Organogenesis and long-term micropropagatlon of Polish pea cultivars

The complete protocol for regeneration and long-term micropropagation of several Polish cultivars of pea (Pisum sativum L.) has been elaborated. The shoots were the most likely regenerated via de novo organogenesis. The adventitious buds formed in callus derived from cotyledons tissue adjacent to the axillary meristems of immature embryos. All cultivars' calli regenerated several shoots per explant on the MS medium supplemented with B5 vitamins and 4.5 mgl-1 of BAP, however some differences in regeneration capacity among cultivars were observed. The plantlets were subsequently micropropagated with slightly higher efficiency and preserving a good viability over the long-term culture on a medium containing 2.0 mgl-1 than one with 4.5 mgl-1 of BAP. The additional step of the pre-conditioning culture of multiplicated shoots on a medium with very low BAP concentration i.e. 0.02 mgl-1 was applied and appeared to be beneficial before rooting in vitro or grafting. The modified MS-derived medium with the half-strength of MS macroelements but with the full original dose of calcium and supplemented with B5 vitamins and 1.0 mgl-1 of NAA was developed for effective rooting. The shoots were also sufficiently transferred into ex vitro conditions using grafting. The majority of the regenerated plants had adapted to in vivo conditions in a greenhouse and subsequently has set seeds. The presented protocol provides relatively efficient rate of de novo pea regeneration and would be useful for Agrobacterium-mediated transformation purposes.

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In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant

Acta Botanica Croatica ◽

10.1515/botcro-2017-0012 ◽

2018 ◽

Vol 77 (1) ◽

pp. 80-87 ◽

Cited By ~ 3

Author(s):

Mahipal S. Shekhawat ◽

M. Manokari

Keyword(s):

Medicinal Plant ◽

Large Scale ◽

Nodal Segments ◽

Ms Medium ◽

Ex Vitro ◽

Culture Bottle ◽

Hybanthus Enneaspermus ◽

Half Strength

AbstractHybanthus enneaspermusis a rare medicinal plant. We defined a protocol for micropropagation,ex vitrorooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L−1BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L−1each of BAP and Kin within 4–5 weeks. The shoots were rootedin vitroon half strength MS medium containing 2.0 mg L−1indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rootedex vitroin soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. Theex vitrorooted plantlets showed a better rate of survival (92%) in a field study thanin vitrorooted plantlets (86%). A comparative foliar micromorphological study ofH. enneaspermuswas conducted to understand the micromorphological changes during plant developmental processes fromin vitrotoin vivoconditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report onex vitrorooting inH. enneaspermusand the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.

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Long-term in vitro Storage Technique of Valuable Birch Genotypes and Plant Production on its Basis

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-3-57-67 ◽

2019 ◽

pp. 57-67

Author(s):

T.M. Tabatskaya ◽

N.I. Vnukova

Keyword(s):

Plant Production ◽

High Survival ◽

Ex Vitro ◽

Regenerative Capacity ◽

In Vitro Storage ◽

Interspecific Differences ◽

Regenerated Plants ◽

Growing Conditions

A technique for the long-term (up to 27 years) in vitro storage of valuable birch genotypes under normal (25 °C, 2.0 klx, 16-h day and 8-h night) and low temperature (4 °C, 0.5 klx, 6-h day and 18-h night) growing conditions on hormone-free media has been described. The study explored for the first time the influence of different strategies to store the clones of Betula pubescens and B. pendula var. сarelica (6 genotypes) on the regenerative capacity of collection samples, adaptive potential of regenerated plants and plant production by the in vitro and ex vitro techniques. It was established that both storage strategies provided a persistently high survival rate (82-100%) and regenerative capacity of in vitro shoots (the multiplication coefficient of 4.2-6.3 and rhizogenic activity of 90-100%). The clones retained their characteristics of height growth under the in vitro and ex vitro conditions, and demonstrated intraclonal homogeneity and lack of signs of somaclonal variability. The plants showed substantial interspecific differences at the stage of multiplication and transfer to the greenhouse. The highest percentage of acclimated plants (75-98% depending on the clone genotype) was obtained after planting of micro plants straight in the greenhouse, which simplified the technology and made plant production less costly. long-term in vitro storage, birch, species, genotype, micropropagation, ex vitro adaptation, plant material

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Micropropagation of Achillea millefolium L. on half-strength ms medium and direct rooting and acclimatization of microshoots in hydroponic culture

Glasnik Sumarskog fakulteta ◽

10.2298/gsf1511099m ◽

2015 ◽

pp. 99-112

Author(s):

Marija Markovic ◽

Dragana Skocajic ◽

Mihailo Grbic ◽

Matilda Djukic ◽

Dragica Obratov-Petkovic ◽

...

