Rooting Of Adventitious Shoots Research Articles

Establishment of clary sage (Salvia sclarea L.) regeneration system and development of glyphosate-tolerant clary sage plants through Agrobacterium-mediated genetic transformation

Clary sage (Salvia sclarea L.) is well known as a highly aromatic herbaceous plant and is commonly used in aromatic products and medicine. The biggest problem in the cultivation of clary sage is weeds, which not only entails a great cost of labor for weeding, but also leads to a great fluctuation in the yield and quality of its products. Therefore, there is a need to breed herbicide-resistant clary sage using genetic engineering. However, the lack of suitable regeneration and genetic transformation systems for clary sage has limited the process of molecular breeding. In this study, we firstly reported the regeneration system of clary sage, where explants from hypocotyls of sage induced adventitious shoots at a rate of 22.22% when cultured on MS medium supplemented with 2.5mg·L-1 6-BA, 0.15mg·L-1 IAA, and 0.25mg·L-1 VC. Rooting of adventitious shoots was achieved at a rate of 94.44% on 1/2 MS medium containing 0.125mg·L-1 NAA, 0.5mg·L-1 IAA, and 0.1mg·L-1 IBA. Secondly, based on the regeneration system, an efficient and stable Agrobacterium-mediated genetic transformation system was established in this paper, and the rate of genetic transformation was determined to be in the range of 3%-5% by transforming the hypocotyls of clary sage with pCAMBIA1301 vector and pCAMBIA1301-GR79-GAT vector. Finally, glyphosate tolerance was obtained from GR79 and GAT co-expressed clary sage plants in this study. This study not only lays the foundation of genetic transformation for the functional gene research and in-depth investigation of various biological problems of clary sage, but also provides a feasible genetic engineering solution for its industry to solve the weed problem, as well as a promotional role for precise molecular breeding of clary sage.

Genotype-dependent mass somatic embryogenesis: a chance to recover extinct populations of Pulsatilla vulgaris Mill.

The paper presents a technique for micropropagation of endangered in Europe and extinct in Poland Pulsatilla vulgaris for ex situ conservation of the genetic resources. Genotype-dependent induction of somatic embryogenesis and rooting was revealed in series of two experiments (I and II) performed under the same experimental conditions. Shoot tips of seedlings were the best explants in both experiments and Murashige and Skoog (MS) medium supplemented with 0.25 or 0.5 mg L−1 BAP was suitable for induction of somatic embryos (SE) and adventitious shoots. Mass SE was obtained in experiment I after explants transfer on ½ MS (2% sucrose) + 0.45 mg L−1 B1 and extending culture to 2–3 months without passages. Rooting of adventitious shoots was a critical point. Out of seven rooting media used in experiment I, only two, ½ MS hormone free (2% sucrose) + 0.45 mg L−1 B1 or MS + 5 mg L−1 NAA + 3.76 mg L−1 B2 resulted in altogether 36.4% rooted shoots. In experiment II, somatic embryogenesis, rooting and acclimatization of adventitious shoots failed. Regenerated plantlets and seedlings converted from SE from experiment I were acclimatized to ex vitro conditions. Both genome size, determined by flow cytometry, and genetic diversity analyzed by ISSR markers, confirmed the compatibility of regenerants from experiment I with P. vulgaris initial seedlings and commercial cultivar. Regenerants obtained in experiment II differed genetically from the regenerants of experiment I and cultivar. Propagated in vitro tissues/organs (SE, adventitious shoots) of P. vulgaris could be a source of material for cryopreservation, artificial seed production and/or for acclimatization of regenerated plantlets and could be used for restoration of the extinct populations.Key MessageThe micropropagation technique via organogenesis and somatic embryogenesis of endangered in Europe pasqueflower was developed as a tool for species recovery. The critical point is that somatic embryogenesis is genotype-dependent, which affects the repeatability of the experiments and also imposes applying molecular techniques to confirm the genetic fidelity of the regenerants with the initial material and other genotypes.

Non-aerated liquid culture promotes shoot organogenesis in Eucalyptus globulus Labill

Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.

