Maximum Callus Research Articles

Comparative Efficiency Analysis of Different Explants and Contribution of Polyamines on in vitro Propagation of Dendrobium Hybrid Sonia

An efficient method of plant regeneration and subsequent growth and development of Dendrobium hybrid Sonia was developed using intact seedling, shoot tip and thin cell layer. Maximum number of PLBs was obtained in MS liquid medium supplemented with 0.5 mg/l NAA and 1mg/l BAP through shoot tip and thin cell layer culture (TCL). Highest number of adventitious shoot formation was recorded in 0.5 mg/l of NAA and 2 mg/l of BAP through shoot tip culture. In shoot tip and TCL cultures, the necrosis was checked in presence of NAA (0.5 mg/l) and BAP (1 mg/l). Maximum callus frequency was recorded in NAA and BAP (0.5 mg/l) through thin cell layer culture. Direct PLB formation was better at 1.0 μM concentration in all the three polyamines tested. Three polyamines tested were effective in direct PLB formation as well as adventitious shoots.
 Plant Tissue Cult. & Biotech. 30(1): 77-86, 2020 (June)

Qualitative and Quantitative Evaluation of Active Constituents in Callus of Lavandula angustifolia plant in Vitro

This study was conducted to describe a protocol for the callus establishing culture of Lavandula angustifolia plant and estimating their content of volatile oil. The quantity of volatile oil callus tissues was compared with that of leaves production. Callus was induced from leaf explants on Murashige and Skoog medium (MS) supplemented with Naphthalene acetic acid (NAA) and Benzyl adenine (BA) in different concentrations. Maximum callus fresh weight was obtained in the combination of 10 mg/L BA and 3 mg/L NAA which reached 18 g after four weeks. The results of this work showed that the quantity of volatile oil from the highest fresh weight callus was 6 ml compared with quantity of 18g of leaves which gave 0.5 ml. Volatile oil of leaf and callus extracts were analyzed using gas chromatography mass spectrometry method (GC-MS) which showed linoleic acid (56.61%) and oleic acid (57.93%) as main components.

MICROPROPAGATION OF AN ENDANGERED AND ENDEMIC MEDICINAL PLANT CAYRATIA PEDATA VAR. GLABRA

Objective: The objective of the present study was to develop standardization protocol for the successful in vitro mass propagation of Cayratia pedata var. glabra through leaf and stem explants, since it is a rare, endangered, and endemic medicinal plant using biotechnological involvements and to conserve this endangered species.
 Methods: The application of biotechnological principles for the establishment of micropropagation under in vitro conditions has been studied by following the methods. The explants, namely, leaf and stem harvested from in vivo plants were thoroughly washed and properly sterilized with sterilients. The explants were transferred to Murashige and Skoog (MS) medium supplemented with growth regulators 6-benzyladenine (BAP) and naphthalene acetic acid (NAA) in the concentration range of 0.5–3.0 mg/l which were tested for callus induction and morphogenesis. The elongated shoots were transferred to MS medium supplemented with NAA at different concentrations for root induction.
 Results: The explants collected from the field (shola) were treated in different steriliants with various concentrations at different time for sterilization. Among the various combinations tried, the Teepol treatment for 10 min followed by bavistin 20 min, antibiotics, namely, ampicillin and rifampicin for 20 min, 70% alcohol for 30 s, and 0.12 % HgCl2 for 3 min was found to be effective. The explants were cultured in MS medium supplemented with various concentrations of BAP and NAA. The results noted that an increase in the concentration of BAP concomitantly reduced the frequency of callus formation. The maximum callusing frequency and more number of shoot formation was observed in the lower concentration of BAP (0.5 mg/l) in combination with NAA (0.2 mg/l). The callus obtained from all the above combinations was sub-cultured on MS medium with same combinations of BAP and NAA. The maximum frequency of root formation in leaf callus was 85% and 75% in stem callus and both were achieved on MS medium with NAA (1 mg/l) after 2 weeks.
 Conclusions: The current investigation provides a competent in vitro propagation method for C. pedata var. glabra which could be commercialized for developing identical plants with high-quality mass multiplication rate and for better conservation of the germplasm. Both the methods described here are well suited for the mass multiplication of this critically endangered and endemic climber species.

