Plantlet regeneration of adult Pinus massoniana Lamb. trees using explants collected in March and thidiazuron in culture medium

A protocol for micropropagation using nodal explants from mature Pinus massoniana trees has been developed. Time of explant collection is crucial for the initial success of aseptic culture. Explants collected in early March gave the highest percentage of explant survival(64.5%) and shoot-forming percentage(52.3%). Thidiazuron(TDZ) concentration significantly influenced shoot formation; 4 μM TDZ was optimum, with 4.8 shoots produced per explant with a mean length of 7.1 cm after 120 days of culture. Regenerated shoots rooted for 60 days in basic medium with 1μM NAA were ready for growth in pots. This is the first report on plantlet regeneration in vitro from mature trees of P. massoniana that provides a reliable method for propagating selected elites.     

Factors influencing direct shoot regeneration from leaves,petioles,and plantlet roots of triploid hybrid Populus sect.Tacamahaca

Since the generation of full-sib artificial triploid families, rapid clone establishment and genetic improvements have been needed. Here, we report an in vitro method of direct shoot regeneration of a triploid hybrid poplar[(Populus simonii × P. nigra 'Italica')×(P. × 'popularis')]. Using different randomized block designs, we selected one triploid to evaluate the explant type, optimal concentrations of plant growth regulators and agar, and culture time under light or dark conditions over 60 days. The highest rate of shoot induction, 80.0%, was obtained using Murashige and Skoog(MS) medium supplemented with 0.2 mg/L benzyladenine, 0.04 mg/L naphthaleneacetic acid(NAA), and 5.5 g/L agar for the first 30 days in the dark,then 3 g/L agar for the next 30 days in light. This last medium yielded the best rate of shoot induction(6.32 shoots/explant). These three media were also used to evaluate the influence of the genotypes of the parents and hybrid triploids on regeneration. Two parents and three of the four full-sib triploids were regenerated successfully;different genotypes and explant types significantly affected the rate of shoot induction and average number of shoots.Leaves but not petioles were a suitable explant. One genotype produced the highest rate of shoot induction of 96.67%.Half-strength MS medium supplemented with 0.2 mg/L indole butyric acid and 0.04 mg/L NAA was the most effective for rooting; rooting rate was 96.67%, survival rate of transplants was 73.33%, and rooting frequency surpassed 85% for each genotype. Overall, this in vitro regeneration system will be useful for the propagation and genetic modification of triploid poplars.     

Different culture conditions applied to in vitro shoot multiplication of two Eucalyptus benthamii explant sources

Eucalyptus adult material requires more successive subcultures in the in vitro multiplication phase for increased vigor and cellular activity. This study evaluated the endophytic manifestation and shoot multiplication of one 13-year-old Eucalyptus benthamii clone under different culture conditions and used canopy branches(CB) and trunk base material as explant sources. The culture media were wood plant medium(WPM), Murashige and Skoog medium(MS) and JADS(Correia and co-authors medium).Based on the results of the initial multiplication experiment, further tests examined sucrose concentrations and p H. Morphophysiology, dry mass production, endophyticmanifestation and histochemical were determined. Explant sources responded differently to MS and JADS media, but the WPM medium promoted homogeneous development.The responses were similar for both explant sources when sucrose concentrations varied. Shoots died in the absence of sucrose, showed high oxidation at 60 g L-1 and optimal development at 30 g L-1. Endophytes were more evident for shoots from the CB origin. Explant sources responded distinctively to treatment due to physiological and intrinsic genetic factors. Therefore, explant sources, different culture media, sucrose concentration and p H may determine micropropagation success and influence the presence and/or intensity of endophytic manifestation.     

Effect of auxins on axillary and de novo shoot regeneration from in vitro shoot cultures derived from forced epicormic buds of teak (Tectona grandis L.)

