Adventitious Bud Regeneration Research Articles

Anatomical observations of adventitious bud regeneration from leaf explants of Ziziphus jujube Mill. ‘Huizao’

The histological process of adventitious shoot regeneration from the leaf explants of Zizyphus jujuba ‘Huizao’ was reported in this study. This is the first report on the detailed histological process of direct shoot regeneration from leaf explants in Z. jujube. Shoot regeneration was obtained from woody plant medium (WPM) supplemented with 2.27 μM thidiazuron (TDZ), 1.07 μM α-naphthalene-acetic acid (NAA) and 2.94 M silver nitrate (AgNO3) for 10 days in the dark followed by 3 weeks at a 14 hours photoperiod. The adventitious buds mostly formed from leaf veins and petioles, and the further histological studies revealed that there were multiple vascular bundles around leaf veins and the adaxial side of explants, and the adventitious buds directly originated from the parenchymatous cells around the vascular bundles without the intervening callus phase. After 3 days of culture, the parenchymatous cells started dividing and meristemoids formed thereafter. The meristematic cells continued division and subsequently gave rise to bud primordia. Well-developed shoot buds through direct organogenesis was achieved after 20 days of culture.

Factors Affecting Regeneration of Tomato Cotyledons

In this research, we employed the tomato cultivar named Zhongshu Si Hao as research material to study the influences on the regeneration of adventitious buds under the different conditions with hormone combinations, various explants, different parts of cotyledons and cotyledon inoculatingmethods; we also screened the best method for seed surface disinfection and the most appropriate medium for the rooting of adventitious buds. The results showed that the best procedures for surface disinfection of tomato seeds would be as follows: firstly, immersing the seeds in 70% ethanol for 30 second, then rinsing them in 20% NaClO with tween-20 for 20 min. The most ideal explant for inducing buds is the middle part of the cotyledon, followed by the stem and the leaf. the best medium formula for cotyledon regeneration is MS+ZT 2 mg/L+IAA 0.1 mg/L, and placing the cotyledon upside down is better to induce the adventitious buds than that placing upside. The best medium for rooting of adventitious buds is 1/2MS+IAA 0.3 mg/L. This research would lay a foundation establish transformation system of tomato in our future research.

Adventitious bud regeneration from the stigma of Sinapis alba L.

Stigmas isolated from flower buds of 'Nakielska' variety of <i>Sinapis alba</i> were used to develop a micropropagation method suitable for breeding of new cultivars. The origin of adventitious bud regeneration was studied on MS medium, under stimulation by bezylaminopurine (BAP) in combination with 2,4-D - dichlorophenoxyacetic acid (2,4-D). Histological analysis showed the structure of Sinapis stigma (composed from four types of tissue: papillae, transmitting tissue, parenchyma and vascular bundles) and revealed that numerous meristematic centers developed from parenchyma cells in close vicinity of vascular bundles. Buds very quickly appeared on the surface of initial explants and later formed multiplantlets that were easily rooted in the soil.

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l −1 α-naphthaleneacetic acid (NAA) and 1.5 mg l −1 2,4-D with 0.5 mg l −1 NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l −1 NAA and 0.5 mg l −1 1-phenyl-3-(1,2,3-thiadiazol-5- yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l −1) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.

Micropropagation of Uraria picta through adventitious bud regeneration and antimicrobial activity of callus

Uraria picta is extensively used in the Asian traditional systems of medicine. Overexploitation of the species for preparation of the drug Dashmula has led to the plant becoming rare and endemic. In the present investigation, an efficient micropropagation protocol has developed from leaf-derived callus of U. picta. Among the various concentrations of cytokinins (6-benzyladenine—BA; kinetin—Kin; and thidiazuron—TDZ) used, a significantly higher number of shoots per culture (58.8 ± 0.8) was observed on Murashige and Skoog (MS) medium fortified with 4.44 μM BA. The shoot regeneration frequency was sustained upon transfer to the same fresh medium at 4-wk intervals over a period of 2 yr. The medium containing various concentrations of auxins (α-napthalene acetic acid (NAA) or indole-3-acetic acid (IAA)) showed callus interspersed root formation; however, MS basal medium containing 3% sucrose revealed direct root induction from in vitro raised shoots. The acclimatized in vitro grown plants showed almost 98% survival upon transfer to soil in earthen pots and grown ex vitro. Randomly amplified polymorphic DNA analysis of 25 arbitrarily selected regenerants and mother plants revealed 100% uniformity and true-to-type nature of the regenerants. Methanolic extracts of callus showed strong antibacterial activity against pathogenic bacteria as compared to leaf and root extracts of in vitro raised plants and wild plants, suggesting the presence of higher concentrations of active chemical constituents (isoflavanoids) in callus cultures of U. picta.

Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type

In order to evaluate the formation of adventitious buds and in vitro regeneration of sour orange plants (Citrus aurantium L.) two organogenesis-inducing experiments were conducted. In the first experiment, the induction and in vitro regeneration of adventitious buds were tested on epicotyl and internodal segments under the influence of BAP or KIN associated with NAA. The second experiment evaluated the in vitro regeneration of sour orange plants related to different explant types (epicotyls segments, internodal segments of in vitro germinated plantlets and internodal segments of greenhouse cultivated plants). Data collected on both experiments included the percentage of responsive explants (explants that formed buds), and the number of buds per explant. The addition of BAP showed the best organogenic response. In vitro germinated epicotyl segments and internodal segments are recommended as explants for sour orange in vitro organogenesis. Rooting of regenerated shoots was achieved without the need of auxin in the medium.

In vitro multiple shoot proliferation and plant regeneration in rose(Rosa hybrida L.)

This study was conducted to investigate an optimal condition for shoot proliferation and regenerate shoots from in vitro leaflet and embryogenic calli from in vitro roots in rose. The effect of BAP on shoot proliferation was somewhat different depending upon genotypes or gelling agents. Leaflets with petiole cut from donor shoots which had been cultured in MS medium supplemented with 0.1 <TEX>$mg{\cdot}L^{-1}$</TEX> NAA for six weeks was effective for regeneration of adventitious buds(ABs) as well as shoot elongation of Rosa hybrida cv. Sweet Pink. Culturing seven leaflet explants per petri plate(<TEX>$100mm{\times}15mm$</TEX>) was effective for regeneration of ABs. Embryogenesis was shown in the calli induced from roots of Rosa hybrida cv. Sweet Pink cultured in the SH medium supplemented with 11 <TEX>$mg{\cdot}L^{-1}$</TEX> 2, 4-D for four weeks. Color of calli induced from roots was yellow although their color was a little different as type of basal medium.

Germination and conversion of somatic embryos derived from mature Quercus robur trees: the effects of cold storage and thidiazuron

Somatic embryos of Quercus robur L. belonging to lines derived from mature trees were germinated in experiments designed to determine the effects of pregermination chilling and the temporary inclusion of thidiazuron (TDZ) in the germination medium. Germination response was evaluated in terms of the percentages of embryos exhibiting root-only elongation, shoot-only elongation, or both (conversion to plantlets). Conversion rates depended on genotype and were lower than those of oak zygotic embryonic axes germinated in vitro. Two months’ storage of somatic embryos at 4°C between maturation and germination treatments did not influence their conversion frequency, although root-only germination was promoted in the three embryogenic lines investigated. Addition of 0.01–5.0 µM TDZ to the germination medium for 7 or 18 days induced multiple shoot formation in the epicotyl region of germinating embryos, although the 18-day treatment was detrimental with TDZ concentrations of 0.5–5.0 µM. Exposure to 0.05–0.1 µM TDZ for 7 days increased the frequency of shoot-only elongation and decreased that of root-only elongation in embryogenic lines with both high and low conversion ability, but whereas the conversion rate of the hard-to-convert line Sainza was significantly increased, that of the readily converted line B-17 was negatively affected. As a result, with 0.1 µM TDZ the total potential plant recovery of Sainza (the conversion rate plus the percentage of embryos exhibiting shoot-only germination) was 64%, approximately the same as that of B-17. Shoots excised from shoot-only germinating embryos can be elongated and rooted by proven micropropagation procedures. An additional result was the finding that adventitious bud regeneration was induced on the cotyledons of TDZ treated embryos (69% with 0.5 µM TDZ), which may prove useful for genetic transformation. This study is the first to have shown that TDZ can increase the germination efficiency of oak somatic embryos.

Adventitious bud regeneration from leaf explants of Platanus occidentalis L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l−1 6-BA and 0.49 mmol l−1 IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l−1 6-BA, 0.16 mmol l−1 NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l−1 IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%.

FEASIBILITY OF ALTERNATIVE SELECTION METHODS FOR TRANSGENIC APPLE AND PEAR USING THE DETOXIFICATION GENE VR-ERE

[...] Our preliminary experiments with apple and pear have demonstrated the following: 1) among the various substrates of VR-ERE, benzaldehyde (BD), a naturally occurring compound, is able to inhibit adventitious bud regeneration from apple as well as pear in vitro leaves, at concentrations ranging from 1 to 1.5 mM; 2) constitutive expression of Vr-ERE in transgenic 'Greensleeves' apple does not provoke any abnormal phenotype after one year of growth in greenhouse, but aldehyde reductase activity is reduced in all transgenic clones; 3) production of transgenic pear using BD instead of kanamycin as selection pressure is feasible, but the efficiency of the selection pressure seems low. In conclusion, the efficiency of Vr-ERE as a selective gene to produce apple and pear transgenic plants is not yet proven and further research is needed.

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.

Efficient selection of a high-yield line by using somaclonal variation in Japanese butterbur (&lt;i&gt;Petasites japonicus&lt;/i&gt;)

To induce somaclonal variations related to plant yield, adventitious buds were directly regenerated from immature flowerheads of a selected line A of the 'Aichi Wase Fuki' of Japanese butterbur (Petasites japonicus). Among the 280 independent regenerants obtained from 300 immature flowerheads, about 2/3 were regenerated within 90 d of culture. The total yield of the randomly selected 50 lines among the early regenerants ranged from 13.8 to 21.6 kg m � 2 (average: 18.2 kg m � 2 ) in the field, and a high correlation (r�� 0.869) was observed between the total yield and the period required for the third leaf initiation in flowerhead culture. A high correlation (r� 0.923) was also observed between the yield in the field and the fresh weight of rhizomes at 3 months of culture in the greenhouse. Since the yield of the original line A was 16.4 kg m � 2 , direct adventitious bud regeneration is a useful technique for inducing high yielding variants in Japanese butterbur, which might be efficiently selected by monitoring the period required for third leaf initiation and the weight of rhizomes.

