Adventitious Bud Production Research Articles

Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

Toona ciliata has considerable economic value, providing high-quality materials and medicines. However, the growth and sustainable development of T. ciliata is limited due to environmental changes, poor natural regeneration ability, and excessive logging. Therefore, this study used sterile seedling leaves from a good provenance of T. ciliata as explants to establish a highly efficient regeneration and genetic transformation system. The effects of different hormones and concentrations on leaf callus induction were investigated. A genetic transformation system using kanamycin (Kan) as the selective agent was established based on the regeneration system. The results showed that Murashige and Skoog (MS) medium supplemented with 3 mg/L N-(Phenylmethyl)-9H-purin-6-amine (6-BA), 1 mg/L kinetin (KT) and 0.05 mg/L 1-naphthlcetic acid (NAA) (T1 medium) were suitable for callus formation and adventitious bud production from whole leaves at 45 days. In addition, pCAMBIA2301-EYFP was introduced into competent cells of Agrobacterium tumefaciens strain GV3101 and used for genetic transformation. Healthy leaves pre-cultured on T1 medium for three days were directly infected with A. tumefaciens infection solution (OD600 = 0.6) for 20 min through the addition of 50 mmol/L of Acetosyringone (AS). Thereafter, the infected leaves were co-cultivated for one day on T1 medium supplemented with 150 mmol/L AS, and the infected leaves were transferred to fresh T1 medium containing 10 mg/L Kan and 100 mg/L Cefotaxime (Cef). Twenty-four positive transformed plants were selected through histological staining and PCR, laser confocal microscopy of EYFP from 259 putative transformants was conducted, and the transformed plants were obtained after approximately three months.

A new scaly tree fern (Cyathea: Cyatheaceae) from Colombia

Based on recent collections, we describe a new species of scaly tree fern, Cyathea giraldoi, from northern Colombia and discuss its ecology. The new species differs from putative close relatives by a character combination including a small size, often decumbent trunk, strongly arching fronds with drooping tips, nearly triangular lamina, thick scurf of matted arachnoid squamules, and frequent production of adventitious buds.

Factors influencing the induction of adventitious bud and callus in the brown alga Sargassum horneri (Turner) C. Agardh

A high frequency of callus induction and propagation from leaf explants of the brown alga Sargassum horneri was achieved within 2 months of culture when grown in medium supplemented with 5 μM uniconazole, which is a triazole-type inhibitor of cytochrome P450 enzymes. Adventitious buds were efficiently formed from the pigmented callus after transfer to medium without uniconazole, indicating that treatment with uniconazole was more beneficial for regeneration of the alga. Favorable culture conditions for induction of adventitious buds and calli included temperatures of 15 to 25 °C and light levels of 20 to 200 μmol photons m−2 s−1. Blue light promoted the production of adventitious buds and calli. The frequency of formation of adventitious buds and calli in explants from thalli with one leaf was more than 90 %, while it was 10 % only when explants were sourced from thalli with 9 to 11 leaves. These findings will be useful for clonal propagation and storage of seed materials for mariculture of Sargassum species.

Adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides) cultured under photoautotrophic conditions

Effects of photoautotrophic and photomixotrophic growth conditions on adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides Bartr. ex Marsh.) were investigated. Rooting and proliferating shoot cultures (Stage I) were grown in either an elevated (1500 ppm) CO(2) concentration ([CO(2)]) at high photosynthetic photon flux (PPF; ~ 150 micromol m(-2) s(-1)) (photoautotrophic condition) with 0, 10 or 30 g l(-1) sucrose or under standard conditions (ambient (360 ppm) [CO(2)] at low PPF (~ 60 micromol m(-2) s(-1)) with 30 g l(-1) sucrose). Leaves harvested from these cultures were analyzed for soluble sugars and were used as explants for adventitious shoot regeneration (Stage II), which was also carried out under photoautotrophic and standard conditions. Photoautotrophic conditions during Stage I promoted growth of rooting shoots but inhibited axillary shoot proliferation. Photoautotrophic conditions during Stage II suppressed callus and adventitious bud production from leaf explants compared with standard conditions. The regeneration environment appeared to be more important in controlling bud formation than the conditions under which the donor shoots were grown. Regardless of Stage I treatment, bud production was up to 100-fold higher for leaves cultured under standard conditions than under photoautotrophic conditions. Once adventitious buds were differentiated from the leaf tissues, however, their elongation was faster under photoautotrophic conditions than that under standard conditions, with some shoots reaching 10 mm in length on leaf explants cultured under photoautotrophic conditions. Because total leaf soluble sugar concentration was always lowest in shoots under standard conditions, which also yielded the highest bud production, the results suggest that endogenous starvation enhanced shoot production.