In this presentation,we report on de novo and axillary shoot regeneration and rooting of shoots maintained over a long term,from cultures of Tectona grandis L.Shoot-tips of teak shoots forced from epicormic buds were used as the starting material for axenic shoot-culture establishment.Long term maintenance of such axenic shoot cultures was carried out by regular sub-culturing on MS media supplemented with N 6-benzyleadenine (BA,8.8 μmol L-1) and indole-3-butyric acid (IBA,2 μmol L-1) for 24 months.Vigorously growing shoot tips (2 3 cm long) were inoculated on the MS basal medium supplemented with different concentrations (0,1,2,4,6,8 or 10 μmol L-1) of either IBA or α-naphthaleneacetic acid (NAA) for rooting.Axillary and de novo shoots were developed from axillary and cut basal ends of shoots,respectively.Shoots growing on auxins were further sub-cultured (every 15 days) and maintained for 45 days.The greatest number of de novo (5.06) as well as axillary shoots (2.85) was observed on the MS medium supplemented with 10 μmol L-1 NAA or 8 μmol L-1 IBA,respectively,after 45 days.The combinations of both IBA (μmol L-1) + NAA (μmol L-1) were tested at different concentrations (4 + 4,6 + 6,8 + 8) supplemented to a half strength MS basal medium with 0.1% activated charcoal for rooting of decapitated and non-decapitated de novo and axillary shoots.Rooting from non-decapitated de novo shoots was highest (93.33%) with a mean number of roots of 4.61 on this medium,supplemented with 6 μmol L-1 IBA + 6 μmol L-1 NAA,after 36 days of initial culture.Individual auxin,however,was not effective for root induction.Rooted shoots were acclimatized in a green house and after four weeks plantlets were transferred to the field.     

Effects of plant growth regulators,carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog(MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid(NAA), 2,4-dichlorophenoxyacetic acid(2,4-D), and indole-3-butyric acid(IBA), were tested at various concentrations(0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight(DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine(BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass(93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83 mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture.     

Effects of Thidiazuron, basal medium and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba×P. berolinensis

The effect of Thidiazuron (TDZ), basal media and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba×P. berolinensis were determined to establish a high efficiency shoot regeneration system from stem explants of P. alba×P. berolinensis. Stems of Populus alba×P. berolinensis were collected from cultured shoots in vitro derived from dormancy buds of 3-year-old seedlings. The stem explants were cultured on MS medium containing 0.02-mg·L-1 NAA (naphthaleneacetic acid), and 0.1, 0.3, 0.5 and 1.0 mg·L-1 concentrations of TDZ to determine the effect of TDZ on shoot regeneration. Three basal media, i.e. MS, woody plant medium (WPM) and B5, were used to test their influences of different media on adventitious shoot regeneration. Green, red, blue and yellow plastic films in comparison with florescent light as control were used to observe their effects on shoot regeneration. The results showed that different concentrations of TDZ had an evident influence on shoot regeneration. Lower concentration of TDZ (0.1 mg·L-1) resulted in more adventitious shoot regeneration and higher concentration of TDZ (>0.1 mg·L-1) inhibited shoot regeneration. Among different media, MS medium exhibited a high efficiency for shoot regeneration, followed by WPM medium, while B5 medium inhibited shoot regeneration. Normal light and yellow light exhibited better effects on shoot regeneration, compared with other light.     

Effects of Thidiazuron,basal medium and light quality on adventitious shoot regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured stem of <Emphasis Type="Italic">Populus alba</Emphasis>×<Emphasis Type="Italic">P. berolinensis</Emphasis>

The effect of Thidiazuron （TDZ）, basal media and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus albaxP berolinensis were determined to establish a high efficiency shoot regeneration system from stem explants of P. alba~P berolinensis. Stems ofPopulus alba~P berolinensis were collected from cultured shoots in vitro derived from dormancy buds of 3-year-old seedlings. The stem explants were cultured on MS medium containing 0.02-mg·L-1NAA （naphthaleneacetic acid）, and 0.1, 0.3, 0.5 and 1.0 mg·L-1 concentrations of TDZ to determine the effect of TDZ on shoot regeneration. Three basal media, i.e. MS, woody plant medium （WPM） and B5, were used to test their influences of different media on adventitious shoot regeneration. Green, red, blue and yellow plastic films in comparison with florescent light as control were used to observe their effects on shoot regeneration. The results showed that differ- ent concentrations of TDZ had an evident influence on shoot regeneration. Lower concentration of TDZ （0.1 mg·L-1） resulted in more ad- ventitious shoot regeneration and higher concentration of TDZ （〉0.1 mg·L-1） inhibited shoot regeneration. Among different media, MS medium exhibited a high efficiency for shoot regeneration, followed by WPM medium, while B5 medium inhibited shoot regeneration. Normal light and yellow light exhibited better effects on shoot regeneration, compared with other light.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

Assessment of factors affecting in vitro shoot regeneration from axillary bud explant of Camptotheca acuminata

Axillary buds from 3-yr.-old seedlings of Camptotheca acuminata in the greenhouse were cultured on the different basal media with different concentrations of growth regulators for shoot regeneration for studying the effects of different basal media, different concentrations of growth regulators (BA or TDZ), sucrose, agar and pH value on shoot regeneration from axillary bud. The results showed that B5 and WPM media were the optimal basal media and the optimal phyotohormone was BA of 1.0 mg/L or TDZ of 0.1 mg/L; The concentrations of sucrose of 30g/L and agar of 6g/L were most suitable for the shoot regeneration; pH value from 5.8 to 6.6 were broadly effective, but the best at pH 5.8.     

