MSB Medium Research Articles

A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables.

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

Plant regeneration from embryogenic callus-derived from immature leaves of Momordica charantia L

Bitter melon (Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as β-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L−1 NAA augmented with 0.5 mg L−1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L−1 GA3. This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production.

Shoot Induction, Multiplication, Rooting and Acclimatization of Black Turmeric (Curcuma caesia Roxb.): An Important and Endangered Curcuma Species

Curcuma caesia Roxb., commonly known as Kali Haldi or black turmeric, is one of the important species in the genus Curcuma. This species has been classified as one of the endangered Curcuma species due to the drastic decrement of this plant in its natural habitat. C. caesia has been overharvested for various purposes, including bioactive compound extraction to fulfill the pharmaceutical industry demand. Hence, this study was conducted to establish a protocol for the propagation of C. caesia via plant tissue culture techniques. In the shoot induction stage, three basal medium formulations, including Murashige and Skoog (MS medium), the combination of Murashige and Skoog macronutrients and B5 micronutrients (MSB5 medium) and woody plant medium (WPM medium) supplemented with 15 μM of 6-benzylaminopurine (BAP), were used. The results found that the MSB5 medium was the most suitable basal medium formulation for shoot induction of C. caesia. In the subsequent experiment, different types of cytokinin, including BAP, kinetin and 2-iP at concentrations of 5, 10, 15 and 20 μM, were fortified in the MSB5 medium for shoot multiplication. The shoot multiplication was further enhanced by supplementing the MSB5 medium with indole-3-butyric acid (IBA) or 1-napthaleneacetic acid (NAA) at the concentrations of 2, 4, 6 and 8 μM. The results showed that a combination of 15 μM of BAP and 6 μM of IBA significantly increased the shoot multiplication with 100% shoot induction, 3.53 shoots/explant, 10.81 cm of shoot length, 9.57 leaves, 0.486 g of leaves fresh weight and 0.039 g of leaves dry weight. After the multiplication, the rooting stage was carried out by altering the basal medium strength into half and full strength and supplementing with 2.5, 5, 7.5 and 10 μM of indole-3-acetic acid (IAA). The full strength of MSB5 medium supplemented with 5 μM of IAA exhibited the highest number of roots and length of roots, with 6.13 roots and 5.37 cm, respectively. After the rooting stage, the plantlets were successfully acclimatized in the potting medium with the combination of cocopeat and peatmoss, and the ratio of 1:1 was found to produce the highest survival rate with 77.78%. In conclusion, the protocol established in this study could be useful for large-scale raw material production, either for conservation or bioactive compound extraction.

Plant Regeneration of Malaysian Aromatic Rice MRQ88 Via Somatic Embryogenesis

Aromatic rice has become one of the most sought-after sources by today's consumers. As a result, by utilising mature seeds, an efficient somatic embryogenesis system for the MRQ88 variety has been constructed. Due to the highest percentage of callus initiation and no browning, MS-B5 medium supplemented with 1 mg/L 2,4-D and 10 mg/L NAA was the best medium for MRQ88 callus induction. Incubation of embryogenic callus on identical medium containing maltose 10 g/L and ABA 10 mg/L for 8 weeks resulted in the highest proportion of somatic embryo initiation (80%). The number of regenerated green plantlets on medium containing NAA and 2,4-D which was previously pre-treated with 5 to 10 mg/L ABA, 10 g/L maltose, showed good results, with 8-9 green plantlets developed.

In vitro selection of blackberry (Rubus fruticosus 'Tupy') plants resistant to Botrytis cinerea using gamma ray-irradiated shoot tips.