In vitro Mutation of Chrysanthemum (Dendranthema grandiflora Tzvelev) with Ethylmethanesulphonate (EMS) in Immature Floral Pedicels

Many new cultivars of chrysanthemum were derived from induced mutation. However, chemical mutagenic agents are not widely used due to their low penetration into plant tissues. The objective of the present study was to induce mutation in chrysanthemum using ethylmethanesulphonate (EMS) in immature floral pedicels, followed by the induction and production of adventitious buds in vitro. Preliminary studies on the sensitivity of pedicels to EMS revealed that the LD50 was close to 0.82% (v/v). Immature pedicels of chrysanthemum cv. Ingrid (dark pink color) were treated with 0.77% (0.075 M) EMS solution for 1 h and 45 min, which was followed by rinsing in water for 15 min and surface disinfection. Afterwards, they were cultivated in Murashige and Skoog medium (salts and vitamins) amended with 1 g l−1 of hydrolyzed casein, 1 mg l−1 6-benzylaminopurine (BAP) and 2 mg l−1 indole-3-acetic acid (IAA). A total of 910 plants were obtained from the pedicels treated with EMS and were evaluated at the flowering stage. Forty-eight mutants (5.2%) were obtained, deviating in petal color (pink-salmon, light-pink, bronze, white, yellow and salmon color). Most of them (89.6% of the total) were phenotypically uniform. The results showed the efficiency of EMS to induce in vitro mutation of chrysanthemum.

Production of Adventitious Buds on the Leaves in Dicksonia sellowiana

Production of Adventitious Buds on the Leaves in Dicksonia sellowiana

322 In Vitro Propagation of the Ornamental Bromeliad, Tillandsia eizii

Many bromeliad species indigenous to the rain forests of Central and South America are threatened because of over-collection and habitat destruction. Studies were conducted to develop propagation protocols for Tillandsia eizii, a rare ornamental bromeliad of ceremonial significance to the Highland Maya communities in Chiapas, Mexico. We anticipate using in vitro propagation for the conservation of this species with the potential of utilizing bromeliads as an alternative and sustainable forest resource. Protocols were developed for the sterilization and germination of axenic seed. Seedling growth in vitro was assessed and outplanting studies were conducted. Media were evaluated to promote adventitious bud production in experiments using the plant growth regulators naphthaleneacetic acid and benzylaminopurine. Pulse time and duration, as well as the stage of seed development, had a marked effect on bud production. The effects of various potting media on plant growth and survival were assessed. A pure pine bark medium elicited over 95 percent survival. Plants exhibited a “tank-like” morphology characteristic of plants in the wild.

Analysis of aMusa spp. somaclonal variant resistant to yellow Sigatoka

Somaclonal variant CIEN BTA-03 resistant to yellow Sigatoka was obtained from a susceptible banana clone (Williams clone), by increasing the production of adventitious buds using 6-Belcilaminopurine at high concentrations. This somaclone has exhibited yellow Sigatoka resistance in the field for five consecutive years of asexual reproduction. We used RAPD markers to generate characteristic “fingerprints” for each probe, concluding that they are reliable tools for evaluating the genetic variability ofMusa regenerants obtained byin vitro culture.

In vitro formation of adventitious buds on mature embryos of Pinus elliottii Engelm. × P. caribaea Morelet hybrids

Four nutrient media and several growth regulator concentrations were used to formulate a nutrient medium for the in vitro production of adventitious buds on mature embryos of Pinus elliottii × P . caribaea hybrids. A modified medium of Gresshoff and Doy supported adventitious bud production on mature embryos better than modified media of Murashige and Skoog, Risser and White or Schenk and Hildebrandt. Benzyladenine (5 μM) with 1 μM indolebutyric acid was more effective than either 5 μM zeatin or 5 μM kinetin to induce adventitious buds on mature embryos. Adventitious shoots elongated on half-strength Gresshoff and Doy medium with 5 μM kinetin. Shoots rooted best on half-strength Gresshoff and Doy medium supplemented with 32 μM indolebutyric acid. Plants were successfully transferred to soil and hardened under mist spray. Differences were observed in the morphogenic potential of embryos, growth and rooting of adventitious shoots of different P . elliottii × P . caribaea hybrid seed families.

