μM BAP and combination of 0.57 μM IAA + 0.55 μM BAP stimulated the formation of MPLBs, which later differentiated into shoots and roots. Four potting substrates were evaluated for Ex vitro seedling survivability of which brick: charcoal: decaying litter: coconut husk (1 : 1 : 1 : 1) gave maximum survival rate (89.0%). Plant Tissue Cult. & Biotech. 29(1): 49-62, 2019 (June)

Download Full-text

In vitro answer of Bulgarian pepper (Capsicum annuum L.)

Genetika ◽

10.2298/gensr0602129r ◽

2006 ◽

Vol 38 (2) ◽

pp. 129-136

Author(s):

Velichka Rodeva ◽

Stanislava Grozeva ◽

Velichka Todorova

Keyword(s):

Culture Medium ◽

Culture Media ◽

Casein Hydrolysate ◽

Basal Medium ◽

Regeneration Ability ◽

Stem Explants ◽

Media Composition ◽

Breeding Lines ◽

High Level

Callusogenesis and regeneration ability of cotyledon and hypocotyl explants from three Bulgarian pepper varieties in MS basal medium supplemented with l-3mg/l BAP. l.0mg/1 IAA and 0.5mg/l GA3 was studied. In the different variants of culture medium was registered high level of callusogenesis and organogenesis in both type of explants from the all varieties. The highest percentage of plant-regenerants is established in cotyledon explants (from 3.3 to 18.3) in variant 3 of the culture medium containing 3mg/l BA. In the process of micropropagation by stem explants of the same studied pepper varieties the addition of the vitamins C. B12. Casein hydrolysate and Ferulic acid had a stimulation effect on the plant growth in height and rooting. In result of anther cultivation from three pepper varieties and four breeding lines the highest percentage of embryo structure formation was registered in varieties Albena and Strjama (12.0 and 13.8 respectively). The Bulgarian peppers are recalcitrant and their in vitro answer is different depending from the explants type, genotype and the culture media composition.

Download Full-text

In vitro propagation of the bay laurel (Laurus nobilis.L) in Morocco

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.4(3).p96-103 ◽

2014 ◽

Vol 4 (3) ◽

pp. 96-103

Author(s):

Abdelali Chourfi ◽

Tajelmolk Alaoui ◽

Ghizlane Echchgadda

Keyword(s):

Seed Germination ◽

In Vitro Culture ◽

In Vitro Propagation ◽

Aerial Part ◽

Basal Medium ◽

Axillary Buds ◽

Laurus Nobilis

Laurus nobilis L. is among the species which are most threatened by massive degradation in Morocco. The multiplication by seed or by cuttings gives very low percentages of recovery that is insufficient to meet the demand of growing market. In vitro culture proves to be a tremendous asset to solve this problem. Our work has focused on the study of seed germination of this species and its multiplication from microcuttings. Finally, we studied the ac-climatization ability of the plantlets resulting from this germination. The study of the germination, via the further measurement of the length of the aerial part and the roots and the number of axillary buds for nine weeks, showed that the MS basal medium was more efficient than media 1/2M.S and WPM. Among the eight tested hormones, IAA yielded the best growth of the plantlets. Hormonal combination of NAA and kinetin resulted into a per-centage of the greatest success in reaching 67 % micropropagation. The study also revealed that the MS basal medium in the presence of the IAA plants can acclimate most easily in two types of substrates with improved development in the peat alone.

