Establishment of an Efficient Micropropagation System for Humulus lupulus L. cv. Cascade and Confirmation of Genetic Uniformity of the Regenerated Plants through DNA Markers

The demand for virus-free hop planting material has increased in the last few years due to its multipurpose uses. The present study aimed to establish an effective protocol for clonal propagation of cv. Cascade using only the cytokinins as PGRs in all stages of micropropagation: (i) in vitro culture initiation using single-node micro-cuttings inoculated on modified Murashige and Skoog (MSm) medium solidified with Plant agar and supplemented with 0.5 mg L−1 6-benziyladenine (BA) with 76% recorded viability of nodal explants; (ii) in vitro multiplication of multinodal shoots on MSm medium gelled with Plant agar and supplemented with different types and concentrations of cytokinins: 2 mg L−1 kinetin (KIN), 0.7 mg L−1 1-(2-Chloro-4-pyridyl)-3-phenylurea) (1 CPPU), 2 mg L−1 meta-topoline (mT) and 0.5 mg L−1 BA, which was the best variant for shoot proliferation (9.48 ± 0.78 shoots/explant); (iii) rooting and acclimatization with the best results obtained by ex vitro rooting and acclimatization of plants in the same stage in perlite (96.00 ± 0.60% acclimatized rooted plants with 100% survival under greenhouse conditions). The true-to-type nature of in vitro raised plants with the mother plant was assessed by Random Amplified Polymorphic DNA (RAPD) and Start Codon Target Polymorphism (SCoT) molecular markers, and then their genetic uniformity were confirmed.

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In Vitro Propagation of an Endangered Helianthus verticillatus by Axillary Bud Proliferation

Plants ◽

10.3390/plants9060712 ◽

2020 ◽

Vol 9 (6) ◽

pp. 712

Author(s):

Marzena Nowakowska ◽

Žaklina Pavlović ◽

Marcin Nowicki ◽

Sarah L. Boggess ◽

Robert N. Trigiano

Keyword(s):

Endangered Species ◽

Shoot Proliferation ◽

Genetic Fidelity ◽

Induction Medium ◽

Axillary Shoot ◽

Axillary Shoot Proliferation ◽

Regenerated Plants ◽

Micropropagated Plants ◽

Ssrs Markers

Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species.

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Micropropagation of Genista aetnensis [(Raf. ex Biv.)DC]

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha43210036 ◽

2015 ◽

Vol 43 (2) ◽

pp. 542-546 ◽

Cited By ~ 1

Author(s):

Giovanni IAPICHINO ◽

Marcello AIRÒ ◽

Emilio LO PRESTI ◽

Leo SABATINO

Keyword(s):

Acetic Acid ◽

Shoot Proliferation ◽

Shoot Development ◽

In Vitro Rooting ◽

Nodal Segments ◽

Axillary Shoot ◽

High Plasticity ◽

Single Node ◽

Indole 3 Acetic Acid

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

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Molecular markers based genetic relatedness studies in tissue culture propagated Japanese plum cultivars Santa Rosa and Frontier

10.21203/rs.3.rs-885836/v1 ◽

2021 ◽

Author(s):

Manisha Thakur ◽

Rakshandha Luharch ◽

Vishal Sharma ◽

Dharam Paul Sharma

Keyword(s):

