Cryopreservation of Arecanut (Areca catechu L.) Embryogenic Calli by V Cryo-plate Method

Aims: Arecanut, a perennial palm species of Arecaceae family, has huge commercial value, and is grown mainly for its masticatory nuts. The ever-increasing demand for uniform quality plantlets from growers necessitates putting in place In vitro mass multiplication and other crop improvement programmes. The present study was carried out to standardize the procedure for cryopreservation of embryogenic calli of arecanut, derived from immature inflorescence cultures, by vitrification based cryo-plate technique. Study Design: Completely randomized design (CRD) with three replications. Place and Duration of Study: ICAR-Central Plantation Crops Research Institute, Kerala, India during 2019. Methodology: The embryogenic calli were precultured in Eeuwen's Y3 basal medium supplemented with sucrose (0.2, 0.3 and 0.4 M) for three days. Explants were affixed on cryo-plates and later dehydrated using plant vitrification solution 3 (PVS3) for 30 min. Cryoplates were inserted in cryovials and cryopreserved. Explants with no cryostorage served as control. Explants were rewarmed quickly in a water bath (40ºC) for 2 min and treated with unloading solution and cultured on recovery medium. Results: The results showed 8-10 % recovery of embryogenic calli that resulted in normal plantlet production. The clonal fidelity studies, using Start Codon Targeted (SCoT) marker, showed no variation of cryopreserved calli in comparison to the original calli. Conclusion: This preliminary study demonstrated the successful use of vitrification (V) cryo-plate technique in cryopreservation of embryogenic calli of arecanut. With better recovery percentage, the optimal concentration of sucrose in the preculture medium was found to be 0.3 M. Desiccation in PVS3 solution for 30 min had no adverse effect.

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Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.)

Plants ◽

10.3390/plants9060789 ◽

2020 ◽

Vol 9 (6) ◽

pp. 789 ◽

Cited By ~ 1

Author(s):

Temitope Jekayinoluwa ◽

Jaindra Nath Tripathi ◽

George Obiero ◽

Edward Muge ◽

Leena Tripathi

Keyword(s):

Cell Suspension ◽

Culture Media ◽

Crop Improvement ◽

Genotypic Variation ◽

Cell Suspensions ◽

Phytochemical Analysis ◽

Embryogenic Calli ◽

Embryogenic Cell ◽

Agrobacterium Mediated Transformation

Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l’Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l’Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants.

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In vitro plant propagation and crop improvement in Lisianthus (Lisianthus Russelianus Hook.)

Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Horticulture ◽

10.15835/buasvmcn-hort:12367 ◽

2016 ◽

Vol 73 (2) ◽

pp. 168

Author(s):

Rodica Pop ◽

Maria Cantor ◽

Erzsebet Buta ◽

Iudita Csete

Keyword(s):

Crop Improvement ◽

Ornamental Plants ◽

Basal Medium ◽

International Market ◽

Nodal Segments ◽

F1 Hybrids ◽

Vase Life ◽

Eustoma Grandiflorum ◽

Planting Material

Romania assists at the present time to an increase of production crops for ornamental plants and as a consequence an increased demand of planting material. Thus, improvements of the current multiplication methods are sought after. Lisianthus russelianus Hook. (Eustoma grandiflorum Grise.) is a relatively new floral crop to the international market, known for beautiful flowers of various colors and for having a long vase life. This study focused on the development of an efficient protocol for rapid regeneration of this species following known basic and applied aspects of lisianthus biotechnology but exploring new potentials. In the course of experiments conducted, for in vitro multiplication there were used nodal segments (1.5 cm) with axillary buds from three F1 hybrids ‘Echo Lavender’, ‘Flamenco White’, ‘Mirage Pastel Pink’ that were inoculated on MS basal medium supplemented with 0.50 mg 1-1 TDZ, 1.0 mg 1-1 BAP and 0.50 mg 1-1 AIA. The results show that the medium with BAP was most effective for obtaining the highest shoots number compared to medium containing TDZ. For rooting induction, two different concentrations of auxin IBA 0.5 mg 1-1 and 1.5 mg 1-1 were used simultaneously on MS basal medium. The highest roots number occurred when using 1.5 mg 1-1 IBA. Both the number of shoots and rooting regeneration were dependent on the cultivar. The highest shoots number was achieved for ’Mirage Pastel Pink’ hybrid (6.91) on the medium containing 1.0 mg 1-1 BAP and 0.50 mg 1-1 IAA.

