Strength Murashige And Skoog Medium Research Articles

Otimização da micropropagação de maçã silvestre ‘Zi Dieer’ através da proliferação de brotos axilares

ABSTRACT: Malus is an important ornamental plant around the world and widely used in commercial production due to its diversity and wide adaptability. ‘Zi Dieer’ crabapple has good adaptability to a wide temperature range (high and low), disease resistance, and high commercial value. To satisfy the market demand, we established ‘Zi Dieer’ crabapple micro-regeneration system for large-scale reproduction. Induced buds were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP: 0.5, 1.0, 1.5 mg/L) and 1-naphthaleneacetic acid (NAA: 0.05, 0.1, 0.3 mg/L) for 35 days. The highest bud induction rate (93.3%) was observed on MS medium supplemented with 1.0 mg/L BAP and 0.1 mg/L NAA, with average micro-shoot length of 1.32 cm. The germinated buds (1 cm in length) were propagated and cultured on MS medium supplemented with BAP (1.0, 1.5, 2.0 mg/L) and NAA (0.1, 0.3, 0.5 mg/L), after 35 days, on the MS basal medium supplemented with 1.5 mg/L BAP and 0.3 mg/L NAA, the best average number of new shoots is 8.56±0.13 (P<0.05) and good shoot length (4.94±0.09 cm), reproduction coefficient higher than MS medium (0) without PGRs. In order to induce rooting, the shoots (length 2 cm) are inserted in half strength MS (1/2 MS) medium containing 0.1, 0.3 and 0.5 mg/L of 3-indole butyric acid (IBA) or NAA, and 1 g/L activated carbon (AC) was added on the basis of NAA 0.3 mg/L to study the effect of activated carbon on the rooting of crabapple. After 38 days of culture, compared with other treatments, among the explants treated with IBA, the rooting rate was higher in the 1/2 MS medium containing 0.1 mg/L (82.1%), and the highest root number (11.29±0.52) was observed, and the root length was the other treatments 2-3 times. Although, the highest rooting rate was observed on 1/2 MS medium containing 0.1mg/L NAA (87.8 %), but the bottom of these buds formed large callus, which was not conducive to survival, and the root length was short. In summary, a feasible and complete rapid propagation program for ‘Zi Dieer’ crabapple has been developed.

Establishment of An in Vitro Mass Propagation System for Dendrocalamus asper.

Dendrocalamus asper is a species of bamboo that has high commercial value and is the bamboo of choice for large scale agro-forestry plantations in the tropical regions of the world. Micropropagation using tissue culture is essential to generate uniform clones that are amenable to establishment in industrial agro-forestry projects for bamboo biomass, habitat restoration or in carbon sequestration. This paper reports on the establishment of D. asper invitro using commercially available seeds. The seeds were surface sterilized using three different chemical agents which were Sodium Hypochlorite (20%), Mercuric Chloride (0.1%) and Ethanol (70%) followed by shoot initiation on Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) with a concentration ranging from 1.0 - 5.0 mg/L. Propagules were multiplied on MS media supplemented with different concentration of IBA Indole-3-Butyric Acid (IBA) and Naphthalene Acetic Acid (NAA), and finally rooted and hardened in peat moss. The findings of our study indicate that the sterilization protocol eliminated all the plant pathogens, resulting in an axenic culture. Full strength MS medium supplemented with 5 mg/L BAP yielded the highest number of shoots (11.46 per explant) after four weeks of inoculation. The highest multiplication rate (3.95 shoots per explant) was obtained on MS medium supplemented with 3 mg/L BAP. The time required from initiation to hardening was 70 to 90 days, following which the plantlets were ready for field trials. The findings of this study will facilitate the establishment of plant tissue culture programmes dedicated to the production of D. asper locally, thus eliminating the need for imports and the possible entry of plant pathogens that can be detrimental to the local agro-forestry industry.

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant
 Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and BAP (3.0 mg/L) in combination with IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively.
 Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded.
 Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period.

Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass.

