Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers (original) (raw)
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The present investigations were made attempting to develop a rapid, reliable, and reproducible in vitro regeneration protocol for Artemisia absinthium L., a medicinal plant of Kashmir Himalayas. Out of several auxin-cytokinin combinations tested, Murashige and Skoog's (MS) medium supplemented with 0.5 mgL −1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mgL −1 kinetin (Kn) was found to be the best for the callus induction. On the other hand, 4.5 mgL −1 6-benzylaminopurine (BAP) and 0.5 mgL −1 1-naphthaleneacetic acid (NAA) in the medium resulted in maximum shoot induction from the callus. Similarly, BAP and NAA at a concentration of 1.5 mgL −1 and 0.5 mgL −1 , respectively, proved to be the best for the multiple shoot induction from nodal explants. Numerous shoots were obtained from nodal explants after third subculture. In vitro rooting was maximum on medium containing indole-3-butyric acid (IBA) at 0.5 mgL −1. The genetic stability of the in vitro raised plants of Artemisia absinthium was assessed using the intersimple sequence repeat (ISSR) and sequence-specific amplification polymorphism (SSAP) molecular markers. Both markers were able to detect the somaclonal variations in the callus regenerated plants, while no variation was detected in the plants regenerated from the nodal explants. SSAP has been found to be more useful in detection of variability as compared to ISSR molecular marker. The results of present study concluded that the direct regeneration protocol will be useful for the production of true to type plants of this medicinally important plant. This will go a long way in reducing the pressure on the natural populations for the secondary metabolite production, especially for extraction of essential oils.
Botanical Studies, 2013
Background Aloe vera (L.) Burm.f is an important industrial crop, which has enormous application in pharmaceutical, cosmetic and food industries. Thereby, the demand for quality planting material of A. vera is increasing worldwide. Micropropagation is the widely accepted practical application of plant biotechnology that has gained the status of a multibillion-dollar industry throughout the world and this techniques can be used to meet the industrial demand of A. vera. Present studies aim to develop a proficient methods of high-frequency true-to-type plantlet regeneration without intermediate callus phase for A. vera. Results Nodal portion of rhizomatous stem of A. vera were cultured on Murashige and Skoog (MS) medium (Physiol. Plant. 15:473 – 497, 1962) supplemented with various cytokinin and A. vera leaf gel (AvG) as organic supplement. Number of proliferated shoots per explant was increased along with the regeneration cycles and on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine and 10.0% (v/v) AvG, only 17.8 ± 0.35 shoots per explant were induced on 1st regeneration cycle whereas on 3rd regeneration cycle these number increase to 38.5 ± 0.44 shoots per explant on the same medium composition. AvG have an encouraging role to increase the proliferation rate and on 3rd regeneration cycle 27.6 ± 0.53 shoot per explant induced on 2.5 mg/L BAP, but these number increase to 38.5 ± 0.44 shoots per explant when 10.0% (v/v) AvG was added along with 2.5 mg/L BAP. After transfer of individual excised shoots to a one-third strength MS medium containing 20.0% (v/v) AvG, all the shoots formed whole plantlets with maximum number (9.6 ± 0.29) of roots per shoot. 95.0% of the regenerated plantlets survived on poly-green house. Normal flower appeared in 84.2% field growing micropropagated plants after 18 to 20 months of field transfer. Further, the of clonal fidelity of the two years old micropropagated plants was established by studying mitotic and meiotic chromosomal behavior and also considered the chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic haploid (pollen mitosis) and diploid chromosome count (n = 7; 2n = 14), or meiotic behavior. Randomly amplified polymorphic DNA analyses revealed there were no somaclonal variations among these regenerants. Conclusions These results confirm the very reliable method for large scale production of true-to-type plantlets of A. vera, which can be used for commercial purpose.
Indian Journal of Agricultural Sciences, 2016
Plant regeneration was achieved from axillary bud, callus and somatic embryos of Dianthus caryophyllus cv.Yellow Dot Com from node and leaf explants cultured on Murashige and Skoog (MS) medium supplemented with plant growth regulators. The explant nodal cutting responded well for direct regeneration while leaf explant was selected for callus induction. The highest number of direct adventitious shoots were achieved with 9.08 µM TDZ and 5.37 µM NAA. The maximum callus induction was achieved with 2.06 µM 2, 4-D and 2.68 µM NAA. The calli derived from MS medium supplemented with 5.37 µM NAA and 2.27 µM TDZ were found to have maximum shoot regeneration potential. The highest number of calli producing shoots and average number of shoots per callus was recorded on MS medium supplemented with 9.12 µM zeatin and 5.07 µM IAA. The calli derived from leaf explants cultured on medium containing 2.06 µM 2, 4-D and 2.68 µM NAA were highly friable, had poor regeneration potential and were selected for cell suspension studies. The cultures were allowed to grow into micro-colonies in liquid medium and subsequently into embryogenic calli on semi-solid and solid MS medium which later differentiated into somatic embryos without growth regulators. Plantlets were obtained from in vitro derived shoots/somatic embryos with 60-80% survival after 30 day of transfer to pots. The plants regenerated from axillary buds, callus and somatic embryos were compared with mother plant to assess genetic fidelity using RAPD and 2.94%, 26.47% and 20.58% variation was observed, respectively.
