An efficient cryopreservation protocol was established for embryogenic cell suspension cultures o... more An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cultures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium containing 4.52 μM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pretreatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryogenic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 μM 6-benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology.
A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rootin... more A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m−3). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET1 and ET2) comparatively to control and other (ET3, ET4 and ET5) lines. Calli of ET1 line showed high regeneration potential on MS medium with N 6-benzylaminopurine (BAP; 1.75 g m−3). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA3; 0.4 g m−3). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m−3).
Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It... more Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.
An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don... more An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l−1) and BA (1.5 mg l−1). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.
In the present study, the regeneration pathway, especially the different events of somatic embryo... more In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.
Garlic extract contains alliin (S-allyl-l-cysteine sulfoxide), an organosulphur compound that con... more Garlic extract contains alliin (S-allyl-l-cysteine sulfoxide), an organosulphur compound that contributes to its therapeutic value and pharmacological importance. Alliin production in plant organs (leaf and root), plantlet, callus (non-embryogenic and embryogenic), embryo (proliferated, matured and germinated embryos), grown under in-vitro conditions was evaluated after 8 weeks. Highest alliin content was recorded in leaves. Clove callus and germinated embryos also showed alliin production. Evaluation of alliin content of in-vitro grown tissues both in normal (control) and sulphur supplemented conditions (4, 8, 16, 32 mg l−1) showed that sulphur treatment at supply of 16 mg l−1 gypsum (CaSO4) significantly enhanced the production of alliin content in all in-vitro grown tissues and organs. These findings suggest that production of alliin is dependent upon type and age of tissue and can be altered by growth conditions.
Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. Th... more Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. The process of embryogenesis has been categorized into a few distinct stages (induction, proliferation, maturation and germination) in which liquid overlaying at varying levels 0 ml (T0), 0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and 1.0 ml (T4) was applied on solid medium. It was found that liquid overlaying improved proliferation; maturation, germination of embryos in C. roseus. In proliferation stage, particularly in T2, torpedo embryo number increased significantly (i.e. 129.6%) as compared to control. Liquid overlaying (T2, T3 and T4) also improved embryo maturation and showed early germination even in maturation medium. It also accelerated normal embryo germination frequency particularly in treatment with T2 and shortened ‘embryo—plantlet’ recovery time. Biochemical analyses revealed more proline, protein and amino acid with increasing level of liquid overlaying as it improved embryo induction, development and faster germination.
Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot deriv... more Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.
In the present study an efficient somatic embryogenesis method has been developed in Catharanthus... more In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l−1). However, only NAA (1.0 mg l−1) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l−1) added medium following a treatment with gibberellic acid (1.0 mg l−1) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.
The therapeutic potency of garlic leaf extract obtained from normal and sulphur treated plants wa... more The therapeutic potency of garlic leaf extract obtained from normal and sulphur treated plants was compared. Alliin, the active compound of garlic leaf extract showed 32% increase in yield under sulphur treated conditions. Alliin obtained from leaf extract of plants brought a significant reduction in serum glucose, triglycerides, total lipids, total cholesterol, LDL- and VLDL-cholesterol levels than glibenclamide in alloxan-induced diabetic rats. Alliin from sulphur treated plants was more effective in comparison to that obtained from plants raised in normal conditions. Serum glucose levels showed significant reduction of 50% in rats administered with leaf extract from sulphur treated plants in comparison to 37% noted in rats administered with leaf extract from normal plants. No alteration in HDL-cholesterol was noted. Similarly, alliin obtained from leaf extract of plants lowered the serum enzyme (ALP, AST and ALT) levels towards normal than glibenclamide. The reduction in serum enzyme levels was significant in rats administered with leaf extract of plants raised under sulphur treated conditions in comparison to that raised under normal conditions. The present findings suggest that leaf extract from sulphur treated garlic possess more antidiabetic potential and hence show more therapeutic potency in comparison to extract obtained from normal plants.
in Vitro Cellular & Developmental Biology-plant, 2010
Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anti... more Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.
