Full-strength Murashige And Skoog Medium Research Articles

Effect of Gibberellic Acid (GA<sub>3</sub>) and Sugar on In <i>Vitro Cormlet</i> Formation, Multiplication and <i>Ex Vitro</i> Sprouting of <i>Gladiolus hybrida</i> Variety Princess Lee

Calli were initiated from corms of <em>Gladiolus hybrida</em> variety Princess Lee by culturing on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l napthaline acetic acid (NAA) and 20 g/l sucrose<strong>. </strong><em>In vitro</em> shoots were regenerated from the calli by transferring into MS medium containing 30 g/l sucrose without growth regulators. Regenerated shoots produced cormlets at the base of the shoot on full or half strength MS medium supplemented with 40-60 g/l sucrose. Highest number of large corms (diameter&gt;1.5 cm) was obtained in the half strength MS medium containing 60 g/l sucrose. Multiplication of <em>in vitro</em>-grown cormlets was achieved by transferring them on to half strength MS medium containing 60 g/l sucrose with 5 or 10 mg/l gibberellic acid (GA₃). Multiplied corms were successfully acclimatized on a medium containing soil: sand: brick pieces at 1:1:1 ratio. Acclimatized cormlets were established on two different potting mixtures i.e. sand: leaf mould: brick pieces (1:1:1) or sand: compost: brick pieces (1:1:1) after pre-treating with or without 100 mg/l GA₃ for 24 hours. Early sprouting (13 days) was observed in cormlets treated with 100 mg/l GA₃ compared to cormlets without any GA₃ treatment (28 days) and, the highest sprouting percentage (83.3 %) was observed on potting mixture containing sand, leaf mould and brick pieces (1:1:1) under greenhouse conditions. <em>Tropical Agricultural Research Vol. 23 (1): 1–10 (2011)</em> <a href="/index.php/TAR/editor/viewMetadata/DOI: http:/dx.doi.org/10.4038/tar.v23i1.4626"> DOI: http://dx.doi.org/<span>10.4038/tar.v23i1.4626</span></a>

Somatic embryogenesis, regeneration and in vitro production of glycyrrhizic acid from root cultures of Taverniera cuneifolia (Roth) Arn.

Taverniera cuneifolia (Roth) Arn. or Indian licorice is considered to be a substitute for Glycyrrhiza glabra L. owing to an equivalent content of glycyrrhizic acid (GA). GA recognized as the main active ingredient of T. cuneifolia, GA imparts several medicinal properties to these plants. However, research on this plant is scanty with no published record on tissue culture studies. Present study demonstrates a method for (1) induction and successful development of somatic embryos from the root culture, (2) regeneration of plants, and (3) GA production by root cultures of T. cuneifolia. Shoot initiation frequency of cultured roots ranged from 52.57% to 97.71%. Plant regeneration frequency (germination) from somatic embryos was 88.66% in 1/4-strength Murashige and Skoog (MS) medium supplemented with 2% sucrose, as against 68% in half 1/2-strength MS containing 2% sucrose. Glycyrrhizic acid of 1.32 mg/gm of dry weight was obtained in full-strength MS medium supplemented with 3% sucrose, through high-performance thin layer chromatography analysis with standard GA in root cultures. The present study provides an efficient method for the mass production of plants from a single mother plant as well as the potential root culture system to study the GA production pathways in T. cuneifolia.

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

SummarySecondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.

FIRST REPORT ON CALLUS INDUCTION IN ASHOKA (SARACA INDICA): AN IMPORTANT MEDICINAL PLANT

Leaves cultured on Murashige and Skoog (MS) media supplemented with BAP (2 mg/L) and NAA (0.5 mg/L) showed calli formation in 29.4% explants. When cultured on half strength MS media (2 mg/L BAP and 0.5 mg/L NAA), 13.3% explants could produce calli. Hypocotyls segments cultured on full strength MS media showed good callusing response (52.6%). Culture of explants on the half strength MS media produced calli in 77.8% of explants. When segments of epicotyls were placed on full strength MS media, a callusing response of 83.3% was observed. Segments when were placed on half strength MS media, a maximum response of 88.9% was observed. The calli failed to differentiate into shoots when sub-cultured on shoot induction media.