Industrial Crops and Products, 2020
There are several studies about the phytochemical investigations and pharmacological activities of Andrographis echioides (L.), but there is scanty information about its in vitro propagation. Hence the present study is undertaken for in vitro micropropagation of Andrographis echioides (L.) through direct organogenesis. Initially, aseptic leaf explants of in vitro seedlings were excised and inoculated on to plant growth regulators (PGR's) supplemented in the Murashige and Skoog (MS) media. Among the individual and combinations of PGR's tested, the combination of thidiazuron (TDZ) along with indole-3-acetic acid (IAA) at 0.5 and 0.2 mg/L concentrations respectively, was proved to be best for high frequency (24.48 ± 1.54) of direct shoot regeneration of A. echioides. Rooting of in vitro raised shoots with the highest rooting percent of 90.42 was carried on IAA (1.0 mg/L), and indole-3-butyric acid (IBA) (1.0 mg/L) amended MS media. in vitro plantlets of A. echioides were hardened in poly cups possessing a 1:2 ratio of compost and garden soil. Initially, hardened plantlets were maintained in a greenhouse for four weeks by irrigating with half-strength MS salt solution and later transferred to field conditions with 80 % survivability. Genetically clonal nature of regenerated plantlets was confirmed by carrying polymerase chain reaction (PCR) based molecular analysis using inter simple sequence repeats (ISSR) and ribulose bisphosphate carboxylase large chain (rbcLa) primers.
Medicinal and Aromatic Plant Science and Biotechnology, 2007
An efficient protocol was developed for the regeneration of plantlets via direct and indirect organogenesis from stem nodal segments of Naravelia zeylanica (L.) DC. (Ranunculaceae). The interaction of 6-benzyl aminopurine (BAP) with D-naphthalene acetic acid (NAA) in Murashige and Skoog (1962) medium induced a varied morphological response. A maximum of 17.6 ± 1.5 shoots per explant were obtained in the presence of 4.0 mg l-1 BAP and 0.6 mg l-1 NAA on MS medium. A higher concentration of BAP (5.0-8.0 mg l-1) and NAA (1.0-2.0 mg l-1) favoured callogenesis while a lower range (2.0-3.0 mg l-1 BAP and 0.25-5.0 mg l-1 NAA) induced adventitious shoot buds and 11.7 ± 1.3 plantlets were obtained from stem calli. The excised microshoots rooted well on MS basal medium without plant growth regulators. Morphologically direct organogenesis regenerants were similar to in vivo plants while the callus regenerants exhibited abnormal development after acclimatization. The survival rate of the plantlets derived from direct organogenesis was 96% whereas in callus regenerants it declined to 72%. Random Amplified Polymorphic DNA (RAPD) markers were employed to analyze the level of genetic variation among the plantlets of direct and indirect organogenesis. Five groups of Operon primers-B 11-20, C 01-20, D 01-10, E 01-10, and F 11-20-were screened, from which six were selected, among which OPF-17 and OPF-19 gave clear and distinguishable bands. A dendrogram was constructed using Euclidean distances by Ward's method. RAPD marker fingerprinting allowed a rapid assessment of the level of genetic variation among the regenerants.
Plant Cell, Tissue and Organ Culture, 2006
Development of suitable strategy to overcome genotypic limitations of in vitro regeneration in sorghum would help utilize high yielding but poor tissue culture responsive genotypes in genetic manipulation programmes. A factorial experiment was conducted with two explants (immature embryos and inflorescences), eight genotypes (five Sorghum sudanense and three Sorghum bicolor genotypes), three levels of 2,4-D (1 mg l -1 , 3 mg l -1 , and 5 mg l -1 ), and two levels of kinetin (0.0 mg l -1 and 0.5 mg l -1 ). The induced callus was transferred to the regeneration media with factorial combinations of IAA (1.0 mg l -1 and 2.0 mg l -1 ) and kinetin (0.5 mg l -1 and 1.0 mg l -1 ). S. sudanense regenerated at significantly higher frequency (38.91%) and produced shoots more intensely (2.2 shoots/callus) than S. bicolor (26.93%, 1.26 shoots/callus). Immature inflorescences regenerated at a much higher frequency (46.48%) and produced significantly more number of shoots (2.71 shoots/callus) than immature embryos (22.35%, 0.99 shoots/ callus). Moreover, differences for plant regeneration between genotypes of the same species were minimal when using immature inflorescences. Increase in the 2,4-D concentration in callus induction media exhibited inhibitory effect on callus induction, growth, shoot induction and number of shoots/callus but inclusion of kinetin in callus induction media improved these responses. Use of immature inflorescence explant and inclusion of kinetin in callus induction media could overcome genotypic limitations of plant regeneration to a large extent. The extent of variability, heritability and expected genetic advance was more in plant regeneration traits than in callus induction traits. This indicated that the variability in respect of these attributes in the genotypes may be due to the additive gene action and selection of genotypes for these characters would be rewarding.
