Callus Proliferation Research Articles

Biomechanics of Far Cortical Locking

The development of far cortical locking (FCL) was motivated by a conundrum: locked plating constructs provide inherently rigid stabilization, yet they should facilitate biologic fixation and secondary bone healing that relies on flexible fixation to stimulate callus formation. Recent studies have confirmed that the high stiffness of standard locked plating constructs can suppress interfragmentary motion to a level that is insufficient to reliably promote secondary fracture healing by callus formation. Furthermore, rigid locking screws cause an uneven stress distribution that may lead to stress fracture at the end screw and stress shielding under the plate. This review summarizes four key features of FCL constructs that have been shown to enhance fixation and healing of fractures: flexible fixation, load distribution, progressive stiffening, and parallel interfragmentary motion. Specifically, flexible fixation provided by FCL reduces the stiffness of a locked plating construct by 80% to 88% to actively promote callus proliferation similar to an external fixator. Load is evenly distributed between FCL screws to mitigate stress risers at the end screw. Progressive stiffening occurs by near cortex support of FCL screws and provides additional support under elevated loading. Finally, parallel interfragmentary motion by the S-shaped flexion of FCL screws promotes symmetric callus formation. In combination, these features of FCL constructs have been shown to induce more callus and to yield significantly stronger and more consistent healing compared with standard locked plating constructs. As such, FCL constructs function as true internal fixators by replicating the biomechanical behavior and biologic healing response of external fixators.

In vitro morphogenic events in culture of Lotus corniculatus L. seedling root explants

The experiments were carried out on <em>Lotus corniculatus</em> (L.) seedling root explants of the cultivar varieties Skrzeszowicka, Caroll A10 and strain 175. Callus formation and shoot regeneration were the major explant response depended mainly on of the studied genotype and used plant growth regulators (PGRs). Primary cortex of proximal and distal end of explant was the most active tissue for callus proliferation. For shoot primordia differentiation deeper zones of cortex took a part. The process of meristematic centre initiation was not uniform and various level of shoot differentiation events were observed not earlier than 3 weeks of culture. Usually, the shoot primordia regeneration began on proximal rather than distal end of the explant. BAP rather than urea derivatives stimulated shoot proliferation in extended cultures. Increasing of BAP and TDZ concentrations brought about the explant polarity and expansion of the meristematic zones. The explant position in root did not have significant influence on the number of regenerated shoots. The cultures only had better bud formation by TDZ when compared to BAP. BAP stimulated bud formation and development of the shoots from them. Short term of TDZ treatment of explants stimulated meristem formation which developed into buds and shoots. CPPU stimulated callus proliferation and bud formation when explants pretreatment was prolonged from 12 to 36 hrs.

Somatic embryogenesis of selected coniferous tree species of the genera Picea, Abies and Larix

Experiments on somatic embryogenesis in selected spruce, fir and larch species were performed to determine if this method of micropropagation enables production of quality seedlings for forest nurseries. High frequencies of embryogenic callus in spruce (23-31%) and fir (29%) were achieved when mature zygotic embryos were used as explants, while in larches (36%) only when megagametophytes (endosperms with immature embryos) were used. The possibility of somatic embryogenesis initiation also from somatic embryos (resulting in secondary and third generation of embryogenic callus) indicate high efficiency of this method of micropropagation. The best results at all stages of somatic embryogenesis (good proliferation of embryogenic callus, high rate of embryo regeneration and their survival) were obtained for Norway spruce, European larch and some hybrid larches.

Effects of cytokinin on callus proliferation associated with physiological and biochemical changes in Vitis vinifera L.

The essential role of 6-benzylaminopurine (BA) in plant tissue culture has been widely known; however, physiological and biochemical mechanisms behind BA requirement have not been fully understood yet. BA may have an important role on callus growth by regulating antioxidant enzyme activities and acting as an effective free radical scavenger. To test this hypothesis, the impact of exogenous BA concentrations on antioxidative system in Vitis vinifera L. cv. ‘Bogazkere’ callus was investigated under in vitro conditions. Relative fresh weight growth (RFWG) of calli, total phenolics (TP) content, endogenous hydrogen peroxide (H2O2), malondialdehyde (MDA), proline concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), and guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were measured. Inhibitory effect of high concentrations of BA on antioxidant enzyme activities and RFWG was found. In the presence of BA at 0.1 mg L−1, SOD, POD, and APX activities decreased, while CAT activity increased in comparison with the controls. Exogenous BA treatments higher than 0.1 mg L−1 resulted in an increase in cellular TP, H2O2, MDA, proline contents, and percentage of EL, while RFWG of calli decreased. Based on the findings, it may be concluded that only 0.1 mg L−1 BA concentration combined with NAA could play a direct role in reducing the level of free radicals and phenolic production associated with proliferation capacity of grape cells under in vitro conditions. Furthermore, cytokinin was effective in the antioxidative enzyme system, lipid peroxidation, and electrolyte leakage.