EFFECT OF SINGLE-WALL CARBON NANOTUBE ON SOYBEAN (GLYCINE MAX) REGENERATION FROM MATURE COTYLEDONARY NODE EXPLANTS

One of the major limitations in producing transgenic soybeans using the agrobacterium-mediated cotyledonary-node method is low regeneration frequency. An improved highly efficient regeneration system of soybean was established herein. Cotyledonary node explants were placed in shoot initiation medium with single-wall carbon nanotube (SWNT) for adventitious shoots regeneration, and adventitious shoots were subcultured in shoot elongation medium with SWNT for shoot elongation and rooting. 40 mg/L SWNT supplemented in shoot initiation medium was found to be the optimal concentration with shoots regeneration frequency significantly increased by 21.5% compared with the control treatment, while for 4 mg/L and 400 mg/L, the increase was 4.6% and 6.5%, respectively. Faster elongation and rooting of adventitious shoots was observed in shoot elongation medium with 40 mg/L SWNT. Soybean plantlet formation frequency within the limited four weeks supplemented with 40 mg/L SWNT reached 48.4% while in the other three treatments: 4 mg/L, 400 mg/L and control, 0% were observed. These results indicate that supplement of SWNT in the soybean medium can efficiently promote adventitious shoots formation frequency, increase plantlet formation frequency and shorten the regeneration period.

Effects of growth regulators on plant to meristem culture and set up to complete plant in physic nut plant (Jatropha curcas L.)

Physic nut plant (Jatropha curcas L.) is considered as a potential source for a non-edible biofuel-producing energy crop throughout the world. This optimized meristem culture technique would be useful for developing uniform and virus-free clones of physic nut plant. Physic nut plant shoot apical meristems with 2 leaf primordia were aseptically isolated to sterilize and culture on Murashige and Skoog (MS) medium supplemented with kinetin (0; 0.1; 0.5; 1.0; 1.5; 2.0 mg.l-1), BA (0; 0.1; 0.5; 1.0; 1.5; 2.0 mg.l-1), TDZ (0; 0.01; 0.05; 0.10; 0.50; 1.0 mg.l-1) individually. Primary shoot induction was most effectively promoted by MS medium supplemented with 0.1 mg.l-1 TDZ. In this concentration, more than 95% of the shoots were regenerated without intervening any callus formation. The use of TDZ in combination with GA3 did not have good effects on the formation of shoots from meristem, in that only long meristems resulted. Also, when surveying the state of culture medium, that semi-liquid MS medium supplemented with 0.1 mg.l-1 TDZ will be suitable for the development of meristems. Rooting of adventitious shoots was achieved on MS medium supplemented with 1.0 mg.l-1 IBA and 3.0 mg.l-1 thiamin.

Root induction in radiata pine using Agrobacterium rhizogenes

Root induction using Agrobacterium rhizogenes was conducted in hypocotyl explants, intact seedlings, de-rooted seedling cuttings and adventitious shoots of radiata pine ( Pinus radiata D. Don). Use of two A. rhizogenes strains (A4T and LB9402), with or without application of IBA, can trigger root formation in different explants. Strain LBA9402 was more effective than A4T in increasing rooting percentage and root number. Addition of 4.4 uMIBA to the medium further enhanced rooting from the cultured hypocotyl segments inoculated with the two A. rhizogenes strains. Strain LBA9402+IBA induced in about 75% of the cultured hypocotyl segments to form roots. In contrast the controls failed to initiate roots on intact seedlings or cultured segments in presence or absence of IBA. Rooting of adventitious shoots from 3 year-old radiata pine was improved following inoculation with LBA9402, suggesting that this rooting treatment has potential to aid clonal propagation of radiata pine.

Root induction in radiata pine using Agrobacterium rhizogenes

Root induction using Agrobacterium rhizogenes was conducted in hypocotyl explants, intact seedlings, de-rooted seedling cuttings and adventitious shoots of radiata pine ( Pinus radiata D. Don). Use of two A. rhizogenes strains (A4T and LB9402), with or without application of IBA, can trigger root formation in different explants. Strain LBA9402 was more effective than A4T in increasing rooting percentage and root number. Addition of 4.4 uMIBA to the medium further enhanced rooting from the cultured hypocotyl segments inoculated with the two A. rhizogenes strains. Strain LBA9402+IBA induced in about 75% of the cultured hypocotyl segments to form roots. In contrast the controls failed to initiate roots on intact seedlings or cultured segments in presence or absence of IBA. Rooting of adventitious shoots from 3 year-old radiata pine was improved following inoculation with LBA9402, suggesting that this rooting treatment has potential to aid clonal propagation of radiata pine.

