Age Of Donor Plant Research Articles

Optimizing the cutting production of Greyia radlkoferi

Greyia radlkoferi leaf extracts has been proven to treat skin hyperpigmentation without any side effects (De Canha et al., 2013). Propagation by seed is fairly easy, but seed germination is associated with the variation of the bioactivity between plants, making it difficult to attain uniform and quality plant material. Clonal propagation using stem cuttings has been reported but need optimization. A randomized complete block design, with three replicates was used to investigate different seasons (winter, spring, summer and autumn), cutting positions (basal, median and apical) and cutting lengths (5, 10, 15 and 20 cm) on various parameters (survival percentage, rooting percentage, number of buds, number of leaves, number of roots per cutting, leaf length and root length). The best treatment combination was then applied to investigate the effect of different ages of donor plants (1–4 year old) on cutting success. Data was analysed using ANOVA and means with significant differences were separated with a t-test at 5% level of significance. Cuttings collected from the basal position and median position produced significantly higher survival and rooting percentages, more roots, buds and leaves as compared to the apical cuttings. Overall, the cuttings made in winter performed better as most parameters measured were significantly higher than the cuttings from spring, summer and autumn. Age of donor plants did not affect the rooting percentage, however, a significantly lower survival percentage, number of buds and leaves were observed in cuttings from three years old trees in comparison to other donor ages. Based on the findings, the optimization of G. radlkoferi stem cutting can be achieved when using 15 or 20 cm basal cuttings during the winter season, taken from any donor ages.

A role of age-dependent DNA methylation reprogramming in regulating the regeneration capacity of Boea hygrometrica leaves.

Plants can regenerate new individuals under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, the role of age-dependent DNA methylation status in the regulation of explant regeneration remains practically unknown. Here, we established an effective auxin/cytokinin-induced shoot regeneration system for the resurrection plant Boea hygrometrica via direct organogenesis and observed that regeneration was postponed with increasing age of donor plants. Global transcriptome analysis revealed significant upregulation of genes required for hormone signaling and phenylpropanoid biosynthesis and downregulation of photosynthetic genes during regeneration. Transcriptional changes in the positive/negative regulators and cell wall-related proteins involved in plant regeneration, such as ELONGATED HYPOCOTYL5 (HY5), LATERAL ORGAN BOUNDARIES DOMAIN, SHOOT-MERISTEMLESS, and WUSCHEL, were associated with the regeneration process. Comparison of DNA methylation profiling between leaves from young seedlings (YL) and mature plants (ML) revealed increased asymmetrical methylation in ML, which was predominantly distributed in promoter regions of genes, such as HY5 and a member of ABA-responsive element (ABRE) binding protein/ABRE binding factor, as well as genes encoding glycine-rich cell wall structural protein, CENTRORADIALIS-like protein, and beta-glucosidase 40-like essential for shoot meristem and cell wall architecture. Their opposite transcription response in ML explants during regeneration compared with those from YL demonstrated the putative involvement of DNA methylation in regeneration. Moreover, a significant lower expression of DNA glycosylase-lyase required for DNA demethylation in ML was coincident with its postponed regeneration compared with those in YL. Taken together, our results suggest a role of promoter demethylation in B. hygrometrica regeneration.

Root cryopreservation to biobank medicinal plants: a case study for Hypericum perforatum L.

In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L−1 gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species.

Adventitious shoot induction from leaf segments in Anthurium andreanum is affected by age of explant, leaf orientation and plant growth regulator

Anthurium is one of the main potted plants on the flower market, and micropropagation is the most used technique for clonal and large scale propagation of this species. Calli, adventitious shoots and regenerated plantlets were obtained from leaf segments of Anthurium andraeanum. The influence of genotype, explant orientation, plant growth regulator, and age of donor plants were tested. We obtained regenerated plants in three (‘Red One’, ‘Red Dark’, and ‘Snow White’) out of four different cultivars. The ‘White Beauty’, however, was considered to be recalcitrant under these conditions. Therefore, the second study was conducted only with this recalcitrant cultivar, and the use of juvenile rather than adult leaf explants enhanced plantlet regeneration at the frequency of 0.3-2.8 plantlets per leaf segment. The use of 1.0 mg·L−1 6-benzylaminopurine (BAP) and no addition of 2,4-D in the culture medium resulted in direct plantlet regeneration of Anthurium. Plantlets obtained from ‘Red One’ and ‘White Beauty’ were successfully micropropagated and acclimatized. In ex vitro phases, leaf-derived plants of these two genotypes maintained their most important horticultural and ornamental characteristics.

