Half-strength MS Medium Research Articles

In vitro rooting and hardening of clonal cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens)

A technique for in vitro rooting and hardening of Gisela 5 (Prunus cerasus x Prunus canescens) cherry rootstock has been developed. In vitro shoot cultures maintained by routine subculturing were used as source of microshoots for rooting experiments. In single step rooting procedure, maximum rooting (100%) was achieved on full strength MS medium fortified with 0.5 mg/l IBA with thin, long roots devoid of callus. In two step procedure of rooting, maximum rooting (60.00%) was observed after 24 hours dark incubation in half strength MS broth fortified with 0.1 and 0.4 mg/l IBA, followed by transfer to semisolid half strength MS basal medium. Addition of activated charcoal (0.4%) in half strength MS medium fortified with 0.5 mg/l IBA showed 40.06% rooting without callus, whereas in phloroglucinol (0.1%) supplemented medium only callus was observed at the bases of the shoots which did not lead to root formation. The in vitro rooted plantlets were successfully hardened in sand: soil: FYM (1:1:1) with 100 per cent survival in the month of August. After two months these plants were transferred to bigger pots containing sand: soil: FYM (1:1:1) and are showing normal growth without any morphological variations.

Optimization of in vitro Propagation of Purple Passion Fruit (Passiflora edulis), an Important Medicinal and Ornamental Plant

Tissue culture techniques can be beneficial for quick clonal propagation and production of disease-free plants of purple passion fruit as one of the most important medicinal and ornamental plants. These techniques are essential tools for the production of transgenic plants and high-value phytochemicals. The present study was planned to introduce an efficient in vitro propagation for purple passion fruit(Passiflora edulis Sims.). To do so, the effect of different plant growth regulators was investigated on micropropagation medium of purple passion fruit. For proliferation stage, nodal segments were cultured in media supplemented with various combinations of different plant growth regulators including: BA (0, 2.2, 4.4 and 8.9 μM), TDZ (2.3, 4.5 and 9.1 μM), GA3 (0 and 2.9 μM) and IBA (0 and 0.5 μM). For rooting, shoots with about 1.5 cm long originating from explants were removed and cultured in half-strength MS medium containing different concentrations of auxin-based plant growth regulators including: IAA (0, 1.1, 2.9, 5.7 and 11.4 μM), IBA (1, 2.5, 4.9 and 8.9 μM), and NAA (1.1, 2.7, 5.4, 10.7 μM). For the proliferation stage, the best plant growth regulator combination was 8.9 μM BA+2.9 μM GA3+0.5 μM IBA, resulting in the maximum shoot proliferation, number of shoots per explants, and shoot length. Half-strength MS medium supplemented with 5.4 μM NAA or 8.9 μM IBA was the most effective treatment for the rooting of shoots. Gradual acclimatization of the rooted plantlets was performed and the plantlets were established in the soil successfully. The micropropagated plants did not exhibit any visually detectable variation to their mother plants.

Lotus maritimus L.'nin (Leguminosae) in Vitro Rejenerasyon Potansiyeli

To investigate the regeneration potential of Lotus maritimus L., the stem, hypocotyl, cotyledon, root, and epicotyl were cultured in MS media containing different combinations of α-naphthalene acetic acid (NAA) concentrations (1, 2, and 4 mg L-1) and cytokinin types [0 (control), 1 mg L-1 of 6-Benzilaminopurin (BAP), and 0.5 mg L-1 of kinetin]. The highest callus induction was achieved with the stem and epicotyl explants at 1 or 2 mg L-1 concentrations of NAA plus 0.5 mg L-1 of kinetin. The best plant regeneration values (3.56 and 3.20 shoots per callus) were determined with the epicotyl and hypocotyl cultured in MS media containing 1 mg L-1 of NAA. The obtained shoots were easily rooted in half-strength MS media containing 1 mg L-1 NAA and substantially adapted to the external environment.