Molecular Markers ◽

Genetic Relatedness ◽

Polymorphic Information Content ◽

Random Amplified Polymorphic Dna ◽

Start Codon ◽

Japanese Plum ◽

Percent Similarity ◽

Allelic Variations ◽

Relationship Of

Abstract Santa Rosa and Frontier are the major Japanese plum cultivars grown throughout the world. The present investigation was performed to understand the genetic relatedness among in vitro propagated plum cultivars Santa Rosa and Frontier using PCR based molecular markers. For the study, three arbitrary markers viz. RAPD (Random amplified Polymorphic DNA), ISSR (Inter-Simple Sequence Repeats) and SCoT (Start Codon Targeted) were used. In RAPD analysis, 18 primers out of 28 amplified and generated 33 scorable bands. The allelic variations when analysed, revealed 84 percent similarity between these two cultivars with highest polymorphic information content of 0.78. Similarly, 15 ISSR primers produced 73 amplicons with an average of 4.86 amplicon per primer and similarity coefficient ranging from 62 to 67 percent. Seven SCoT primers out of 26 resulted in a total of twenty- six scorable bands with 24 polymorphic bands. Cluster analysis from all the three markers used broadly divided plum cultivars santa rosa and frontier into two major clusters containing in vitro shoots, their progenies and mother trees of respective genotypes. The study concluded that these three marker systems were found to be effective in revealing genetic relationship of these two commercially important plum cultivars.

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In vitro multiplication protocol for Curcuma mangga : Studies on carbon, cytokinin source and explant size

Journal of Horticultural Sciences ◽

10.24154/jhs.2021.v16i01.008 ◽

2021 ◽

Vol 16 (1) ◽

pp. 69-76

Author(s):

A A Waman ◽

P Bohra ◽

R Karthika Devi ◽

J Pixy

Keyword(s):

Carbon Source ◽

Large Scale ◽

Shoot Proliferation ◽

Ex Vitro Rooting ◽

Tropical Species ◽

Scale Production ◽

Ex Vitro ◽

In Vitro Multiplication ◽

Planting Material

Mango ginger (Curcuma mangga Valeton & Zijp.) is an underutilized rhizomatous species that has been valued in tropical Asian countries as a source of vegetable, spice, salad, medicine, and essential oil. This species is hardy and requires less care for obtaining good yields. Rhizomes are the commonly used propagules for the species, which are also the economic part of the crop. Huge quantity of seed rhizomes is required to promote this crop in larger areas. An efficient in vitro multiplication protocol is one of the options to meet the planting material requirement. Effects of carbon source (glucose, fructose and sucrose) and concentration (1 and 3%, w/v), cytokinins (BAP and meta topolin) and concentration (1 mg/L and 2 mg/L), size of explants (one/ two/ three bud) and IBA treatment (0, 250, 500 and 1,000 mg/L) for concurrent ex vitro rooting cum hardening were studied. Results revealed that for facilitating efficient multiplication, the medium should be supplemented with glucose (3%) as a carbon source and meta topolin (1 mg/L) as cytokinin. Two-bud explant should be used for subculture as it promoted superior shoot proliferation. Concurrent ex vitro rooting cum hardening was possible even without auxin treatment. The present protocol could be useful for large-scale production of quality planting material of this underexploited tropical species.

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Clonal Propagation and Karyomorphological Study of Solanum torvum Swartz: An Ethnobotanical Species of Tripura, India

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v28i1.37199 ◽

2018 ◽

Vol 28 (1) ◽

pp. 69-76

Author(s):

H Reshmi Singha ◽

Sangram Sinha ◽

Rabindra Kumar Sinha

Keyword(s):

Clonal Propagation ◽

Culture Media ◽

Shoot Proliferation ◽

Induction Phase ◽

Root Induction ◽

Metaphase Chromosomes ◽

Solanum Torvum ◽

Regenerated Plants ◽

Bud Induction

An efficient method of clonal propagation through nodal culture of Solanum torvum Swartz. is described. Different concentrations of BAP/Kn alone or in combination with IAA were tested for direct shoot bud induction and proliferation. Lower concentration of BAP/Kn alone produced better shoot proliferation and elongation. Maximum number of shoot proliferation was achieved from MS supplemented with Kn 0.5 mg/l with an average 4.0 ± 1.41 shoots during 28 days of culture. Addition of IAA to the culture media in combination with BAP/ Kn significantly reduced the number of shoot formation. Regenerated plants also produced roots during subsequent culture in the same media supplemented with BAP/Kn alone or in combination with IAA. The easy nature of in vitro rooting of S. torvum was recorded without any separate root induction phase. Regenerated plants were successfully transferred to the field condition. Clonal feature was cytologically confirmed through the study of mitotic metaphase chromosomes of regenerated plants which reveals 2n = 24 somatic chromosomes. Comparative karyomorphological details between the mother and regenerated plants of S. torvum revealed close similarity in their chromosomal complements and falls under the category of "1B" Stebbin’s symmetric index suggesting true to type nature of the regenerated plant.Plant Tissue Cult. & Biotech. 28(1): 69-76, 2018 (June)

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Optimization of biotechnological process clonal micropropagation in vitro of Asparagus officinalis L.