SHOOT ORGANOGENESIS AND PLANT REGENERATION FROM LEAF CALLUS CULTURES OF BLACK BARK SILVER BIRCH (BETULA PENDULA ROTH. ‘MELANOCORTEA’)

A 50-year-old specimen was used for in vitro cloning of silver birch dark bark form (Betula pendula 'Melanocortea'). For callus induction leaves were cultivated on Simola (S) medium. The effects of different media (MS, WPM and S) in combination with different cytokinin levels (BA and zeatin) on adventitious bud formation from callus segments were tested. The best effect was obtained on medium S containing 5 and 10 mg l -1 zeatin. The effect of different auxin concentrations (IBA and NAA) on the rooting of adventitious shoots indicated that highest percentage of rooting (100%) was achieved on half-diluted MS medium in combination with 0.5 mg l -1 NAA + 0.5 mg l -1 IBA. The high percentage (97%) of acclimatized plants obtained, showed that the practice of opening the test tubes some days before pricking out under ex vitro conditions and high humidity facilitated acclimatization. The successful acclimatization was probably due to the better aeration of the rooting system in a substrate of peat and zeolite.

In vitro induction of multiple buds in tissue culture of Juniperus excelsa

In the present experiment adventitious buds were induced on excised cotyledon segments and embryo explants of Juniperus excelsa on both Eriksson (1965) and Murashige and Skoog (1962) basal media containing either 0.5 or 1 mg 1 −1 BAP (6-benzylaminopurine) with or without NAA. When 0.02 mg 1 −1 NAA was added to Eriksson basal medium containing 0.5 mg 1 −1 BAP as many as 92 adventitious shoots were formed per cotyledon segment. Embryo explants showed the best response when the same concentration of NAA was added to Murashige and Skoog medium containing 1 mg 1 −1 BAP. The presence of NAA in Murashige and Skoog basal medium suppressed the morphogenetic response of cotyledon explants and successive growth of the buds. When the proliferated explants with numerous scale-like organs were transferred to respective basal media containing neither BAP nor NAA and when all explants with overcrowded propagules were divided into two or more sections, the growth of the propagules was further stimulated. Better rooting of adventitious shoots was achieved when shoots were pre-treated with IBA (1 mg 1 −1) and NAA (0.5 mg 1 −1) or activated charcoal (1% w v ) for three weeks and incubated in a covered seed tray filled with non-sterile compost for four months. Untreated shoots also rooted after four months when maintained on the same basal medium. These aseptically rooted shoots needed more careful acclimatisation.

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10-14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 μM 2,4-D and 5.0 μM TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (1∶20 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.

In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos

Organogenesis and somatic embryogenesis were induced from megagametophyte and zygotic embryo explants of 2 cycad species, Ceratozamia hildae and C. mexicana, cultured on modified B5 medium containing kinetin (0–13.9 μM) and 2,4-d (0–9.0 μM). Organogenesis occurred from megagametophyte explants of both species on the range of media tested. Somatic embryogenesis was largely restricted to zygotic embryo explants. Somatic embryos germinated in vitro: however, rooting of adventitious shoots was unsuccessful.

Factors influencing formation, development and rooting of adventitious shoots from embryos of picea abies (L.) Karst

A method is described for multiple shoot and plantlet formation on embryos of Picea abies (L.) Karst. Adventitious bud primordia were induced by culturing newly isolated embryos on media containing cytokinin. Further development of the bud primordia required transfer to cytokinin-free media. Adventitious shoots with a distinct stem were isolated from the embryos. After a couple of months the shoots were given a root-inducing treatment. When the roots had started to elongate the shoots were transferred to unsterile conditions. The capacity for bud production varied depending on the background of the seeds. The results showed that the method used for induction of adventitious buds strongly affected the ability of the induced buds to develop into vigorous adventitious shoots which could be rooted. The most critical factors which are discussed are: basal media concentration, cytokinin concentration, choice of cytokinins, and photoperiod.

Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non‐sterile conditions in a humid environment with occasional misting. An intervening 5‐day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO2 during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.

Formation and Rooting of Adventitious Shoots in Hevea brasiliensis

Formation and Rooting of Adventitious Shoots in Hevea brasiliensis