In vitro: Influence of Various Concentrations of Plant Growth Regulators (BAP & NAA) and Sucrose on Regeneration of Chenopodium quinoa Willd. Plant

In vitro: regeneration of Chenopodium quinoa Willd. was achieved from cotyledonary nodes explants. In this study, used 6-Benzylaminopurine (BAP) and α-Naphthalene Acetic Acid (NAA) of plant growth regulators with different concentrations individually as well as in combination and used different concentrations of sugar (sucrose) with different concentrations. For was rooting, used half strength (½MS), full-strength MS and ½ MS supplemented with 0.2 mg/l of NAA. The results mentioned, explant responding (%) to multiplication was about 73% for all BAP treatments compared with control and average numbers of shoot increased with increased BAP concentration except 5 mg/l of BAP. The highest explant responding (%) was in media supplemented BAP without NAA compared other treatments noted that the media with combination of BAP and NAA gives formation of callus in bases of the plantlets. Also, the result inducted the combinations between (BAP–NAA) was highly significantly (P≤ 0.001) and less effective on number of shoots where the highest number of shoot was 3.40 in media with 3 mg/l BAP compared other treatments. The highest of explant responding 93.33% was in media supplement with 10 g/l sucrose and (10 g/l sucrose + 3 mg/l BAP), but sucrose level for good greening and developed shoots (4 shoots) was in medium supplement with 10 g/l sucrose. The shoots rooted well on half-strength MS medium with 60% percentage of root. The rooted shoots were acclimatized and transferred to green house to follow their development.

In vitro organogenesis and plant regeneration of Thymus serpyllum L.: an important aromatic medicinal plant

An efficient in vitro propagation system has been developed for the rapid micropropagation of Thymus serpyllum L. (Banajwain), an aromatic medicinal herb from nodal explant on MS medium. Phenolic leaching and high rate of contamination was the most significant problem in establishing in vitro culture of Thymus serpyllum which was overcome by preparing explants in an antioxidant ascorbic acid (1000 ppm) at 6°C for 45 min and addition of the same antioxidant (50 mgl−1) to the MS medium. The frequency of shoot production was influenced by different cytokinins (Kn, BAP, and Kn + BAP) and 95.56% shoot induction was observed when MS medium was supplemented with 1.0 + 2.0 mgl−1 (Kn + BAP). The maximum average number of shoots 16.93 ± 2.15 and average length (3.98 ± 0.55) was recorded when MS medium have 0.5 + 2.0 mgl−1 (Kn + BAP). The in vitro regenerated microshoots were rooted on MS and half strength MS medium and there was significant difference in root induction on both media under the influence of auxins (IAA, IBA, and NAA). The maximum average number (11.67 ± 3.03) and average root length (3.88 ± 0.71) was reported in half MS medium having 1.0 mgl−1 IBA. The complete regenerated plantlets were acclimatized under growth chamber before transferring to the earthen pots and showed 90% survival.

Evaluation of root growth, antioxidant enzyme activity and mineral absorbability of carnation (Dianthus caryophyllus “Express golem”) plantlets cultured in two culture systems supplemented with iron nanoparticles

In this study, different concentrations (0, 0.025, 0.05, 0.075, 0.1 and 0.2 mM) of iron nanoparticles (FeNPs) were used to replace 0.1 mM Fe-EDTA in half-strength MS medium to assess their effect on root growth, antioxidant enzyme activity and mineral absorbability of carnation plantlets cultured in in vitro system (MO) and microponic system (MP). The results showed that MP supplemented with 0.075 mM FeNPs was the most optimal culture condition for not only rooting stage, but also antioxidant activities and mineral absorption of carnation. The 3-week-old shoots cultured in this condition obtained the highest growth values with plant height (6.67 cm), root number (9.00 roots/plantlet), average root length (1.80 cm), fresh weight (70.33 mg), dry weight (5.67 mg), dry mass ratio (8.05 %), chlorophyll a (1.96 mg g−1 FW) and chlorophyll b (0.87 mg g−1 FW). Furthermore, the antioxidant activity analysis results showed that the activity of superoxide dismutase (28.52 U g-1 prot), catalase (540.98 U g-1 prot), and ascorbate peroxidase (0.55 U g-1 prot) in plantlets grown in MP system supplemented with 0.075 mM FeNPs were higher than those in MO system (14.15, 426.66 and 0.46 U g-1 prot; respectively) and also in the control (7.84, 309.08, 0.35 U g-1 prot; respectively). In addition, the absorption of potassium (205.51 mg g−1 DW), calcium (27.70 mg g−1 DW), magnesium (25.55 mg g−1 DW) and the iron-uptake rate (79.37 %) were highest in the MP system added with 0.075 mM FeNPs, as compared with those of the others. Plantlets derived from culture in MP system supplemented with FeNPs gave higher survival rate, better growth and development than that of the control treatment (used 0.1 mM Fe-EDTA). The plantlets were investigated after six weeks transplanting into the greenhouse.