Download Full-text

Daya hidup planlet karet asal in vitro microcutting pada berbagai periode penutupan sungkup plastik dan komposisi media tumbuh Survival rate of in vitro microcutting-derived rubber plantlets on various plastic cover closed periods and medium compositions

E-Journal Menara Perkebunan ◽

10.22302/iribb.jur.mp.v80i1.46 ◽

2016 ◽

Vol 80 (1) ◽

Author(s):

. SUMARYONO ◽

Masna Maya SINTA ◽

. NURHAIMI-HARIS

Keyword(s):

Survival Rate ◽

Hevea Brasiliensis ◽

Tree Canopy ◽

Ex Vitro ◽

Media Composition ◽

Transparent Plastic ◽

Growing Medium ◽

Major Bottleneck ◽

Better Than

AbstractIn vitro culture through microcutting technology can be used for clonal propagation of rubber (Hevea brasiliensis Muell. Arg.) rootstocks. Acclimatization of in vitro plantlets to ex vitro conditions is a major bottleneck in the micropropagation of many plants.This research was conducted to study the effect of plastic cover closed period and media composition on the survival rate of rubber plantlets. Plantlets derived from microcutting were planted on plastic pots containing a mixture of soil, cocopeat, dung manure, and sand or zeolite. The plantlets were then placed inside a closed transparent plastic cover that opened after 2, 3, 4 and 6 weeks. The cover was placed under tree canopy. The second experiment used the same media composition with or without cocopeat and with sand or zeolite. At 1.5 month after culture, observation was done on the number of survived plantlets, plantlet height and the percentage of rooted plantlets. The results show that the best coverclosed period was six weeks and the best growing medium was a mixture of soil, cocopeat, dung manure, and zeolite (6:2:1:1v/v). On the two combined treatments, the survival rate was 73.3% after 1.5 month of acclimatization. The use of zeolite and a higher soil percentage gave positive influences on rubber plantlet survival rate. The second experiment results confirmed that the use of zeolite was better than sand and the use of cocopeat was definitely needed. It can be concluded that the best of acclimatization of rubber plantlets from microcutting was on a medium mixture of soil, cocopeat, dung manure, and zeolite (6:2:1:1) and placed inside a closed plastic cover for six weeks before the cover was opened gradually. AbstrakKultur in vitro melalui teknologi microcutting dapat digunakan untuk perbanyakan klonal batang bawah tanaman karet (Hevea brasiliensis Muell. Arg.). Aklimatisasi planlet in vitro ke kondisi ex vitro merupakan hambatan utama pada mikropropagasi berbagai jenis tanaman. Penelitian ini dilakukan untuk mempelajari pengaruh lama penutupan sungkup plastik dan komposisi media tumbuh terhadap daya hidup planlet karet. Planlet karet asal microcutting ditanam pada pot plastik berisi media dengan berbagai campuran tanah, cocopeat, pupuk kandang, dan pasir atau zeolit. Planlet selanjutnya diletakkan di dalam sungkup plastik transparan tertutup rapat yang dibuka setelah 2, 3, 4 dan 6 minggu. Sungkup plastik diletakkan di bawah tajuk pepohonan. Percobaan kedua menggunakan komposisi media serupa dengan atau tanpa cocopeat dan dengan pasir atau zeolit. Pada umur 1,5 bulan, pengamatan dilakukan terhadap jumlah planlet yang hidup, tinggi planlet, dan persentase planlet yang berakar. Hasil penelitian menunjukkan bahwa lama penyungkupan terbaik adalah enam minggu dan media tumbuh terbaik adalah campuran tanah, cocopeat, pupuk kandang, dan zeolit (6:2:1:1 v/v). Pada kombinasi kedua perlakuan tersebut, daya hidup planlet karet mencapai 73,3% setelah 1,5 bulan aklimatisasi. Penggunaan zeolit dan persentase tanah yang lebih tinggi berpengaruh positif terhadap daya hidup planlet karet. Hasil percobaan kedua menegaskan bahwa penggunaan zeolit lebih baik daripada pasir dan penggunaan cocopeat mutlak diperlukan. Dapat disimpulkan bahwa aklimatisasi planlet karet asal microcutting terbaik dilakukan pada media campuran tanah, cocopeat, pupuk kandang, zeolit (6:2:1:1) dan diletakkan di dalam sungkup plastik tertutup selama enam minggu sebelum sungkup dibuka secara bertahap.