The present study describes an efficient protocol for the micropropagation of Centratherum punctatum Cass. through transverse thin cell layers (tTCLs). The effect of plant growth regulators (PGRs), thickness of tTCLs, and source of tTCLs were evaluated. The tTCLs of varying thickness (0.5 to 5.0 mm) were excised from 10-d-old leaf and 45-d-old node and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of PGRs (BAP, TDZ, KN; 0.1 to 2.0 mg L−1), alone or in combination with NAA (0.1 to 1.0 mg L−1) for shoot induction. The leaf and node tTCLs responded with direct shoot regeneration. A significant effect of thickness or width on shoot induction was observed. For leaf, 1.0-mm-wide and for node 2.0-mm-thick tTCLs showed a maximum response. MS medium supplemented with 1.5 mg L−1 TDZ and 1.0 mg L−1 BAP in combination with 0.2 mg L−1 NAA was found to be optimum for shoot induction from tTCLs of leaf (96% response with 41.5 shoots per explant) and node (87% response with 11.9 shoots per explant), respectively. Rhizogenesis was obtained when micropropagated shoots were transferred to half strength MS medium supplemented with various concentrations (0.25 to 2.0 mg L−1) of IBA. The rooted plantlets were eventually acclimatized and transferred to soil. The clonal fidelity assessment by SCoT markers of the mother plant and micropropagated plants revealed their genetic similarity. Chlorophyll content estimation indicated lower levels in in vitro plants, which increased to a comparable level to that of field-grown plants after 3 mo of acclimatization, showing absence of stress during acclimatization. The procedure described here is a promising tool for micropropagation of C. punctatum as it produces high-frequency healthy shoots from a minimum explant source.

Optimization of Hormonal Compositions of Media for In vitro Propagation of Apple (Malus ×domestica Borkh.) Cultivars

Background: Apple (Malus × domestica Borkh.) is one of the most important fruit crops in the world. Traditionally, vegetative propagation methods (including cutting, budding, and layering) are time-consuming (about three years), with low production rates and low success in obtaining virus-free plants. Objective: The present study was planned to investigate the in vitro propagation of apple (M. domestica) cultivars from nodal segments. Methodology: The Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of plant growth regulators (PGRs) were used for shoot proliferation and root induction. The optimal concentrations of PGRs in the MS medium were assessed. The effect of full and half-strength MS medium on root induction was investigated. Results: Examination of the effects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin revealed that the significantly highest shoot response was recorded for the ‘Princess’ cultivar with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), and leaf number per shoot (6.15) after four weeks of culture. The root induction in micro shoots of three apple cultivars was observed after 20 days of culturing. The strength MS medium (full and half) containing 1.5 mg/L IAA significantly affected (at P<0.05, chi-square test) root induction in all three apple cultivars, especially rooting rate. However, there was no significant difference in root number and root length per micro shoot among the apple cultivars. Among the cultivars, significantly the highest rooting rate(48.30%), root number (6.25), and root length (4.15cm) were recorded for cultivar ‘Princess’ on full-strength MS medium. Conclusion: PGR combination of IAA (1.0 mg/L) and kinetin (3.0 mg/L) was found to be the best for shoot proliferation. The shoot responses were found to increase with an increase in kinetin concentration combined with IAA at 1.00 mg/L.

A standardized protocol for genetically stable artificial seed production of Ficus religiosa L. using ISSR fingerprinting

Ficus religiosa LQuery. has several ornamental, medicinal, and economical applications. The in vivo propagation of this species has shown various limitations. Due to this reason, the present study efforts on genetically uniform artificial seed production from in vitro developed shoot tips of this species. The in vivo shoot tips were cultivated on Murashige and Skoog (MS) media containing different growth regulators. The maximum shoot response (93.67%) and the longest shoot length (3.85cm) were exhibited with 0.5mg L-1 6-furfuryl-amino purine (Kn), 0.2mg L-1 benzyladenine (BA) and 0.1mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) in combination. A treatment of 3% sodium alginate and 75mM calcium chloride having a polymerization time of 15min was exhibited to be superior for artificial seed production of these in vitro grown shoot tips. Artificial seed-derived micro shoots yielded the highest root response (94.44%) and roots per shoot (4.61) with 0.5mg L-1 indole-3-butyric acid (IBA) and 0.1mg L-1 BA in combination on ½-strength MS media. In comparison to 4°C-kept artificial seeds, 24°C-stored artificial seeds had superior germination potential across all storage times. The soil:organic manure (1:1) generated 90% of plantlet survival after 28days of primary hardening than other mixtures tested. The secondary hardening displayed 92% of plant survival after 60days. The banding patterns of ISSR analysis between the mother plant and hardened plants were discovered to be monomorphic. This methodology provides a promising and affordable approach to the large-scale plant productionof this significant species.