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PENINGKATAN KERAGAMAN GENETIK PURWOCENG MELALUI IRADIASI SINAR GAMMA DAN SELEKSI IN VITRO

Jurnal Penelitian Tanaman Industri ◽

10.21082/jlittri.v19n2.2013.88-98 ◽

2020 ◽

Vol 19 (2) ◽

pp. 88

Author(s):

IKA ROOSTIKA ◽

IRENG DARWATI ◽

YUDIWANTI YUDIWANTI

Keyword(s):

Genetic Variation ◽

High Temperature ◽

Gamma Irradiation ◽

Somatic Embryos ◽

In Vitro Selection ◽

Basal Medium ◽

Root Induction ◽

Embryogenic Calli ◽

Low Altitude

ABSTRAK Peningkatkan keragaman genetik purwoceng memerlukan aplikasi teknologi alternatif yang mampu membentuk keragaman baru. Tujuan penelitian adalah untuk meningkatkan keragaman genetik dan toleransi purwoceng terhadap cekaman suhu tinggi melalui iradiasi dan seleksi in vitro. Tahapan penelitian meliputi induksi mutasi kalus embriogenik dengan sinar gamma, seleksi in vitro dengan cekaman suhu tinggi, induksi perakaran somaklon putatif, analisis keragaman genetik secara flowcytometry, dan aklimatisasi somaklon putatif. Iradiasi dilakukan pada dosis 0, 1, 2, 3, 4, dan 5 Krad sedangkan seleksi in vitro dilakukan pada tiga level suhu (20, 25, dan 30 0 C). Induksi perakaran dilakukan dalam dua tahap, dengan menggunakan media DKW atau MS yang mengandung sukrosa 3-6% dengan penambahan IBA atau NAA taraf 0,5-1,5 ppm. Hasil penelitian menunjukkan bahwa kalus purwoceng mampu bertahan hidup pada dosis iradiasi tertinggi (5 Krad). Meningkatnya dosis iradiasi cenderung meningkatkan pendewasaan embrio somatik. Pada tahap seleksi in vitro, kalus purwoceng mampu tumbuh pada kondisi suhu tertinggi (30 0 C). Tingkat proliferasi kalus yang tinggi dan jumlah embrio somatik terbanyak diperoleh dari perlakuan suhu 25 0 C. Embrio somatik yang terbentuk dari perlakuan suhu tinggi tersebut merupakan kandidat somaklon yang toleran suhu tinggi pada lingkungan dataran rendah. Diantara embrio somatik yang terbentuk, hanya embrio yang berasal dari perlakuan suhu 20 0 C saja yang berhasil membentuk planlet. Media yang terbaik untuk induksi perakaran adalah media MS yang mengandung sukrosa 4% dengan penambahan NAA 1,5 ppm. Analisis ploidi pada daun embrio somatik menunjukkan terbentuknya varian yang bersifat tetraploid (4x). Kata kunci: Pimpinella pruatjan, iradiasi sinar gamma, seleksi in vitro, keragaman genetik, suhu tinggiABSTRACT To improve new pruatjan genetic variations, the alternative technology should be applied. The objective of the research was to increase pruatjan genetic variation and tolerance to the high temperature through induced mutation and in vitro selection. The steps of this study were induced mutation of embryogenic callus by gamma irradiation, in vitro selection, root induction of putative somaclones, genetic variation analysis by flowcytometer, and putative somaclones acclimatization. The dosages of gamma irradiation were 0, 1, 2, 3, 4, and 5 Krad. In vitro selection was conducted at three temperatures (20, 25, and 30 0 C). The root induction was conducted in two steps by using DKW or MS media containing of 3-6% sucrose with addition of 0.5-1.5 ppm IBA or NAA. The result showed that embryogenic calli could survive after treatment of the highest gamma irradiation dose. It tends to increase the maturation of somatic embryos. During in vitro selection, embryogenic calli could grow at the highest temperature but the highest callus proliferation and the number of somatic embryos were obtained from 25 0 C. The somatic embryos survived and grew at the high temperature are assumed as somaclones which considered as the candidates of tolerant plants to high temperature that can be developed in the of low altitude area. Among the regenerated somatic embryos, only the 20 0 C-derived embryos were successfully form plantlets. The best medium for root induction was MS basal medium containing of 4% sucrose supplemented with 1.5 ppm NAA. The ploidy analysis of somatic embryos leaf showed a tetraploid (4x) variant. Key words: Pimpinella pruatjan, gamma irradiation, in vitro selection, genetic variation, high temperature

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Micropropagation of Homalomena pineodora Sulaiman & Boyce (Araceae): a new species from Malaysia

