Explant Age Research Articles

REGENERATION OF JOJOBA BY SOMATIC EMBRYOGENESIS: CONTROL OF EMBRYO FORMATION, POLYAMINE CONTENT AND ROLE OF THE AGE OF EXPLANTS AND GROWTH REGULATORS

ISHS I International Symposium on Acclimatization and Establishment of Micropropagated Plants REGENERATION OF JOJOBA BY SOMATIC EMBRYOGENESIS: CONTROL OF EMBRYO FORMATION, POLYAMINE CONTENT AND ROLE OF THE AGE OF EXPLANTS AND GROWTH REGULATORS

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

AbstractThe effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l−1 6‐benzylaminopurine and 0.15 mg l−1 1‐naphthaleneacetic acid. The addition of 2.5 mg l−1 AgNO3 was very beneficial to shoot regeneration in B. napus and Ag2S2O3 (10 mg l−1) was even superior to AgNO3 (2.5 mg l−1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.

Root specific elicitation and exudation of fluorescent β-carbolines in transformed root cultures of Oxalis tuberosa

Stable transformation was achieved in oca ( Oxalis tuberosa L.) using an Agrobacterium rhizogenes-mediated system. Transformation frequencies varied with the use of different types of strains of A. rhizogenes and the age of explants. The transfer of rol A gene into the oca genome was confirmed by PCR analysis. In vitro transformed root cultures of oca grown in sterile liquid media induced purplish-blue fluorescence of the culture flask medium when irradiated with UV light. We have previously observed a similar phenomenon, the exudation of fluorescent compounds by the roots of in vitro and field-grown oca plants. Hairy root cultures of O. tuberosa transformed with A. rhizogenes (ATCC-15834) exuded constitutive levels of harmine (7-methoxy-1-methyl-β-carboline) and harmaline (3,4-dihydroharmine), the main fluorescent compounds detected from oca’s root exudates. Transformed roots showed better growth and exudation of harmine and harmaline compared to the untransformed normal roots. Upon elicitation with fungal cell wall elicitors from Phytophthora cinnamoni, the production and exudation of harmine/harmaline was enhanced in both transformed and non-transformed roots. Harmine and harmaline showed a wide range of antimicrobial activity against soil-borne microorganisms. Biologically, these findings suggest that in nature β-carbolines are constitutive antimicrobial compounds released into the rhizosphere upon microbial challenge. Transformed root cultures of oca make a simple, reliable and well-defined model system to investigate the molecular and metabolic exudation of fluorescent β-carboline biosynthesis, and to evaluate the biological significance of the phenomenon of root exudation of fluorescent metabolites.

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower.

A number of factors that are known to influence genetic transformation were evaluated to optimize Agrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the synthetic cryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae

An Improved Protocol for Regeneration of Sorghum bicolor from Isolated Shoot Apices.

An improved and producible protocol for in vitro regeneration of sorghum [Sorghum bicolor (L.) Moench] from shoot meristem explant is reported. By striking an optimal balance between a weak auxin like naphthalene acetic acid (NAA) and the cytokinin thidiazuron (TDZ), the isolated shoot meristems were manipulated to follow either organogenic or embryogenic pathway. There was no intermediate callus formation. Multiple buds were induced on enlarged meristems using MS medium with 5.0μM of TDZ, 17.72μM Benzylaminopurine (BAP), and 1.074μM NAA. To maximize the number of bud initials per explant, three parameters (seed size, germination technique, and age of explant) were studied. Five to 7-day-old shoot meristems responded best with ≥80% induction of bud initials. Seed size was not significant in influencing induction potential of the shoot meristems. Six weeks after in vitro culture, each meristem produced 35-40 shoot buds. Direct somatic embryogenesis (≥80% induction) was accomplished following a two-step culture procedure consisting of induction of multiple buds and formation of somatic embryos. A high frequency (700-1000) of somatic embryos per explant was obtained on MS medium with 17.72μM BAP and 2.69 μM NAA. Shoots from both organogenic and embryogenic pathways rooted on half-strength Murashige and Skoog (MS) medium with 8.28μM Indole 3-butyric acid (IBA) and 1.14μM Indole acetic acid (IAA). After one month on rooting medium, plants with well-developed roots were transferred to jiffy cups. Such plants were subsequently acclimatized in the glasshouse, and were grown till maturity; they showed normal seed set. Random amplified polymorphic DNA (RAPD) analysis of regenerants did not detect any DNA polymorphism.