STANDARDIZATION OF MICROPROPAGATION TECHNIQUE OF MULBERRY (MORUS ALBA L.)

A standardization of protocol for rapid multiplication of mulberry (Morus alba L.) was established in vitro using nodal explant excised from hydroponically grown mature mulberry plants. Shoot initiation was induced on Murashige and Skoog (MS) medium supplemented with different concentrations of (BAP) 6- Benzyl amino purine. Effect of different concentration of 6- Benzyl amino purine was studied on multiplication of shoot, revealed that elongated shoots (2-3 cm) were cultured on half strength MS medium supplemented with (IBA) indole-3-butyric acid. Higher percentage of shoot initiation (90%) was observed on MS medium supplemented with 2 mg/l 6-bezyl amino purine. Initiated shoots gave a higher average number of shoots and elongation was observed when it was subcultured on MS medium supplemented with 1.4 mg/l 6-bezyl amino purine. A maximum percentage of root formation (70%) was observed when elongated shoots were subcultured on half strength MS medium containing 1.3 mg/l indole-3-butyric acid. Rooted plants transferred to pots containing sterile FYM, garden soil and sand (1:1:1) for acclimatization in green house showed (75%) survival capacity.

<i>In vitro </i>Propagation of <i>Carica papaya</i> L. Variety ‘Horana Papaya Hybrid 01’ Using Shoot Tip

In vitro plantlet regeneration ability of the local hybrid papaya variety Horana Papaya Hybrid 01 was evaluated using shoot tips from greenhouse-raised seedlings. Shoots tips surface sterilized in 20% Sodium Hypochlorite (Clorox®) for 20 min were established in Murashige and Skoog (MS) basal medium containing 6-Benzylaminopurine (BAP; 0.0,0.5,1.0,1.5 mg/L) in combination with 1-Naphthalene Acetic Acid (NAA; 0.0,0.1,0.5 mg/L) for shoot multiplication. Considering the high mean number of shoots per explant (4.8 ± 0.5) and absence of calli, 1.0 mg/L BAP was the best treatment for direct organogenesis. Proliferated shoots were transferred to a 1.5 strength MS medium containing 0.25 mg/L BAP and Gibberellic Acid (GA3; 0.0, 0.15, 0.30 mg/L) for further elongation. Elongated shootlets were placed in half-strength MS medium supplemented with Indole-3-Butyric Acid (IBA; 0.0,1.0, 2.0, 3.0 and 4.0 mg/L) for rooting. The highest root induction response (86%) and roots suitable for acclimatization were observed with 2.0 mg/L of IBA. Plantlets were acclimatized in poly cups containing a sterile potting mixture (soil, sand, compost, and coir dust; 1:1:1:1) and a survival rate of 75% was achieved under in vitro conditions. The findings of the present study can be optimized to develop a suitable in vitro micropropagation protocol for rapid clonal propagation of this papaya variety for producing true to type planting material.

Effect of Plant Growth Regulators on in Vitro Propagation of Paris Polyphylla

ABSTRACT In vitro propagation using plant growth regulators (PGRs) was evaluated in Paris polyphylla. Thin cell layers (0.5–1 mm) of rhizomes (explants) were sliced and inoculated on half-strength MS (Murashige and Skoog) medium with 3% (w/v) sucrose and solidified with 0.3% (w/v) clerigel. In this medium, 50% of the explants were treated with (0.5 µg mL−1) activated charcoal (AC) and 50% without AC. The half strength MS medium was supplemented with different concentrations of 6-benzylaminopurine (BAP) either individually or in combinations with auxins. The optimal percentage (80%) of shoot response was induced by the synergistic action of BAP (0.5 µg mL−1) and α-naphthalene acetic acid (NAA) (0.2 µg mL−1) in the medium containing AC. After sub-culturing, the highest number (4.00 ± 1.00) of shoot buds per explant was achieved in the MS medium with AC, which was augmented with a combination of BAP (0.5 µg mL−1) and NAA (0.2 µg mL−1). The medium containing AC showed greater shoot induction than the non-AC medium. Up to 94.4% of the regenerated shoots successfully established in the greenhouse soil.