Blackberry is an economically important crop in Mexico, and its yield is substantially reduced by gray mold, a disease caused by Botrytis cinerea. One of the means to obtain B. cinerea-resistant plants is gamma irradiation. Shoot tips of in vitro-micropropagated blackberry plants (Rubus fruticosus 'Tupy') were irradiated with five doses of Cobalt-60 gamma radiation (0, 15, 30, 45, and 60 Gy) and cultured on Murashige and Skoog basal medium containing 1.0 mg l-1 benzylaminopurine and 0.06 mg l-1 indole-3-butyric acid (MSB medium). After 28 days of culture, survival was evaluated to determine mean lethal dose (LD50), and 200 shoots were further irradiated at the determined LD50 (30.8 Gy). After 28 days, the surviving shoots were micropropagated on MSB medium for 60 days. Non-irradiated shoots were screened for the in vitro selection of resistant B. cinerea, exposing them to different concentrations of sterile culture filtrate of B. cinerea (0, 2, 4, 6, 8, and 10 g l-1) for 28 days to determine mean lethal concentration (LC50), and the irradiated surviving shoots were further exposed to the determined LC50 (4.6 g l-1). Three surviving lines (rfgum5, rfgum6, and rfgum17) that did not present changes compared with the control shoots were micropropagated to obtain plantlets, which were further subjected to in vitro resistance assays using detached leaves inoculated with B. cinerea (1×103 spores ml-1). Plants of rfgum5 and rfgum6 mutant lines were highly resistant and presented similar growth to control plants. Therefore, this methodology is useful to obtain B. cinerea-resistant blackberry plants.

Isolation and identification of beneficial orchid mycorrhizal fungi in Paphiopedilum barbigerum (Orchidaceae)

ABSTRACT Seed germination and seedling development in nearly all orchid species rely on a symbiotic relationship with mycorrhizal fungi; however, this is not the case with all mycorrhizal fungi. This study aims to provide an understanding about the important role of mycorrhiza in seed germination and growth of Paphiopedilum barbigerum. Therefore, we isolated and identified endophytic fungi from the roots of wild P. barbigerum. The beneficial mycorrhizal fungi Epulorhiza sp. FQXY019 and Tulasnella calospora FQXY017 were screened by seed symbiotic germination tests and found to promote seed germination. However, only the seeds inoculated with FQXY019 progressed from the seed germination to rooting stage. This shows that mycorrhizal fungi and P. barbigerum have a specific relation at different growth phases. In addition, we selected FQXY019 and inoculated it into MS medium, B5 medium, OMA medium, and PDA medium. The results showed that FQXY019 co-cultured on PDA significantly promoted the increase in seedling fresh weight, leaf length, and root length (p < .01). Furthermore, it significantly promoted the root number and leaf number of seedlings compared with those co-cultured on MS, B5, and OMA media and control (p < .05). Thus, this study demonstrated the promoting effect of Epulorhiza sp. FQXY019 on seed germination and seedling development, making it an alternative method for the artificial propagation of P. barbigerum.

Breaking seed dormancy and regeneration in Cannabis sativa L.

Cannabis sativa L. is an important medicinal plant species that grow under natural conditions and has been legalized in 20 out of 81 Turkish provinces. The female inflorescence is a highly branched compound raceme with indeterminate habit of growth. This results in different maturing of seeds on the inflorescence and induce physiological dormancy on seeds. The study aimed to improve seed germination percentage using various concentrations of GA₃, GA₃ + BAP, germination on water and water solidified with agar, MS or Gamborg B5 medium. The results showed that the best seed germination was noted on Gamborg B5 medium. Different explants were used to regenerated plantlets on Gamborg B5 medium. All explants were suitable for callus regeneration variably. Only the stem nodes of Samsun Vezirköprü were suitable to induce shoots and plantlets. These plantlets were acclimatized on clay loam soils and transferred to field condition during October 2020, where they acclimatized successfully. These studies provide an effective insight into the mechanism seed dormancy in C. sativa. Further studies using other plant growth regulator concentrations will improve shoot regeneration and aid in utilizing the methods for breeding purpose.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton species. As a prerequisite for this, a reliable and efficient method of high frequency plant regeneration in G. arboreum must be established. In this study, callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB medium (MS salts and B5 vitamins) with 0.09 µM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 2.32 µM KT (kinetin). During propagation, callus was effectively selected for subculture on different media based on cell morphology to induce embryogenic callus and somatic embryos. Embryogenic callus was induced on MS5 medium (MSB, 37.59 mM KNO3, 62.47 µM NH4NO3, 3% (w/v) glucose, 6.8 mM glutamine, 3.8 mM asparagine) from suspended cultures after several cycles of alternate solid MS3 medium—liquid MS4 medium culture, a key step in somatic embryogenesis. Solid MS3 medium was supplemented with 2.46 µM IBA (īndole-3-butyric acid), 0.93 µM KT, 6.8 mM Gln, 3.8 mM Asn; liquid MS4 medium was supplemented with 0.1 g/L NaCl, 0.1 g/L KCl and 0.1 g/L CuSO4. The solid–liquid alternate culture was effective for embryogenic callus induced in G. arboreum. MS5 medium with maltose (3% w/v) or glucose (1.5% w/v) + maltose (1.5% w/v) performed better than that added single glucose (3% w/v) for somatic embryo maturation and germination. The regenerated plants with well-developed roots were directly transferred to the soil or grafted onto germinated cotton plants. Plant regeneration via somatic embryogenesis in diploid cultivated species was established, but there is a need for improvement and optimization for gene functional analysis and gene editing in the diploid cotton species. Regenerated plants of G. arboreum, a cultivated diploid cotton, were obtained via somatic embryogenesis by adjusting the culture mode and medium compositions.

Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens

BackgroundUrsolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA.MethodsLeaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h.ResultsHigh frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation.ConclusionThis is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

In vitro plant regeneration of some recalcitrant indica rice (Oryza sativa L.) varieties

Rice is the staple diet for the greater part of the world’s population and there is a regular expanding need of rice production. The plant development and productivity are decreased by numerous factors. Utilizing biotechnological instruments is the most useful alternative to create rice varieties with incredible grain quality, improved productivity and protection from different stresses. Nonetheless, the deficiency of a simple, speedy and competent protocol for embryogenic callus induction and plant recovery is a significant draw back and there is substantial genotype-dependence. The current study explores the impact of plant growth regulators on the callus induction, proliferation and recovery from developed seeds in three rice varieties—SR 1, Jehlum and K 332. Greatest callus induction (82.33 ± 2.51% in SR1, 99.66 ± 0.57% in Jehlum and 93.33 ± 2.88% in K332) was seen in type 2 media containing 3% maltose, 500 mg/l casein hydrolysate, 500 mg/l l-proline, 5 mg/l l-glutamine, 40 mg/l cysteine, 5 mg/l l-asparagine, 100 mg/l ascorbic acid, 4 mg/l AgNO3, 600 mg/l MgCl2 enhanced with 4 g/l gelrite, 0.2 mg/l BAP and 2.5 mg/l 2, 4-D. Most noteworthy recurrence of shoot recovery (75.0 ± 3.0% in SR1, 86.66 ± 2.88% in Jehlum and 88.33 ± 2.88% in K332) was seen in the MS B5 medium containing 3 g/l phytagel enhanced with 4% sucrose, 2.5 mg/l BAP, 1 mg/l zeatin, 0.2 mg/l NAA and 0.5 mg/l TDZ. The current protocol gives a quick callus induction and recovery system, which could be utilized in effective genetic transformation and for creating genetically altered plants.

Gene Transformation of Alfalfa Plants (Medicago varia L.) DomesticCultivar andlsquo;Burgaltaiandrsquo; for Increasing Leaf Size

‘Burgaltai’ cultivar of alfalfa is the most important forage in Mongolia and its yield had decreasing last decades due to cross pollination. This study was conducted for increasing leaf size of ‘Burgaltai’ cultivar through Agrobacterium mediated genetic transformation transferring growth regulating factor AtGRF2 gene of Arabidopsis thaliana. In vitro regeneration was derived from seven day old hypocotyl and cotyledon explants for callus induction. Induced calli were used for shoot induction and proliferation. Proliferated shoots carried to root induction medium. The best calli induction medium was MSB5 (Murashige Skoog’s medium with Gamborg’s vitamin) medium supplemented with 2 mg/L 2,4-D, 0.2 mg/L BAP and 1 mg/L NAA, shoot induction and proliferation media from calli were MSB5 medium supplemented with 1mg/L BAP, 0.1 mg/L NAA, proliferated shoots were rooted on half strength MSB5 medium supplemented with 0.5 mg/L NAA. For the genetic transformation, hypocotyl explants used as an initial source. Totally 235 explants were tested to transformation and 30 explants (12.7%) were resistant to hygromycin antibiotics and regenerate plantlets. Plantlets were transferred to the soil and fresh young leaf used for GUS histochemical assay and RT-PCR for confirmation. Among them fully regenerated seven independent transgenic plantlets (T6, 8, 11, 12, 13, 15, 22) were confirmed. Transgenic ‘Burgaltai’ cv. of alfalfa leaf size was increased by 0.7 cm2 comparing to the wild type. T6 and 11 lines showed 3:1 segregation ratio in T1 progenies (X2=0.04-0.42, p ≤ 0.52-0.84).