A Comparative Study of in vivo Plant and in vitro Callus Extracts of Centratherum punctatum Cass

The present study aims at comparative study between plant and callus extract, with respect to analysis of phytochemical constituents, antioxidant, antibacterial activity and cytotoxicity properties of Centratherum punctatum using aqueous medium and different solvents such as, methanol and ethyl acetate. In vitro studies in MS media supplemented with BAP 4.5 mg/L + Kn 4.0 mg/L has shown the high callus induction percentage of 92.33% with a maximum callus weight of 1.08 g. The phytochemical analysis of aqueous, methanol and ethyl acetate extract of C. punctatum in vivo plant and in vitro callus showed the presence of alkaloids, flavonoids, phenols and carbohydrates. The aqueous extract of both plant and callus showed the presence of tannins, proteins and steroids whereas the methanol extract showed the presence of tannins, amino acids and terpenoids. The ethyl acetate extract showed terpenoids and protein. FTIR analysis of plant and callus aqueous extract had a maximum characteristic band at 3399.87 cm-1 and 3412.73 cm-1 respectively indicating the presence of N-H stretching. GC-MS analysis revealed the presence of 11 different compounds in ethyl acetate extracts of plant and the callus extract revealed the presence of 15 different compounds which was absent in the plant extract. Plant extract exhibited maximum total phenol content than callus extract. The in vitro callus extract showed higher DPPH radical scavenging activity with lower inhibition percentage than in vivo plant extract. A maximum zone of inhibition was observed in methanol extract of in vivo plant and in vitro callus (15 mm and 14 mm respectively) against Bacillus subtilis. The ethyl acetate extract of in vivo plant and in vitro callus had a zone of 14 mm and 12 mm against E. coli. A maximum zone of inhibition (12 mm and 11 mm respectively) was observed in both methanol and ethyl acetate of in vivo plant and in vitro callus against Staphylococcus aureus. Antiproliferative analysis revealed that in vivo plant has inhibitory percentage of 23.6 whereas callus exhibited 28.5% against HeLa cells.

Response of some durum wheat (Triticum durum Desf.) genotypes on tissue culture parameters

Bu araştırmada Türkiye makarnalık buğday tarımında öneme sahip ve biyotik-abiyotik stres faktörlerine yüksek adaptasyon özelliklerinden dolayı ıslah programlarında ebeveyn olarak yararlanılan 13 makarnalık buğday genotipi (Altın 40/98, Ankara 98, Berkmen-469, Çeşit 1252, Eminbey, İmren, Kızıltan-91, Kunduru 414/44, Kunduru 1149, Meram 2002, Mirzabey 2000, Selçuklu 97, Yelken 2000) kullanılmıştır. Bu genotiplerin, kallus kültürü parametrelerine olan tepkileri belirlenerek biyoteknolojik karakterizasyonları yapılmıştır. Olgun embriyoların kullanıldığı araştırmada, steril koşullarda tohumlardan çıkartılan embriyolar 2 mg l-1 2,4-D ve MS mineral maddelerini içeren katı besin ortamında kültüre alınmıştır. Gelişen kalluslar, sürgün ve kök oluşturabilmeleri için hormon içermeyen MS ortamına transfer edilmiştir. 13 makarnalık buğday genotipinin olgun embriyoları karşılaştırıldığında; kallus ağırlığı, rejenerasyon kapasitesi ve kültür etkisi parametrelerinde genotipler arasındaki farkın P<0.01 düzeyinde önemli olduğu belirlenmiştir. Test edilen 13 makarnalık buğday genotipinden Berkmen-469, Kızıltan-91 ve Eminbey en yüksek rejenerasyon kapasitesi ve kültür etkisi değerlerinin oluşturarak, rejenerasyon yeteneği en yüksek kallusları meydana getirmişlerdir. Bu genotiplerin doğrudan gen aktarılabilme potansiyellerinin diğer genotiplere göre daha yüksek olduğu ve önümüzdeki dönemlerde biyoteknolojik yöntemlerle dayanıklı çeşit geliştirilmesine taban oluşturabilecek genotipler olduğu belirlenmiştir.