In vitro propagation of an economically important medicinal plant Lawsonia inermis L. through nodal segments

The present investigation aimed to standardize efficient plant regeneration protocol through in vitro culture by using nodal segment for mass multiplication of Lawsonia inermis an economically important medicinal plant species. Mass multiplication of shoots induced on Murashige and Skoog (MS) medium supplemented with different growth regulators like auxins and cytokinins separately and in different combinations. The medium fortified with 6-Benzylaminopurine ( BAP) 1.0 mg/l + kinetin (KN) 1.5mg/l explained best compared to all other combinations. In vitro raised plantlets were excised and transferred in half strength MS medium supplemented with different growth regulators like Indole Butyric acid ( IBA) and naphthalene acetic acid (NAA ) (0.5-3.0 mg/l) in an experiment that gave rise to rooting. The half strength of MS medium additive with IBA in separate and in different combinations with NAA concentrations (0.5-3.0 mg/l) supported root development. The best response of rooting was obtained on half MS medium fortified with 1.0 mg/l IBA. The regenerated plantlets were successfully transplanted to pots. Regenerants were transferred to the field conditions and recorded the survival rate.. Among all the carbon sources and gelling agents used, sucrose (3%) in combination with 0.8 per cent agar-agar has proved significantly better. Multiple shoots formation with longer shoots were achieved on medium with 1.0mg/l BAP and 1.5mg/l Kn. Thus, it is possible to develop a large number of plants of L. inermis through shoot bud regeneration which can cater for the need of pharmaceutical as well as other industries.

In Vitro Micropropagation of Andrographis macrobotrys

ABSTRACT In vitro propagation method was developed for Andrographis macrobotrys for large-scale multiplication. Nodal explants were cultured on Murashige and Skoog (MS) medium containing cytokinins including 6-benzyl amino purine (BAP), kinetin (KN), 2-isopentyl adenine (2-iP) and thidiazuron (TDZ) at 2.5, 5, 7.5 or 10 μM L−1. Multiple axillary shoots (32 shoots per explants) were regenerated from nodal explants on MS medium supplemented with 7.5 μM L−1 BAP. Quarter strength MS medium supplemented with 5 μM L−1 indole-3-butyric acid (IBA) was effective for induction of roots from the regenerated shoots and on this medium optimal of 49 roots were regenerated from shoots. Micropropagated plants were transplanted to pots containing soil and coco-peat (1:1) mix and maintained in growth chamber at 25 ± 2°C, relative humidity 80%, irradiance of 100 µmol m−2 s−1 for 16-h photoperiod. After eight weeks, plants were transferred to field where soil was mixed with farmyard manure and sand.

Estimation of endogenous indole-3-acetic acid in leaf explants and a protocol for direct regeneration in Lilium cv. Pavia

A method for regeneration via direct organogenesis in Lilium cv. Pavia using leaf primary explants has been established. Outer leaves from in vitro plantlets of 10 to 12 cm were used as the primary explant for direct organogenesis. Shoots were directly regenerated from leaf explants within 25 d of culture on Murashige and Skoog (MS) augmented with 4.5 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 5.4 μM α-naphthaleneacetic acid (NAA), and 2.2 μM 6-benzylaminopurine (BAP). Different regions of the same leaf explant exhibited varied responses. The acropetal 3 to 4 cm of the leaf (L1) produced the maximum number of shoots (75%) indicating its high regeneration ability whereas the response of middle portion (L2) and basipetal portion of the leaf (L3) exhibited a significantly decreasing trend of shoot induction with 37.5 and 12.5%, respectively. HPLC analysis of endogenous indole-3-acetic acid (IAA) concentration established significantly higher concentration in the acropetal segments (L1). The in vitro regenerated plants were rooted in half strength MS media and were successfully hardened and field transferred. Our study established that different regions of the same leaf produce significantly different responses for shoot induction and this is attributed to the variable levels of endogenous IAA. This quick method of direct organogenesis can be applied for commercial multiplication of this important hybrid.

