Callus Clumps Research Articles

Indirect plant regeneration in Dioscorea rotundata and Dioscorea alata yam genotypes using different explants

Yam (Dioscorea sp.) is an important staple crop for millions of smallholder farmers in West Africa. In this study, we aimed to develop an efficient indirect plant regeneration system for three important yam genotypes “Kukrupa,” “Mankrong Pona” and “Matches.” The potential of leaf primordia, petioles and axillary bud explants for callus induction on Murashige and Skoog (MS) medium supplemented with different concentrations (0, 3, 5, 7 and 9 mg/l) of 2,4-dicholorophenoxy acetic acid (2,4-D) or picloram were tested. Callus induction was influenced by auxin concentration as well as explant. The highest callus induction was achieved using leaf primordia (“Kukrupa” and “Mankrong Pona”) explants on 3 mg/l picloram or axillary buds (“Matches”) on 5 mg/l picloram. Transfer of callus to 6-benzylaminoapurine (BAP) amended medium resulted in the formation of at least two shoots per callus clump. Plantlets were obtained on basic yam medium supplemented with 0.2 mg/l 1-naphthalene acetic acid (NAA), 3 mg/l kinetin and 0.5 mg/l BAP after four weeks. Post acclimatization plantlet survival was high (75 − 82.5%). The indirect plant regeneration protocol reported through this study helps in the improvement of important yam genotypes through tissue culture-oriented genome editing, in vitro mutagenesis and also for its mass propagation.

Auxin resistant 1 gene (AUX1) mediates auxin effect on Arabidopsis thaliana callus growth by regulating its content and distribution pattern

Callus sustained growth relies heavily on auxin, which is supplied to the culture medium. Surprisingly, there is a noticeable absence of information regarding the involvement of carrier-mediated auxin polar transport gene in callus growth regulation. Here, we delve into the role of the AUXIN RESISTANT 1 (AUX1) influx transporter in the regulation of callus growth, comparing the effects under conditions of light versus darkness. It was observed that callus growth was significantly enhanced under light illumination. This growth-stimulatory effect was accompanied by a decrease in the levels of free auxin within the callus cells when compared to conditions of darkness. In the aux1–22 mutant callus, which lacks functional AUX1, there was a substantial reduction in IAA levels. Nonetheless, the mutant callus exhibited markedly higher growth rates compared to the wild type. This suggests that the reduction in exogenous auxin uptake through the AUX1-dependent pathway may prevent the overaccumulation of growth-restricting hormone concentrations. The growth-stimulatory effect of AUX1 deficiency was counteracted by nonspecific auxin influx transport inhibitors. This finding shows that other auxin influx carriers likely play a role in facilitating the diffusion of auxin from the culture medium to sustain high growth rates. AUX1 was primarily localized in the plasma membranes of the two outermost cell layers of the callus clump and the parenchyma cells adjacent to tracheary elements. Significantly, these locations coincided with the regions of maximal auxin concentration. Consequently, it can be inferred that AUX1 mediates the auxin distribution within the callus.

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023

Shoot organogenesis and somatic embryogenesis from leaf and petiole explants of endangered Euryodendron excelsum

Euryodendron excelsum H.T. Chang is a rare and endangered woody plant endemic to China. It is very important to conserve and propagate this species from extinction. In this study, leaves and petioles from the axillary shoots in vitro were used as explants to culture on the different plant growth regulator (PGR) woody plant medium (WPM) and establish an efficient shoot proliferation and plant regeneration system. WPM supplemented with 1.0 mg/L 2,4-D induced callus dedifferentiated into buds and somatic embryos on various media,including PGR-free WPM. However, only adventitious shoots formed on WPM with 1.0 mg/L of cytokinins such as 6-benzyladenine (BA), kinetin (KIN) or thidiazuron (TDZ). When another cytokinin, zeatin, was used, somatic embryos were induced directly from From cut surface of these explants. Adventitious roots could be induced from both explants on WPM with 1.0 mg/L α-naphthaleneacetic acid (NAA). Somatic embryos cultured in PGR-free WPM or WPM with 0.2 mg/L NAA developed roots. Plantlets derived from somatic embryos were transferred to a peat: sand (1:1, v/v) substrate, and showed survival rates of 64.3% at 30 days and 54.6% at 90 days. Callus clumps with adventitious shoot buds that were transferred to WPM containing 1.0 mg/L BA and 0.2 mg/L NAA generated a mean 3.3 multiple shoots. Callus-derived shoots regenerated and rooted successfully (100%) on agar-free vermiculite-based WPM with 0.5 μM NAA after 30 d. Plantlets transplanted to peat soil: vermiculite (1:1, v/v) displayed the highest survival (96.7%) after three months.