Callus Induction and Virus Free Potato Mini-Tuber Production through Meristem Culture

The experiments were conducted with a view to establish a protocol for virus free mini-tubers production, in vitro, using potato meristem as explant, collected from potato sprouts of three popular potato varieties Diamant, Cardinal, Granola. In the present study four levels of GA3 (100, 200, 300 and 400 ppm) were used to assess the influence on sprouting abilities in three selected potato verities. The maximum sprouting efficiency was observed in 400ppm GA3 treatment within short period of time in Granola variety. The effect of different combination and concentration of IBA, 2, 4-D and BAP was used along with fresh MS media to inoculate meristems of potato sprouts. IBA concentration were 0.25mg/L, 1.5mg/L; 2, 4-D concentration were 0.125mg/L, 0.25mg/L, 0.37mg/L, 0.50mg/L, 1.0mg/L 2.0mg/L and BAP concentration were 0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2mg/L. The effect of combined hormones was also studied. The Diamant variety showed the maximum callus size (0.74cm) within a very short period of time (2 days) while treated at the concentration of 0.25 mg/L IBA, 0.25 mg/L 2, 4-D and 1.00 mg/L BAP. Granola meristem inoculated in hormonal treatment showed the best results regarding minimum days required to shoot initiation (5.34 days) followed by Diamant (6.68) days and Cardinal (7.64 days). The highest number of shoots/plantlet (4.33 shoots/plantlet) and the longest plantlet (9.32 cm) was found in Granola variety followed by Diamond (3.67 shoots/plantlet; 7.53cm) and Cardinal (4.0 shoots/plantlet; 7.62 cm). Maximum numbers of leaves were also found in Granola variety (15 leaves) in the treatment combination of 0.25 mg/L IBA, 0.125 mg/L 2,4-D and 0.5 mg/L BAP. Granola gave maximum performance in respect of maximum length of root (6.55 cm roots/plantlets) within a short time of root initiation (20 days), followed by Diamant (4 cm roots/plantlets) of root initiation (4.47 days) and Cardinal (4.53 cm roots/plantlets) of root initiation (26.39 days). Rooted plantlets were gradually acclimatized and successfully established in the field. However, in overall observation in this study, Granola showed better performance from meristem tissue culture and all plants were found normal and free from potato viruses that were tested through ELISA test.

Physicochemical factors modulate regeneration and Agrobacterium-mediated genetic transformation of recalcitrant indica rice cultivars - ASD16 and IR64

Abstract A comprehensive study of various components of rice tissue culture media and physical parameters was carried out to characterize the factors affecting in vitro plantlet regeneration and subsequent transformation from two recalcitrant indica rice cultivars, ASD16 and IR64. The choice and concentrations of carbon sources, light/dark incubation, gelling agents, amino acid supplementation and hormone combinations in the regeneration medium greatly influenced the regeneration potential. Among four different carbon sources tested maltose (3%) was identified as the best for callus and shoot induction. Among different formulations of hormones tested, both cultivars recorded the highest regeneration frequencies when the medium was supplemented with 3.0 mg/L BAP+1.0 mg/L NAA. Partially desiccating the calli for 48 h resulted in a higher frequency of regeneration and among different gelling agents, gelrite induced maximum callus induction while agarose was found to be a better choice in regeneration. Callus induction and embryogenic calli initiation were higher when the calli were incubated in the dark, whereas high shoot numbers and regeneration efficiency in both cultivars were enhanced when incubated in the light. The optimized conditions yielded higher regeneration frequencies during transformation experiments and the transformants were analyzed by PCR, Southern blotting and GUS histochemical staining for asserting successful transformation. The results indicated that manipulation of media supplements and partial desiccation reported here used either individually or in combination can greatly enhance plantlet regeneration which is essential for an efficient genetic transformation system in recalcitrant indica rice cultivars.