Organogenesis and somatic embryogenesis in Codiaeum variegatum (L.) Blume cv. “Corazón de Oro”

Adventive organogenesis and somatic embryogenesis were induced from leaf explants taken from in vitro or in vivo plants of Codiaeum variegatum cv. “Corazon de Oro.” Shoot multiplication occurred with N6-benzyladenine (BA) alone, where the simultaneous production of adventitious buds and somatic embryos occurred at the fourth subculture, and on leaves not in contact with the medium. A medium with BA and 2,4 dichlorophenoxy acetic acid (2,4-D) produced the largest organogenic response, for both in vivo- and in vitro-produced explants. Somatic embryogenesis was only induced when such explants were transferred to a medium lacking 2,4-D. Thus, a medium with BA only produced the largest percentage of explants with shoots and embryos. Replacing BA with thidiazuron induced up to 100% bud regeneration on in vitro-produced explants by 60 d, but was slower for in vitro-grown explants. Both types of embryos exhibited growth arrest that was partially overcome by transfer to hormone-free basal medium with activated charcoal. Rooted plants from all explants were successfully obtained on a medium with indole-butyric acid (IBA).

In vitro plantlet regeneration from embryonic explants ofPinus pinea L

Clonal propagation ofPinus pinea L. was achieved by organogenesis on cotyledon explants and the influence of several factors on adventitious bud production and development was investigated. Gupta and Durzan (DCR) medium with benzyladenine (5 µM) induced higher bud production. Bud development and shoot elongation required subcultures on medium with activated charcoal. Rooting was obtained after 10 d culture on medium containing IBA (10 µM).

Towards a Protocol for the Micropropagation ofPinus patula

SYNOPSIS In this investigation, different approaches to the micropropagation of P. patula were attempted and assessed in order to identify the most promising one for commercial application. Of the regenerative routes investigated, viz. axillary bud proliferation (dwarf shoot formation) from juvenile shoots, adventitious bud production and somatic embryogenesis from young seedlings, the first appears to have the most potential in terms of yield, time in culture and availability of explant material.

Continuousin vitro multiplication of shoot buds of Norway spruce (Picea abies L.) by intermittent application of growth regulators

A culture system was developed which allows continuous production of adventitious buds. Segments of seedlings germinated on media supplemented with different growth regulator combinations were used as explants. Cultivation was carried out in two phases, which alternate permanently: (I) without and (II) with growth regulators. Thus it was possible to establish meristematic tissue cultures from 5-10% of the seedlings tested. They have produced buds now for over 3 years with multiplication rates of about 1.5 within 5-6 weeks.

Micropropagation of Pinus caribaea Morelet

Adventitious shoot formation was induced in excised mature embryos of Pinus caribaea using a modified Murashige and Skoog medium (MSM) supplemented with 6-benzyladenine. The highest frequency (96%) of adventitious bud production was observed when embryos were exposed to 8.9 μM BA for one week prior to transfer to a growth regulator-free medium. Increased BA concentration and longer exposure to BA significantly reduced survival rates of explants. Dilution of the basal medium to 1/4× and 1/8× decreased shoot formation but 1/2× was just as effective as full-strength. Addition of auxins, glyphosate and coconut water to the rooting medium did not improve rooting success beyond that of spontaneous rooting. Sucrose at 1.5% significantly increased rooting of shoots. Plantlets were successfully transferred to the soil after preincubation in liquid medium.

Plant regeneration of Populus alba "Siberia extremeña" from ament

Populus alba "Siberia Extremeña", a clone of great rusticity and high interest for poplar culture, is difficult to propagate by conventional cutting methods. Plant regeneration from ament callus has been achieved in Murashige-Skoog (MS) medium supplemented with 2 mg/l of 2,4 dichlorofenoxyacetic acid (2,4-D) y 1 mg/l of kinetin. Optimal adventitious bud production arised from callus treated with 1 mg/l of thidiazuron and 0,1 mg/l of α-naphtaleneacetic acid (NAA). Induced buds were subcultured in modified MS mediun plus 0,1 mg/l of 6-benzyladenine (BA) and 0,002 mg/l of NAA. Shoots were rooted in Woody Plant Medium (WPM) plus 0,2 mg/l of indole-3-butyric acid (IBA), reaching a 95 p. 100 rooting. Acclimation of plantlets to glasshouse and nursery conditions rendered a 96 p. 100 survival tax.