Foliar micro-morphology: a promising tool to improve survival percentage of tissue culture raised plantlets with special reference to in vitro propagation of Vitex negundo L.

The in vitro induced anomalies during micropropagation affect acclimatization process of the regenerants, which influence the survival success of tissue culture raised plantlets. The comparative foliar micro-morphological analysis of in vitro developed; ex vitro adapted and field transferred plantlets could help in understanding the acclimatization of micropropagated plants. An enhanced shoot regeneration system for Vitex negundo L. was developed through axillary shoot regeneration and ex vitro rooting methods in present study. Murashige and Skoog’s medium (MS) incorporated with 1.5 mg L−1 6-benzylaminopurine (BAP) was recorded optimum for shoot regeneration from nodal segments. The shoots were further amplified (34.5 ± 0.21 shoots per culture vessel with 11.9 ± 0.10 cm average length) on full strength MS medium containing 0.5 mg L−1 BAP and 0.25 mg L−1 indole-3 acetic acid (IAA). The in vitro regenerated shoots were rooted under laboratory conditions (on half strength of MS medium incorporated with 1.0 mg L−1 IBA) as well as in greenhouse (ex vitro) when the shoots were treated with 300 mg L−1 IBA for 5 min. The hardened plantlets were successfully transferred to the field. The micro-morphological changes were observed in stomatal structure, index, veins and trichome density on the leaves developed in each stage of propagation of V. negundo. In vitro induced micro-morphological anomalies reduced stability of plant health and survival percentage which ultimately lead to the reduction in number of plantlets. The keen foliar micro-morphological analysis at subsequent stages assisted in developing plantlets with 100% survival success in V. negundo.

Young capitulum as important explant in in vitro mass propagation of gerbera (Gerbera jamesonii)

A new route of in vitro propagation of gerbera selected clones was successfully established using young capitula in tight buds and buds that were started to unfold stage as explant source. The one-fourth pieces of young capitula of tight flower stage and half-strength MS medium containing 0.25 mg/l BAP was the suitable for initiation and produced higher number of shoots per explant up to 3.8 shoots. The results were improved by culturing the one-fourth piece of 01.092 capitulums on MS medium fortified by 0.2 mg/l BAP and 0.02 mg/l NAA producing the highest shoot formation up to 8.5 shoots per explant with 28.7 leaves per explant and 2.1 cm leaf length. High multiple shoots were determined in third to fourth subculture periods and reduced thereafter with high multiplication rate noted on 01.092 clone. Shoots were easily rooted on half-strength MS medium supplemented with 2 g/l activated charcoal. Plantlets were transferred to ex vitro condition with 96.4% survivability of 03.045 clone using Cycas rumphii bulk and cocopeat (1:1, v/v) under spraying 1 g/l Growmore (32N:10P:10K) solution once week periodically. The route has high potential applied in qualified plantlet production for other Gerbera’s due to high shoots produced up to 35 shoots per whole young capitulum used.

CHIA SEED AS A SOURCE OF IN VITRO ESTABLISHMENT OF Salvia hispanica L. PLANTS

Technique of tissue culture for Chia (Salvia hispanica) micropropagation was achieved, this study investigated the impact of various concentrations of plant growth regulators on shoot multiplication and root induction with the Chia’s mature seed as a source explant. The highest percentage of shoot formation (80%), shoots number per explant(3.20) and shoot length(3.26 cm), were recorded on MS medium enriched with BAP(1.0 mgl-1) after eight weeks of seed culture. The optimal medium for the rhizogenesis was achieved on half strength MS medium fortified with 1.0 mgl-1 IBA after four weeks of culture, which had the highest rooting percentage (100%) with highest mean of roots number (5.6 roots per shoot) with (3.40 cm root length). The rooted plants were successfully adapted ex vitro with a survival rate of 85%.

