in vitro plants
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Plants ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 226
Author(s):  
Marwa Hanafi ◽  
Wei Rong ◽  
Lucie Tamisier ◽  
Chadi Berhal ◽  
Nicolas Roux ◽  
...  

: The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflexiviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation.


2021 ◽  
Vol 28 ◽  
pp. 58-65
Author(s):  
L. R. Hrytsak ◽  
M. Z. Prokopiak ◽  
O. Yu. Mayorova ◽  
Kh. M. Kolisnyk ◽  
N. M. Drobyk

Aim. Study of the dynamics Gentiana lutea L. plant growth processes in vitro depending on the light regime changes of their cultivation in order to develop a scheme to increase their adaptive potential. Methods. Methods of plant cultivation in vitro, biometric method, as well as ANOVA variance analysis and middle group analysis in pairs using the Tukey test (Tukey test) were used. Results. It is shown that the cultivation of G. lutea plants in vitro using 25 W/m2 light flux intensity in the region of photosynthetically active radiation and the ratio of blue (Eb): green (Eg): red (Er) ranges = 41.8%: 42.7 %: 15.5% triggers non-specific photomorphogenesis reactions for intact plants, which lead to poor root system development, stem elongation, formation of small leaves with a thin leaf blade, overall low productivity and low adaptation potential of G. lutea plants to ex vitro and in situ conditions. Increasing the light flux intensity to 44 W/m2 and increasing the red wave proportion up to 20.3% allows not only to improve the bioproductivity of G. lutea plants which are cultivated in vitro, but also to increase the coefficient of microclonal reproduction without the additional use of exogenous growth regulators. The greatest growth of the aboveground and underground parts, increase in effective leaf surface are observed in vitro plants during cultivation at 135 W/m2 light flux intensity and spectral composition Eb: Eg: Er = 29.5%: 32.5%: 38.0%. Conclusions. It is possible to improve plant bioproductivity by changing the light conditions of plant cultivation in vitro, and, accordingly, to increase the adaptive potential to ex vitro and in situ conditions. Keywords: Gentiana lutea L., in vitro plants, light flux intensity, spectral composition, growth parameters.


Author(s):  
Gisela Manuela de França Bettencourt ◽  
Juliana Degenhardt ◽  
Germana Davila dos Santos ◽  
Vânia Aparecida Vicente ◽  
Carlos Ricardo Soccol

Author(s):  
M. Nieto-Soriano ◽  
María E. Galindo-Tovar ◽  
Miriam C. Pastelín Solano ◽  
Luis A. Solano Rodríguez ◽  
Otto R. Leyva Ovalle ◽  
...  

Objective: To identify the sex of in vitro plants of papaya (Carica papaya L.) MSXJhybrid obtained via somatic organogenesis, through SCAR type molecular markers. Design/Methodology/Approach: Eight-month old MSXJ papaya hybrid plants in thefructification stage were collected in Cotaxtla, Veracruz, Mexico. They weresuperficially disinfected with abundant running water, detergent during 30 min, andthen alcohol at 70% was added for one minute, commercial chlorine at 30% for 30min, and they were rinsed with sterile distilled water; then the meristems werecultivated in MS medium and after 30 d a subculture was made. The DNA extractionwas made with the CTAB method, and the DNA PCR was done with the Deputy et al.(2002) method, and the primers T1, T12 and W11 were used.Results: The T1 primer was the positive control and the T12 and W11 primersallowed the amplification of fragments that identify hermaphrodite, feminine and maleplants, while the T12 and W11 primers were specific for hermaphrodite plants.Study Limitations/Implications: It is required to standardize the method for it to beinexpensive.Findings/Conclusions: The sexuality of papaya plants can be differentiated until thestage of flowering, which is why the implementation of molecular markers wouldfacilitate plant selection if it is implemented at a large scale. Costs, maintenance timeand elimination of plants of unwanted sex are reduced this way.


Author(s):  
Dulce M. Ramírez-Hernández ◽  
Odón Castañeda-Castro ◽  
María Elena Galindo-Tovar ◽  
Luis A. Solano Rodríguez ◽  
Otto R. Leyva Ovalle ◽  
...  

Objective: To analyze the genetic uniformity of MSXJ hybrid papaya in vitro plants, obtained by direct organogenesis.Design/Methodology/Approach: The MSXJ papaya hybrid demonstrates quality characteristics for the national and exports market. In vitro culture of plant tissues represents a useful tool for their multiplication and conservation, but somaclonal variation can diminish their genetic and agronomic uniformity. In order to analyze the genetic uniformity of in vitro plants of this hybrid, ten ISSR primers were used for in vitro plants micropropagated during nine subcultures. DNA was extracted using the CTAB method. Data were analyzed using the program PopGene v 1.3.1.Results: Eighty-five loci of 200 to up to 2000 pb were generated, with 37 polymorphic loci. In the cluster analysis, three groups were observed which separate subculture one, subcultures two to eight, and subculture nine; the Gst value of 0.87 indicated genetic uniformity as far as subculture eight.Study Limitations/Implications: Papaya is one of the most important tropical fruits worldwide; however, these plants need to be healthy and genetically uniform to guarantee commercial success. In vitro propagation allows obtaining healthy and uniform plants, but it is necessary to study genetic uniformity during their micropropagation.Findings/Conclusions: The in vitro multiplication of the MSXJ papaya hybrid permitted the regeneration of vigorous plants in 30 d. Molecular profiles indicate that as far as subculture eight, there is genetic uniformity. As such, no more thaneight subcultures are recommended during micropropagation.