Comparative studies of organogenesis and plant regeneration in various soybean explants

Various explants were compared for organogenesis and plant regeneration in soybean ( Glycine max Merr.). Young cotyledons produced organogenic calli, from which adventitious buds and shoots were produced by culture in vitro. Flowering and pod development were observed on regenerated shoots even in vitro, but the recovery of plants was very inefficient. Histological studies revealed weak connections of the regenerated bud primordia with other differentiated tissues, which might be the main handicap to plant recovery in this culture system. Plant regeneration could also take place on the plumule of young embryo explants. The regeneration process started with the enlargement of the plumule followed by the production of adventitious buds. Adventitious buds regenerated much more readily from cotyledonary nodes and some from the plumules in mature embryo explants. An improved culture protocol for efficient plant regeneration in soybean by culturing explants from mature embryos and acclimatizing regenerated plantlets at the early stage is proposed.

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.

SUMMARYAdventitious bud formation on Sitka spruce [Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.

Improved Micropropagation of Begonia × hiemalis by Maintaining Donor Plants in Long-day Conditions

Abstract The effect of donor plant photoperiod on subsequent in vitro adventitious bud production of Begonia × hiemalis (Fotsch) cv. Schwabenland Pink was monitored by sampling petiole explants from plants maintained in either short- (8 hr, SD) or long-day (16 hr, LD) photoperiods. Adventitious bud production on SD and LD explants was similar for the first 28 days of treatment. A subsequent loss of regenerative capacity occurred in SD explants and was associated with the changing physiological status of the donor plant. Maintaining donor plants in LD increased bud production and extended the time during which the donor plants could be used effectively in commercial micropropagation programs.

Controlling vitrification in Larixdecidua via culture media manipulation

Vitrification rates obtained from our inverted embryo system were significantly decreased by lowering the cytokinin concentrations in Brown and Lawrence medium containing 10 mM L-glutamine from 10 to 1 mg/L, or by replacing 10 mM L-glutamine with equimolar concentrations of Ca(NO3)2 or by adding 1 g/L Gelrite to the normal 10 g/L Difco–Bacto agar in the culture media. In all treatments, decreased vitrification was accompanied with decreased adventitious bud production. With reduced N6-benzylaminopurine, vitrification decreased from 77 to 29%, but bud production decreased from 61 to 17 buds per explant and mortality increased from 3 to 33%. Incorporation of Ca(NO3)2 into the media decreased vitrification from 87 to 21%, but the number of adventitious buds per embryo decreased from 75 to 42. Vitreous shoots were reverted to normal development with an 81% reversion frequency and a 6% mortality rate by culturing these shoots on Gresshof and Doy medium with one-half total nitrogen. Elongation of previously vitreous shoots was best when these shoots were cultured on Gresshof and Doy medium + 0.5 mg/L N6-benzylaminopurine for 2 weeks, followed by subculture on Gresshof of and Doy medium + 10 g/L charcoal.

Adventitious bud production on explants of Begonia×hiemalis depends on the developmental state of the donor plant

In vitro responses can be influenced by the developmental status of the donor plant tissue. The effects of the donor plant photoperiod and the developmental stage of the plant on organogenesis of petiole explants of Begonia×hiemalis cv. Schwabenland Pink are reported. Long day plants had progressively more branches, total stem length, leaf area, and branch and shoot mass. In short days, flowering was earlier and a greater proportion of dry weight accumulation was allocated to reproductive structures. Similar explant responses were obtained from all developmental stages until flowering was well advanced in short days and then the regenerative capacity diminished. Primary measurements of donor leaves (length, area and weight) at the time of explant removal were not well correlated with adventitious bud production. Loss of regeneration was not determined by tissue or plant age but was associated with the progressive development of flowers. Thus the donor plant photoperiod only indirectly affected bud production. Organogenesis decreased with duration of short days but increased in long days and thus both the duration of the multiplication phase and the intensity of the in vitro response was enhanced by maintaining donor plants in long days.