Evaluation of explants for in vitro propagation of Citrus indica Tanaka - An Endangered Species

Of the five explants viz., shoot tip, nodal segment, leaf disc, cotyledon and root tip excised from exegenic seedlings of Citrus indica Tanaka shoot tip cultured in MS supplemented with 0.5 mg/l of BAP generated shoots within 4.74 days, exhibiting highest percentage of response (85.82%) with highest number of shoots (8.9) and shoot length (3.04 cm). On the other hand nodal segment cultured in MS supplemented with 1.0 mg/l BAP showed 80% response in 5.16 days with a shoot number of 5.41 and shoot length of 2.43 cm. Cotyledon explants inoculated on MS supplemented with1.0 mg/l of TDZ produced shoots in 20 days with the highest response of 69.88%, with 3.77 shoots per cotyledon and shoot length of 2.03cm. Viable callus was obtained from leaf disc cultured on half strength MS medium with less Ca++ with 2, 4-D 0.5 mg/l + Kn 0.25 mg/l. This callus when inoculated on half strength MS medium with Kn 1.5 mg/l showed highest shoot bud proliferation of 66.66% with 10.06 shoots per callus. Root tip explant failed to produce any shoots. In vitro raised shoots of Citrus indica when cultured on half strength MS medium supplemented with NAA (1.0 mg/l) showed 80 % rooting in 5.66 days, with highest number of roots (6.16 per shoot) and longest root (3.78cm). Ninety per cent of in vitro rooted plantlets of Citrus indica survived in open conditions.
 Plant Tissue Cult. & Biotech. 30(1): 87-96, 2020 (June)

In vitro isolation of a gamma ray induced mutant in chrysanthemum (Dendranthema grandiflora)

In the present investigation, an effort was made to develop an efficient ray floret regeneration protocol in order to isolate, purify and establish a novel gamma ray induced mutant in the form of brick red flowers in chrysanthemum (Dendranthema grandiflora Tzvelev.) cv. Tata Century (pink). Maximum survival (60.00%) and callus formation (56.60%) in minimum duration (10.20 days) were recorded when the ray florets were pre-treated with carbendazim (0.2%) + ridomil (0.2%) + 8-HQC (200 mg/l) for 2 h followed by surface sterilization with HgCl2 (0.1%) for a duration of 4 min and cultured on MS medium supplemented with BAP (4.0 mg/l) and NAA (1.0 mg/l). The maximum regeneration of microshoots (66.00%) from the ray floret induced callus was recorded on MS medium fortified with BAP (4.0 mg/l) + NAA (1 mg/l). MS medium supplemented with BAP (4.0 mg/l) + NAA (0.05 mg/l) + GA3 (0.1 mg/l) was found to be best for highest micro-shoot proliferation (80.00%). Highest rooting (87.60%) was induced after inoculating the microshoots individually on half-strength MS medium fortified with 0.5 mg/l NAA and 60 g/l sucrose. Successful acclimatization of in vitro raised plantlets was done in glass jar with polypropylene cap each filled with a mixture of sterilized cocopeat, soilrite and perlite (1:1:1 v/v) supplemented with half-strength MS inorganic salts. After 3-4 weeks of acclimatization the plantlets were successfully transferred to field conditions.

Regeneration of shoots via direct somatic embryogenesis from the leaf surface of Scaevola taccada (Gaertn.) roxb. – a climate resilient species of coastal areas

Scaevola taccada (Gaertn.) Roxb. (family Goodeniaceae) is a large littoral shrub of great economic and ecological importance. Plantlets were regenerated from the in vitro raised leaf explants via direct somatic embryogenesis in this study. Nodes were cultured on Murashige and Skoog's (MS) medium augmented with 1.5 mg L −1 6-benzylaminopurine (BAP) and the in vitro raised leaves used as secondary explants to induce somatic embryogenesis directly from the leaf surface on the MS medium supplemented with two kinds of cytokinins, namely BAP and N6-furfuryladenine (Kinetin). The highest percentage of explants (92%) responded with globular somatic embryos after 4 weeks of incubation on 2.0 mg L −1 BAP. The anatomical observations of somatic embryos (SE) revealed that continuous cell divisions lead to the formation of proembryos and followed by globular somatic embryos. The bipolar structures with the shoot apical meristems formation observed after 4 weeks of cultures. Light microscopic evaluations showed that direct somatic embryogenesis from leaf explants were nonsynchronous. Culturing of four weeks old somatic embryos (SE) on MS medium fortified with 0.25 mg L −1 BAP and 0.15 mg L −1 IAA induced substantial changes in formation of multiple shoots. It was observed that full strength MS medium retarded the germination frequency of SE, and tip browning and vitrified shoots were detected. The reduced concentrations of ammonium nitrate salt (half) in the MS medium increased number of healthy shoots (36 shoots per culture vessel). Similarly, increased quantity of calcium chloride (CaCl2) in the medium (1.5 times) alleviated tip browning of the shoots. The shoots were successfully rooted in vitro on half strength MS medium + 2.0 mg L −1 IBA with 12.6 roots per shoot. The rooted shoots were hardened in the soilrite® in the greenhouse and finally transferred to the field. After 3 months, 100% survival rate was recorded under in vivo conditions. The developed protocol could be used for genetic transformation systems in S. taccada as leaves might be the better choice of explants.