The effect of genotype and explant age on somatic embryogenesis of coffee

We demonstrate that embryogenic capacity of coffee (Coffea arabica) depends on the genotype, and that it is a character fixed at the early generations F3 or F4. Therefore, the embryogenic capacity F5 lines can be predicted in a breeding program if it is possible to evaluate the F3 of F4 ancestors, or lines with the same parents on those generations. Regeneration via somatic embryogenesis of genotypes from a C. arabica cv. Caturra by Timor Hybrid cross were evaluated, to determine the effect of the pedigree, month of leaf collection, as well as its interaction with the genotype. Large variations in embryogenic capacity among genotypes were detected with rates ranging from 4.8 to 72.7. There was association between embryogenic response of the progenies and the progenitor from which they proceeded. Significant differences were also found in embryogenic responses depending on the month of leaf explant collection, as well as a significant interaction of genotype by planting month. The results are relevant for breeding and tissue culture purposes of coffee because they will allow speed up of the breeding program and save effort by being able to predict embryogenic capacity of a line, based on the response of either its progenitors or the F3 or F4 lines of the same origin. In addition, high embryogenic capacity lines can be considered directly for genetic transformation or for massive multiplication (bioreactors).

Factors Affecting Somatic Embryogenesis from Cotyledonary Explants of Safflower

Frequency of safflower (Carthamus tinctorius L.) somatic embryogenesis, number of somatic embryos per responding explant and somatic embryo maturation and germination were affected by genotype, explant age, carbon source, and ethylene. Among 8 cultivars tested, 7 were embryogenic with varying frequencies. The best response was obtained with cv. Girna. Whole cotyledonary explant from 10-d-old plants was best responding compared to 5- or 15-d-old ones. Among different carbon sources, sucrose at 87.6 mM concentration was most suitable for embryo induction, maturation and germination. Of the different ethylene inhibitors, silver nitrate at 50 [micro ]M concentration significantly increased the embryogenic frequency and also the number of embryos per responding explant. Silver nitrate has pronounced effect on embryo maturation but had no effect on germination.

Effects of explant age, growth regulators and carbohydrates on sugar accumulation in Citrus juice vesicles cultured in vitro

Juice vesicle explants of satsuma mandarin cultured on MS solid medium grew expansively and their weight increased 250–400% over a month and accumulated sugar (50–90 mg/g juice). However, sugar accumulation did not occur when one-third of fruit was cultured. Sugar accumulation only occurred in the juice vesicle explants excised from fruitlet later than 51 days after full bloom and cultured on a medium containing 10% sucrose. Supplements of benzyladenine (BA), gibberellic acid (GA3) or naphthalene acetic acid (NAA) in the medium promoted the growth of juice vesicles, but these growth regulators did not stimulate sugar accumulation. Effects of sucrose, glucose and fructose on vesicle growth and sugar accumulation were determined. Fructose was not effective for vesicle growth. Total sugar content in juice vesicles was highest on the medium containing glucose. Sugar composition in vesicles cultured on various carbohydrates indicated that sugar degradation and conversion occurred in vesicle explants.