Hypericum adenotrichum SPACH‘UN KALLUS İNDÜKSİYONU VE ADVENTİF SÜRGÜN REJENERASYONU

In this study, A standart protocol to callus formation and adventitious shoot regeneration of Hypericum adenotrichum Spach via direct or indirect organogenesis has been described.Callus induction was carried out by using leaves which were collected from their native environment. The maximum callus induction frequency has been observed on MS media containing 4 mgL-1 BA and 0.2 mgL-1 NAA. These calli were not induced to shoot regeneration. Shoot formation was obtained by transferring the calluses from BA-containing media to MS medium containing 0.5 mgL-1 KIN. Maximum shoot number was observedin transferred calluses from callus induction medium containing 3 mgL-1 BA to media containing 0.5 mgL-1 KIN.Direct shoot formation developed on leaf explants of H. adenotrichum on MS media containing KIN alone. The highest direct shoot development was observed on MS medium with 1 mgL-1 KIN.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5mgl-1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25mgl-1 2, 4-D and 1.0mgl-1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5mgl-1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0mgl-1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45pg and 33.56pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."

Impact of seasonal variation on ‘daidzein’ accumulation in callus and in vivo parts of Pueraria tuberosa (Willd.) DC

Daidzein-natural isoflavones found in Pueraria tuberosa (Willd.) DC. (Fabaceae) which is a potent medicinal plant. It acts as signal carriers and responds to the pathogenic attacks and reduces pain during menopause, osteoporosis and also antidiabetic in nature. In the present work, in vitro callus development from tuber explants during both rainy and summer seasons was conducted. Further, a simple unambiguous and rapid high-performance thin-layer chromatography method was established for quantitative estimation of daidzein in callus and in vivo parts to assess the impact of seasonal change on valuable phytochemical accumulation. Maximum callusing (90%) was obtained on MS medium fortified with a combination of 6N-Benzylamino purine (2 mg/l) and 2,4-dichlorophenoxyacetic acid (2 mg/l) during the summer season from the tuber explant. During the HPTLC method validation, the linearity range obtained was 100–1000 ng/spot with a regression value (r) value of 0.99845. All the parts were found to contain a significant amount of daidzein. The maximum daidzein (2112.567±0.35 ng/g) content was obtained from a young tuber bark followed by callus (171.903±0.33 ng/g) during the summer season compared to rainy season parts and callus. Thus, it can be concluded that in vitro callus is an alternative source of daidzein without destroying the natural plant and the developed HPTLC method could be used for quality control analysis and recommended for daidzein quantification for different herbal formulation and drug preparation.

Callus mediated organogenesis in Lilium polyphyllum D. Don: A critically endangered Astavarga plant from North Western Indian Himalaya

Lilium polyphyllum D. Don (Liliaceae) is a critically endangered herbaceous perennial, commonly known as snow lily or Ksheerkakoli. Bulbs of the plant are of immense medicinal use and have astringent and anti-inflammatory properties. Over exploitation of the species from wild and degradation of habitats are posing threats to its existence. Therefore, an attempt has been made to develop a propagation protocol and promote mass multiplication from bulb scales of L. polyphyllum. Maximum callus was induced from the bulb scales in basal MS medium containing 2,4-D (0.5, 1, 1.5, 2 and 3 mg/l), BAP (0.5, 1, 1.5 and 2 mg/l) and NAA + BAP (0.5+2 mg/l). Maximum shooting (98.75%) was achieved in the callus when shifted to MS medium supplemented with BAP (1 and 2 mg/l) and NAA (0.5 mg/l), with an average of 1-2 cm shoot length per culture. The well developed in vitro regenerated shoots were transferred to the rooting medium and 100% rooting was achieved in half-strength MS basal medium supplemented with IBA and IAA (1 and 2 mg/l). The in vitro regenerated plantlets were shifted to a glasshouse in the sterilized soil and vermiculite mixture for acclimatization. After survival, plantlets were finally transferred to the open environment with 90% survival success.