Large-scale in vitro propagation of Impatiens repens Moon., a critically endangered medicinal plant in Sri Lanka

SummaryImpatiens repens Moon. (Ceylon Balsam: Family Balsaminaceae) is an endemic, critically endangered, medicinal plant in Sri Lanka. Habitat destruction and over-exploitation have resulted in the virtual extinction of this species. To grow this plant sustainably, a micropropagation protocol was developed using nodal explants for rapid, large-scale propagation. Nodal segments were established on Murashige and Skoog (MS) medium supplemented with different combinations of 6-benzylaminopurine (BAP) and Kinetin for shoot induction. Shoots were established on MS medium supplemented with different combinations of BAP and indole-3-acetic acid (IAA) for shoot multiplication. Micro-shoots were established on full-strength or half-strength MS medium supplemented with different concentrations of α-naphthalene-acetic acid (NAA) for root induction. MS medium supplemented with 2.0 mg l–1 BAP and 1.0 mg l–1 Kinetin gave the highest mean number of shoots per nodal explant (4.7), the highest mean shoot length (3.2 cm), and the highest frequency of shoot regeneration (100%). Addition of a combination of BAP and IAA significantly influenced shoot multiplication. The highest mean number of shoots per shoot explant (18.8) was observed on MS basal medium supplemented with 2.0 mg l–1 BAP and 0.1 mg l–1 IAA. The highest mean number of roots per shoot, mean root length, and frequency of rooting were observed on half-strength MS medium supplemented with 1.0 mg l–1 NAA. Plantlets were acclimatised, with a 96% survival rate, on a 3:1 (v/v) mix of sand: compost (leaf mould).

English

&nbsp; A prolific plant regeneration system using scalps derived from shoot tips of&nbsp;Musaspp. cv. Tanduk was developed. Highly proliferating scalps, produced after four monthly subcultures of shoot tip explant on Murashige and Skoog (MS) medium supplemented with 100&nbsp;mM BAP and 1.0&nbsp;mM IAA, were placed on MS basal medium supplemented with 1.0, 2.5 and 5.0&nbsp;mM BAP. Rooting of shoots was assessed on hormone-free half strength and full strength MS media and on MS medium supplemented with 1.0, 5.0 and 10&nbsp;mM IBA. Four types of potting media comprising of sand, peat, sand + top soil + goat dung (3:2:1 v/v) and top soil + sand (1:1 v/v) were evaluated during acclimatization of the plantlets. Prolific shoot regeneration from scalps was obtained on MS medium containing 2.5&nbsp;mM BAP, at 9.61 and 40.6 shoots per explant after 4 and 8 weeks of culture, respectively. Meanwhile, the highest mean shoot height of 2.19 cm was attained on MS medium with 1.0&nbsp;mM BAP after 8 weeks of culture. Full-strength MS medium supplemented with 5.0&nbsp;mM IBA produced the highest mean number of roots per explant at 15.08, while the highest mean root length of 11.07 cm was obtained on hormone-free half strength MS medium at week 4 of culture. The highest plant survivability of 77.5% was achieved in potting medium consisting of top soil + sand + goat dung after 6 weeks of acclimatization. The plants were morphologically normal with vigorous stems and broad green leaves. &nbsp; Key words:&nbsp;In vitro, shoot tip, scalps, regeneration.

An improved micropropagation of Spilanthes acmella L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm−3 BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.

Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella.

In vitro regeneration of Leucaena leucocephala by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 µM N6-benzylamino-purine (BAP) and 5.37 µM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 µM indole-3-butyric acid (IBA) and 0.23 µM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 µM NAA and 12.24 µM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 µM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 µM BAP, 2.5 to 5.0 µM IBA and 0.5 mM spermidine.

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N6-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.

Micropropagation of Pongamia pinnata through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA3) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA3 during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA3 in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.

Liquid culture system for shoot multiplication and secoiridoid production in micropropagated plants of Centaurium erythraea Rafn

An efficient shoot multiplication method from shoot tips of Centaurium erythraea using liquid Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (0.1 mg l −1) and 6-benzylaminopurine (BAP) (1.0 mg l −1) was developed. Under these conditions, almost 60 microshoots per explant were produced within 4 weeks. This is three times more as compared to shoots obtained on agar-solidified medium. Shoots taken from liquid culture were rooted with frequency 85% and 77% on hormone-free full-strength and half-strength MS medium, respectively within 6 and 4 weeks. The plantlets were transferred into soil and they survived acclimatization with 90% success, producing healthy plants, morphologically similar to plants derived from shoots grown on solid culture during their multiplication stage. The shoots of 10-week-old micropropagated plants of C. erythraea accumulated secoiridoid glucosides (gentiopicroside, swertiamarin and sweroside) up to 149 mg g −1 dry weight. The value was significantly higher than those achieved for other tested plant materials, such as shoot cultures and aerial parts of wild-grown plants. It is hoped, that the system using liquid shoot culture could be useful for large-scale micropropagation of C. erythraea plants with high production of pharmacologically important products.