Download Full-text

Clonal propagation of Trichocentrum stramineum (Orchidaceae), a threatened species endemic to Mexico

Botanical Sciences ◽

10.17129/botsci.2468 ◽

2020 ◽

Vol 98 (2) ◽

pp. 355-365

Author(s):

Selene Ramos-Ortiz ◽

Luz Ma. Rangel-Guerrero ◽

Martha Elena Pedraza-Santos ◽

Víctor Manuel Chávez-Ávila ◽

José Guadalupe Martínez-Ávalos ◽

...

Keyword(s):

Survival Rate ◽

Clonal Propagation ◽

Basal Medium ◽

Botanical Garden ◽

Genetic Characteristics ◽

Orchid Species ◽

Apical Bud ◽

Whole Plants ◽

Study Species

Background: Cloning techniques are applied to an endangered orchid species in order to reproduce individual plants and to preserve their genetic characteristics. Objectives: To establish a new protocol for clonal propagation of a threatened orchid of horticultural importance. Study species: Trichocentrum stramineum, a threatened orchid endemic to Mexico. Study site and duration: Totutla, Veracruz, Mexico. All experiments were designed and carried out at the Botanical Garden-UNAM and the IIAF-UMSNH over a course of six years. Methods: Seeds were germinated in a modified KC basal medium; protocorms and apical bud explants were obtained from the resulting in vitro plants and cultivated with or without plant growth regulators (PGRs). Both experimental groups were subcultured in order to evaluate the number of protocorm-like bodies (PLBs) and buds per explant. Results: On average, protocorms generated 51.2 and 54.1 PLBs in the absence or presence of 1 mg l-1 6-benzyladenine (BA), respectively, while 13.1 and up to 23.7 PLBs and / or shoots were observed on the apical bud explants in the absence or presence of 1 mg l-1 kinetin, respectively. In both cases, responses were direct, without the formation of an intervening callus. Approximately 200 PLBs were subcultured and developed into whole plants within 14 weeks. These were acclimatized to greenhouse conditions with a 90 % survival rate after 12 weeks. After 44 weeks, flowering was observed (3 %) individuals measuring at least 12 cm in height. Conclusions: The developed protocol proved to hold great potential for commercial propagation and conservation programs.

Download Full-text

In vitro seed germination in Cymbidium elegans Lindl. and Dendrobium densiflorum Lindl. ex Wall. (Orchidaceae)

Botanica Orientalis Journal of Plant Science ◽

10.3126/botor.v6i0.2917 ◽

2010 ◽

Vol 6 ◽

pp. 100-102 ◽

Cited By ~ 5

Author(s):

Shreeti Pradha ◽

Bijaya Pant

Keyword(s):

Seed Germination ◽

Basal Medium ◽

Naphthalene Acetic Acid ◽

Genetic Constitution ◽

Ms Medium ◽

Orchid Species ◽

C Elegans ◽

Protocorm Formation ◽

Dendrobium Densiflorum

A comparative study of in vitro seed germination of two endangered orchid species, viz. Cymbidium elegans Lindl. and Dendrobium densiflorum Lindl. ex Wall., was carried out on Murashige and Skoog's (MS) medium, supplemented with different concentrations and combination of 6-benzylaminopurine (BAP) and á-Naphthalene acetic acid (NAA). The hormone-free MS medium and MS medium supplemented with various growth hormones were found effective for in vitro seed germination of both species. However, the seeds of these two species showed variation in their germination behavior. Hormone-free MS basal medium was found most effective for seed germination of D. densiflorum; whereas, basal medium supplemented with BAP (1mg/l) was effective for C. elegans. The seeds of D. densiflorum showed quick response in earlier germination, protocorm formation and further development into seedlings in comparison to C. elegans. In C. elegans, germination of immature seeds started after nine weeks of inoculation; whereas in D. densiflorum, the initiation of germination started after five weeks of culture. The variations in seed germination, protocorm formation and seedling differentiation in the two orchid species might be due to the differences in their genetic constitution and the presence of different endogenous growth stimulating substances present in their seeds. The present study has provided useful information for in vitro clonal mass multiplication of these commercially important orchid species. Key-words: growth hormone; in vitro study; orchid.DOI: 10.3126/botor.v6i0.2917 Botanica Orientalis - Journal of Plant Science (2009) 6: 100-102