Effects of donor plant age and explants on in vitro culture of Cedrela montana Moritz ex Turcz

To evaluate the organogenic potential of Cedrela montana Moritz ex Turcz, explants from mature (10-20 year-old) and juvenile (7-18 month-old) trees were collected. The first grouping included buds, leaves, and nodes derived from juvenile basal offshoots and rejuvenated shoots from cuttings. The second, included leaves, petioles, nodes, internodes and nodes of in vitro elongated shoots. The highest organogenic potential was observed in nodes from juvenile trees: 45.8% of explants presented axillary bud elongation, while 56.2% presented rooting in a growth regulator free culture medium. Fifty- one percent of elongated shoots produced adventitious shoots with 0.5 µM NAA and 0.5 µM BA; 30% with 0.5 µM NAA and 1 µM BA; and 30% with 1 µM BA. Twenty percent presented roots with 0.5 µM NAA. Root formation was stimulated in a medium supplemented with activated charcoal (5 gL -1 ). The acclimatization of eighty percent of plantlets regenerated from nodes, and of 72.5% in vitro generated shoots was successful. On the contrary, mature trees material presented low organogenic response. Axillary bud elongation was recorded just in 10.7% of explants from juvenile shoots and in 6.7% of explants from rejuvenated shoots. In conclusion the age of donor plant and type of explant affect the organogenic potential of C. montana. This study contributes to the understanding of this species' response under in vitro conditions.

Changes of Soluble Sugars and Enzymatic Activities During Adventitious Rooting in Cuttings of Grewia optiva as Affected by Age of Donor Plants and Auxin Treatments

Changes of Soluble Sugars and Enzymatic Activities During Adventitious Rooting in Cuttings of Grewia optiva as Affected by Age of Donor Plants and Auxin Treatments

Optimization of Brassica napus (canola) explant regeneration for genetic transformation

Brassica napus (canola) is the second largest oilseed crop in the world. It is among the first crops to be genetically transformed, and genetically modified cultivars are in commercial production at very significant levels. Despite the early lead with respect to transgenesis, there remain cultivars that are recalcitrant to transformation. To address this, we have conducted an elaborate investigation of the conditions for regenerating shoots from hypocotyl explants from four genetic lines: Invigor 5020, Westar and Topas as well as a microspore culture derived line of Topas (Line 4079). We analyzed the effect of hormonal combinations in regeneration medium, donor plant age and explant type on the regeneration capacity of these plants. The analysis showed that hypocotyls of eight-day-old seedlings grown on media supplemented with 1mg/L dinitrophenylhydrazine (2,4-D) produced the most shoots. Globular somatic embryos emerged following two weeks of 2,4-D treatment. When transferred to the medium containing 5mg/L benzyladenine (BA), approximately 82% of embryos produced shoots within six weeks. Invigor plants were shown to regenerate more efficiently than Topas; the number of plantlets regenerated from Invigor was approximately 40-50% more as compared to Topas or Line 4079. When hypocotyl explants were co-cultivated with the Agrobacterium strain GV3101 harboring a binary vector carrying a firefly luciferase reporter gene (LUC), significant numbers of plantlets were LUC-positive in a luciferase assay. Frequency of such plants were: Invigor 5020 (54.2 ± 2.5%), Westar (53.7 ± 5.3), Topas (16.0 ± 0.24) and Line 4079 (13.4 ± 4).

