Plant Growth Regulator-free MS Research Articles

Silver nitrate and 2-(N-morpholine) ethane sulphonic acid in culture medium promotes rapid shoot regeneration from the proximal zone of the leaf of Capsicum frutescens Mill.

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO3) and 1.98 mg L−1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10 ± 1.7 and 8 ± 1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA + 22.20 µM BA and 10 µM AgNO3. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA3) and 10 µM AgNO3. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.

In Vitro Cryopreservation of Date Palm Caulogenic Meristems.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulation-vitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.

Somatic embryogenesis and in vitro plantlet regeneration in Salicornia brachiata Roxb.

Salicornia brachiata Roxb. (Amaranthaceae) a leaf-less annual succulent halophyte, grows under extremely saline conditions and is an important resource for salt stress responsive genes. Here we report somatic embryogenesis and in vitro plantlet regeneration in S. brachiata for the first time. In vitro-grown seedlings were used as explants and 86.0 ± 1.7 % explants produced embryogenic callus on 0.75 % agar-gelled Murashige and Skoog’s (MS) medium containing 2.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-d) in 16 weeks. Somatic embryogenesis was achieved on MS medium supplemented with 0.25 mg l−1 2,4-d. On this medium sustained cell division and growth within the first 8 weeks resulted in the formation of cell aggregates in 73.65 ± 0.44 % cultures producing globular somatic embryos (SEs). Gradually these converted into heart, torpedo and cotyledonary shaped SEs following subsequent 16 weeks subculture on the same medium. Around 35 % SEs germinated on plant growth regulator free MS medium. The somatic seedlings were acclimatized on sterile soil with 40–45 % survival.

In Vitro Plant Regeneration of Cymbopogon jwarancusa (Jones) Schult from Meristematic Base of Spikelet

Cymbopogon jwarancusa is a multiple stress (es)-tolerant, C4 and aromatic grass of extremely arid parts of the Thar Desert. Genetic amelioration of this cross pollinated species necessitates development of an effective in vitro plant regeneration protocol. Therefore base of the spikelet comprising a zone of actively dividing meristematic cells was used as source(s) of totipotent/pluripotent cells for callogenesis. These explants produced competent and highly regenerative cultures on agar gelled MS medium containing 22.62 μM of 2, 4-D. The cultures multiplied optimally on MS medium with 18.1 μM of 2, 4-D, 4 % sucrose and 0.5 % glucose. The callus organized to globular embryos on plant growth regulator-free MS medium followed by emergence of coleoptile and rhizogenesis. Incorporation of 4.65 μM of Kinetin in the regeneration medium enhanced the rate of plant regeneration. About 40–45 plantlets were obtained from a single explant derived cultures. Of the in vitro raised plantlets, 98 % survived under the greenhouse conditions. The plants matured and set viable seeds.

Conservation and storage of Curcuma amada Roxb. synseeds on Luffa sponge matrix and RAPD analysis of the converted plantlets

Curcuma amada Roxb. (family Zingiberaceae), is gaining global attention as potential source of new drug molecule(s) as it is credited with compounds having remarkable pharmacological properties. Recognizing the risk of loss of genetic variability of C. amada in response to imprudent exploitation of the natural resources coupled with unsystematic cultivation practices, preservation of its genetic resources has attracted the current research attention. The present study exploited the multifarious properties of Luffa sponge (i.e. sound physical strength, stability in texture/shape over wide pH range and repeated autoclaving, cost effective availability, etc.) as a novel matrix for synthetic seed conservation. In this study, we have evaluated the consequences of the presence of two fungicides, namely – bavistin and rosebengal, in the gel matrix as well as in the Luffa sponge for conservation of contamination free germplasm of C. amada. A maximum of 50% and 75% regrowth could be recorded amongst rosebengal and bavistin containing synthetic seeds respectively after 4 weeks of plating on plant growth regulator-free MS medium. Further growth of the encapsulated microshoots was significantly impeded by the presence of rosebengal within the synthetic seeds. On the contrary, the bavistin containing synthetic seeds demonstrated five times better regeneration ability to contamination-free shoots. A total of 78% survival was achieved upon transfer of these healthy plantlets to glass-house. RAPD fingerprinting revealed 84.62% genetic similarity between randomly selected synthetic seed derived plantlets. This report strengthened the vital conservation approach of C. amada using inexpensive Luffa sponge as storage matrix and bavistin for eradication of contaminations.

In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.