TDZ induced plant regeneration from immature cotyledons of Pterocarpus marsupium Roxb. and validation of genetic homogeneity using ISSR markers

Pterocarpus marsupium Roxb. (Indian kino) is a threatened leguminous tree species well known for its therapeutic properties. A genetically stable plant regeneration system in P.marsupium has been developed using immature cotyledon (IC) explants excised from 12-days old in vitro grown seedlings. ICs were cultured on Murashige and Skoog (MS) media augmented with various concentrations of plant growth regulators to induce multiple shoots. Among the various concentrations and combinations used, MS medium augmented with thidiazuron (TDZ; 4.45 µM) and naphthalene acetic acid (NAA; 1.07 µM) showed the highest regeneration frequency (95.83%) with a maximum number of adventitious shoot buds (25.4 ± 0.55) with 22.7 shoots per explant. The harmful effects of extended exposure to TDZ at long-term regimen was prevented by transferring the organogenic cultures to MS medium fortified with 6- benzylaminopurine (BAP) for further multiplication and elongation. The highest number of shoots (36.0 ± 0.74) and shoot length (5.8 ± 0.10) were observed on 4.44 µM BAP augmented media after the third subculture. The elongated shoots were treated for 24 h with 19.60 µM indole-3-butyric acid (IBA) solution and rooted on half strength MS medium amended with 2.85 µM IBA. The plantlets were acclimatized successfully in the greenhouse with a 90% survival rate. Genetic homogeneity of regenerants were analyzed by inter simple sequence repeat (ISSR) markers. The comparative amplified products across the in vitro developed plantlets and the mother plant showed good genetic consistency. The protocol mentioned herein would be beneficial for raising genetically uniform plantlets and commercial cultivation of P. marsupium.

In vitro propagation and DNA barcoding of the rare medicinal plant Aerva Lanata (L.) Juss. ex Schult grown in Gebel Elba

Aerva Lanata (L.) Juss. ex Schult is an important medicinal multipurpose plant that belongs to the family Amaranthaceae. An efficient protocol for In vitro propagation of A. Lanata was established. Nodal segment explants collected from mature shrubs from the Gebel Elba, South Egypt. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyl adenine (BA) (2.22, 4.44, 8.90 and 17.80 M) and Kinetin (Kin) (2.32, 4.60, 9.30, and 18.6M) individually for shoot induction. The highest shoot induction percentage (93.33%) with maximum number for shoots (4.07 shoots per explant) was noticed on MS medium supplemented with 4.44 M BA. The maximum number of multiple shoots per explants (9.13) was obtained in the combination BA (4.44 M) and NAA (2.14 M). For root induction, the highest rooting percentage (80%) with mean root number (4.33 roots per shoot) and length (3.83 cm) of roots were noticed on half- strength MS medium augmented with 4.90 M IBA. Rooted plantlets were transferred into plastic pots containing sand, peat moss and garden soil mixture at equal volumes, and successfully acclimatized in the greenhouse with a survival rate of 73.3%. Plant identification is a crucial aspect to understand and conserve plant diversity from extinction. DNA barcode analysis of A. Lanata was carried out using. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scal production of this important medicinal plant. We therefore, used the rbcL gene to identify A. lanata. Maximum likelihood tree analysis was performed to evaluate the discriminatory power of the rbcL gene. Our findings showed that using rbcL gene sequences enabled identifying the majority of the samples (90%) to the genus and species level.