Micropropagation of sea grape (Coccoloba uvifera (L.) L.)

A method for micropropagation of Coccoloba uvifera (L.) L. has been developed through bud proliferation from surface-sterilized nodal explants collected from a 9 yr old mature tree. Ninety six percent of the explants exhibited bud development on agar-gelled Murashige and Skoog's (MS) medium supplemented with 3.0 mg L−1 6-benzylaminopurine (BAP) if incubated at 26 ± 2 °C, under 40–50 µmol m−2s−1 spectral photon flux density (SPFD) light intensity for 16/8 h light/dark photoperiod and 55–60% relative humidity. The cloned buds multiplied (12 ± 1.30 shoots per inoculum), and grew on MS medium + 1.0 mg L−1 BAP + 0.5 mg L−1 of α-Naphthalene acetic acid (NAA) and additives, 50 mg L−1 ascorbic acid, 25 mg L−1 each of adenine sulphate, citric acid and arginine. Higher concentrations of auxins in the culture medium induced callus formation. The additives improved quality of shoots as these prevented deformation of shoots. The shoots rooted (7.2 roots per shoot) on half-strength MS medium with 2.5 mg L−1 NAA. Ex vitro rooting was achieved by treating the basal parts of the in vitro shoots with 400 mg L−1 NAA (14.6 roots per shoot) and subsequent planting in pots filled with 1:1:1 mixture of soilrite® (soil conditioning mixture), cocopeat and garden soil. The cloned plantlets were acclimatized in greenhouse; 93.8% of these were survived. This tissue culture method can be applied for large scale production and sustainable conservation of C. uvifera, a vulnerable species for agroforestry of the coastal regions for windbreak and soil reclamations.

In vitro propagation, genetic stability and alkaloids analysis of acclimatized plantlets of Thalictrum foliolosum

Thalictrum foliolosum is an endemic herb known for its medicinal properties and used for various clinical applications including ophthalmic, skin disease and dyspepsia. Due to its medicinal properties, the plants are uprooted hence can be prone to extinction. In the present study, a reproducible in vitro propagation protocol has been developed using axillary shoot buds and nodal segments. Seedling derived axillary shoot buds were cultured in Murashige and Skoog’s (MS) medium supplemented with 2.24 µmol of 6-benzylaminopurine (BAP) and readily produced maximum shoot (7.2 ± 0.40) with the highest percentage of response (91.42%). Also, nodal explants (field-grown plant) developed maximum shoots (3.2 ± 0.48) on MS medium containing 4.49 µmol BAP with a combination of 0.54 µmol α-naphthaleneacetic acid (NAA). Best growth and foliage development was achieved at 2.24 µmol BAP with 0.54 µmol NAA in presence of 0.3% activated charcoal and 113.4 µmol ascorbic acid. Micropropagated shoots showed maximum percentage (63.30%) of rooting in half-strength MS medium containing 1.23 µmol indole-3-butyric acid (IBA) and acclimatized in soilrite and leaf manure (2:1) during 4 weeks. Monomorphic bands developed by random amplification of polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers confirmed the genetic stability of in vitro established plants. Additionally, HPLC analysis showed higher benzylisoquinoline (BIQ) alkaloids content in in vitro established plant root extracts. The micropropagation protocol developed in this study provides an alternative strategy for germplasm conservation and protection which at the same time can also be exploits for the production of pharmacologically active compounds. An efficient protocol for the production of genetically stable tissue-raised planting material of Thalictrum foliolosum has been developed for its conservation and pharmaceutical uses.

Genetic stability analysis using DNA barcoding and molecular markers and foliar micro-morphological analysis of in vitro regenerated and in vivo grown plants of Artemisia vulgaris L.