Rapid plant regeneration, analysis of genetic fidelity and essential aromatic oil content of micropropagated plants of Patchouli, Pogostemon cablin (Blanco) Benth. – An industrially important aromatic plant

Rapid and prolific shoot regeneration via direct organogenesis was induced from leaf explants of Patchouli, Pogostemon cablin (Blanco) Benth., an aromatic plant of immense industrial value, on Murashige and Skoog (MS) medium with benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) within 4 weeks. The adventitious shoot bud induction and plant regeneration were greatly influenced by the origin, age of donor plant, and leaf position on stem. Leaf explants prepared from in vivo plants of different ages showed higher regeneration response as compared to the explants from in vitro plantlets of respective age. The shoot regeneration ability of explants was significantly related to the age of the donor plants as well as the leaf position on the stem. The highest number of shoots (94.6/explants) was obtained from 96.2% of leaf explants derived from the leaves located on the second node of the 3-month-old in vivo plants on Murashige and Skoog's (MS) medium containing 2.5 μM benzylaminopurine (BAP) and 0.5 μM naphthaleneacetic acid (NAA). Incorporation of 1.0 μM gibberellic acid (GA 3) in MS medium significantly improved the shoot elongation (1.8-fold) within 2 weeks in 95% of the cultures. Regenerated shoots rooted spontaneously on growth regulator-free half strength MS medium and were successfully hardened and transferred to nursery with 96–100% survival rate. Genetic fidelity of the in vitro derived plants was assessed using random amplified polymorphic DNA (RAPD). Fourteen arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor plant. The molecular analysis complemented and compared well and showed genetic stability in the plants regenerated through direct shoot bud differentiation from leaf explants. The gas chromatogram of the extracted oils from in vitro derived plants and the mother stock plant showed similar essential oil profiles. Rapid and high multiplication frequency, molecular, genetic and essential oil content stability ensure the efficacy of the protocol developed for the production of this industrially important aromatic plant.

Vegetative propagation of African Blackwood (Dalbergia melanoxylon Guill. & Perr.): effects of age of donor plant, IBA treatment and cutting position on rooting ability of stem cuttings

A study to determine the best vegetative propagation protocol was carried out for Dalbergia melanoxylon. Natural regeneration of D. melanoxylon is limited due to poor seed viability and germination. The effects of age of donor plant, indole-3-butyric acid (IBA) treatment and cutting position on rooting of stem cuttings were investigated. The effect of age of donor plant was significant (P < 0.001) only for percent rooting, position effect was significant for all studied parameters, and IBA treatment at 300 ppm was insignificant only for percent callusing. Interactive effects of cutting position × age of donor and cutting position × IBA treatment were significant for percent rooting and root number per cuttings while interactive effect of age × IBA was significant only for percent rooting. Observed difference in rooting performance could be due to uneven distribution of root promoting and inhibiting substances with respect to age of donor plant and IBA which intensified root formation. Cuttings from juvenile donor plant performed better in all rooting parameters than mature donor plant. The highest percentages in rooting were (71.11%) and (24.42%) for cuttings from juvenile and mature donor plants, respectively. IBA treated cuttings produced higher percentage rooting, number of roots and root length than untreated cuttings which revealed a strong IBA influence on rooting ability of stem cuttings in D. melanoxylon. It was concluded that stem cuttings should be taken from juvenile donor plants and basal cutting position should be used to produce high quality planting stock material for the afforestation and conservation programme of D. melanoxylon.

In vitro plant regeneration from seedling-derived explants of ramie [Boehmeria nivea (L.) Gaud

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B5 vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.

Metabolic changes during adventitious root primordium development in Tectona grandis Linn. f. (teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment

Aging of the donor tree decreased adventitious root formation in shoot cuttings of Tectona grandis Linn. f. (teak). Exogenous application of auxins, i.e., a-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) has a significant positive effect on the percentage of rooting. The maximum percent rooting was obtained with 4,000 ppm IBA as compared to other treatment. Significant increase in root number was recorded in shoot cuttings treated with 4,000 ppm NAA. The overall rooting response was better in the treatment with IBA rather than with NAA. Further periodic samples (0, 10, 20, and 30 days) were taken to assess the total soluble sugar, starch, protein, and peroxidase (PER) activity in the rooting zone of shoot cuttings of teak during adventitious root formation. Application NAA and IBA to shoot cuttings resulted in an increase in the level of total soluble sugar, starch, protein, and PER-activity in the rooting zone. The stored carbohydrates were utilized during adventitious root formation. Hence, total soluble sugar and starch contents of cuttings, irrespective of age of donor plants, decreased with the passage of time in cuttings planted for rooting. Significant fluctuations were observed in the protein content of cuttings during the time of root induction. There was an increase in the protein content with the passage of time from the day of planting up to its 20th day, followed by a sharp decline in the protein content of cuttings at the 30th day of planting, irrespective of the age of donor plants or the treatment of cuttings with auxins. Irrespective of donor plant age, PER-activity in the cuttings increased from the day of their planting for rooting up to the 20th day, and then declined at its 30th day of planting. It was interesting to note that PER-activity remained higher at all stages in the cuttings of 2-month-old seedlings which rooted profusely as compared to the cuttings of 15- and 30-year-old donor plants those rooted poorly. This study

Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.)