Phytochemicals and antioxidant capacity of wild growing and in vitro Hypericum heterophyllum

In vitro cultures of Hypericum heterophyllum Vent. was established by using MS and MS-B5 medium contained plant growth regulators such as kinetin, BAP, IAA, TDZ and picloram. Flower, leaf, stem and in vitro samples (callus and in vitro plantlets) of H. heterophyllum were analysed by LC-MS/MS for their chemical contents such as quinic acid, gallic acid, (+)-catechin, ferulic acid, vanillic acid, p-coumaric acid, caffeic acid, and quercetin; moreover their radical scavening activities conducted by DPPH and ABTS methods were evaluated. Among all the analysed samples, the in vitro plantlets shown the highest antioxidant activity (IC50, 220 μg/mL for DPPH and 254 μg/mL for ABTS), probably due to the presence of phenolic acids and flavonoids, specifically the higher total phenolic content (64.4 mg GAE/g extract) than other samples. The phytochemical variation among all samples was discussed through principal component analysis (PCA) and hierarchical cluster analysis (HCA). The in vitro plantlets might offer possibilities for the production of high-value secondary metabolites as pharmaceuticals and food preservatives. This study is the first report on analyses and comparison of secondary metabolites and antioxidant activities in different plant parts and in vitro samples of endemic H. heterophyllum.

D-Tagatose Effectively Reduces the Number of Streptococcus mutans and Oral Bacteria in Healthy Adult Subjects: A Chewing Gum Pilot Study and Randomized Clinical Trial.

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases.

The effect of different hormone combinations on direct and indirect somatic embryogenesis in Agave americana

Direct and indirect somatic embryogenesis in Agave Americana through basal parts of micropropagated shoots were considered in this study. For micropropagation, 2 factors (BA and NAA) were utilized in different concentrations. A factorial experiment based on a completely randomized design (CRD) was accomplished with four replicates in a modified Murashige and Skoog medium (MSB). BA in 3 mg/L along with NAA in 0.1 mg/L was the best treatment for multiplication. Callus and embryo induction were evaluated using different auxins in a CRD experiment with three replicates in MS medium supplemented with L2 vitamins. Picloram 2.5 and 2 mg/L and also Dicamba 1 mg/L induced 66.7%, 60%, and 53.3% of explants to somatic embryo, respectively. Also, 2,4-D 2 mg/L had the highest impact on callus induction (95.6%). Embryogenesis was studied using Dicamba and Picloram in the same culture medium in a factorial experiment based on CRD with three replicates. The results of orthogonal polynomial analaysisindicated that high concentrations of Dicamba from 0 to 1.5 mg/L had a significant effect on somatic embryo induction. However, higher concentrations had the opposite effect on somatic embryo numbers. Germination of somatic embryos was performed in MSB medium without growth regulators.

Highly competent in vitro propagation of Thrixspermum japonicum (Miq.) Rchb.f., a rare epiphytic orchid

An efficient protocol for asymbiotic seed germination and multiple shoot production of Thrixspermum japonicum has been established. Seeds were cultured on MSB (Murashige and Skoog, (MS) macro- and micro-nutrients, 0.1% (w/v) activated charcoal (AC), and 3.0% (w/v) sucrose) medium fortified with different concentrations of coconut water (CW), indole-3-acetic acid (IAA), or α-naphthaleneacetic acid (NAA), and gibberellic acid (GA3) to promote seed germination. The highest seed germination percentage (88.1%) was observed on MSB medium fortified with 0.2% (v/v) CW, 0.2 mg L−1 NAA and 0.5 mg L−1 GA3. Optimized shoot induction (94.3%) and maximum shoot numbers (8.3) were obtained on protocorm cultured on MS medium amended with 0.1% (w/v) AC, 3.0% (w/v) banana pulp, 2.0% (w/v) potato homogenate, 0.3 mg L−1 KN, 0.2 mg L−1 NAA, and 0.5 mg L−1 GA3. Rooting of the shoots was best achieved on MS medium supplemented with 0.1% (w/v) AC, 3.0% (w/v) banana pulp and 2.0% (w/v) potato homogenate, and 0.8 mg L−1 indole-3-butyric acid. Plantlets were acclimatized in the greenhouse with 88.1% survival rates. The protocol proposed in this study could be useful for conservation of S. japonicus, an important and rare epiphytic orchid.