2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Tran Tu Khoa ◽  
Pham Minh Duy ◽  
Tran Thi Huong ◽  
Nguyen Thi Quynh

The Indian leadwort (Plumbago indica L.) of the family Plumbaginaceae is a plant with high pharmaceutical value, as it contains plumbagin, a naphthoquinone with antibacterial, antifungal and anticancer properties. Among the propagation methods for the Indian leadwort, in vitro propagation is considered an effective method in producing disease-free transplants in a short period of time with high propagation rate. When plants grown in vitro are transferred to ex vitro condition, the environmental factors in the nursery house such as light, temperature, humidity and microorganism in the soil will affect their growth. Characteristics of transplants themselves is also critical for the subsequent growth. It is, thus, essential to establish the standards to evaluate and qualify in vitro plants for transplanting to ex vitro condition. Among these standards, the culture age of in vitro plants affects the maturations of their root, stem and leaves, which can in turn influence the acclimating ability and growth of in vitro plants after transplantation. The purpose of this study is to investigate the effects of the culture age of in vitro Indian leadwort plants on their performance during ex vitro stage. For this purpose, three different culture ages of uniform in vitro plants, 35, 42 and 49 day-old, were studied. After 28 days of cultivation in the nursery house under the light intensity of 70 ± 10 µmol m-2 s-1, temperature of 35 ± 4 oC and relative humidity (RH) of 60 ± 10%, all three treatments achieved 100% survival rate. Increased fresh and dry weights and percentage of dry matter after cultivation in ex vitro condition were not statistically different between 42 day-old and 49 day-old in vitro plants, but were significantly different between these plants and 35 day-old in vitro plants. The development of shoot and root in ex vitro stage of 42 day-old and 49 day-old in vitro plants was more balanced, as shown by the higher ratio of shoot/root dry weight, than 35 day-old in vitro plants. The results of this study showed that for this Plumbago species, bigger in vitro plants led to better growth during ex vitro stage. These results also indicated that it was possible to transfer in vitro Plumbago plants to ex vitro condition after 5 weeks of in vitro culture stage.  


Bionatura ◽  
2021 ◽  
Vol 6 (1) ◽  
pp. 1462-1465
Author(s):  
Liliana Villao ◽  
José Flores ◽  
Efrén Santos-Ordóñez

Bananas and plantains (Musa spp.) are among the most critical socioeconomic crops globally, being a staple food for millions of people in the tropics and an essential component for the export market, including the subtropics. Besides conventional breeding, genetic improvement of bananas and plantains could be performed through genetic engineering and new breeding techniques. Furthermore, plant tissue culture is essential for these technologies, including developing embryogenic cell suspensions and in vitro plants. The transient and stable genetic transformation could be performed from in vitro plants, shortening Musa transgenic lines development compared to genetic transformation while using embryogenic cell suspension. In this study, a genetic transformation protocol was established from banana apical meristems for the ‘Williams’ cultivar (genotype AAA). The protocol was based on the co-cultivation of the explants (whole in vitro plants or bisected meristematic tissues derived from in vitro plants) with Agrobacterium tumefaciens strain LBA4404 harboring two binary vectors denominated pLVCIBE1 (cassette: MabHIPP promoter::luc2::Tnos, P35S::hpt::Tnos) and pLVCIBE2 (cassette: P35S::luc2::Tnos, P35S::hpt::Tnos), independently. The stable genetic transformation was obtained by subculturing in vitro banana plants in selection medium (12.5µg/mL of hygromycin) for 8 weeks from bisected meristematic tissue transformation. Genetic transformation was confirmed in vivo with the use of the luciferase reporter gene system. Furthermore, PCR was performed on DNA extracted from leaves of regenerated transgenic in vitro plants after 8 weeks of selection, confirming stable genetic transformation. Therefore, genetic transformation was achieved in the apical meristematic tissue of in vitro banana plants with co-cultivation of Agrobacterium tumefaciens.


2020 ◽  
Vol 26 ◽  
pp. 183-189
Author(s):  
L. R. Hrytsak ◽  
N. M. Drobyk

Aim. To analyze the experience of Ukrainian and foreign scientists on technologies to increase the adaptive potential of cultivated in vitro plants to ex vitro conditions. Results. Modern acclimatization technologies are mainly aimed at improving the methods of adaptation of planting material of in vitro collections to ex vitro conditions. Much less attention is paid to technologies to increase plant resilience at the stage of their multiplication and growth in vitro. Integration and systematization of research results of a large number of scientists is allowed to describe the main strategies and methodological techniques, which implementation can significantly increase the adaptive potential of in vitro plants. Conclusions. Optimization of physical and chemical conditions of plant cultivation in vitro can induce changes in their phenotype, intensity of photosynthetic reactions, water balance, which increases the adaptive potential of plants and facilitates the process of their acclimatization to ex vitro conditions. Key words: in vitro plants, acclimatization to ex vitro conditions, adaptive potential, technology.


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