Biological Systems: Theory and Innovation ◽

10.31548/biologiya2021.03.003 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Y Kolomiiets ◽

◽

A Skuba ◽

Keyword(s):

Culture Medium ◽

Asparagus Officinalis ◽

Skoog Medium ◽

Clonal Micropropagation ◽

Planting Material ◽

Regenerated Plants ◽

Murashige And Skoog Medium ◽

Macro And Micronutrients ◽

Indole 3 Acetic Acid

The study presents the results of obtaining regenerated plants of asparagus from seeds. Surface sterilizing the seeds by 0,75% sodium hypochlorite for 30 min is effective, during this obtained 83% viable sterile plants. The Murashige and Skoog medium supplemented with 6‑benzylaminopurine (2 mg/L), inositol (100 mg/L) and thiamine (0,4 mg/L) was found to be the best for seed germination. The expediency of using kinetin (1 mg/L) as a growth regulator to obtain a homogeneous plant material was established. The reproduction coefficient was 6,0. Only 11% of the explants formed callus. For the selection needs and production of somaclonal variants, the use of the culture medium with indole-3-acetic acid (0,2 mg/L) and 6‑benzylaminopurine (1 mg/L) is justified. In this condition reproduction coefficient was 3,7, and the level of different intensity callusogenesis was 59%. The rooting of obtained plants was performed in Murashige and Skoog medium supplemented with a half dose of macro- and micronutrients and growth regulators. Rooting frequency was up to 63%. The knowledge of hormonal requirements helps to promote isolated tissue and cells technologies of asparagus with purpose of rapid propagation and obtaining healthy, high-quality planting material.

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In vitro conservation of centennial Austrian Cornelian cherry accessions

Plant Biotechnology Reports ◽

10.1007/s11816-021-00678-1 ◽

2021 ◽

Author(s):

Margit Laimer ◽

Maria Zeiser ◽

Veronika Hanzer ◽

E. G. Borroto Fernandez

Keyword(s):

Future Generations ◽

Single Node ◽

Breeding Programs ◽

Cornelian Cherry ◽

Planting Material ◽

Cornus Mas ◽

Medicinal Use ◽

Health Promoting ◽

Superior Clones

AbstractCornelian cherry (Cornus mas) appears in a list of fruit and nut species growing in Europe considered neglected and underused economically. Although C. mas has a long-standing traditional medicinal use, only in recent years interest in products and food made from Cornelian cherries, said to have health-promoting effects, increased. This in turn raises the demand for improved planting material. In the Pielach Valley Region, Lower Austria, hundreds of centenary specimens of Cornus mas, but even a few millennial plants can still be encountered. The occurrence of these plants requested an active intervention to genetically characterize and preserve this valuable biodiversity, particularly in the light of changing environmental conditions. Efforts for the establishment of an in vitro collection of this valuable germplasm of centenary cornelian cherries yielded 193 mericlones initiated from single node explants from 41 selected plants. The selected donor plants were grouped by estimated age ranging from 10 years, > 50 years, > 100 years, > 200 years, > 400 years and 1000 years. The final goal of our efforts is to preserve these genetic resources, also checked for genetic and phytosanitary quality, for future generations and to use the superior clones for further breeding programs.

