Efficient in vitro establishment and multiplication of Picrorhiza kurroa Royle ex Benth through in vitro raised seedlings (original) (raw)
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Propagation of Picrorhiza kurroa Royle ex Benth: An important medicinal plant of Western Himalaya
Journal of Medicinal Plants Research, 2012
This study is aimed at developing propagation methods and ex situ conservation for Picrorhiza kurroa, an endangered medicinal plant of western Himalaya. Regeneration using leaves from mature plant of characterized germplasm is beneficial because the source plant is not damaged. A regeneration protocol was standardized by using leaves from aseptic shoot cultures, raised from ex vitro leaves. Maximum regeneration percent (94.33) and significantly higher shoot number (38.0) was evident in middle portion of leaf at 2.32 µM of kinetin (Kn). Abaxial surface that was in touch with the medium was more responsive as compared to adaxial surface. The time of exposure to thidiazuron (TDZ) was emphasized as 15 days interval, gave the best response in terms of shoot number (42.0). For shoot multiplication, Kn at 2.32 µM was optimum. Microshoots with well developed root system were obtained in MS basal medium after 4 weeks. Incubation of cultures at low temperature (15°C) for ten days enhanced the survival percent under green house conditions and could be correlated with the development of thick cuticle and well differentiated leaf tissues (palisade and spongy parenchyma). Flow cytometric analysis was performed to check the genetic stability of in vitro plantlets. In a parallel study, seed progenies of these germplasm were raised under ex situ conditions. Its reproductive cycle was also studied for successful domestication.
Satureja khuzistanica Jamzad is an important multipurpose medicinal plant in Iran. The essential oil of S. khuzistanica is characterized by high concentration of carvacrol (93%). The seeds of S. khuzistanica are dormant and have a very low germination under normal conditions. This is the first protocol for in vitro seed germination and plantlet regeneration of this plant. Experiments were done to determine the effects of cold stratification (two, four and six weeks), chemical treatments including various levels of gibberellic acid (GA 3 ) (50, 100, 200 and 500 ppm), potassium nitrate (0.2, 0.4 and 0.6%), sulfuric acid (50%) and also the synergistic effect between cold stratification and other factors on seed germination of S. khuzistanica. The most germination percentage was achieved when six weeks stratification was followed by 200 ppm GA 3 . For shoot proliferation, the node explants were cultured on Linsmaier and Skoog (LS) medium supplemented with different concentrations of benzylaminopurine (BAP) and kinetin (Kn) within the range of 0.5 to 2 µM and combinations of indole-3-butyric acid (IBA: 2 and 5 µM) with BAP (2 and 5 µM). Multiple shoots were obtained from the nodal explants, the higher frequency (77%) of shoot formation was observed in the LS that contained BAP (5 µM) in combination with IBA (2 µM). The best condition for rooting were LS medium plus 2.5 µM of IBA. The rooted plants were successfully transferred to garden soil, exhibiting a normal development.
In vitro regeneration of an endangered medicinal plant Picrorhiza scrophulariiflora
Biologia plantarum, 2011
A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants.
2016
Background: Gentiana kurroo is a critically endangered herb which is known as a bitter drug in Indian medicine. The present study was conducted to optimize the production and germination of artificial seeds in G. kurroo for its mass propagation. Methods: This study utilized somatic embryogenesis as a source for the artificial seed production of G. kurroo royle. Morphogenetic capabilities of leaf explants have been optimized on Murashige and Skoog (MS) media containing different concentrations and combination of kinetin (KN)+2,4dichlorophenoxyacetic acid (2,4-D). Upon callus induction, torpedo shaped somatic embryos were selected for artificial seed production. The artificial seed were produced using the different concentrations of sodium alginate and calcium chloride solution followed by germination on MS media supplemented with indole-3-butyric acid (IBA)+KN+gibberrelic acid (GA3). Results: The study revealed that KN (1 mg\L) and 2,4-D (0.5 mg\L) are most appropriate for callus ini...