Effect of plant growth regulator, light and media formulation on initiation and growth of Parkia biglobosa (Jacq.) R. Br. Ex Don callus cultures from cotyledons and mature zygotic embryos

Cotyledons from 14-days old seedlings and mature zygotic embryos (MZE) of Parkia biglobosa were cultured on either Murashige and Skoog (1962) medium (MS) or woody plant medium (WPM) supplemented with a range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations and incubated in either 24-hour darkness or 16-hour daylight to induce callus formation. Cotyledon explants exhibited 100% callus formation while MZE gave 66.3%. 0.55mg l-1 2,4-D gave the highest size and weight of callus formed in both explants. WPM favored callogenesis better than did MS, and incubation in the dark promoted callus formation better than a 16-hour light regime. Analysis of variance revealed varying degrees of interaction between the tested factors on callus induction (p< 0.05). Induced calluses were sub-cultured on callus proliferation medium consisting WPM supplemented with 0.55mgl-1 2,4-D and 0.1mg l-1 benzylaminopurine (BAP) singly or in combination for growth. Callus growth after 3 weeks was highest (9.2mg) in media supplemented with 2,4-D only. Results of the present study are novel because callus induction in Parkia biglobosa was quantified in terms of size and weight for the first time. It was also found that WPM may be the medium of choice (rather than MS which has hitherto been used) for initiating callus P. biglobosa callus cultures using 2,4-D. These findings will enhance efficient production of P. biglobosa callus for somatic embryogenesis, callus-mediated regeneration, genetic transformation, and as raw material for secondary metabolite (thioproline) production from cell cultures.Keywords Parkia biglobosa, Leguminosae, callus induction, woody plant medium, plant genetic resources.

Genotypic variation and aging effects on the embryogenic capability of Kalopanax septemlobus

In an attempt to obtain insight into the differential responsiveness of different genotypes of Kalopanax septemlobus regarding their embryogenic capacity, several parameters such as endogenous hormonal levels, DNA content, embryogenic callus proliferation and somatic embryogenesis were studied in several genotypes of this plant. Also, to understand the effect of the age of the explants on their embryogenic capacity, the same parameters were studied in two embryogenic cell lines of different ages in the selected genotype. In the present study, it was observed that the cytokinins/abscisic acid (ABA) ratio plays an important role in embryogenic capacity in the studied genotypes of K. septemlobus species. A decrease in embryogenic capacity of callus was observed with increasing age, along with a marginal decrease in the DNA content of nuclei. Further, it can be suggested from our results that young embryogenic callus is a better choice for somatic embryo formation than the long-term-maintained callus in K. septemlobus.

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11-15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.

IN VITRO CULTURE OF Pogostemon cablin BENTH. (NILAM PLANT): THE EFFECT OF NAA AND BAP ON EMBRYOGENIC CALLUS PROLIFERATION AND SUBSEQUENT SOMATIC EMBRYOGENESIS