Productive method for commercial propagation through direct embryogenesis and organogenesis in Ledebouria revoluta—an underutilized medicinal plant with cardioprotective properties

The bulbaceous herb Ledebouria revoluta (family Asparagaceae) is an important cardioprotective plant. Two different pathways of in vitro direct regeneration system consisting of somatic embryogenesis and shoot organogenesis have been established for high-frequency micropropagation. A rectangular piece (6 × 10 mm2) of leaf lamina was used as an explant to study the morphogenetic regulation by exogenously supplied auxins, cytokinins, and polyamine within controlled in vitro environment. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose and various concentrations and combinations of plant growth regulators (PGRs), as per experimental needs. Both β-naphthoxyacetic acid and α-naphthalene acetic acid (NAA) auxins individually induce somatic embryogenesis. Combined application of auxins and cytokinins further improves the embryonic response. The synergistic effect of NAA (2.0 mg L–1), kinetin (1.0 mg L–1), and spermidine (SDN, 217.88 mg L–1) provided the best result with 96.7 ± 1.8 % response and highest 63.4 ± 0.8 embryos per explant. The highest (94.5 ± 2.5 %) conversion of embryos to complete plantlets was noted in presence of 1.0 mg L–1 kinetin. The morphogenic path altered from embryogenic to organogenic when auxin was totally withdrawn, and a higher concentration of cytokinin was applied. The synergism between 6-benzylaminopurine (3.0 mg L–1) and SDN (217.88 mg L–1) proved the best PGR combination for the direct shoot (or bulblet) organogenesis with maximum 96.6 ± 2.2 % response and highest 48.6 ± 0.5 bulblets per explant. Total withdrawal of auxin was essential for direct bulblet organogenesis because the presence of any auxin always shifts the morphogenic path from organogenic to embryogenic. Histological study confirms the bipolar structure of somatic embryo and unipolar bulblets. Highest 12.7 ± 0.54 roots per bulblet were induced on ½ strength MS medium containing 2.0 mg L–1 indole-3-butyric acid. Finally, the plantlets were acclimatized with 96.7 % survival rate and all of the survived plants produced bulbs and 92.5 % of them flowered normally in field conditions. This in vitro system permits a high-frequency regeneration through embryogenic and organogenic paths, which can be used for large-scale commercial micropropagation for round-the-year supply of this important cardioprotective medicinal plant.

Micropropagation protocol for Stevia rebaudiana through axillary shoot proliferation

The present investigation was carried out at Anand Agricultural University, Gujarat during the year 2016 to develop a micropropagation protocol for mass multiplication of medicinally rich stevia plants. Axenic culture was established by sequential application of 200 mg/l Kanamycin, 200 mg/l Carbendazim-50% and 1000 mg/l HgCl2. Out of two different basal media, viz. Gamborg (B5) and Murashige and Skoog (MS) used, full strength MS gave highest (98%) culture establishment. Out of three different basal media, viz. Gamborg’s (B5), Linsmair and Skoog (LS), Murashige and Skoog (MS) and four different cytokinins, viz. 6-benzylaminopurine (BAP), Kinetin (Kn), Thiadiazuron (TDZ), Zeatin (Zn) tested for multiple shoot induction, full strength MS medium and 2 mg/l BAP, respectively, promoted maximum shoot formation. In the experiment to study the effect of varying strength of MS media and various plant growth regulators, viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), Naphthalene acetic acid (NAA) on in vitro rooting, ½ strength MS and 1 mg/l NAA, respectively, gave superior response. Among the 26 different combinations of potting mixtures used for primary acclimatization of in vitro rooted plants, cocopeat based substrates yielded highest survival rate (75.06%). Secondary acclimatization was carried out in the soil bags in the poly house. Thus, a reproducible and reliable micropropagation protocol for mass multiplication of Stevia rebaudiana (Bertoni) Bertoni using nodal segments has been developed.

Effect of Cytokinins on In Vitro Propagation of Ajuga xylorrhiza Kit Tan (Critically Endangered), Endemic to Turkey