Factors Influencing the in Vitro Cultivation of Common Bean (Phaseolus Vulgaris L.) Leaf Petioles

ABSTRACTThe influence of genotype, explant's age and medium on in vitro cultivation of common bean (Phaseolus vulgaris L.) leaf petioles was investigated. Bulgarian cultivars Doubru-djanski 7; Plovdiv 11M and Plovdiv 10 were used in the experiments. Leaf petiole explants arising from in vitro seedlings were cultivated on modified MS media containing different amounts of TDZ, Paclobutrazol, NAA, BAP, IBA and GA3 in different combinations. Explant's development in different ages (5-; 7- and 14-days old) on the investigated media was genotype dependent. Bi-factorial ANOVA and three-factorial analyses were used for evaluation of the results that precisely disclosed the factor's interactions. The applied 3-factorial dispersion analysis revealed that the interaction between factors genotype-explant age (AB) and the threefold interaction genotype-explant age-media (ABC) exerted the strongest influence on the explant development and callus formation. The influence of interaction between factors genotype-explant's age (AB) increase along with cultivation time. No dependence was found between the size of initial plants, leaf petiole position (near to the leaf or to the steam), explant fresh weight, their development and quantity of formed callus. An additional experiment with precultivated sterile seeds on media containing 1 μM IAA or 1 μM BAP (on the base of MS) contributed. Better explant development was observed in comparison to their cultivation only on MS medium.

A Two-Step Approach to Scale Up Green Plant Regeneration Through Somatic Embryogenesis from in vitro Cultured Immature Inflorescences of a Male Sterile Line and a Maintainer Line of Rice

ABSTRACT A novel, two-step protocol for rapid propagation of cyto-plasmic male sterile (CMS: A line) and maintainer (B line) line of rice through somatic embryogenesis was developed and more than 100-170 green plantlets from A and B lines were regenerated per single immature inflorescence after 8 weeks of culture. Immature inflorescence explants of IR58025A and IR58025B were cultured on an induction medium supplemented with 2 mg l−1 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l−1 6-benzyl amino purine (BAP) for a period of 2 weeks. The medium under appropriate conditions of explant age and hormones allowed predetermination of partially differentiated cells of the individual spikelet into an embryogenically competent proembryogenic group. Differentiation of these proembryogenic groups was achieved by transferring them to embryo proliferation medium containing a high concentration of BAP, proline and maltose for a period of 2 weeks. About 800-1200 green plants could be regenerated after 8 weeks of cu...

A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus annuus L.)

We describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 μM BA and without auxin. Addition of 1 μM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.

Hormonal regulation of lipogenesis in cultured adipose tissue from perinatal and month-old goats

Perinatal (1–2 days old) and month-old (24–32 days old) male goats were used to investigate the effect of age and chronic culture (24 h) of perirenal and omental adipose tissue explants in the presence of insulin, cortisol and recombinant bovine somatotrophin (bGH), alone or in different combinations, on lipogenic responses in the absence and in the presence of catecholamines in acute incubations (2 h). Addition of insulin alone or bGH alone or insulin plus bGH had no effect on the rate of fatty acid synthesis in tissues of both groups. The presence of cortisol alone decreased ( P<0.05) and the combination of insulin plus cortisol increased ( P<0.05) lipogenesis in perirenal adipose tissue explants from month-old goats. No such effects of these hormonal combinations were noted in omental adipose tissue from perinatal and month-old goats or in perirenal adipose tissue from perinatal goats. When perirenal adipose tissue explants from month-old goats were cultured in the presence of insulin plus cortisol plus bGH the rates of lipogenesis were lower ( P<0.05) than those in cultures with insulin plus cortisol. Isoprenaline (Iso), but not noradrenaline (Ne), inhibited lipogenesis in freshly prepared adipose tissue explants; however, when maintained in tissue culture for 24 h, a response to Ne was induced. In cultured perirenal adipose tissue explants of both perinatal and month-old goats, the mean values of fatty acid synthesis were decreased ( P<0.05) by Ne and Iso: Ne and Iso were equally effective. However, in cultured omental adipose tissue explants, Iso was more effective than Ne.

Embryo rescue of interspecific hybrids of Brachiaria spp.