An experiment to investigate the somatic embryogenesis from shoot-derived callus of Pogostemon cablin (nilam plant) has been conducted at the Plant Biotechnology Laboratory, Agricultural Faculty, University of Jambi from January through to July 2004. Callus proliferation was induced on explants taken from young shoots cultured on solid MS medium supplemented with phytohormones NAA (0.8, 1.1, 1.4, and 1.7 ppm) and BAP (1.1, 1.4, 1.7, and 2.0 ppm) under in vitro conditions. Cultures were maintained at 25 􀂱 1 oC, light intensity 50 􀁭mol m-2 s-1, and 16 hours photoperiod. The results indicated that all cultured explants showed positive responses on callus proliferation on all treatments within two weeks of culture initiation. The effect of phytohormones, however, was unspecific as all callus showed similar properties, from non-embryogenic to embryogenic. The addition of NAA and/or BAP to the culture medium was not significantly affected the number of days to callus proliferation. Callus fresh weight was significantly affected by NAA (P = 0.01) or BAP (P = 0.05), but the interaction of these phytohormones resulted in a non-significant effect on callus fresh weight (P = 0.18). Also, BAP significantly affected callus dry weight (P =0.03). However, neither NAA nor its interaction with BAP significantly affected callus dry weight (P = 0.07 and 0.16, subsequently). Embryogenic and non-embryogenic callus were subcultured separately onto new fresh media with the same composition as for callus induction. Following this subculture, embryogenic callus regenerated somatic embryos within ten days, whereas non-embryogenic callus did not show any symptom of embryogenesis, and lost their proliferative capacity after six weeks of subculture. The regenerated somatic embryos continued to grow to form profuse mass of young plantlets ready for in vivo acclimatization. Keywords: Pogostemon cablin, nilam, patchouli oil, tissue culture, in vitro culture, NAA, BAP

In vitro hormone-regulated growth and floral induction of Cuscuta reflexa: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscutareflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L−1 benzyl adenine (MMS1c). Supplementation of 2 mg L−1 naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L−1 NAA and 2 mg L−1 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L−1 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.

Micropropagation of Pogostemon cablin Benth. through Direct Regeneration for Production of True to Type Plants

Multiple shoots from nodal explants of Pogostemon cablin Benth. MS supple-mented with 0.5 mg/l BAP initiated a mean of 45.66 shoots/nodal explant. Within four weeks of initiation, regenerated multiple shoots attained a height of 3.6 cm. Subsequent transfer of these in vitro derived nodal segments onto MS supplemented with BAP and Kn ( 0.5 mg/l), induced a mean of 62.45 shoots. Higher concentration of either BAP or Kn more than 0.5 mg/l resulted in callus proliferation and showed hyperhydric shoots with morphological abnormalities. Rooting was readily achieved upon transfer of shoots on half strength MS supplemented with 100 mg/l activated charcoal. Rooted shoots, following acclimatization in greenhouse, were successfully transferred to soil with 91% survival. Also shoots regenerated in vitro were directly transplanted to soil and acclimatized. Tissue cultured plants were analyzed for oil content by employing gas chromatography and found that the patterns were similar to mother plants. This confirms the true to type nature of micropropagated plants. Key words: Micropropagation, Direct regeneration, Pogostemon cablin, organogenesis D.O.I. 10.3329/ptcb.v20i1.5971 Plant Tissue Cult. &amp; Biotech. 20(1): 81-89, 2010 (June)

Plant regeneration from in vitro leaf tissues of Viburnum dentatum L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.

Callus culture of Cyclocarya paliurus (Batal.) Iljinskaja leaves

The hormone combinations and dark culture of the callus induction for Cyclocarya paliurus (Batal.) Iljinskaja leaves were optimized by an orthogonal experiment. The effects of hormones on callus proliferation were also studied. The results show that the optimum medium for callus induction of C. paliurus leaves was MS + 1.0 mg·L−1 6-BA + 0.5 mg·L−1 NAA + 1.0 mg·L−1 2,4-D. The combination of 6-BA and NAA significantly affected the induction of callus, while the effect of 2,4-D was less. The maximum rate of callus induction was 95.4%. A 10-day darkness period was beneficial for callus induction. The best proliferation medium was 1/4 DKW + 0.5 mg·L−1 6-BA + 1.0 mg·L−1 2,4-D.

In vitro regeneration of Persian poppy (Papaver bracteatum)

AbstractPersian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.

Culture of Immature Embryos of Coconut, Cocos nucifera L: Callus Proliferation and Somatic Embryogenesis

The culture of immature embryos of coconut (Cocos nucifera L var. typica) was attemptedwith a view to developing a technique for clonal propagation. Embryos, 6-7 monthspostanthesis produced whitish, compact, embryogenic callus tissues in the presence of 12-20uM 2,4-D. Callus was maintained by subculture on a medium of the same composition.About 50% of the callus cultures produced globular embryos when transferred to 8 pM 2,4-D.Embryos in 22% of these cultures germinated and produced shoots, 6 mm long, when 6-benzylamino purine and kinetin (10 p.M each) were incorporated into the culture medium.The age of the embryo was an important factor determining callus proliferation andsubsequent embryogenesis. More than 50% of embryos excised from 6-7-month-old nutsproduced embryogenic callus tissues. Five to six-month-old nuts were not mature enough forexcising the embryos. Embryos from older nuts (8 months and above) germinated in culture.Only a small portion of the immature embryo produced embryogenic callus tissues. Thefuture root region proliferated into a brownish callus which in turn produced roots profusely.The cotyledon of the immature embryo expanded and developed into a haustorium withoutany sign of callus formation. The shoot apex produced a few plumular leaves but no callusingwas observed.