In vitro propagation and genetic fidelity evaluation in LA Lilium

The present experiment was conducted at the Division of Floriculture and Landscaping, IARI, New Delhi, Indiaduring 2015–17 to develop a protocol for callus induction, PLB formation and plant regeneration from LA LiliumBrindisi using in vitro leaf segments, and to assess the genetic stability using SSR marker. Micropropagation of LALilium Brindisi led to compact calluses of dark brown to black colour. The in vitro regenerated leaves were inoculated at different concentration of 6-BAP and 2,4-D. Along with calluses, protocorm like bodies were also induced from the surface of cultured leaf segments, which further developed into shoots. MS medium fortified with 6-BAP (0.25 mg/l) and 2, 4-D (5 mg/l) recorded maximum callus formation. The mean number of shoot per callus clump ranged from 1.12 to 3.88, maximum number of shoots were recorded with 6-BAP (4 mg/l) and NAA (0.25 mg/l). Rooting ranged from 72–100% in IBA medium. Twenty regenerates were randomly selected for testing the fidelity. Out of 18 screened markers, only 10 produced clear and reproducible bands. A total of 244 bands were generated from 10 SSR primers in which seven primers were found polymorphic. Dendrogram generated by data analysis using Darwin 6 software package clearly indicated that the in vitro raised plants through leaf explant via callus phase were divided into three main clusters. The result of cluster analysis was supported by principal coordinate analysis (1/2 axis) where all the genotypes were distributed over different quadrangles. The total somaclonal variation was estimated to be 1.9% which indicated that even the plantlets raised through callus phase exhibited low frequency of somaclonal variation in case of LA hybrids of Lilium.

Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera).

A highly efficient genetic transformation system of Liriodendron hybrid embryogenic calli through Agrobacterium-mediated genetic transformation was established and optimized. The Agrobacterium tumefaciens strain EHA105, harboring the plasmid pBI121, which contained the ß-glucuronidase (GUS) gene and neomycin phosphotransferase II (npt II) gene under the control of the CaMV35S promoter, was used for transformation. Embryogenic calli were used as the starting explant to study several factors affecting the Agrobacterium-mediated genetic transformation of the Liriodendron hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, Agrobacterium optical density, infection duration, co-cultivation period, and delayed selection. Transformed embryogenic calli were obtained through selection on medium containing 90 mg L−1 G418. Plant regeneration was achieved and selected via somatic embryogenesis on medium containing 15 mg L−1 G418. The optimal conditions included a pre-culture time of 2 days, a co-culture time of 3 days, an optimal infection time of 10 min, and a delayed selection time of 7 days. These conditions, combined with an OD600 value of 0.6, remarkably enhanced the transformation rate. The results of GUS chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the GUS gene was successfully expressed and integrated into the Liriodendron hybrid genome. A transformation efficiency of 60.7% was achieved for the regenerated callus clumps. Transgenic plantlets were obtained in 5 months, and the PCR analysis showed that 97.5% of plants from the tested G418-resistant lines were PCR positive. The study of the Liriodendron hybrid reported here will facilitate the insertion of functional genes into the Liriodendron hybrid via Agrobacterium-mediated transformation.