Ajuga xylorrhiza Kit Tan is a rare endemic perennial species growing in the stony habitats in Southeastern Anatolia region, Turkey. An efficient and reproducible protocol has been developed for micropropagation of endangered Ajuga xylorrhiza. Shoots were cultured on Murashige and Skoog (MS) basal medium, supplemented with various concentrations of 6-benzyl amino purine (BAP), thidiazuron (TDZ), and kinetin (Kin). The optimal shoot formation was with the medium containing 0.125 mg L−1 BAP (7.91 shoots per explant), and 0.25 mg L−1 Kin (4.9 shoots per explant) was recorded. The best result in terms of shoot length (3.43 to 3.63 cm) was obtained from the Kin treatments. Kin parameters were more effective on shoot length and multiple shoot production than BAP and TDZ. For rooting, regenerated shoots were transferred to 1/4 strength MS medium containing two different concentrations of indole acetic acid (IAA) and α-naphthalenetic acid (NAA) at 0 (control), 0.5, or 1.0 mg L−1.The best results in terms of the number of roots per shoot were obtained from the medium supplemented with 0.5 mg L−1 IAA (3.10 roots per explant). Rooted plantlets were transferred to soil for acclimization with 100% success rate.

Micropropagation and essential oil characterization of Plectranthus amboinicus (Lour.) Sprengel, an aromatic medicinal plant

Plectranthus amboinicus (Loureiro) Sprengel is a therapeutically valued herb highly sought by various industries. This has led to its uncontrolled harvest from the wild leading to the risk of extinction. As an alternative, commercial propagation of P. amboinicus is desirable to avoid further depletion of wild populations. This paper describes the establishment of an efficient protocol for the multiplication, rooting and acclimatization of P. amboinicus. The greatest frequency of shoot multiplication of P. amboinicus was on semi-solid Murashige and Skoog (MS) medium supplemented with 0.4 mg L−1 6-benzylaminopurine (BAP). Media supplemented with higher concentrations of BAP or used in combination with gibberellic acid (GA3) produced abnormal plantlets. Optimal rooting was observed on plantlets cultured in half strength MS medium. Acclimatization was achieved by transferring the rooted plants onto sterile peat-moss moistened with MS medium and exposed to the glasshouse environment gradually over a period of 2 months. P. amboinicus responded readily to tissue culture, but careful attention was required to media formulation to avoid abnormal plantlet development. The composition of essential oils extracted from field grown plants and in vitro microshoots were comparable.

Improved Culture Media for Embryogenic Callus Generation in Sorghum [Sorghum bicolor (L.) Moench

Many attempts on optimization of sorghum [Sorghum bicolor (L.) Moench] tissue culture induction media have been made, but the culture system remains with some bottlenecks compared to that of other crops. This study aimed at assessing the suitability of various induction media to produce embryogenic callus (yellow and friable) with high induction rates and reduced phenolic exudation. The six culture medium modifications: 3 based on Murashige and Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes. Although there was a genotype influence on the attainment of a yellow callus, friability of the callus was determined to be dependent on the culture medium and not the genotype. Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH2PO4, 1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO4·5H2O (type E) was found to be the most effective resulting in about 60% yellow coloured callus induction with 25% friability. Addition of CuSO4·5H2O, KH2PO4, L-proline and L-asparagine significantly reduced the phenolic production. Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds. The half strength MS medium was also observed to suppress phenolic production. Medium 190-2 produced the highest regeneration frequency (40%) among the 3-regeneration media tested. The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.

Callus induction and high frequency organogenesis in saffron (Crocus sativus L.)

Saffron (Crocus sativus L.) is an economically important plant with a very slow natural multiplication rate and plant tissue culture is expected to enhance its reproductive capacity. In present study, corm explants were used for micro-propagation. Auxins and cytokinin were found important for in vitro propagation of explants. Overall, 93% calli were induced from meristematic region of corm explants propagated on Murashige and Skoog (MS) medium supplemented with 2 mg L−1 2,4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg L−1 kinetin (Kn), 2% sucrose and 100 mg L−1 ascorbic acid after 6 weeks inoculation. Somatic embryogenesis was observed on half strength MS medium supplemented with 2 mg L−1 indole-3-acetic acid, 2 mg L−1 Kn and 100 mg L−1 ascorbic acid. Organogenesis from calli was observed with 89.6% shooting on MS medium containing 2 mg L−1 Kn and 2 mg L−1 IAA. Adventitious rooting of 91.6% calli was obtained on MS medium supplemented with 2 mg L−1 indole-3-butyric acid. There are plenty of protocols available for tissue culture of saffron but this protocol provides an easy and time efficient method for micropropagation of saffron in vitro.