SOMATIC EMBRYOGENESIS IN MINIATURE POTTED ROSE (ROSA HYBRIDA L.)

Regeneration via somatic embryogenesis was established for the miniature rose cultivar (Rosa hybrida L. 'Linda'). Initially, in vitro cultures of eight miniature rose cultivars were established and then sufficient amounts of callus were produced on MS-media containing high levels of 2,4-D. Embryogenic callus was induced on calli of six cultivars incubated on media containing either zeatin or thidiazuron (TDZ). Although the frequency of somatic embryogenesis in 'Linda' was only 3.3%, further work on transformation was continued with this cultivar, because of promising results on plant regeneration. ABA was used for proliferation of embryogenic callus, and high concentrations of BAP combined with low concentrations of NAA and GA 3 were used for shoot regeneration and elongation. The observed high demand of auxin for callus induction and of BAP for shoot regeneration appeared to be due to low endogenous concentrations of these hormones in miniature roses. Shoots were rooted on 1/2 MS medium supplemented with IAA and IBA and transferred to the greenhouse. Plants flowered after about 10 weeks and were morphologically indistinguishable from the original plant.

In vitro morphogenesis in leaf sheath explants of sugarcane hybrid var. CoS 99259

Leaf sheath explants of sugarcane variety CoS 99259 were inoculated on agar gelled Murashige and Skoog’ (MS) medium containing different auxins (NAA, IBA and 2,4-D) for callus proliferation. Maximum 70.7±8.1% explants showed callus initiation on MS medium containing 3.0 mg/l of 2, 4-D followed by 68.3±5.3% in presence of 4.0 mg/l. At lower concentrations of 2, 4-D (1.0–2.0 mg/l), the frequency of callus induction was significantly low. The frequency of callus formation in presence of NAA and IBA was significantly lower than that obtained in presence of 2,4-D at a particular concentration. The growth of callus was also inferior in presence of NAA and IBA than 2,4-D. The growth of callus was also influenced by the type and concentration of auxin. A good growth of callus was obtained in presence of 3.0–4.0 mg/ l of 2,4-D. For shoot regeneration, callus cultures were transferred on MS medium containing BAP and Kinetin with or without NAA and GA3. Maximum 68.5 ± 4.6% callus cultures showed shoot proliferation on medium containing BAP, Kinetin and NAA (0.5mg/l each) which was significantly higher than the responses obtained on other hormonal treatments tried. The rate of shoot multiplication in terms of number of shoots per culture was also influenced by the growth regulators. Maximum 22.5 ± 1.9 shoots per culture could be counted on medium containing BAP, Kn and NAA (0.5 mg/l each) followed by 16.3 ± 1.2 shoots/culture in presence of BAP, Kn, NAA and GA3 (0.5 mg/l). The frequency of root formation increased significantly with the increasing concentration of NAA from 2.0 to 7.0 mg/l at a particular sucrose concentration. Similarly, an increase in sucrose level from 30 g/l to 50 g/l further enhanced the rooting percentage irrespective of NAA concentration. Maximum (85.5 ± 7.6%) rooting was recorded in presence of 5.0 mg/l NAA in combination with 50 g/l sucrose.

Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain)

An efficient protocol for plant regeneration through somatic embryogenesis was developed for the first time in five autochthonous grapevine cultivars (Treixadura, Torrontés, Mencía, Merenzao and Brancellao) from Galicia (north-western Spain). Improvements of the induction protocol for the cv. Albariño in respect to previously reported data were also made. Media containing NN salts and MS vitamins supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) were effective in inducing somatic embryogenesis. The addition of casein hydrolysate produced the best results for up to four cultivars (Albariño, Treixadura, Merenzao and Brancellao). Somatic embryogenesis was also induced in explants collected during the binucleate pollen microsporogenesis stage (R3 flower stage) of all cultivars with the exception of Treixadura, suggesting that under appropriate conditions explants can display longer windows of competence. Transfer of embryogenic callus to differentiation medium produced callus proliferation and somatic embryo development proliferation by secondary embryogenesis. However, an extensive process of precocious embryo germination was observed reducing the efficiency of secondary embryo proliferation. This situation was overcame by the use of differentiation medium lacking growth regulators (DM1 medium), which allowed reducing precocious germination by half on average and improving embryo proliferation by secondary embryogenesis. Transfer of normally developed, ungerminated isolated embryos to germination medium allowed obtaining very high percentages of embryo germination (up to 97% in Mencía, more than 87% averaging all cultivars). A comparison of plant conversion between precociously and normally germinated embryos showed that precocious germination in differentiation medium reduced plant conversion, even at high rates depending on the cultivar (from 93% to 39% in Brancellao, from 86% to 61% averaging all cultivars).

High-frequency somatic embryogenesis from germinated zygotic embryos of Schisandra chinensis and evaluation of the effects of medium strength, sucrose, GA3, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l−1 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA3), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA3 negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l−1 BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.

In vitro multiplication of Pogostemon cablin Benth. through direct regeneration

An efficient method was developed to initiate multiple shoots from nodal explants of Pogostemon cablin Benth. MS medium supplemented with 0.5 mg/L BA initiated a mean of 45.66 shoots/nodal explant. Within 4 weeks of initiation, regenerated multiple shoots attained a height of 3.6 cm. Subsequent transfer of these in vitro derived nodal segments onto MS medium supplemented with BA and KN ( 0.5 mg/L), induced mean of 62.45 shoots. Higher concentration of either BA or KN more than 0.5 mg/L resulted in callus proliferation and showed hyperhydric shoots with morphological abnormalities. Rooting was readily achieved upon transfer of shoots on half strength MS medium supplemented with 100mg/L activated charcoal. Rooted shoots, following acclimatization in green house, were successfully transferred to soil with 91% survival. Also shoots regenerated in vitro were directly transplanted to soil and acclimatized. Tissue cultured plants were analyzed for oil content by employing gas chromatography and found that the patterns were similar to mother plants.

Effect of Plant Growth Regulators on Callus, Cell Suspension and Cell Line Selection for Flavonoid Production from Pegaga (&lt;i&gt;centella asiatica&lt;/i&gt; L. urban)

Problem statement: Considering pegaga medicinal properties and over-exploitation, the requirement for a tissue culture technique as an alternative production system was crucial. Approach: Investigation of cell suspension culture response to different plant growth regulators (PRGs) for flavonoid production from elite cell line was carried out. Callus cultures were initiated from the leaf explants of Centella asiatica on Murashige and Skoog (MS) medium containing B5 vitamins and 30 g L−1 sucrose supplemented with different concentrations (0.5-2.5 mg L−1) of 2,4-D, NAA, Dicamba, Picloram and IBA supplied singly and in combination with different concentrations (0.5-1.5 mg L−1) of kinetin, BAP and TDZ. Results: Callus induction was observed for all the PGRs tested. The highest callus induction frequency (86.67%) was observed in MS medium containing 2.0 mg L−1 2,4-D while the combination of 2.0 mg L−1 2,4-D and 1 mg L−1 kinetin in MS medium gave the highest biomass yield (0.27 g dry weight culture−1). This combination was also found to be best for callus proliferation for all the accessions investigated. Among the four accessions tested, UPM03 was found to have the highest biomass yield (0.041 g DW culture−1) and hydrolysed flavonoid content (10.75 mg g−1 DW) after the 12th day of culture. The flavonoids present in the four accessions were quercetin, kaempherol, luteolin and rutin based on high performance liquid chromatography (HPLC) analysis. These results indicated that C. asiatica accession UPM03 was the potential elite cell line in mass production of flavonoid, especially luteolin. Coclusions/Recommendations: In the establishment of cell suspension culture, 2 mg L−1 2,4-D and 1 mg L−1 kinetin were the best PGRs in supporting the cell growth and flavonoid production. This is the first report on the use of PRGs on the establishment of cell suspension cultures in flavonoid production of C. asiatica.