Download Full-text

88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab88 ◽

2011 ◽

Vol 23 (1) ◽

pp. 149

Author(s):

E. Y. Herrera ◽

C. de Frutos ◽

R. Laguna-Barraza ◽

A. Gutierrez-Adan ◽

D. Rizos

Keyword(s):

Survival Rate ◽

Survival Rates ◽

Calf Serum ◽

Basal Medium ◽

Bovine Embryos ◽

Oviduct Fluid ◽

Bovine Oocytes ◽

Cryopreservation Method ◽

Hatching Rates

Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.

Download Full-text

Ovule and Embryo Culture of Arachis hypogaea and Interspecific Hybrids1

Peanut Science ◽

10.3146/i0095-3679-15-2-14 ◽

1988 ◽

Vol 15 (2) ◽

pp. 98-104 ◽

Cited By ~ 1

Author(s):

H. T. Stalker ◽

M. A. Eweda

Keyword(s):

Embryo Culture ◽

Interspecific Hybrids ◽

Shoot Growth ◽

Basal Medium ◽

Lower Percentage ◽

Naphthaleneacetic Acid ◽

Wide Crosses ◽

In Vitro Techniques ◽

Basal Media

Abstract Interspecific hybridization in Arachis is difficult between species within sectional groups and nearly impossible among more distantly related species. Embryos usually abort early in the reproductive cycle; thus in vitro techniques are necessary to recover many desirable hybrid combinations in the genus. The objectives of this investigation were to develop techniques whereby mature plants could be recovered from otherwise aborting embryos. First, ovule culture was performed using eight genotypes, three levels of kinetin, and the two basal media Murashige and Skoog (MS) and N6. Two-tenths mg/L kinetin in media resulted in 24% of the ovules swelling to a size of 3-4 mm which could be used for excising embryos. Embryo culture was next performed on five genotypes. The transfer series (I) 0.2 mg/L kinetin for 21 days, (2) 0.5 mg/L 6-benzylamino-purine (BAP) for 14 days and, (3) MS without growth regulators resulted in 34.6% of ovules producing plants across genotypes; other transfer series either resulted in a lower percentage of plant recovery and/or tissues of some genotypes which did not survive to maturity. The BAP medium induced shoot growth, while root growth was induced on the MS without growth regulator medium. Approximately 90% of embryos transferred to a mist system after 7-9 weeks in vitro survived transplanting to soil. Two interspecific hybrids were recovered from incompatible hexaploid x diploid crosses, but only after roots were induced using a MS basal medium with 4 mg/L 1-naphthaleneacetic acid:2 mg/L indole-3-butyric acid in a fourth tissue transfer. The experiments illustrated the feasibility of rescuing embryos of A. hypogaea and interspecific peanut hybrids. The process is slow and will be most applicable to wide crosses which cannot be obtained by more conventional methods.

Download Full-text

Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models

10.1101/2021.03.01.433282 ◽

2021 ◽

Author(s):

Emma Neal ◽

Ketaki Katdare ◽

Yajuan Shi ◽

Nicholas Marinelli ◽

Kameron Hagerla ◽

...

Keyword(s):

Stem Cell ◽

Blood Brain Barrier ◽

Pluripotent Stem Cell ◽

Molecular Transport ◽

Culture Conditions ◽

Brain Barrier ◽

Media Composition ◽

Blood Brain ◽

Basal Media

It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principle component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in different basal media with fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.

Download Full-text