Age of explant and six different media were evaluated with the objective of regenerating higher numbers of interspecific hybrids between sexual and apomictic Brachiaria. Immature embryos of 7–8, 9–10 and 11–12 days after pollination (DAP), from artificial hybridization between Brachiaria ruziziensis (R) as female parent, and B. brizantha (B) or B. decumbens (D) as male parent, were cultured in modified MS media (M4) – supplemented with different combinations of growth regulators and vitamins. Embryos cultured 9–12 DAP showed high percentage (85–100%) of germination for all the crosses examined. Germination and survival rates varied according to accessions within crosses. Six different media (all modified MS with different growth regulators and vitamins) were tested with the objective of inducing multiple shoots from 7 to 10 DAP embryos, from crosses between R × B. The media M1, supplemented with Kinetin (13.94 μM) and NAA (5.37 μM), and media M3, supplemented with BA (4.44 μM and IAA 2.85 μM), regenerated adventitious shoots and calli about 30–40 days after inoculation. The highest multiplication rate observed was 2.85 shoots per explant in media M1, 60–70 days after culturing. Two other media, M6, supplemented with 2,4-D (13.57 μM) and M2, with 2,4-D (9.05 μM) and BA (8.87 μM) exclusively induced the formation of calli. The described protocols proved to be efficient in regenerating healthy seedlings from immature embryos of interspecific hybrids in Brachiaria.

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m–2 s–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively.

Improved shoot development and rooting from mature cotyledons of sunflower

Regeneration and development of shoots from sunflower cotyledons were improved by optimizing explant age, plant growth regulator concentrations, and duration of exposure to plant growth regulators during shoot initiation, development and rooting. Shoot initiation required only a brief exposure to auxin and cytokinin, and minimizing the duration of exposure to high levels of plant growth regulators improved shoot development. Rooting was improved in terms of both the number of shoots that rooted and the time required for rooting by incorporating activated charcoal in the lower layer of a 2-layer rooting medium.

GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency.

Several factors were investigated for their influence on the transfer of an intron-containing β-glucuronidase (GUS) gene into blueberry (Vaccinium spp.) leaf explants during the early stages of Agrobacterium-mediated gene transfer, including days of cocultivation, strain of Agrobacterium tumefaciens, explant age and genotype. The number of GUS-expressing leaf zones and calli were counted immediately and 2 weeks after cocultivation, respectively, to evaluate the gene transfer process. Agrobacterium tumefaciens strain EHA105 (pEHA105/p35SGUS-int) was significantly more effective for transformation than strain LBA4404 (pAL4404/p35SGUSint). Four days of cocultivation with A. tumefaciens strain EHA105 yielded about 50-fold more GUS-expressing zones than 2 days of cocultivation. Significant differences among cultivars were observed for both GUS-expressing leaf zones and calli. For some cultivars, explant age influenced the number of GUS-expressing leaf zones and calli. In most cases, the number of GUS-expressing calli was highest in those cultivars where GUS expression in the leaves was high.

Agrobacterium tumefaciens-mediated transformation of cauliflower, Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.

Temporal effect of 2,4-D on induction of embryogenic nuclellar cultures and somatic embryo development of ‘Carabao’ mango

Embryogenic nucellar cultures were established on B5 major salts, MS minor salts and organics, 400 mg/l−1 glutamine, 60 g/l−1 sucrose, 2 g/l−1 gellan gum, and 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D). There was no clear relationship between developmental age of the nucellar explants and induction of embryogenic cultures. The temporal requirements for culture initiation and for induction of embryogenic competence from nucellar explants were determined by pulsing the cultures for 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 d. Culture initiation required a minimum 7–14 d pulse with 2,4-D, and was maximum after a 56-d pulse; however, embryogenic competence was optimum after a minimum of 28 d exposure to 2,4-D. Somatic embryogenesis occurred directly from the nucellar explants at low frequencies. Somatic embryo maturation only occurred following plating of suspensions onto semisolid medium, and was stimulated by 2.4–4.8 µM kinetin and 4.4 µM 6-benzyladenine.

Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering.

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees.

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.