In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

In vitro regeneration, antioxidant potential, and genetic fidelity analysis of Asystasia gangetica (L.) T.Anderson

An effective protocol for the plant regeneration via direct and indirect organogenesis has been developed from leaf explants of Asystasia gangetica (L.), cultured on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of auxin and cytokinins. Approximately 86% of explants produced direct shoots on MS medium containing 0.5 mg L−1 6-benzyladenine (BA) and 10 μg L−1 Triacontanol (TRIA) with a maximum of 4.82 ± 0.29 shoots per leaf segment. For production of callus-mediated plantlets (indirect), primarily callus was induced on MS medium containing 2 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), which was then subcultured on medium with 0.1 mg L−1 naphthaleneacetic acid (NAA), 0.5 mg L−1 BA, and 1 to 8 mg L−1 2-isopentenyl adenine (2iP) in order to develop organogenic callus and subsequent shoot induction. A maximum of 6.84 ± 0.05 shoots per callus clump was obtained on MS media supplemented with 4 mg L−1 2iP, 0.5 mg L−1 BA, and 0.1 mg L−1 NAA. The shootlets produced roots when cultured on half-strength MS media supplemented with 2 mg L−1 indole-3-butyric acid (IBA). In vitro propagated plantlets were hardened on soil rite and acclimatized to field condition with 85% survivability. The chlorophyll content of acclimatized plants was comparable with that of the mother plant, while stomatal micromorphology of regenerated plants exhibited no abnormalities. The radical scavenging and antioxidant activity of methanolic extract of leaves were measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP), and phosphomolybdenum test. In all experiments, regenerated plants exhibited enhanced antioxidant potential indicating micropropagated plants could be exploited for isolation of novel biomolecules. Further, the genetic homogeneity of acclimatized plants was confirmed by PCR-based start codon targeted (SCoT) markers and ycf1b DNA barcoding primers which exhibited monomorphic bands identical to the normal mother plant and no variations were observed.

Elimination of phytoplasmas in brinjal by meristem-tip culture incombination with thermotherapy

The main aim of the study was to eliminate the phytoplasmas in brinjal using meristem-tip culture in combination with thermotherapy. Brinjal shoot tips were taken from field grown plants and subjected to different temperature/time treatments followed by meristem tip excision and culture in a callus inducing medium. The 45°C for 2 minutes showed the minimum number of days for callus induction and the maximum survival percentage of explants. MS medium with BAP 1 mgl−1 and kinetin 1 mgl−1 recorded the maximum shoot initiation percentage at all temperature treatments. The MS medium with BAP 2 mgl−1, kinetin 1 mgl−1 and NAA 0.5 mgl−1 recorded minimum number of days for multiple shoot formation and produced more shoots per callus clump. For root formation, half concentration in MS medium supplemented with IBA 0.5 mgl−1 and IAA 0.5 mgl−1 recorded lesser number of days for root initiation and produced more roots per shoot. Effective regeneration was recorded at 45°C for 2 minutes. To confirm the elimination of phytoplasmas, DNA was extracted from these plants and from little leaf diseased brinjal plants. The molecular detection was done through PCR and the phytoplasma infected plant sample produced the expected length amplification products while no amplification was observed in the in vitro grown plants.

Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile, Tripogon loliiformis

Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0mgL−1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL−1 6-benzylaminopurine (BAP) and 0.5mgL−1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL−1 BAP but with 0.25mgL−1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36 h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0 μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0 μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5 μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15 μM TDZ for 24 h) explants was achieved on WPM augmented with 6.0 μM TDZ and 0.5 μM NAA after 8 weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (∼0.5 g) and were transferred onto WPM fortified with different concentrations (2.0–9.0 μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0 μM) and gibberellic acid (GA3, 0.5–3.0 μM). Maximum shoot number (10.4 ± 0.74) and average shoot length (4.75 ± 0.71 cm) were observed in WPM enriched with 2.0 μM BAP, 1.0 μM IAA and 1.5 μM GA3 after 8 weeks of incubation. The developed shoots (4 cm) were excised, pulse-treated for 24 h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0 μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60 days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.

High frequency plant regeneration from leaf culture of Neolamarckia cadamba.