Horticultura Brasileira ◽

10.1590/s0102-05362012000100007 ◽

2012 ◽

Vol 30 (1) ◽

pp. 39-43 ◽

Cited By ~ 2

Author(s):

Christine Stanly ◽

Arvind Bhatt ◽

Baharuddin Sulaiman ◽

Chan Lai Keng

Keyword(s):

Large Scale ◽

Ornamental Plants ◽

Basal Medium ◽

Fluorescent Light ◽

Scale Production ◽

Shoot Cultures ◽

Ms Medium ◽

Plantlet Production ◽

Mother Plants

Homalomena pineodora (family Araceae) is a species found to have impressive foliage characteristics which remain evergreen throughout the year. Therefore, H. pineodora can be grown as an ornamental plant. Generally H. pineodora needs 3-5 years to propagate and multiply. However, the demand for new ornamental plants is increasing worldwide and the quality of planting material is a basic need for boosting productivity. Therefore an efficient micropropagation protocol for large-scale production of H. pineodora was developed. In vitro shoot cultures were initiated from the rhizomatous buds on MS basal medium. The best conditions for propagating H. pineodora was found to be MS medium supplemented with 3% sucrose and 0.5 mg L-1 BA (6-benzyladenine) under 24 h of cool fluorescent light which produced an average of 3.8 shoot per explant. Presence of an auxin was not necessary for plantlet production. Liquid MS medium supplemented with 0.5 mg L-1 BA, enhanced the shoot production of H. pineodora as compared to agar-gelled medium with same composition. All the in vitro plantlets of H. pineodora were successfully acclimatized with 100% survival rate. Scanning electron microscopy confirmed the similarity of leaf microstructures between the in vitro and mother plants of H. pineodora.

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Development of In vitro Tetraploid Plants of Hevea brasiliensis

International Journal of Plant & Soil Science ◽

10.9734/ijpss/2019/v28i630125 ◽

2019 ◽

pp. 1-12

Author(s):

U. K. Divya ◽

S. Sushama Kumari

Keyword(s):

Hevea Brasiliensis ◽

Chromosome Doubling ◽

Crop Improvement ◽

Embryo Germination ◽

Regeneration Frequency ◽

Immature Inflorescence ◽

In Vitro Techniques ◽

Flow Cytometric ◽

Study Development

Increase in global consumption of natural rubber necessitates crop improvement of Hevea aimed at increased productivity. As conventional breeding of Hevea is very elaborate and time consuming. Hence in the present study development of tetraploids through chromosome doubling of diploid callus obtained from cultured immature inflorescence of Hevea using colchicines were attempted. Chromosome doubling of the diploid callus occurred when treated with 1.25 µM colchicine for 3 days. In higher concentrations as well as at longer exposure periods, the callus texture and viability were affected. 48 % embryo induction and a maturation frequency of 45 % were obtained. Embryo germination and plant regeneration with a germination frequency (30 %) and a regeneration frequency (20 %) were obtained. Cytological and flow cytometric analyses confirmed the tetraploid nature of the colchicines treated callus. In vitro tetraploid plant developed through these in vitro techniques can be further used in Hevea brasiliensis breeding.

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An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis)

Frontiers in Plant Science ◽

10.3389/fpls.2021.773656 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wan-Chin Yeap ◽

Norkhairunnisa Che Mohd Khan ◽

Norfadzilah Jamalludin ◽

Muhammad Rashdan Muad ◽

David Ross Appleton ◽

...

Keyword(s):

Oil Palm ◽

Gene Editing ◽

Elaeis Guineensis ◽

Crop Improvement ◽

Dna Delivery ◽

Nucleotide Substitutions ◽

Cleavage Efficiency ◽

Palm Species ◽

Short Palindromic Repeat

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

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Peningkatan Toleransi Alumunium pada Jeruk Batang Bawah dengan Teknik Seleksi In Vitro Berulang

Jurnal AgroBiogen ◽

10.21082/jbio.v6n1.2010.p33-39 ◽

2016 ◽

Vol 6 (1) ◽

pp. 33

Author(s):

Mia Toruan Kosmiatin ◽

Rosa Yunita ◽

Ali Husni

Keyword(s):