Keyword(s):

Medicinal Plant ◽

Nutrient Solution ◽

Ph Value ◽

Ex Vitro Rooting ◽

In Vitro Rooting ◽

Efficient Production ◽

Ms Medium ◽

Ex Vitro ◽

Half Strength

The aim of this study was to determine the possibility of micropropagation of the medicinal plant A. millefolium on half-strength MS medium and ex vitro rooting and acclimatization of the obtained microshoots in hydroculture in order to establish an efficient production method. Two explant types were used: basal and terminal cuttings, and better results were achieved when terminal cuttings were used. The development of shoots in the multiplication phase was successful with a regeneration percentage of 100%. Ex vitro rooting in a modified Hoagland nutrient solution was successful (83%), but the percentage of in vitro rooting on half-strength MS medium without hormones was higher (95%). However, bearing in mind that mass production of A. millefolium is more efficient when the phase of in vitro rooting is excluded, this method could be recommended for commercial propagation of this medicinal plant. It is necessary to conduct additional research in order to optimize the composition, EC and pH value of the hydroponic nutrient solution.

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Adjustment of Mineral Elements in the Culture Medium for the Micropropagation of Three Vriesea Bromeliads from the Brazilian Atlantic Forest: The Importance of Calcium

HortScience ◽

10.21273/hortsci.44.1.106 ◽

2009 ◽

Vol 44 (1) ◽

pp. 106-112 ◽

Cited By ~ 15

Author(s):

Alice Noemí Aranda-Peres ◽

Lázaro Eustáquio Pereira Peres ◽

Edson Namita Higashi ◽

Adriana Pinheiro Martinelli

Keyword(s):

New Media ◽

Mineral Composition ◽

Culture Media ◽

Dry Weight ◽

Defined Medium ◽

Ms Medium ◽

Media Formulation ◽

Half Strength

Many different species of Bromeliaceae are endangered and their conservation requires specific knowledge of their growth habits and propagation. In vitro culture of bromeliads is an important method for efficient clonal propagation and in vitro seed germination can be used to maintain genetic variability. The present work aims to evaluate the in vitro growth and nutrient concentration in leaves of the epiphyte bromeliads Vriesea friburguensis Mez, Vriesea hieroglyphica (Carrière) E. Morren, and Vriesea unilateralis Mez, which exhibit slow rates of growth in vivo and in vitro. Initially, we compared the endogenous mineral composition of bromeliad plantlets grown in half-strength Murashige and Skoog (MS) medium and the mineral composition considered adequate in the literature. This approach suggested that calcium (Ca) is a critical nutrient and this was considered for new media formulation. Three new culture media were defined in which the main changes to half-strength MS medium were an increase in Ca, magnesium, sulfur, copper, and chloride and a decrease in iron, maintaining the nitrate:ammonium rate at ≈2:1. The main difference among the three new media formulated was Ca concentration, which varied from 1.5 mm in half-strength MS to 3.0, 6.0, and 12 mm in M2, M3, and M4 media, respectively. Consistently, all three species exhibited significantly higher fresh and dry weight on M4, the newly defined medium with the highest level of Ca (12 mm). Leaf nitrogen, potassium, zinc, magnesium, and boron concentrations increased as Ca concentration in the medium increased from 1.5 to 12 mm.