Comparison of genetic transformation in Morus alba L. via different regeneration systems

Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.

Direct shoot regeneration from leaf and internode explants of Aloysia polystachya [GRIS.] mold. (Verbenaceae)

Adventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l−1) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m−2 s−1 photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27±2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5μM induced the highest regeneration rate (87±8.8%) after 20 d of culture, yielding 5.9±0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 µM sucrose, 0.8 % agar, 3.62 µM 2,4-dichlorophenoxy acetic acid and 2.22 µM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant-1) was achieved on MS medium supplemented with 8.88 µM BA, 2.5 µM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 µM) favoured shoot elongation and indole 3-butyric acid (7.36 µM) induced rooting. Rooted plants were hardened and successfully established in soil.

Effects of antibiotics on plantlet regeneration via organogenesis in Populus euphratica

Four antibiotics, kanamycin, geneticin, carbenicillin and cefotaxime, were evaluated for their effect on the regeneration of adventitious buds, shoot differentiation, rooting from regenerated shoots of Populus euphratica as well as on their control of Agrobacterium-mediated transformations. Results showed that the optimum concentration ranges of kanamycin and geneticin were 15–20 and 10–15 mg·L−1 at the stage of transgenic plantlet selection. The inhibitory effects of cefotaxime and carbenicillin varied among different genotypes of Agrobacterium. The inhibition of cefotaxime on Agrobacterium C58 was stronger than that of carbenicillin. The optimum concentration of cefotaxime was 100 mg·L−1. Cefotaxime and carbenicillin had similar effects on Agrobacterium LBA4404 and their optimum concentrations were both 150 mg·L−1.

Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L. Millsp.) expressing the synthetic BT cry1Ab gene

Summary Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeonpea, due to the limited genetic variation in cultivated germplasm. We have developed an efficient method to produce transgenic plants of pigeonpea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol is based on the direct regeneration of adventitious shoot buds in the axillary bud region ofinvitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration ofN 6 -benzyladenine (22.0mM). The tissue with potential to produce adventitious shoot buds can be explanted and used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeonpea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transformants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of singlecopy inserts of the introduced genes. Reverse transcription– polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests.

Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens (D.Don.) Endl.)

A rapid and effective system of somatic embryogenesis and organogenesis from the in vitro needles of redwood (Sequoia sempervirens (D.Don.) Endl.) had been established. The influences of plant growth regulators (PGRs) and days of seedlings in vitro on adventitious bud regeneration and somatic embryogenesis were studied. The process of somatic embryo formation was also observed. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l). SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l). The optimal days of seedling in vitro for adventitious bud and somatic embryogenesis were 30 days and 30-40 days, respectively. The developments of somatic embryos were similar to that of zygotic embryogenesis. The result of histocytological studies indicated that proteins were gradually accumulated in the process of somatic embryo formation and there were two peaks of starch grains accumulation that one was in the embryogenic callus and the other was in the globular embryos. These results indicated that starch and protein were closely related with the energy supply and the molecular base of somatic embryogenesis, respectively.

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic Jatropha curcas L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.

In vitro adventitious shoot bud differentiation and plantlet regeneration in Feronia limonia L. (Swingle)

In vitro adventitious shoot bud regeneration systems are considered most suitable for Agrobacterium-and biolisticsmediated genetic transformation to obtain transgenic plants. In the present investigation, multiple adventitious shoot buds could be induced directly from Feronia limonia hypocotyl explants inoculated on Murashige and Skoog (MS) medium containing different growth regulators. During the initial phase, the hypocotyl segments nearer to the cotyledons responded quickly compared to those closer to the root. The response, however, was comparable in both the segments in subsequent subculture. Of the various cytokinins, 2.22 μM 6-benzylaminopurine (BA) proved to be more effective compared to kinetin (Kn). The two-way interaction of BA and Kn significantly influenced shoot regeneration and contributed the most among the interactions studied. The best response, however, was obtained when 2.22 μM BA and 2.32 μM Kn were combined. Although the effect of auxins like α-naphthaleneactic acid (NAA) combined with cytokinins evoked a significant responsein terns of number of shoot buds, this response did not supersede the effect of combined cytokinins. Vone of the polyamines tested induced shoot buds on hypocotyl segments. Adventitious shoots were multiplied on MS medium containing 2.22 μM BA, 6.96 μM Kn, and 0.05 μM NAA. More than 60% of the shoots produced roots when cultured on medium containing one-quarter strength MS salts, 10% suerose, 0.6% agar, and 7.36μM indole-3-butyric acid. The adventious origin of shoot buds showing continuous vascular connections was confirmed through histological investigations.