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Comparative Rooting of Shoot Tips of Four Japanese Persimmon Cultivars vs. Shoots Regenerated from Roots Cultured In Vitro

HortScience ◽

10.21273/hortsci.35.5.940 ◽

2000 ◽

Vol 35 (5) ◽

pp. 940-944 ◽

Cited By ~ 4

Author(s):

Takuya Tetsumura ◽

Hisajiro Yukinaga

Keyword(s):

Histological Study ◽

Adventitious Shoots ◽

Shoot Proliferation ◽

Shoot Tips ◽

Diospyros Kaki ◽

Japanese Persimmon ◽

Rooting Ability ◽

Regenerated Plants ◽

In Vitro Roots

When cultured in vitro, roots of four Japanese persimmon (Diospyros kaki L.) cultivars formed adventitious shoots on MS medium with 10 μm zeatin and 0.01 μm indole-3-acetic acid, although their organogenetic capacities varied. Histological study revealed that the origin of the adventitious shoots was the pericycle. The regenerated shoots grew well on the shoot proliferation medium (MS with 5 μm zeatin). Final rooting percentages of shoots regenerated from roots of three of the four cultivars were greater than those of shoots that originated from shoot tips and that had been subcultured >50 times. Shoots regenerated from `Jiro' roots rooted 10 days earlier, had more roots than those from shoot tips, and maintained higher rooting ability over ten subcultures. Rooted `Hiratanenashi' shoots regenerated from roots survived better after acclimatization than those from shoot tips. No obvious variants were observed either in vitro or in the field. The trees regenerated from roots flowered within 4 years. These findings suggest that partial rather than true rejuvenation was responsible for both the early flowering and the juvenile characteristics, i.e., the enhanced rooting ability, observed in the regenerated plants. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

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Micropropagation of the Aquatic Plant Cryptocoryne lucens

HortScience ◽

10.21273/hortsci.25.6.687 ◽

1990 ◽

Vol 25 (6) ◽

pp. 687-689 ◽

Cited By ~ 8

Author(s):

Michael E. Kane ◽

Edward F. Gilman ◽

Matthew A. Jenks ◽

Thomas J. Sheehan

Keyword(s):

Polyurethane Foam ◽

Aquatic Plant ◽

Shoot Proliferation ◽

Shoot Multiplication ◽

Naphthaleneacetic Acid ◽

Shoot Cultures ◽

Ex Vitro ◽

Single Node ◽

In Vitro Establishment

Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).

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(288) Determination of Genetic Stability of In Vitro-derived Birch Plants

HortScience ◽

10.21273/hortsci.41.4.1077c ◽

2006 ◽

Vol 41 (4) ◽

pp. 1077C-1077

Author(s):

Wenhao Dai ◽

Victoria Magnusson ◽

Andrea Swanberg

Keyword(s):

Genetic Stability ◽

Random Amplified Polymorphic Dna ◽

Genetic Fidelity ◽

Genetic Variations ◽

Meristem Culture ◽

In Vitro Techniques ◽

Somaclonal Variations ◽

Regenerated Plants ◽

Leaf Tissues

Many woody plants, including some birch species, can be cloned using such in vitro techniques as pre-existing meristem culture, organogenesis, and embryogenesis. However, clonal fidelity of in vitro-derived plants is always a big concern because somaclonal variations may be induced during the entire in vitro process. To address this issue, we used random amplified polymorphic DNA (RAPD) markers to determine the genetic stability of in vitro-propagated plants of Betula platyphylla `Fargo'. Forty-two greenhouse-grown birch plants derived from a 10-year shoot tip culture (shoot-derived) and 42 in vitro plants regenerated from leaf tissues (regenerated) were randomly selected and evaluated for their genetic fidelity by RAPD. To date, 20 primers (C1-C20, Operon Technologies) were screened for all 84 plants. Only strong bands that are conservative were scored. Each primer generated a unique set of amplification products. Most of scoreable bands are ranged from 350 to 1800 bp. A total of 3696 fragments were amplified from 42 shoot-derived plants by all 20 primers with an average of 4.4 bands per primer, in which 6 primers produced polymorphic bands, indicating some genetic variations within shoot-derived plants. Nineteen out of 20 primers yielded 2772 clear and reproducible bands (an average of 3.47 per primer) from 42 regenerated plants with no significant variations being detected. Our preliminary results showed that in vitro regenerated plants are genetically uniform. However, a long-term tissue culture might result in a few genetic variations of birch species.

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