Neolamarckia cadamba is a miracle tree species with considerable economic potential uses as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish a highly efficient and robust protocol of plant regeneration for N. cadamba. Greenish callus was induced from very young leaf explants of sterile in vitro plantlets cultured on Murashige and Skoog's (MS) medium supplemented with 3 mg l-1 thidiazuron (TDZ), 0.1 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l-1 α-naphthaleneacetic acid (NAA). The callus could differentiate into nodular embryogenic structures or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. The micro-shoots developed roots in MS supplemented with 0.05 mg l-1 NAA and 0.05 mg l-1 indole-3-butyric acid (IBA), while the nodular embryogenic structures germinated directly and developed into plantlets on induction medium contained with 0.5 mg l-1 (or 1 mg l-1) 6-benzyladenine (6-BA) and 0.05 mg l-1 NAA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 92.0% survival. This regeneration protocol can be used in large scale cultivation needs and may be useful for future genetic modifications of N. cadamba.

Plantlet regeneration from primary callus cultures of Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, a rare bulbous germplasm

Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, an endangered valuable genetic resource, was used to establish and optimize a callus propagation system and to investigate the effects of internal and external phytohormones for the purpose of germplasm conservation. Of the combinations and concentrations of auxins and cytokinins examined, Murashige and Skoog (MS) medium supplemented with 8 g L−1 agar, 30 g L−1 sucrose, 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.69 to 5.37 μM α-naphthaleneacetic acid, and 0.44 μM 6-benzyladenine, 0.45 μM thidiazuron, and 0.28 μM zeatin riboside generated the best results, effectively promoting callus proliferation. Four callus types could be discriminated, of which type A (yellowish, granular) and type B (yellow, medium-granular) were dry, friable, and grew well. Periodic acid-Schiff staining revealed small and regular cells, with numerous starch granules surrounding each nucleus. In culture, callus clumps produced an average of 14.33 shoots under “MS + 7-d-dark–light” treatment with 100% regeneration frequency. Bulblets formed within 60 d after shoot transfer to bulblet formation medium. Type A and B callus was likely to be embryogenic, according to morphology, cytology, and high shoot regenerating capacity. Examination of endogenous phytohormone levels showed that the abscisic acid to indole-3-acetic acid (ABA/IAA) ratio gradually increased with increasing diameter of callus clumps treated with all exogenous phytohormones, except zeatin riboside, leading to the hypothesis that callus induction competence was closely associated with endogenous ABA/IAA ratio. This first report should assist further genetic studies of this rare Lilium and other bulbous plants.

Plant regeneration and Agrobacterium-mediated transformation of the miracle tree Neolamarckia cadamba

Neolamarckia cadamba is a miracle tree species with considerable economic potential used as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish an efficient protocol of plant regeneration and Agrobacterium-mediated genetic transformation for N. cadamba. Greenish callus were induced from different type explants when cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) and kinetin (KT). The callus could differentiate into somatic embryos or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. It was found that MS medium supplemented with 2 mg/L TDZ, 0.2 mg/L KT provided the most suitable medium for leaf explants to induce adventitious shoot, delivering a maximum regeneration frequency with a mean of 8.2 shoots per explant. The micro-shoots developed roots in MS supplemented with 0.05 mg/L α-naphthaleneacetic acid (NAA), and could be successfully acclimatized to a greenhouse with more than 90% survival. Based on this regeneration protocol, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for N. cadamba. Genetic transformation was obtained through the infection of leaf with A. tumefaciens strain GV3101 harboring a modified binary vector pCAMBIA1305 containing the marker genes β-glucuronidase (GUS) and fluorescent protein YFP. The presence of GUS in transformed plants was confirmed using PCR and histological stain, while the expression of YFP was confirmed through fluorescent microscopy and RT-PCR. Out of 276 putative transformants, 15 plants were found to be positive transgenic. Nevertheless, by adopting this protocol, positive transformed plants could be obtained within three months.