International Journal of Pharmaceutical Sciences Review and Research
The present study evaluates the effect of plant growth regulators (2, 4-D and Kinetin) and on two types of explants (leaf and node) on induction and growth of friable callus in Rauvolfia serpentina, a commercially important medicinal plant. Friable callus are the most suitable source for cell suspension culture that would enable the production of active principles at the in vitro level. For callusing, explants were obtained from shoots raised in vitro. Percentage response from nodal explant with respect to callus induction was comparatively higher (89.7 %) than that of leaf. While 2, 4-D (2 mg L-1) alone appeared to be effective but with Kinetin (1.6 mg L-1) proved to be the best PGR combination. Callus obtained were whitish to whitish green in color and friable in texture. Proliferation of callus significantly increased at 0.5 mg L-1 followed by 0.25 mg L-1 of 2, 4-D concentration alone. For shoot regeneration, calli were cultured in MS basal media supplemented with 2 mg L-1 BAP. Histological evaluation of paraffin sections of various stages starting from the callus initiation up to shoot regeneration were prepared, to study the developmental pattern of callus formation and shoot organogenesis in R. serpentina.
Digitalis purpurea L. (foxglove; Scrophulariaceae) is an herbaceous medicinally important cardiac glycoside-producing plant. The aim of the present study was to access the seed viability and influence of plant growth regulators on in vitro seed germination and seedling development. The 2,3,5-triphenyl tetrazolium chloride (TTC) test showed that 100% of seeds were viable while a direct germination test in soil and in Petri dishes showed only about 20% germination ability. The surface-sterilized seeds were cultured in vitro on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar and different concentrations (0 to 15.0 μM) of cytokinins (6-benzyladenine-BA; kinetin-Kin and thidiazuron-TDZ) and auxins ((-naphthaleneacetic acid-NAA; indole-3-acetic acid-IAA and 2,4-dichlorophenoxy acetic acid-2,4-D) alone and in combination. Addition of all types and concentrations of cytokinins and auxins stimulated the rate and percentage of seed germination. Significantly higher seed germination (65.5 ± 1.2% and 63.1 ± 3.2%) was observed on MS medium containing 10.0 μM BA and Kin, respectively than control (16.7 ± 3.1%). Addition of 10.0 μM IAA in the MS medium was most effective for significantly highest (81.0 ± 3.1%) germination percentage. This was evident by significantly higher germination speed (GS; 2.70 ± 0.1), germination value (GV; 31.3 ± 2.4) and vigor index (VI; 259.1 ± 10.1) on MS medium fortified with 10.0 μM IAA as compared with control (GS: 0.56 ± 0.1; GV: 01.4 ± 0.5 and VI: 50.0 ± 09.4). Addition of cytokinins and auxins to the culture medium significantly increased the growth of seedlings. The protocol developed in the present study can be used for large-scale seedling formation and biomass production of this important medicinal plant. It also used to obtain sterile and uniform starting material for various in vitro studies for the improvement of this plant.
American Journal of Plant Sciences, 2015
G. lotoides L. is a threatened plant that is frequently harvested for medicinal purpose. However, its distribution in the world is limited because of short period of seed viability and poor seed germination. The objective of this study was to develop in vitro propagation protocol for G. lotoides through callus induction. For callus induction, different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid) and BAP (6-benzyl amino purine) were used. Seeds were sown on growth regulator-free MS medium and shoots from the in vitro germinated seedlings were excised and cultured on MS medium containing 0.5 mg/l BAP. Young leaves from these shoots were used as explant for callus induction and shoot regeneration. Maximum callus induction (100%) was observed on medium containing 2,4-D (0.5, 2.0, 3.5 mg/l) or NAA (2.0, 2.5 mg/l) in combination with 0.5 mg/l BAP. However, 2,4-D was the best in overall callus induction. The highest regeneration (20%) frequency was achieved on the medium containing 0.5 mg/l BAP. Highest number of shoot (2.83 ± 1.22) and shoot length (2.16 ± 0.87 cm) per explant were obtained in the presence of 0.25 mg/l BAP + 0.5 mg/l KIN (Kinetin). In shoot multiplication media, maximum mean (6.43 ± 0.87) of shoot was observed on MS medium containing 0.5 mg/l BAP. The best shoot length (1.70 ± 0.14 cm) was recorded on control medium. The highest (95%), maximum root number (14.10 ± 1.47) and root length (1.01 ± 0.10 cm) were obtained on a medium supplemented with 1.5 mg/l Indole-3-butyric acid (IBA). All the plants (100%) were survived after acclimatization in greenhouse. The present study can be useful for callus induction and indirect shoot regeneration form G. lotoides.