The current study provides an enhanced and reproducible micropropagation system in Artemisia vulgaris L. using nodal explants of mature plants. The explants were cultured on basal Murashige and Skoog’s (MS) medium augmented with 6-benzylaminopurine (BAP) and Kinetin (Kin) concentrations individually. Direct regeneration of shoots (7.7 ± 0.36 shoots per explant) from nodal meristems was attained on MS medium combined with 1.5 mg L−1 BAP alone. Sub-culturing of these cultures on MS medium supplemented with 0.5 mg L−1 BAP and 0.5 mg L−1 inddole-3 acetic acid (IAA) helped in further multiplication of shoots (75.8 ± 1.00 shoots per culture vessel). Elongated shoots were transferred to half strength MS medium contained 1.0 mg L−1 IBA for roots formation. Rooting was also stimulated from the sliced ends of the shoots via ex vitro rooting method by pulse treating the shoots with 300 mg L−1 IBA for 5 min. Well rooted plants were shifted into small paper cups having manure: sand: soil in 1: 1: 1 proportion for hardening. The acclimatized plants were shifted to the natural field conditions for complete plant growth. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) markers and universal DNA barcoding (rbcLa gene primers) were applied to analyze the genetic stability of micropropagated plants with the mother plant. The banding patterns of regenerants were found to be monomorphic in nature, representing the similarity with the mother plant and no somaclonal variations were observed. Similarly, foliar micro-morphological characters like stomata, trichome and vein pattern of leaves were studied from in vitro and acclimatized plants to focus on the developmental adaptation of micropropagated plantlets towards survival in field conditions. This study explores the highly reproducible procedure for A. vulgaris through nodal propagation without any somaclonal variations. It is the first report on foliar micro-morphological analysis at subsequent stages of in vitro propagation and genetic homogeneity of micropropagated plants with the mother plant. The current study paves the way for the development of genetically uniform A. vulgaris plants regenerated from nodal shoot segments and successful survival in field.

Somatic embryogenesis and plant regeneration in Betula platyphalla

Betula platyphylla is a native tree species in northern China that has high economic and medicinal value. We developed an efficient protocol for the induction of somatic embryogenesis in B. platyphalla from immature zygotic embryos and assessed the effects of explant type, genotype, and plant growth regulators (PGRs) on embryogenic callus induction. Among the various explants evaluated, embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid (2,4-D). Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction. On the 20 days after pollination, immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency (16.8%) when cultured on half-strength MS medium supplemented with 2.0 mg L−1 2,4-D and 0.2 mg L−1 6-benzylaminopurine (6-BA). The process of proliferation of embryogenic callus, somatic embryo formation, and subsequent plantlet conversion occurred under optimal culture conditions. When regenerated plants were transplanted to soil, 95% of them developed normally and grew vigorously. This somatic embryogenesis system required 3–4 months for the regeneration of B. platyphalla plantlets from immature zygotic embryos.

Differential Influence of Growth Regulators during Somatic Embryogenesis of Gynodioecious Papaya Varieties‘CO.7’ and ‘Red Lady

The study involved two auxins viz., 2,4-D (2,4-Diclorophenoxyacetic acid) and picloram at three different concentrations (1,2, 3 mg/L) in full strength MS media to study their comparative influence on induction of somatic embryogenesis from immature zygotic embryos of two gynodioecious varieties of papaya ‘CO.7’ and ‘Red Lady’. In papaya cultivar ‘CO.7’, 2,4-D at 2 mg/L gave the highest callus induction frequency of 90.93%, whereas comparatively higher concentration of 3 mg/L 2,4-D was found suitable for ‘Red Lady’ (87.26%). Although there was profuse callus formation, 2 mg/L 2,4-D recorded comparatively higher frequency of embryogenic calli in ‘Red Lady’ (51.67%) when compared to ‘CO.7’ (30.00%). Somatic embryo maturation was achieved upon transfer of embryogenic calli exhibiting globular stage embryos on to maturation medium (MS medium + ABA (Abscisic acid) and BAP (Benzyl amino purine) in different concentrations + glutamine 400 mg/L). In the maturation medium, the combination of 1.5 mg/L ABA and 0.4 mg/L BAP registered better conversion of the globular embryo to cotyledonary embryos than other levels. The frequency of somatic embryo germination was higher in ‘Red Lady’ (50.00%) as compared to ‘CO.7’ (31.67%) on half-strength MS medium devoid of growth regulators.