The effects of growing season and donor plant age on anther culture in Capsicum were investigated. Pepper cultivars Kekova and Sera Demre 8 were grown under unheated greenhouse conditions in winter season and field conditions in summer season. Flower buds were collected and cultured in weekly intervals from November to May in winter and from April to December in summer. The results of this study indicated that anthers from these two pepper genotypes gave different embryogenic response to seasonal effects. Kekova cultivar gave the highest embryogenic yield in summer season while this was occurred in winter season for Sera Demre 8 cultivar. The results of the study revealed that the effect of donor plant age was also an important factor in anther culture of pepper. Since 4-month-old plants gave the highest embryo yield in both cultivars in each seasons, it is possible to say that anthers collected from old plants have sufficient embryogenic response when the optimum developmental stage is selected.

Variation in Shoot Anatomy and Rooting Behaviour of Stem Cuttings in Relation to Age of Donor Plants in Teak (Tectona grandis Linn. f.)

Shoot anatomical features viz., number of vessel elements, width and length of vessel and fibre elements were studied in relation to juvenility/maturity of teak (Tectona grandis Linn. f.). The findings show significant differences in these features, which indicates that variation in shoot anatomy can be used as a reliable indicator of juvenility/maturity in teak. The effect of age of donor plants and application of auxins on adventitious root formation in single noded leafy stem cuttings of teak was also investigated. Aging of donor trees suppressed rooting and sprouting of cuttings, but increased callus formation at the base of cuttings. Per cent rooting and per cent sprouting in cuttings decreased as age of donor plants increased from 2-months to 15-years and up to 30-years. IBA treatment promoted per cent rooting as well as per cent sprouting, NAA promoted only rooting and it suppressed sprouting. The effect of auxin treatment on rooting and sprouting of teak stem cuttings varied with the age of donor plants. Auxin and its concentration, which suppressed rooting and sprouting in cuttings of younger donor plants, actually promoted rooting when cuttings were taken from more mature donor plants. It appears that the auxin requirement for causing and promoting rooting in teak cuttings increased with increasing age of donor plants. The higher auxin requirement for causing and promoting rooting in cuttings of older trees may be due to a decrease in the content on endogenous auxins or decreased sensitivity of aging tissues to rooting promoters and /or accumulation of inhibitory substances which inhibit rooting.

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.

Effects of donor plant temperature, photoperiod, and age on anther culture response of Capsicum annuum L.

Effects of temperature and photoperiod for donor plant growth on embryo formation in Capsicum annuum anther culture were investigated. Donor plants were grown in glasshouses at minimum temperatures between 16 and 30° C and at photoperiods between 11 and 19 h. Anthers were collected from individual plants over five to nine week periods to test the significance of donor plant age. Embryos were obtained at all temperature regimes with a calculated optimum temperature of 26.4° C. Embryo formation was unaffected by the photoperiods tested. Embryo formation varied among successive samplings. However, a significant decline in anther culture response with increasing donor plant age was observed in all three experiments.

Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L.

Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.

Dimorphism in bud regeneration in flax (Linum usitatissimum)

The study of the expression of morphogenetic potential in stem segments of flax (Linum usitatissimum L.) was undertaken as a function of donor plant age and level (basal, median, or apical) on the stem axis and in the absence of growth regulatory substances. Neoformation of normal buds (resembling seedlings) gave rise to shoots with no visible axillary buds, and a few roots were obtained from explants taken from young, vegetative plants; these organs were more numerous on basal than on apical explants. If the donor plant was in a prefloral or floral state, thus older and at a different physiological stage, a "modified" bud was formed, characterized by its slow growth, short thick stem and leaves, and sometimes with visible axillary buds. This type of bud was more numerous in apical than in basal segments. Callus was also formed and was reduced in size and limited to the cut extremities of explants when the donor plant was young, but was large and covered almost all the explant when the plant was older.