TDZ-induced plant regeneration in Brassica oleracea L. var. botrytis: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB5) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB5 medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB5 medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB5 medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB5 medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA3). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.

Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties

Rice is a staple food whose productivity is affected by multiple environmental stresses. The use of biotechnological tools is the best possibility to develop rice varieties with increased productivity, exceptional grain quality and resistance to various stress. Several parameters in the genetic transformation (co-cultivation period, acetosyringone concentration, temperature of the co-cultivation medium, antibiotic concentration etc.) of rice varieties—Shalimar Rice 1, Jhelum and K 332 were optimized for the first time using β-glucuronidase (GUS) gene as a reporter. The GUS expression in presence of 100 μM acetosyringone at pH 5.2 was more at 22 °C as compared to 28 °C in all the rice varieties. The selection medium containing 250 mg l−1 of cefotaxime and carbenicillin prevented Agrobacterium overgrowth and the use of 50 mg l−1 hygromycin resulted in killing of the untransformed calli. The regeneration medium containing MS B5 medium supplemented with 4% sucrose, 3 g l−1 phytagel, 2.5 mg l−1 BAP, 1 mg l−1 zeatin, 0.2 mg l−1 NAA and 0.5 mg l−1 TDZ allowed efficient regeneration of the rice calli. The overall regeneration frequency of Shalimar Rice 1, Jhelum and K 332 were 64, 75 and 77% respectively. Putative transformed plants were analyzed for the presence of the transgenes through GUS histochemical analyses and southern blotting. The present protocol provides an efficient and rapid embryogenic callus induction, transformation and regeneration system, which could be conveniently used for producing genetically modified plants and might help in the transformation of other related rice genotypes.

Comparative determination of the essential oil composition in Bulgarian endemic plant Achillea thracica Velen. during the process of ex situ conservation

Ex situ conservation of Bulgarian endemic plant Achillea thracica Velen. was achieved by successful in vitro cultivation of mono-nodal segments on MS-B5 medium supplemented with 1.0mg/L BA for 20days and subsequent transferring of regenerated plants on hormone free basal MS-B5 medium for root development and accumulation of leaf biomass. In vitro multiplicated plants were successfully acclimated in a growth chamber with 100% survival. GC–MS analysis of the essential oils resulted in the identification of 30, 10 and 28 compounds in in situ grown, in vitro cultivated and ex vitro adapted plants, respectively, constituting 77.7%, 99.9% and 84.1% of the total oils. The wider variety of compounds was found in the essential oils of in situ and ex vitro adapted plants where santolina alcohol, β-eudesmol, 1,8-cineole, germacrene D, α-cadinol and artemisia alcohol were the principal components comprising 68.7% and 69.3 of the oil, respectively. In vitro cultivated plants consist of mainly 1,8-cineole, germacrene D and artemisia alcohol representing 87% of the oil. Different growth conditions affect the composition of essential oils, suggesting their possible involvement in the process of adaptation and surviving in changing environmental conditions.

Optimization of Agrobacterium-mediated genetic transformation of shoot tip explants of green gram (Vigna radiata (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100 % shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5 + 2.46 µM IBA and about 90 % of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301 + AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23 °C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T0 plants. Stable GUS activity was detected in flowers of T0 plants and leaves of T1 plants. PCR analysis of T1 progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.

High frequency induction of multiple shoots and plant regeneration from zygotic embryo axis explants of cotton cultivar L-604 (Gossypium hirsutum L.).

An efficient, robust regeneration protocol has been developed using zygotic embryo axis explants of an elite cultivar L-604 (Gossypium hirsutum L.). Explants regenerated on MSB5 medium supplemented with 0.5 mg-l Thidiazuron, 0.1 mg-l Naphthalene acetic acid, 5.0 mg-l Silver nitrate and 1.0 g-l activated charcoal, was identified as the best combination for producing the maximum number (12.67) of multiple shoots per explant. In addition, the highest shoot elongation (5.30 cm) and maximum regeneration frequency (89.0%) was also recorded in the same hormonal combinations. In vitro shoots were successfully rooted on ½ MSB5 medium containing 1.5 mg-l IBA. Well rooted plants were subsequently transferred to polyhouse and greenhouse conditions for primary and secondary hardenings respectively and about 95% of the plants were established successfully. The conditions optimized in this study for inducing multiple shoots could be best suitable for development of transgenic L-604 variety for agronomically important traits.