Effect of photoperiod <em>In vitro</em> callus induction and regeneration of Guggul [<em>Commiphora wightii</em> (Arnott)

The impact of various photoperiod regimes on callus induction from callus culture in guggul leaf explants was carried out in present investigation. The leaf explants showing the maximum callus (0.90g) induction on MS medium supplemented with 2.0 mg/l 2,4-D with 100 per cent frequency were tested for their response to different regimes (10:14, 12:12, 14:10, 16:8) of photoperiod. The cultures were incubated with a light intensity of 3000 lux at 25±2 ° C. Maximum callus induction was observed (0.95 g)in leaf explants incubated at 14:10 hours photoperiod with 100 per cent frequency followed by (0.88 g) with 100 per cent frequency at 10:14 hours photoperiod. Minimum callus induction (0.69g) was observed with 90 per cent at 12: 12 hours photoperiod regimes. Callus induction in leaf explant completely inhibited at 16:8 hours photoperiod. 14:10 was most suitable photoperiod regime for callus induction in leaf explants. Leaf derived callus (2.0 mg/l 2, 4-D, 5.0 mg/l Kn + 0.5 mg/l NAA and 1.0 mg/l Kn) was subcultured at the same level of plant growth regulator to maintain the stock cultures. Shoot/ embryo morphogenesis was not observed on any of the these plant growth regulators except 2.0 mg/l 2, 4-D. Semi compact, light green callus proliferating from cut surface of the leaf explants on the medium supplemented with 2, 4-D (2.0 mg/l) when assessed for its organogenetic capacity. Upon subculture on cytokinin (BAP 0.1-3.0 mg/l) and auxin (2, 4-D 0.1-3.0 mg/l) added singly in the medium and in combination de novo shoot morphogenesis was observed. The reproducibility of this morphogenesis was only 25-30 per cent.

In vitro Plant Regeneration Studies in Brinjal (Solanum melongena L)

The present investigation was undertaken to standardize the protocol for high frequency invitro regeneration of brinjal variety Navkiran which is the essential pre-requisite step of application of biotechnological interventions for crop improvement. Multiple shoot regeneration via callus induction was achieved using epicotyl and hypocotyl as explant at various concentrations and combinations NAA (Naphthalene acetic acid) (0.5mg/L and 1mg/L) and BAP (6-Benzyl Amino Purine) (1.0mg/L, 1.5mg/L and 2.0mg/L) in MS media. Earliest callus induction (6.33±0.33d), was observed when epicotyls was inoculated in MS media fortified with 0.5mg/L NAA and 2.0mg/L BAP. However, maximum callus weight (1.35±0.10g) was obtained in MS media fortified with 0.5mg/L NAA and 2.0mg/L BAP when hypocotyls was used as explant. Ninety percent callus induction was obtained in MS media supplemented with 0.5mg/L NAA and 2.0mg/L BAP in both the explants. Regarding the quality characters of callus, both explants showed variable response in different treatments used under the study. Colour of the callus was dark green, light green and cream, texture of callus was loose, friable and compact and abundance was plenty, moderate and poor. Among the explants used, epicotyl gave the best response to shoot regeneration, it was 52.77±2.61 and took minimum days of shoot regeneration (29.33±0.88), maximum shoot length (3.96 ± 0.06) in MS media fortified with 0.5mg/L NAA and 2.0mg/L BAP. In vitro rooting (68.82±2.70) was obtained within 14.33±1.20 days with 19.10±0.86 number of roots in MS media supplemented with 1.0 mg/L IBA. The rooted plantlets were successfully hardened in a mixture of cocopeat, vermiculite and perlite in ratio of 1:1:1 for 15 days and then transplanted in the main field with survival rate of 95 per cent.