In vitro shoot regeneration from seedling explants in Brassica vegetables: Red cabbage, broccoli, Savoy cabbage and cauliflower

Brassica oleracea varieties (red cabbage, broccoli, Savoy cabbage and cauliflower) were tested for their ability to regenerate shoots in vitro. Cotyledon, hypocotyl and root explants of 7 day-old seedlings were incubated on Murashige and Skoog's (MS) medium supplemented with 1 mg l-1 6-benzyladenine (BA) or 6-furfurylaminopurine (KIN) in combination with 0, 0.1, and 0.2 mg l-1 indole-3-butyric acid (IBA). Hypocotyls showed the best explants in almost all varieties tested with a minimum regeneration potential of 75% and producing 3.5-7.4 shoots per explant. The BA-supplemented media were optimal for both shoot regeneration and multiplication. Shoots rooted maximally (100%) on plant growth regulator-free MS medium containing 2% or 4% sucrose. Increased sucrose content improved plant acclimation in the greenhouse.

Development of triploid daylily (Hemerocallis) germplasm by embryo rescue

Interploidy crosses between diploid daylily (Hemerocallis) cultivars (2n = 22) and tetraploid cultivars (2n = 44) were performed via in vivo hybridization aiming to produce triploid hybrid germplasm (3n = 33). Plant growth regulator-free MS based medium containing 5 levels of sucrose—1, 2, 3, 4 or 5% was used to optimize the embryo rescue medium. It was determined that the medium supplemented with 3% sucrose gave highest rate of immature hybrid embryo rescue. Thirty-seven hypothetically triploid genotypes were subjected to verification of ploidy status by root tip chromosome counting and flow cytometry. Thirty-one of them were confirmed triploids. These genotypes were in vitro propagated, acclimatized to ex vitro conditions, and planted for future field performance evaluation and environmental testing. The newly developed triploid genotypes could open new horizons for further polyploidy breeding.

STIMULATION OF IN VITRO BULBLET GROWTH IN GALANTHUS SPECIES WITH SUCROSE AND ACTIVATED CHARCOAL

We report here a reliable method to stimulate growth of in vitro-produced bulblets of Galanthus species to a size suitable for acclimatization. Proliferating bulblet cultures, from bulb chip explants, were induced and maintained on MS medium supplemented with 30 g/L sucrose, 1 mg/L BA and 0.1 mg/L NAA. The six treatments, prepared in plant growth regulator-free MS medium, were 30 or 60 g/L sucrose in all combinations with 0, 1 or 5 g/L activated charcoal. Fifteen replicates per treatment per plant type (G nivalis, G nivalis 'Flore Pleno' and G. elwesii) were inoculated with bulblet clumps containing 2-4 bulblets. Assessments were made after incubation for 16 weeks at 18°C with 16h photoperiods. Even in the absence of plant growth regulators the cultures continued to form new bulblets in all treatments. Bulblet growth was greatly stimulated by the addition of activated charcoal. In contrast, doubling the amount of sucrose in the medium to 60g/L had only a marginal effect on bulblet growth in the absence of charcoal. Addition of charcoal and sucrose together was found to have a synergistic effect on bulblet growth, in that the benefit of adding the higher rate of charcoal could be seen at only the higher sucrose concentration. Charcoal also induced the formation of large numbers of roots and stimulated root elongation. Bulblets had a normal anatomy in longitudinal section, and, although many of the bulbs sprouted in vitro and lateral bulb units developed in larger bulbs, none formed flowers. Thus it is expected that in vitro-produced bulbs will require either flower initiation treatment(s) or a minimum of one season in field conditions to achieve flowering size.

Efficient Production of Polyploid Plants via Protoplast Culture of Iris fulva

In protoplast culture of the Louisiana iris species, Iris fulva (2n=42), N6 medium supplemented with 1 mg/l 2,4-D, 1 mg/l kinetin, 200 mg/l casein hydrolysate, 250 mg/l proline, 0.3 M glucose and 2.5 g/l gellan gum was suitable for cell division and colony formation. When colonies formed were transferred to plant growth regulator free MS medium, 61 plant lines were regenerated via somatic embryogenesis. Flow cytometric analysis revealed that leaf tissue of I. fulva consisted of 2C, 4C and 6C cells. Consequently, we identified this species as polysomatic in the genus Iris. Such polysomaty was also recognized in the suspension calli. Sixty one plant lines obtained by protoplast culture consisted of 25 diploid (41.0%), 1 triploid (1.6%), 29 tetraploid (47.5%) and 6 hexaploid lines (9.9%). In addition, chromosome analysis showed that somatic chromosome numbers of the diploid, hypertriploid and tetraploid lines were 2n=42, 67 and 84, respectively. On the other hand, all lines regenerated from suspension culture of I. fulva were diploid lines in contrast to the results from protoplast culture. Thus, it can be concluded that protoplast culture of this species is an efficient technique for production of polyploid plants.