In Vitro Selection ◽

Acid Soil ◽

Phase 1 ◽

Aluminum Tolerance ◽

Basal Medium ◽

Culture Technique ◽

Low Ph ◽

Embryogenic Calli ◽

Marginal Soil

Aluminum Tolerance Improvement of Rootstock Citrus through Repeated In Vitro Selection. Mia Kosmiatin, Rosa Yunita, and Ali Husni. National orange productivity was trend to decrease because of pathogen attack and reducing of planting area. One of alternative ways to preserve and increase orange productivity was using marginal soil mainly acid soil. This matter pushed the breeder to prepare tolerant rootstock and stable in the acid soil. In vitro culture technique was effective and efficient methods to produce tolerant and stable rootstock in acid soil through simulation of acid soil with addition of high aluminum and low pH in the medium. By the simulation the selection could be done in cell level, so cell was selected after induction of variation. A rootstock which high compatibility with scion, useful rooting, and aluminum tolerance could be increased orange productivity through acid soil development. The research was conducted in 3 phase: (1) induction of embryogenic calli, (2) improvement of genetic variation through mutation, and (3) In vitro selection with AlCl3.6H2O for aluminum and low pH tolerant. Immature embryos of rootstock were use as explant. The result showed that the best embryogenic calli were induced on MS basal medium with MW vitamin + NAA 7,5 mg/l + kinetin 0,5 mg/l. Before selection, 1.000 rad dosage was the most tolerant dosage to growth embryogenic calli. After selection, 2.000 rad dosage was the best dosage to produce shoots which stable tolerant to aluminum. Selected 88 mutant shoots were produced after three times selection on the same medium which AlCl3.6H2O added at low pH.

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Transient genetic transformation of embryogenic callus of Cocos nucifera

Biologia ◽

10.2478/s11756-011-0104-4 ◽

2011 ◽

Vol 66 (5) ◽

Cited By ~ 9

Author(s):

Antonio Andrade-Torres ◽

Carlos Oropeza ◽

Luis Sáenz ◽

Tomás González-Estrada ◽

José Ramírez-Benítez ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Genetic Transformation ◽

Embryogenic Callus ◽

Cocos Nucifera ◽

Reporter Genes ◽

Embryogenic Calli ◽

In Vitro Morphogenesis ◽

Practical Applications ◽

Palm Species

AbstractCoconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods, and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical applications, such as the improvement of coconut micropropagation via somatic embryogenesis.

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In Vitro Regeneration of Buffalograss [Buchloe dactyloides (Nutt.) Engelm] through Immature Inflorescence Culture

HortScience ◽

10.21273/hortsci.32.3.521c ◽

1997 ◽

Vol 32 (3) ◽

pp. 521C-521

Author(s):

Shuizhang Fei ◽

Paul E. Read ◽

Terrance P. Riordan

Keyword(s):

Embryogenic Callus ◽

Callus Formation ◽

In Vitro Regeneration ◽

Basal Medium ◽

Good Choice ◽

Seasonal Effect ◽

Genotypic Effect ◽

Immature Inflorescence ◽

Callus Production

Buffalograss is native to the Great Plains of North America. Its excellent drought resistance and low growth habit make it a good choice for a low-maintenance turf. A reproducible and efficient regeneration protocol of buffalograss is critical for further genetic transformation. By using immature inflorescences as explants, we have achieved the regeneration of buffalograss of two female clones, `315' and `609', a male clone, NE 84-45-3, and a synthetic cultivar, `Texoka'. Somatic embryogenesis was observed. The medium used for callus initiation was MS basal medium supplemented with various concentrations of 2,4-D and BA. After 4 weeks of dark culture, calli with nodular structures were transferred to the same basal medium supplemented with BA and either a reduced rate of 2,4-D or no 2,4-D. It was demonstrated that 2,4-D at 2 or 3 mg/L is optimal for embryogenic callus production. The presence of BA from 0.1 mg/L to 0.5 mg/L was required for the regeneration of `315', `609', and NE 84-45-3. For `Texoka', 2,4-D at 0.5 mg/L with BA at 0.3 mg/L in the regeneration medium favored normal development of somatic embryos that were capable of germination. A genotypic effect was observed with regard to embryogenic callus production; explants of the male genotype NE 84-45-3 exhibited a higher percentage of embryogenic callus formation than was found for the two female genotypes. A significant seasonal effect was also observed with inflorescences collected in early May exhibiting a higher percentage of callus formation than those collected in the summer and fall.

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Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v19i1.4990 ◽

1970 ◽

Vol 19 (1) ◽

pp. 89-99

Author(s):

K. Choudhary ◽

M. Singh ◽

M. S. Rathore ◽

N. S. Shekhawat

Keyword(s):

Somatic Embryogenesis ◽

Growth Regulators ◽

Somatic Embryos ◽

Basal Medium ◽

Long Term Study ◽

Continuous Application ◽

First Time ◽

Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l of 2,4-D and 0.5 mg/l of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

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