Efficient Shoot Regeneration via Indirect Organaogenesis of a Highly Recalcitrant Species of Cissampelos pareira (L.)

An efficient protocol was standardized for successful regeneration of Cissampelos pareira (L.) through indirect organogenesis. Nodal explants were cultured on MS fortified with 0.5 ± 1.0 mg/l BAP, Kn either single or in combination with NAA 0.5 mg/l. The combinations induced profuse, compact, light green to greenish coloured calli. Some differences in the morphology of callus such as change in the colour and texture was also observed with increasing the concentration of BAP 0.5 ± 2.0 mg/l + NAA 0.5 mg/l. Maximum callus induction was observed on 1.0 mg/l BAP and 0.5 mg/l NAA showed greenish, friable and granular lush colour. The calli were subcultured on fresh MS that contained BAP and Kn single or in combination with NAA (BAP 0.5 ± 2.0 mg/l, Kn 0.5 ± 2.0 mg/l, NAA 0.5 mg/l). The maximum regeneration frequency of shoot organogenesis was recorded on BAP (2.0 mg/l) + NAA (0.5 mg/l). Healthy microshoots were separated and transferred to the rooting medium. Here, MS augmented with IBA 1.0 mg/l showed maximum rooting. Well rooted plantlets were transferred to the field and maximum survival frequency was recorded when BAP (1.0 mg/l) + NAA (0.5 mg/l) for callus induction, for shooting BAP (2.0 mg/l) + NAA (0.5 mg/l) and for rooting IBA (1.0 mg/l) was used. The regenerated whole plants were subjected for hardening where the maximum survival frequency was found to be 80%. This reproducible protocol can be used for regeneration and genetic transformation studies.

PHYTOCHEMICAL AND ANTIOXIDANT ACTIVITY OF BALIOSPERMUM MONTANUM (WILLD.) MUELL. ARG.

Objective: The present study was carried out to evaluate the phytochemical constituents and antioxidant potential of the leaf, stem, root, and stem-derived callus extracts of Baliospermum montanum.
 Methods: An in vitro regeneration protocol was developed for the induction of callus from stem segments cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combination of 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN), 1-naphthaleneacetic acid, and 2, 4-dichlorophenoxyacetic acid. The total phenol, flavonoid, and tannin content were determined according to standard methods. The antioxidant activity of methanolic extract was evaluated using 2, 2-diphenyl-l-picrylhydrazyl (DPPH) and reducing power assays.
 Results: The maximum callus induction was observed on MS medium supplemented with a combination of KIN (0.5 mg/L) + BAP (3.0 mg/L). Methanolic extract of root and aqueous extract of leaf exhibited higher content of phenols. Whereas total flavonoids and tannin were maximum in methanolic extract of leaf compared to other extracts. The methanolic extract of B. montanum leaf had greater antioxidant activity than stem, root, and callus by DPPH and reducing power assays.
 Conclusion: Our study suggests that the methanolic extracts of B. montanum showed potent free radical scavenging activity. Further studies are necessary for isolation and characterization of phytochemical compounds.

Optimized in vitro micro-tuber production for colchicine biosynthesis in Gloriosa superba L. and its anti-microbial activity against Candida albicans