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Phenotypic aberrations during micropropagation of Soymida febrifuga (Roxb.) Adr. Juss

Notulae Scientia Biologicae ◽

10.15835/nsb619202 ◽

2014 ◽

Vol 6 (1) ◽

pp. 99-104 ◽

Cited By ~ 4

Author(s):

Kishore Kumar CHIRUVELLA ◽

Arifullah MOHAMMED ◽

Rama Gopal GHANTA

Keyword(s):

Seed Viability ◽

Ms Medium ◽

Stem Cuttings ◽

Endemic Plant ◽

Seedling Mortality ◽

Root Regeneration ◽

Half Strength ◽

Shoot Necrosis

Like most of the medicinal plants Soymida febrifuga (Meliaceae) possess significance for its valuable secondary metabolites. Multiplication of this endemic plant is limited by difficulty in rooting of stem cuttings, high seedling mortality rates and low seed viability period. Hence efficient protocols for in vitro mass propagation has been established from field grown and aseptic seedlings explants. Strikingly, we observed aberrant structures such as vitrified shoots, faciated shoots, albino shoots as well shoot necrosis during its micropropagation. These phenotypic maladies were observed during organogenesis and rooting. Compared to other abnormalities, shoot necrosis nonetheless was frequent and pronounced leading to plant death. Shoots when subjected to rooting also displayed necrosis which was controlled by transferring to MS medium containing various concentrations and combinations of calcium levels, activated charcoal, glucose, fructose and auxins. Microshoots initiated roots on half strength MS medium with IBA and IAA individually or in combination within two weeks. MS half strength solid medium supplemented with CAN (556 mg l–1), CAP (1.0 mg l–1), IAA (2.0 mg l–1) and IBA (2.0 mg l–1) in combination was found to be more efficient in showing high frequency (95%) of root regeneration. Rooted plantlets were successfully hardened and 70-85% of regenerated plants were successfully acclimatized to natural environment. In vitro derived plantlets were morphologically similar to in vivo plants.

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Improved micropropagation and foliar micromorphological studies in Turnera ulmifolia L. – An important medicinal plant

Folia Horticulturae ◽

10.2478/fhort-2018-0024 ◽

2018 ◽

Vol 30 (2) ◽

pp. 283-294 ◽

Cited By ~ 1

Author(s):

Mani Manokari ◽

Mahipal S. Shekhawat

Keyword(s):

Shoot Proliferation ◽

Nodal Segments ◽

Naphthalene Acetic Acid ◽

Ms Medium ◽

Half Strength ◽

Turnera Ulmifolia ◽

Semi Solid ◽

Number Of Shoots

Abstract The present study reports an efficient in vitro propagation system for Turnera ulmifolia using nodal segments as explants. Turnera ulmifolia (Passifloraceae) is an important garden plant with multipotent medicinal values. Effective shoot proliferation was achieved on agar gelled MS medium (Murashige and Skoog, 1962). The maximum number of shoots (8.3 ± 0.57) per initial explant was obtained on MS medium supplemented with 8.88 mM of 6-benzylaminopurine (BAP) and 0.54 mM of α-naphthalene acetic acid (NAA). The highest number of shoots (59.5 ± 2.10) proliferated on semi-solid MS medium (with agar) augmented with 2.22 mM of BAP and 2.32 mM of kinetin (Kin) along with 0.54 mM of NAA. Longer (4-5 cm) and healthy shoots were rooted (12.0 ± 0.10 roots per shoot) on half-strength MS medium fortified with 9.84 mM of indole-3 butyric acid (IBA). The in vitro regenerated plantlets were hardened in the greenhouse and transferred to the field. Significant developmental changes were observed in the foliar micromorphology of in vitro raised plantlets when these were transferred to the field. The stomatal index was gradually reduced (26.72 to 21.25) in the leaves from in vitro to field environments. But, vein-islets and veinlet terminations (13.4 and 7.6) were increased (39.7 and 18.4) respectively from in vitro to in vivo grown plants. Simple, unicellular, less frequent and underdeveloped trichomes were observed with the leaves of in vitro plants but fully developed trichomes recorded in the field transferred plants. The study could help in understanding the response and adaptation of tissue culture raised plantlets towards changed environmental conditions.

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Preliminary results of in vitro culture of pea and lupin embryos for the reduction of generation cycles in single seed descent technique

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.2013.021 ◽

2013 ◽

Vol 82 (3) ◽

pp. 231-236 ◽

Cited By ~ 12

Author(s):

Maria Surma ◽

Tadeusz Adamski ◽

Wojciech Święcicki ◽

Paweł Barzyk ◽

Zygmunt Kaczmarek ◽

...