Toward Introgression of Ornamental Cucurbita Germplasm: Generation of C. maxima ×pepo hybrids

Cucurbita maxima and C. pepo are difficult to hybridize, and it was our objective to generate F1 hybrids between ornamental cultivars of the two species. C. maxima `Lakota' and C. pepo `Jack O'Lantern'; and `OZ'; were selected as parents. `Lakota' (L) is an heirloom, hubbard-type cultivar producing pear-shaped, red-orange fruit with dark green mottling, `Jack O'Lantern'; (J) is an open-pollinated Halloween-type pumpkin cultivar and `OZ' is a Halloween-type hybrid. Sixteen plants of each cultivar were greenhouse-grown in a CRB design during the period July-Sept. 2003. Interspecific crosses were made in both directions, with intraspecific crosses (J × O) and selfs (L) serving as controls. Fruits were harvested about 20 d after pollination. Embryos were excised under aseptic conditions and grown on either full strength Murashige and Skoog (MS) media with 6% sucrose (S6), full strength MS media with 6% maltose (M6), or half strength MS media with 3% sucrose (S3). Fruit set was generally greater in the intraspecific crosses (33%) and selfs (67%) than in the interspecific crosses (15 %), with the notable exception of the interspecific combination L × J (85% fruit set). Embryos of interspecific and control crosses were about 1.5mm and &gt;1cm long, respectively. Hypocotyl and root growth 10 d after plating was better on S3 (3.2 and 1.7 cm) than on S6 (1.6 and 0.25 cm) or M6 (0.35 and 0 cm), and a greater number of functional hybrids were obtained from embryos grown on S3 (6 plants) than on S6 (2 plants) or M6 (2 plants). The interspecific plants were backcrossed to one of the parents and novel combinations of shape, color and variegation in hybrid fruit were observed.

Micropropagation and hairy root culture of Solanum Laciniatum Ait.

An efficient system for in vitro micropropagation of Solanum laciniatum Ait. has been established. Shoot induction on leaf explants was most successful on Murashige and Skoog (MS) medium supplemented with 10 μM N6-benzyladenine (BA) and 1 μM α-naphthaleneacetic acid (NAA). BA (13 μM) was optimal for further shoot multiplication, and rooting of separated shoots was achieved on medium without plant growth regulators. At each subculture, 20–25 shoots were obtained on each explant, from which six to eight were suitable for separation and further rooting. Leaf explants grown in vitro were successfully infected by Agrobacterium rhizogenes ATCC 15834. The established hairy root culture was, on the basis of dry weight, more productive when grown on half-strength MS medium than on full-strength MS (3% sucrose) and full-strength MS (6% sucrose) medium. The amount of solasodine-containing glycoalkaloids in hairy roots as measured by a colorimetric method was 0.3–1% of dry weight, which is higher than in the shoot culture (0.5% of dry weight) and lower than in leaves of in vivo-grown plants (1.1–1.4% of dry weight). The amount of solasodine-containing glycoalkaloids in leaves of in vivo-grown plants of S. laciniatum was similar to the related species Solanum aviculare Forst. Both species are morphologically similar, therefore we effectively distinguished them by flow cytometry. The genome size of S. laciniatum was determined as 4.03 pg and the genome size of S. aviculare as 1.69 pg.

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz).

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 dimethylaminopurine (2iP) + 1 mg dm-3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm-3 2iP + 0.5 mg dm-3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm-3 speeded up the bud proliferation more than 10, 20 and 40 g dm-3. However, the largest shoot buds were observed with 40 g dm-3 sucrose. Solidification of culture media by 1.75 g dm-3Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm-3Phytagel.

In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants

Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.

Micropropagation of Ulmus pumila L. from mature trees

Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l(-1) BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l(-1) naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.

IN VITRO SHOOT REGENERATION FROM WESTERN SOAPBERRY

Surface sterilized stem nodal sections of western soapberry (Sapindus drummondii Hook. &amp; Arn.) were cultured on Murashige and Skoog (MS) medium. The basal media consisted of one half and full strength MS medium each supplemented with the following (mg-1): Nicotinic acid 0.5, pyridoxine Hcl 0.5, Glycine 2.0, myo-inositol 100, sucrose 30,000 and agar 8000. Each medium also was supplemented with either 0, 0.01, 0.1, 1.0 and 10 mg/l Thidiazuron (TZD) or 0, 0.5, 2.0 and 5 mg/l 6-Benzyladenine (BA). The pH of all media was adjusted to 5.8 ± 0.1. The culture media were autoclaved at 120°C at 1.5 Kgcm-1 pressure for 15 min. The highest percentage of nodal sections resulting in shoot regeneration occurred on 1/2 MS with TZD at 0.01 mg/l and MS medium containing 0.5 mg/l of BA Increasing the TZD concentration above 0.1 mg/l resulted in callus formation on cut surfaces.