Gloriosa superba L. tubers are a rich source of commercially important colchicine and due to overexploitation, the species has become vulnerable. In the present investigation, in vitro tuber productions were carried out for its propagation and conservation. The in vitro and field-grown tubers were assessed for their colchicine content and antimicrobial activities. Maximum callusing was obtained when the medium was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). Among different auxins tested for in vitro tuberous root production, IAA (1.5 mg/L) induced 78.2% tuberous root per callus. In vitro micro-tuber raised in media supplemented with 1.5 mg/L thidiazuron (TDZ) recorded the highest response (72.9%) with 28.4 tubers per explant. Sucrose (6%) with TDZ (1.5 mg/L) produced significantly more micro-tubers per callus. Elicitor treatment with AlCl3 at 125 µM and 150 µM resulted in a significant increase in the micro-tuber and tuberous root production respectively. N6-(2-Isopentenyl) adenine (2ip) (1.0 mg/L) induced the highest frequency of in vitro micro-tuber sprouting and tuber formation compared to 6-benzylaminopurine (BAP) and 1-naphthalene acetic acid (NAA). The elicitor-treatments with AlCl3 significantly increased the colchicine content of in vitro tuberous root and tubers than that of the field grown tubers. The anti-microbial activity of in vitro raised tubers, tuberous roots and AlCl3 treated samples were significantly higher compared to the field grown samples. An optimized tissue culture system for mass propagation of G. superba with conservation aspects and the production of high-value colchicine is presented here, which can be used in various medicinal systems. Manipulation of PGR’S significantly enhanced in vitro tuberization from noncorm bud explants. Elicitor treatment with AlCl3 enhanced the production of colchicine. HPLC analysis revealed significantly higher colchicine content in in vitro raised plants compared to field grown tubers. The study will help in mass propagation, conservation, and commercialization of Gloriosa superba L. for large scale production of colchicine.

Development of a novel murine delayed secondary fracture healing in vivo model using periosteal cauterization

IntroductionDelayed union and nonunion development remain a major clinical problematic complication during fracture healing, with partially unclear pathophysiology. Incidences range from 5 to 40% in high-risk patients, such as patients with periosteal damage. The periosteum is essential in adequate fracture healing, especially during soft callus formation. In this study, we hypothesize that inducing periosteal damage in a murine bone healing model will result in a novel delayed union model.Materials and methodsA mid-shaft femoral non-critically sized osteotomy was created in skeletally mature C57BL/6 mice and stabilized with a bridging plate. In half of the mice, a thin band of periosteum adjacent to the osteotomy was cauterized. Over 42 days of healing, radiographic, biomechanical, micro-computed tomography and histological analysis was performed to assess the degree of fracture healing.ResultsAnalysis showed complete secondary fracture healing in the control group without periosteal injury. Whereas the periosteal injury group demonstrated less than half as much maximum callus volume (p < 0.05) and bridging, recovery of stiffness and temporal expression of callus growth and remodelling was delayed by 7–15 days.ConclusionThis paper introduces a novel mouse model of delayed union without a critically sized defect and with standardized biomechanical conditions, which enables further investigation into the molecular biological, biomechanical, and biochemical processes involved in (delayed) fracture healing and nonunion development. This model provides a continuum between normal fracture healing and the development of nonunions.

Accelerating hybrid rice breeding through the adoption of doubled haploid technology for R-line development

Hybrid rice holds significant yield advantage over inbred lines, but its adoption rate is very slow in India. Development of cytoplasmic male sterile line (CMS) and fertility restorer line with superior grain quality is the prerequisite in three line hybrid rice breeding programme. Minimum of three to four years are required for development of useful R-line which severely delays the pace of hybrid breeding. Application of doubled haploid (DH) technology will be very useful to develop elite R-lines within short period of time. An elite inbred line LAXMI was crossed with the R-line RPR158 and anthers from the resulting F1 population was used for development of DH plants. Eight hormonal combinations and four different cold pre-treatment conditions were tried in tissue culture. Maximum callusing frequency was observed in N6 medium supplemented with 2.0 mg L−1 2, 4-diclorophenoxyacetic acid (2,4-D), 0.5 mg L−1 napthalene acetic acid (NAA), 0.5 mg L−1 benzyl amino purine(BAP) and 6% maltose with 3 days cold pre-treatment. Highest green plant regeneration frequency was found with Murashige and Skoog medium supplemented with 0.5 mg L−1 NAA, 0.5 mg L−1 kinetin, 2 mg L−1 BAP. A total of 280 DH plants were generated and evaluated under field conditions for agronomic characters and fertility restoration capacity in CMS system. Two distinct DH lines were identified as improved R-lines based on high restoration ability (94%), superior grain quality and medium slender grain type. This study successfully demonstrated that superior R-lines can be developed within two crop seasons through the application of DH technology.