Optimisation of the tissue culture protocol for the endangered Aloe polyphylla

An optimised, rapid tissue culture protocol was established for the highly endangered Aloe polyphylla (Schonland ex Pillans). Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (Murashige and Skoog 1962), supplemented with 100mgl - 1 myo-inositol, and 30gl - 1 sucrose. Different cytokinins (kinetin, zeatin and BA) singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins gave good shoot proliferation. The optimal concentrations for shoot proliferation of each of the cytokinins tested were: zeatin (0.5mgl - 1 ), kinetin (1.5mgl - 1 ) and BA (1.5mgl - 1 ). In combination with auxins, the optimal combinations were kinetin/NAA (2.0/0.1mgl - 1 ), kinetin/IBA (1.5/1.0mgl - 1 ), zeatin/IBA (1.0/0.5mgl - 1 ), zeatin/NAA (1.0/1.0mgl - 1 ), BA/IBA (1.0/1.0mgl - 1 ) and BA/NAA (1.5/0.1 mgl - 1 ). Although it gave the highest number of shoots per explant, BA (1.0–3.0mgl - 1 ) induced hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25 °C, while 30gl - 1 was the optimal concentration of sucrose. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil:sand:vermiculite (1:1:1 v/v/v) was the best, with a 98% plantlet survival.

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l−1 of 2,4-D and 30 g l−1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l−1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (67∼77%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.

Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis

Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47/1-150 and 47/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system), inoculated leafexplants were incubated on MS medium supplemented with2,4-D and kinetin and then subcultured onto plantgrowth regulator-free MS medium in order to inducedirect somatic embryogenesis. In the secondregeneration system (the B5h system), the inoculatedexplants were incubated on B5h medium to induceindirect production of somatic embryos viaembryogenic callus. In both systems, an effectivekanamycin selection regime was employed and wasmaintained when the embryos were subcultured onto arecovery medium (Boi2Y) to promote further embryodevelopment. The use of Boi2Y medium was particularlyimportant for shortening the regeneration time andpromoting a higher frequency of healthy plantletproduction from the somatic embryos. The maturesomatic embryos were finally transferred to plantgrowth regulator-free MS medium for plantletformation. Transgenic plantlets were produced within10–14 weeks in the MSH system and 12–16 weeks in theB5h system. The MSH system appears to be the fastesttransformation system reported for leguminous speciesto date. Confirmation of transformation was obtainedusing a re-callusing assay on kanamycin and subsequentSouthern blot hybridisation and PCR analysis. Theability to induce expression of GUS activity in leafexplants containing the cell division cycle genepromoter:gusA constructs by 2,4-D treatment alsoproved to be a reliable indicator of transformation.

Plant regeneration from callus cultures of Lithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines of Lithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 μM NAA and 0.3 μM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

Rapid Micropropagation of xChitalpa tashkentensis

×Chitalpa tashkentensis (Chilopsis linearis × Catalpa bignonoides) is an attractive small tree producing lavender to white orchid like flowers. Micropropagation would allow for the rapid clonal propagation of new hybrids for testing cold hardiness and landscape performance. The rapid growth response of Chitalpa shoot cultures also makes it an excellent subject for the study of in vitro growth parameters of woody plants. Shoot cultures were initiated from shoot tips on Anderson's rhododendron medium with MS vitamins, 3% sucrose, 1 μm BA, pH 5.6 and solidified with 0.6% phytagar. Shoot cultures stabilized rapidly. Two-node microcuttings were placed on modified MS media (200.1 μm Na2 EDTA and 200.5 μm FeSO47H2O), MS vitamins, 3% sucrose, pH 5.6, 0.6% phytagar and supplemented with NAA (0 0.5, 1.5, or 3 μm) in combination with BA (0, 1, 5, 10, 15, 20, 30, or 40 μm). Cultures grown on media supplemented with 1 mm BA produced the longest shoots and the most nodes per shoot. Cultures grown on media supplemented with 10 μm BA produced the most shoots. Microshoots readily rooted on plant growth regulator free MS medium and were easily acclimated.