In vitro regeneration using twin scales for restoration of critically endangered aquatic plant Crinum malabaricum Lekhak & Yadav: a promising source of galanthamine

This study intended to develop a significant in vitro regeneration protocol for sustainable propagation, conservation and re-establishment of critically endangered aquatic plant species Crinum malabaricum Lekhak & Yadav (Malabar river lily). This plant is the natural source of galanthamine, the drug to treat Alzheimer’s disease. We present a scientific understanding, emphasizing the use of twin scales (separated from the large parent bulb) in direct regeneration of new shoots and proliferation of bulblets assisted by nutrients supply. The meristematic region of the bulb plate, present between the scales was activated using cytokinins to produce shoots (maximum 12 shoots per twin scale) on full strength Murashige and Skoog’s (MS) medium augmented singularly with 2.0 mg L−1 6-benzylaminopurine (BAP). Upon subculturing of shoots on diverse concentrations of plant growth regulators (BAP and IAA/NAA), BAP alone at 2.0 mg L−1 was served optimum for the better proliferation of shoots (53 shoots). The regenerated shoots were rooted in vitro on half strength MS medium fortified with various types of auxins. Highest number of roots (11.6 within 4 weeks) and bulblets (after 3 months) resulted with 1.0 mg L−1 Indole-3-butyric acid (IBA) under in vitro conditions. The rooted plants were hardened in the greenhouse and finally transferred to the natural stream with 83% survival rate. The SCoT (start codon targeted) and ISSR (inter simple sequence repeats) marker analysis of in vitro raised and mother plants confirmed the genetic stability of tissue cultured plants and the reliability of present protocol for C. malabaricum. It is the foremost report on in vitro regeneration and genetic fidelity analysis for restoration of this critically endangered aquatic plant using twin scale technique. The study could help in ex situ conservation, reintroduction and restoration of C. malabaricum population in its natural habitat. Crinum malabaricum is a critically endangered aquatic plant species and natural source of galanthamine. It is endemic to India and about 1000 plants only available in a private estate in the Western Ghats of India. In vitro regeneration protocol has been developed utilizing twin scales for conservation of this plant. It is the first report on effective in vitro propagation and bulblets formation in C. malabaricum.

A simple and efficient Agrobacterium-mediated in planta transformation protocol for horse gram (Macrotyloma uniflorum Lam. Verdc.)

BackgroundRecalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized. ResultsGerminated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 μM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T1 plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture. ConclusionsIt concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.

Stimulatory effect of copper and zinc sulphate on plant regeneration, glutathione-S-transferase analysis and assessment of antioxidant activities in Mucuna pruriens L. (DC)

The effect of heavy metals, CuSO4 and ZnSO4 on morphogenic response of cotyledonary node explants of Mucuna pruriens (L.), a fast growing tropical medicinal legume was evaluated. The climber holds economic importance in traditional as well as modern pharmaceutical areas. Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with various strength (0.5, 2.5, 4.5, 6.5 or 8.5 µM) of BA (6-benzyladenine), Kn (Kinetin) and 2-iP (2-isopentenyl adenine) were screened to establish optimum culture conditions for in vitro regeneration using cotyledonary node explant. Optimized medium (MS + BA 2.5 µM) was augmented with different concentration of CuSO4 (0.5–25.0 µM) and ZnSO4 (5–150 µM) to evaluate the effect of heavy metals on regeneration potential of the explants. Among various concentrations tested, MS medium with optimized BA (2.5 µM) along with 5.0 µM CuSO4 or 80.0 µM ZnSO4 proved beneficial, inducing 11.20 and 13.40 shoots per explant, respectively. The results demonstrated that glutathione-S-transferase (GSTs) was involved in fighting heavy metal stress, and might also have a role in in vitro regeneration. Elongated healthy microshoots on transfer to half strength MS medium produced 3.40 roots per shoot after 28 days of incubation without the necessity of exogenous auxin. The in vitro grown regenerants were hardened in Soilrite, and then established in garden and sandy soil (1:1) under natural light where they showed normal growth and development. The work highlights the enhanced multiplication and better growth of the regenerants using elevated concentration of CuSO4 and ZnSO4. GSTs overexpression activity not only confers to overcome the stress but enhanced the in vitro regeneration of M. pruriens also.