Keyword(s):

Root Development ◽

Ms Medium ◽

Ex Vitro ◽

Single Seed Descent ◽

Yellow Lupin ◽

Single Seed ◽

Temperature Regimes ◽

Regenerated Plants ◽

Almost All

The aim of the studies was to establish in vitro conditions for the culture of pea and lupin embryos as the first step in the development of an in vitro assisted single seed descent technique for the attainment of homozygous populations. Materials for the study included of pea, and narrow-leafed and yellow lupin cultivars. Embryos dissected from mature but still-green seeds were cultured in vitro on two modified MS media and under three temperature regimes. Shoot and root lengths of regenerated plants were measured after 7, 14 and 21 days of culture. For pea plants full-strength MS medium with 4 g l−1 agar and temperature 22/ 20°C (day/night) appeared to be the most conducive to shoot and root development, whereas for lupin plants lower temperatures were more propitious: 12°C in the dark for narrow-leafed lupin and 16/ 12°C (day/night) for yellow lupin. Almost all the cultured embryos developed into plants, but not all the regenerated plants survived acclimation to ex vitro conditions.

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In Vitro Long-Term Cultures of Papaya (Carica Papaya L.).

10.21203/rs.3.rs-262638/v1 ◽

2021 ◽

Author(s):

Jose Javier Regalado González ◽

Manuel López Granero ◽

Carlos Lopez Encina

Keyword(s):

Morphological Characteristics ◽

Basal Medium ◽

Multiplication Rate ◽

Ms Medium ◽

High Quality ◽

Poor Growth ◽

Average Multiplication ◽

Half Strength

Abstract We present the data on proliferation corresponding to 10 years of continuous incubation in vitro of papaya shoots, and propose a reliable method for long-term micropropagation for papaya, using two types of explants: Microshoots from somatic embryos, and from axillary buds of papaya. Three different media were assayed. The proliferation medium (PPRM) allowed to maintain papaya shoots under continuous proliferation during 20 years, maintaining a consistent behaviour. Most of the shoots developed in PPRM rooted during the incubation, and after acclimated easily, maintaining the morphological characteristics of the parental plants, flowering and setting fruits normally. The PPRM medium consist in MS medium supplemented with NAA (0.1 mg l-1), BA (0.5 mg l-1), GA3 (0.5 mg l-1) and Adenine sulphate (40 mg l-1). The average multiplication rate was higher than 20 shoots per explant along the long-term assay. The elongation medium (PELM), was designed to recover shoots with a poor growth, and allowed the development of high quality shoots ready for rooting, and consist in a MS basal medium supplemented with NAA (0.1 mg l-1), Kin (0.5 mg l-1) and GA3 (1 mg l-1). The rooting medium (PROM) was designed to induce high quality roots from non-rooted shoots and consist in a half strength MS medium plus IBA (1mg l-1). On PROM, agar can be exchanged for expanded vermiculite. Acclimation took place inside an acclimatization tunnel under progressive hydric stress. After 4 weeks, the plant recovery rate was 90% for plants maintained under continuous proliferation during ten years.

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In vitro and ex vitro rooting of Spiraea betulifolia subsp. aemiliana (Rosaceae), an ornamental shrub

BIO Web of Conferences ◽

10.1051/bioconf/20202400058 ◽

2020 ◽

Vol 24 ◽

pp. 00058

Author(s):

Dinara Muraseva ◽

Vera Kostikova

Keyword(s):

Ms Medium ◽

Pulse Treatment ◽

Root Number ◽

Ex Vitro ◽

Effective Technique ◽

Nutrient Media ◽

Half Strength ◽

Regenerated Plantlets ◽

Ornamental Shrub

Two methods of rhizogenesis – in vitro and ex vitro of Spiraea betulifolia subsp. aemiliana (C.K. Schneid.) H. Hara microshoots have been compared. Pulse treatment of microshoots with aqueous solutions of 4% “Heteroauxin” or 2% succinic acid (ex vitro rooting) did not effective -rooting frequency ranged from 3 to 19%. It was established that the in vitro rooting on nutrient media supplemented with auxins was a more effective technique, providing a high percentage of rooted microshoots. The use of half- strength MS medium supplemented with 0.1 μM indolyl-3-butyric acid (rooting frequency 8 8%, root number 3.5 ± 0.3 per plantlet) was found to be the most efective for in vitro rooting. The in vitro rooted regenerated plantlets were successfully acclimatized with 55% of survival rate.

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