In Vitro Organogenesis of a Slipper Orchid, Paphiopedilum ‘Alma Gavaert’

The aim of the present study was to improve the regeneration efficiency of callus lines in a slipper orchid, Paphiopedilum ‘Alma Gavaert’. Three kinds of vegetative tissues, root, stem and leaf segments, were used as explants to induce callogenesis; out of these, only root explants formed callus and was subcultured in the presence of 5 mg/L dicamba and 5 mg/L 2,4-D combined with 1 or 2 mg/L TDZ. The resulting four callus lines, assigned as 5Di1T, 5Di2T, 5D1T and 5D2T, respectively, were used to test the effect of NAA to BA ratios on re-differentiation, wherein the highest number of shoots (approximately 2 shoots/0.1 g callus clump) were obtained in callus line 5D2T at ratios of 0.001 and 0.002. A largely improvement of shoot regeneration efficiency was obtained by continuous selection of callus lines which derived from different explant positions. Eventually, six callus lines, including 5D2T-T6-G5 to 5D2T-T6-G10, were able to produce approximately 10 times of shoots per callus clump when compared with the parental callus line 5D2T.

Regeneration Rate of Eggplant Somatic Embryogenic In Various Maturation Media

Ralstonia solanacearum is one of the most important pathogen that causes bacterial wilt disease in eggplant and inhibits eggplant production. Improvement of eggplant varieties resistant to bacterial wilt can be accomplished through genetic manipulation. Regeneration of in vitro plants isone of the important tools to supports plant improvementthrough biotechnology. This study was aimed to determine the rate of eggplant regeneration in various maturation media, and to find the best medium for eggplant regeneration based on maturation rate and the number of cotyledon produced. We used resistant eggplant (accession 032) as the material to produce somatic embryogenic.There were 7 types of regeneration media used in this research. MS medium was supplemented with a certain concentration of plant growt regulators , such as: 1 mg / L + BAP 1 mg / L, NAA 4mg / L, TDZ 0.005 mg / L, TDZ 0.001 mg / L, CuSO4 2mM + BAP 1 mg / L, CuSO4 2mM + BAP 2 mg / L and Kinetin 1 mg / L + CuSO4 2mM. Three clumps of callus per plate with three replications were transferred to MS suplemented medium. The parameters observed were the color of callus before and after they were transfered to regeneration medium, the day of formation of globular, heart-shaped, tubular and cotyledonary phase, and the number of cotyledons formed. The results obtained showed the somatic embryogenic color of the 032 genotype was white with friable structure before being transferred to regeneration medium and was turned to yellowish white after being transferred to the regeneration medium. On the day sixth, friable embryogenic somatic of eggplant was developed into nodule on medium MS + NAA 4 mg / L, MS + CuSO4 2mM + BAP medium 1 mg / L, and MS + CuSO4 2mM + BAP 2 mg / L. Somatic embryogenic callus of accession 032 were able to pass complete globular, heart-shaped, tubular and cotyledonary phase. The most responsive medium for somatic embryogenic callus regeneration, based on the days of the callus phases formation and the number of early-phase cotyledons obtained, were MS medium suplemented with CuSO4 2mM + BAP,and CuSO4 2 mM + BAP 2 mg / L.Keywords: eggplant, Ralstonia solanacearum, regeneration, cotyledonary, clump, BAP

Investigations on various methods for cryopreservation of callus of the medicinal plant Satureja spicigera