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 2017
An efficient indirect in vitro plant regeneration protocol for Dianthus caryophyllus L. cv. 'Master' was developed using leaf explants. This study revealed the morphogenetic potential of leaf explant as a source for micropropagation. Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l Naphthalene acetic acid (NAA) resulted in maximum (94.44%) callus induction. MS medium supplemented with 1.5 mg/l thidiazuron, 0.25 mg/l Kinetin and 0.25 mg/l NAA was found to be highest for average shoot regeneration (80.56%), average number of shoots (6.01) and average shoot length (1.93 cm). For in vitro multiplication of shoots, MS medium supplemented with 2.0 mg/l Kinetin and 0.25 mg/l NAA was found to be the best which resulted in 14.64 average number of microshoots. It was observed that the half strength MS basal medium supplemented with 1.5 mg/l Indole-3-butyric acid and 0.02% activated charcoal showed maximum rooting (98.19%) with 9.60 average number of roots per microshoot having 4.24 cm root length. The in vitro rooted plantlets were hardened gradually and were successfully acclimatized under ex vitro conditions. The genetic variation in the in vitro raised plants was confirmed using DNA based markers [Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats] for assessment of genetic stability of plants raised through indirect regeneration.
In vitro plant regeneration from cotyledonary nodes of recombinant inbred lines of lentil
Scientia Horticulturae, 2012
An efficient and reproducible in vitro regeneration protocol for lentil was developed. For shoot regeneration, cotyledonary node explants of ten elite genotypes were cultured in an inverted orientation on different shoot regeneration media that consisted of Murashige and Skoog (MS) medium supplemented with 1 mg L −1 6-benzylaminopurine (BAP) (M1), 1 mg L −1 BAP + 0.45 mg L −1 indole-3-acetic acid (IAA) (M2), and 2 mg L −1 BAP (M3). High percentages of shoot regeneration ranging from 80 to 100% on M1 and M3 media and from 50 to 100% on M2 medium were induced. M1 was the most efficient shoot regeneration medium for most genotypes tested. For rooting, in vitro and in vitro-in vivo methods were used. Low and variable rooting percentages ranging from 0 to 45% were recorded with in vitro-in vivo method. Efficiency of rooting on in vitro medium varied depending on the medium in which shoots had been previously regenerated and the genotype tested. When M1 medium was used, high rooting percentages (over 40%) for most genotypes except for microsperma genotypes were found. When the 10 genotypes were screened for good regeneration performance using M1 medium, 2 main clusters and 3 subgroups within one of the clusters were formed based on similarities respect of the number of regenerated shoots per explant and rooting percentages. Subgroup 1 composed by A1146 genotype produced the highest number of shoots per explant (6.17 shoots) and a high rooting percentage (60%) so was selected for further transformation and use as a potential commercial variety.
In Vitro Cellular & Developmental Biology – Plant, 2016
Simarouba glauca DC. is a multipurpose tree species known for oil, timber, and medicinal properties. The application of biotechnological methods for genetic improvement of this species depends on the availability of an efficient plant regeneration system. In this study, the shoot regeneration potential of various seedling-derived explants was assessed after culturing on Murashige and Skoog (MS) and woody plant (WP) medium containing different growth regulators. The explants differed in their capacity for shoot bud formation and subsequent shoot elongation on the media tested. Shoot bud induction was achieved at a high frequency (44.8-76.2%) from different explants on MS medium with 2 mg L −1 6benzylaminopurine (BAP) as compared to other media tested. Cotyledons exhibited the highest capacity for shoot bud induction (76.2%) and shoot elongation (9.1 elongated shoots per explant). The in vitro-regenerated shoots rooted at a frequency of 66.7% after pulse treatment in 10 mg mL −1 indole-3-butyric acid (IBA) solution for 5 min followed by culture on half-strength WP medium with 0.2 mg L −1 IBA. The regenerated plants were acclimatized and established in the glasshouse with a survival rate of 80%. Molecular characterization of regenerated plants using 14 random amplified polymorphic DNA (RAPD) and 15 intersimple sequence repeat (ISSR) primers revealed a high number of monomorphic bands, with only 1.6-2.6% of the bands being polymorphic. The regeneration system established in the study has the potential to be used for rapid multiplication, conservation, and genetic transformation of this species.