Enhancement of capsaicinoids in vitro production by abiotic elicitors in placenta-derived callus of Capsicum annuum L. Tunisian var. ‘Baklouti Medenine’

Production of natural phytochemicals via tissue culture is an efficient method for both laboratory and industrial-scale production. To evaluate the influence of abiotic elicitation on in vitro production of capsaicinoids (CAPs), callogenesis was induced from the placenta of pepper (Capsicum annuum L.) Tunisian var. ‘Baklouti Medenine’ on MS medium supplemented with 9.04 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 μM kinetin (Kin). Placenta-derived callus was subjected to different concentrations of sucrose, sodium chloride (NaCl), polyethylene glycol (PEG 6000), and silver nitrate (AgNO3). After 4 wk. of culture, callus was harvested and capsaicin (C) and dihydrocapsaicin (DHC) were quantified by liquid chromatography-mass spectrometry (LC-MS). Maximum callus production was obtained with 34.22 mM and 68.44 mM NaCl, with an improvement in growth index (GI) by 3481% and 3736% respectively. In elicitation-free culture, DHC was the major CAPs (79.27%). The highest accumulation of DHC was obtained with 17.11 mM NaCl (39.52 mg g−1 DW; 4% of callus DW). Meanwhile, elicitation raised C content to approximately three times that of DHC. C production increased from 0.17 mg g−1 DW in control culture to 27.88 and 26.79 mg g−1 DW at 17.11 mM NaCl and 14.71 μM AgNO3, respectively. For both treatments, C represented more than 2.6% of callus DW. 17.11 mM NaCl-treated callus, which produced the highest concentrations of both C and DHC, showed the least callus production. Hence, Tunisian var. ‘Baklouti Medenine’ appears an interesting resource for these alkaloids biosynthesis and can be used to develop a large scale production of valuable CAPs compounds.

Feasible production of biomass and natural antioxidants through callus cultures in response to varying light intensities in olive (Olea europaea. L) cult. Arbosana

Light is the most important physical factor in growth and development of plants. Light intensity is directly proportional to the growth and accumulation of natural antioxidants during in vitro cultures of various medicinal plants. The present research study was designed to determine the effect of different light intensities i.e. normal light (2000–2500 lx), diffused light (500–1000 lx) and complete dark (0 lx) on callus growth dynamics and production of natural antioxidants in olive cult. Arbosana. Highest callus induction frequency (50%) was observed in the stem explants pre-treated with silver nanoparticles suspension (AgNPs: 50 ppm) and cultured on MS media supplemented with combination of 6-Benzylaminopurine (BAP: 2 mg/l), Gibberellic acid (GA3: 1.5 mg/l) plus Naphthalene acetic acid (NAA: 0.5 mg/l). Maximum callus biomass (FW = 1414 mg/l) was recorded when the cultured explants were incubated initially for seven days in complete darkness, followed by transference to diffused light for one week and then finally placed under normal light in total fifty six days culture period. Moreover, phytochemical analysis of the callus cultures showed significantly higher activities of antioxidant enzymes i.e. SOD, POD, CAT and APx (2.45, 2.96, 2.57 and 1.67 U/mg. protein) in the callus cultures grown under dark condition as compared with other light treatments. For non-enzymatic antioxidant potential, maximum activity of TPC, TFC, PAL and DPPH (2.42 mg GAE/g, 1.50 mg QAE/g, 3.95 U/mg and 75%) were recorded in the calli raised in vitro under diffused light. This is the first report on the production of natural antioxidants in response to different light intensities in callus cultures of Olea europaea. Future studies should focus on large scale production of callus cultures in order to yield maximum biomass from this high valued plant.