Orthosiphun stamineus is a herbaceous plant that is popularly known as Misai Kucing. It is widely used in traditional medicine as diuretic agent. This study was divided into two parts that was the in vitro production of complete plantlet through axillary branching and callus culture derived from leaf explant. In axillary branching method, sterilization was conducted using 0.02mg/100ml of mercuric chloride followed by rinsing with 20% and 50% of Clorox for 20 minutes and 5 minutes respectively. This sterilization method was able to remove the contaminants from the surface of the axillary stem and almost 70% of the explants were survived. Axillary bud was placed on Murashige and Skoog (MS) basic medium and cultured for 1 month. The in vitro shoot was inoculated on MS medium which was supplemented with different concentrations of BAP and NAA. The medium that contained 1.0mg/L of BAP gave the best shoot multiplication (13.25) and shoot length (6.23cm) after 8 weeks in culture. Root formation in term of percentage of root (70%) and the number of root produced (10.50) were the best when shoot inserted into medium contained 6mg/L IBA after 3 weeks in culture. However, MS medium that was supplemented with 2 mg/L IBA enhanced in the root length (3.85 cm). Meanwhile, in callus culture, the leaf explant was placed on MS medium containing with various concentrations of 2,4-D for induction of callus. The optimum level of callus induction and proliferation rate (0.42) were obtained with 4mg/L 2,4-D. The callus cells were tested in medium with Evan’s Blue staining and the result showed that the cells were embryogenic. However, the shoot induction from the callus was failed in all tested mediums containing different combinations of BAP and 2,4-D.
In Vitro Germination and Direct Shoot Induction of Yeheb (Cordeauxia Edulis Hemsl.)
Agriculture Forestry and Fisheries, 2014
'Yeheb' (Cordeauxia edulis Hemsl) is a multipurpose and evergreen shrub and endemic to southeastern corner of Ethiopia and Somalia. It is adapted to low and irregular rainfall and survives a very long dry season. It has enormous economic and food security role to the pastoralist of Somali in Ethiopia. However, the plant is threatened with extinction due to overexploitation and its' poor natural regeneration capacity. In addition, 'yeheb' is usually reported having limited reproductive capacities and often have very specific and limited conditions for seed germination, flowering and seed shelf life. Therefore, to overcome these propagation challenges, an experiment was conducted with the aim of developing a protocol for the in vitro regeneration of 'yeheb' from cotyledonary node. The result of these studies revealed that seed was washed by 5% sodium hypochlorite for ten min in aseptic condition found to be more effective in surface sterilization. The sterilized seed cultured on half strength of Gamborg (B5) medium was found to be the most suitable medium for germination (26.67%).The highest shoot initiation percentage (89 % of explants produces shoots), number of shoots per explant and number of leaf per shoot were obtained from cotyledonary node explants cultured on Murashige and Skoog (MS) media supplemented with 2.00 mg. l-1 N 6-benzylaminopurine (BAP) within nine weeks. While, the highest shoot length and shoot fresh weight were recorded from control (free BAP) and 6.00 mg. l-1 BAP, respectively. The highest shoot multiplication (4.56 number of shoot induced) and elongation (2.97cm) were obtained from the induced shoot were cut and placed on MS media supplemented with 2.00 mg. l-1 BAP+6.00 mg. l-1 of gibberellic acid (GA 3) and free BAP+6.00 mg. l-1 of GA 3 , respectively. The elongated shoots were transferred to different media supplemented with various types and levels of hormones but none of them induced root. As a conclusion, this is the first attempt for direct in vitro regeneration of C. edulis and permissible result for cryopreservation.
An elite genotype of Picrorhiza scrophulariiflora Pennell was multiplied in vitro for its conservation. Rhizomes of mature plants collected from various locations of the eastern Himalayan region of Indo-China border were characterized morphologically and analyzed by HPLC to determine the content of marker compounds, namely picroside I and II. Amidst the genotypes, one from Ha, Bhutan was found to contain the highest amount of total picroside (7.33% dw). Subsequently, a rapid and highly reproducible method of micropropagation from rhizome or shoot tips was developed. While 100% bud break from rhizomes was achieved on Woody Plant Medium (WPM) containing 0.44 PM BAP (6 benzyl amino purine), 40-fold multiplication was achieved on WPM fortified with 2.3 PM Kn (kinetin) within 12 weeks. The multiplied shoots were elongated on WPM supplemented with 0.44 PM BAP. Around 90% of in vitro shoots were rooted without basal callus formation on WPM supplemented with 5.3 PM NAA (Į-naphthalene acetic acid) within 4 weeks. Following this protocol, 1100 micropropagated plantlets of an elite line (Ha, Bhutan) were hardened in their natural habitat. The present study illustrates the usefulness of additives for mass propagation and germplasm conservation and is, to the best of our knowledge, the first report of in vitro propagation of P. scrophulariiflora.