Catharanthus roseus is an important medicinal plant that contains two well-known anticancerous al... more Catharanthus roseus is an important medicinal plant that contains two well-known anticancerous alkaloids, vincristine and vinblastine. Cell culture technology has been employed for a long time to improve the alkaloid yield. In this chapter, various processes of somatic embryogenesis such as embryo induction, proliferation, maturation and germination are described. In this embryogenic system, embryos showed irregularities in structure and registered poor conversion frequency. Several carbon sources were added in order to improve the embryo quality before germination: 3% fructose or 3–6% maltose were found to be effective during maturation. Plantlet conversion was high on 3–6% maltose and 3% fructose. In addition, suspension culture, indirect embryogenesis and loss of embryogenic potential with time are discussed in brief. The authors felt that the low yields of vincristine and vinblastine may be improved if the single cell embryo origin concept is utilized in a genetic modification program.
We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through... more We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.
High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Sko... more High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm−3 benzylaminopurine and 1.5 mg dm−3 kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm−3 indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.
Genotoxic studies with reference to pollution biomonitoring is a recent approach and can be used ... more Genotoxic studies with reference to pollution biomonitoring is a recent approach and can be used for maintenance of crop productivity. Effect of the cadmium chloride in the pure germ line of broad bean (Vicia faba L.) were evaluated in relation to the chromosomal abnormalities and rate of cell division. Seeds grown in the nutrient medium for 48 hrs containing different concentrations of cadmium chloride (control, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM) showed different genotoxic effects such as polyploidy, multipolarity, chromosomal bridge with fragments, lagging chromosome and micronuclei. Relative division rate (RDR) was decreased with increasing cadmium concentration while relative abnormality rate (RAR) increased suggesting that cadmium may cause genetic changes in pure germ line and produce irrepairable damage in root meristem. Therefore, combining plant bioassays and root analysis of Cd++ content would allow us to define phytotoxicity thresholds in plants.
Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (... more Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm−3 of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA3, 1.0 mg dm− 3) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm−3 BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm−3 BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.
An efficient cryopreservation protocol was established for embryogenic cell suspension cultures o... more An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cultures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium containing 4.52 μM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pretreatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryogenic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 μM 6-benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology.
A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rootin... more A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m−3). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET1 and ET2) comparatively to control and other (ET3, ET4 and ET5) lines. Calli of ET1 line showed high regeneration potential on MS medium with N 6-benzylaminopurine (BAP; 1.75 g m−3). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA3; 0.4 g m−3). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m−3).
Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It... more Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.
An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don... more An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l−1) and BA (1.5 mg l−1). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.
In the present study, the regeneration pathway, especially the different events of somatic embryo... more In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.
Garlic extract contains alliin (S-allyl-l-cysteine sulfoxide), an organosulphur compound that con... more Garlic extract contains alliin (S-allyl-l-cysteine sulfoxide), an organosulphur compound that contributes to its therapeutic value and pharmacological importance. Alliin production in plant organs (leaf and root), plantlet, callus (non-embryogenic and embryogenic), embryo (proliferated, matured and germinated embryos), grown under in-vitro conditions was evaluated after 8 weeks. Highest alliin content was recorded in leaves. Clove callus and germinated embryos also showed alliin production. Evaluation of alliin content of in-vitro grown tissues both in normal (control) and sulphur supplemented conditions (4, 8, 16, 32 mg l−1) showed that sulphur treatment at supply of 16 mg l−1 gypsum (CaSO4) significantly enhanced the production of alliin content in all in-vitro grown tissues and organs. These findings suggest that production of alliin is dependent upon type and age of tissue and can be altered by growth conditions.
Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. Th... more Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. The process of embryogenesis has been categorized into a few distinct stages (induction, proliferation, maturation and germination) in which liquid overlaying at varying levels 0 ml (T0), 0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and 1.0 ml (T4) was applied on solid medium. It was found that liquid overlaying improved proliferation; maturation, germination of embryos in C. roseus. In proliferation stage, particularly in T2, torpedo embryo number increased significantly (i.e. 129.6%) as compared to control. Liquid overlaying (T2, T3 and T4) also improved embryo maturation and showed early germination even in maturation medium. It also accelerated normal embryo germination frequency particularly in treatment with T2 and shortened ‘embryo—plantlet’ recovery time. Biochemical analyses revealed more proline, protein and amino acid with increasing level of liquid overlaying as it improved embryo induction, development and faster germination.
Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot deriv... more Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.
In the present study an efficient somatic embryogenesis method has been developed in Catharanthus... more In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l−1). However, only NAA (1.0 mg l−1) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l−1) added medium following a treatment with gibberellic acid (1.0 mg l−1) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.
The therapeutic potency of garlic leaf extract obtained from normal and sulphur treated plants wa... more The therapeutic potency of garlic leaf extract obtained from normal and sulphur treated plants was compared. Alliin, the active compound of garlic leaf extract showed 32% increase in yield under sulphur treated conditions. Alliin obtained from leaf extract of plants brought a significant reduction in serum glucose, triglycerides, total lipids, total cholesterol, LDL- and VLDL-cholesterol levels than glibenclamide in alloxan-induced diabetic rats. Alliin from sulphur treated plants was more effective in comparison to that obtained from plants raised in normal conditions. Serum glucose levels showed significant reduction of 50% in rats administered with leaf extract from sulphur treated plants in comparison to 37% noted in rats administered with leaf extract from normal plants. No alteration in HDL-cholesterol was noted. Similarly, alliin obtained from leaf extract of plants lowered the serum enzyme (ALP, AST and ALT) levels towards normal than glibenclamide. The reduction in serum enzyme levels was significant in rats administered with leaf extract of plants raised under sulphur treated conditions in comparison to that raised under normal conditions. The present findings suggest that leaf extract from sulphur treated garlic possess more antidiabetic potential and hence show more therapeutic potency in comparison to extract obtained from normal plants.
in Vitro Cellular & Developmental Biology-plant, 2010
Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anti... more Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.
Catharanthus roseus is an important medicinal plant that contains two well-known anticancerous al... more Catharanthus roseus is an important medicinal plant that contains two well-known anticancerous alkaloids, vincristine and vinblastine. Cell culture technology has been employed for a long time to improve the alkaloid yield. In this chapter, various processes of somatic embryogenesis such as embryo induction, proliferation, maturation and germination are described. In this embryogenic system, embryos showed irregularities in structure and registered poor conversion frequency. Several carbon sources were added in order to improve the embryo quality before germination: 3% fructose or 3–6% maltose were found to be effective during maturation. Plantlet conversion was high on 3–6% maltose and 3% fructose. In addition, suspension culture, indirect embryogenesis and loss of embryogenic potential with time are discussed in brief. The authors felt that the low yields of vincristine and vinblastine may be improved if the single cell embryo origin concept is utilized in a genetic modification program.
We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through... more We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.
High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Sko... more High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm−3 benzylaminopurine and 1.5 mg dm−3 kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm−3 indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.
Genotoxic studies with reference to pollution biomonitoring is a recent approach and can be used ... more Genotoxic studies with reference to pollution biomonitoring is a recent approach and can be used for maintenance of crop productivity. Effect of the cadmium chloride in the pure germ line of broad bean (Vicia faba L.) were evaluated in relation to the chromosomal abnormalities and rate of cell division. Seeds grown in the nutrient medium for 48 hrs containing different concentrations of cadmium chloride (control, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM) showed different genotoxic effects such as polyploidy, multipolarity, chromosomal bridge with fragments, lagging chromosome and micronuclei. Relative division rate (RDR) was decreased with increasing cadmium concentration while relative abnormality rate (RAR) increased suggesting that cadmium may cause genetic changes in pure germ line and produce irrepairable damage in root meristem. Therefore, combining plant bioassays and root analysis of Cd++ content would allow us to define phytotoxicity thresholds in plants.
Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (... more Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm−3 of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA3, 1.0 mg dm− 3) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm−3 BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm−3 BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.
Uploads