Satureja spicigera callus initiated from young leaves was successfully cryopreserved by vitrification. Four protocols desiccation, vitrification with PVS2, vitrification with PVS3 and DMSO freezing were tested for suitability. According to the results of the analysis of variance, significant differences were found among four protocols used for callus cryopreservation. The regrowth percentage of cryopreserved callus was significantly highest after treatment with PVS3 (98.7%). Based on this result, vitrification with PVS3 is recommended as the most suitable protocol for cryopreservation of S. spicigera callus. In this protocol, the callus excised from leaves of in vitro-grown plants was precultured on MS medium supplemented with 0.4M sucrose, 0.5mgl−1 2,4-D, 0.6mgl−1 BAP and 6gl−1 agar under a 16h photoperiod at 25°C for two days. After preculture, the callus was transferred to a Petri dish with 2ml loading solution and loaded at 25°C for 25min. Then, the callus was dehydrated with 2ml PVS3 at 25°C for two hours. After transferring to a 1.8ml cryotube containing 1ml PVS3, the cryotube was directly immersed into liquid nitrogen. The fast rewarming procedure consisted of plunging the cryotubes into a water-bath at 40°C for 2min. After removing PVS3, the contents of the cryotubes were poured into a Petri dish with 4ml of a 1.2M sucrose solution. Callus clumps were incubated in this solution at 25°C for 20min. After that, the callus was transferred to recovery medium. Results of measurements by differential scanning calorimetry (DSC) have shown that callus treated by desiccation and vitrification no freezing and melting peaks occurred. These cryopreservation protocols would be useful for the ex-situ conservation of Satureja spicigera callus at very low temperatures.

English

The wild resource of Dendrobium candidum, a well-known epiphytic orchid, is limited. Plant cell culture technology is a promising alternative for production of high-value secondary metabolites .In this research, factors affecting the induction, maintenance, and multiplication of callus from protocorm-like bodies (PLBs) of D. candidum were systematically studied, and a technical callus inducing system with 100% rate was established which higher than previously reported values. The result showed that 2,4-dichlorophenoxy acetic acid(2,4-D) and 1-naphthalene acetic acid (NAA) could of benefit to the callus induction. The optimized medium for callus induction was MS medium containing 0.5 mg/L 2,4-D and 0.25 NAA or 0.5 mg/L kinetin (KT). The callus clump enrichment method was used for directional screening and cell adaptation on MS medium supplied with plant growth regulators (PGR) such as 5 mg/L NAA and 0.5 mg/L 6-benzyladenine (6-BA) or KT for 30 days per cycle. After 6 to 9 months, D. candidum cell lines were obtained and the histological analysis showed that the main differences between callus and PLBs were meristematic cell content and internal cell differentiation degree, and the meristematic cells distributed in the external strip were contributed to the formation of callus. At last, the amount of polysaccharides and total amino acids were compared between the obtained cell lines, tissue culture seedlings and PLBs. The results showed that polysaccharides was 24.67% in callus and higher than that in tissue culture seedlings and PLBs. The amount of total amino acids was 5.94% in callus and higher than that in tissue culture seedlings. So the callus is considered a good choice for the expanding of D. candidum medicine sources. Key words: Dendrobium candidum, Callus induction, tissue culture, natural products, plant growth regulators

An Efficient Regeneration and Genetic Transformation Protocol ofColeus forskohliiusing Biolistic Gun

An efficient selection and plant regeneration protocol for biolistic gun transformation using leaf derived callus of Coleus forskohlii has been developed. Highest regeneration frequency 90% with 50 shoots per callus clump was obtained on Murashige and Skoog (MS) media supplemented with benzylaminopurine (BAP) 2.0 mg L−1 + naphthalene acetic acid (NAA) 0.5 mg L−1 The rate of shoot multiplication was increased with each subculture. Rhizogenesis was obtained on the same media composition. The in vitro raised plants were established successfully in sand and cocopeat (1:1). Callus of C. forskohlii was bombarded using biolistic gun with pABC plasmid DNA which contains β-glucuronidase (GUS) reporter gene and Arabidopsis thaliana white brown complex homologs (AtWBC19) as selectable marker gene. Kanamycin in the shoot induction medium was compared qualitatively and quantitatively for its efficiency as a selection agent for the selection and regeneration of transgenic plants after biolistic gun transformation. Kanamycin levels at or above 50mg L−1 completely inhibited growth of untransformed shoots. The integration of selectable marker gene GUS and AtWBC 19 into the genome of transgenic plants was confirmed using histoenzymatic GUS assay and polymerase chain reaction (PCR) respectively. These results pave the way for the transformation of Coleus forskohlii with desirable genes.

Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.