In vitro regeneration of Populus tomentosa from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ‘fasta-3'. A high frequency of callus induction([97 %) was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on1/2MS basal medium supplemented with 0.25 mg/L BA,0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.     

In vitro establishment of shoot meristems of Ilex paraguariensis and identification of endophytic bacteria

Yerba mate(Ilex paraguariensis A.St.-Hil.)is a species of great economic,social and environmental importance for the southern regions of Brazil,Uruguay and Argentina.Currently the most diverse products are obtained from mate leaves,including mate tea.The objective of this study was to establish shoot meristem cultures(meristematic dome and a few primordia)of elite clones and identify the endophytic bacteria present in the explants.We tested the effect of clones(F1,F2,A03 and A07),culture media(MS,1/2MS,1/4MS and WPM),cytokinins(kinetin,BA and 2iP),activated charcoal(1,2 and 3 g L^-1),and disinfecting agent(sodium hypochlorite and mercuric chloride)on in vitro establishment.F1 and F2 clones were the most responsive for shoot meristem in vitro culture.WPM medium supplemented with 8.8 lM 2iP,0.2 lM NAA and 3 g L^-1 activated charcoal was the most suitable for the in vitro establishment of the F1 clone.No phytotoxic effect of the disinfectant was observed and some meristems sprouted.The isolated endophytic bacterium was identified for the first time in yerba mate as Agrobacterium larrymoorei.To conclude,we were able to establish in vitro culture of yerba mate using meristems as explants but the tissues were not free of endophytic microorganisms which could interfere with explant response.     

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 1/2 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 and16 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.     

Establishment of in vitro culture of Populus euphratica Olivier

The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine (BAP) and gibberellic acid (GA) in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingjiang, west China, on selected media with MP2 0.5 mg·L-1 BA 0.1 mg·L-1 NAA. The shoots were elongated on a medium with 0.25 mg·L-1 BAP, 0.1 mg·L-1 NAA and 2 mg·L-1 GA and were then rooted on a medium with 0.2-0.5 mg·L-1 IBA. All the media were incorporated with 30 g·L-1 sucrose and an adjusted pH at 6.3.     

Rooting of Pinus radiata somatic embryos: factors involved in the success of the process

In vitro conditions of the culture media, plant growth regulators and culture containers may cause anatomical and physiological changes that have negative effects on rooting and ex vitro acclimatization of somatic plantlets. The control of these factors could contribute to the improvement of somatic embryogenesis systems in conifers, especially in pines. The influence of macronutrient concentrations, explant type and culture containers in Pinus radiata D. Don in vitro somatic embryo rooting were analyzed. The highest rooting percentage was observed using half-strength macronutrient concentrations, complete micronutrients and vitamins of Quoirin and Lepoivre medium. Although the use of glass culture vessels was the best to increase the efficiency of the somatic embryogenesis process in terms of rooting, the use of ventilated containers resulted in a significant increase in the percentage of plants able to be planted in the field.     

爬行卫矛下胚轴高频离体再生体系的建立(英文)

In this paper,a protocol for efficient shoot regeneration was successfully developed from hypocotyl explants of Euonymus fortunei var.radicans.Some factors that influenced shoot regeneration such as different combinations of plant growth regulators,types of medium and inoculation ways were studied in order to establish an efficient plant regeneration for transformation.The results showed that hypocotyl explants wero horizontally cultured on a basic medium composed of MS medium supplemented with 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA for induction and development of adventidous shoots.Ninety-four percent of regeneration frequency and 5.1 shoots per explants were obtmned after 30 days of culture.Regenerated shootsproliferated efficiently on a shoot multiplication medium consisting of MS medium containing 1.0 mg·L-1 BAP and 0.1 mg·L-1 NAA.Microshoots were rooted on a rooting medium made up of MS medium enriched with O.5 mg·L-1 IBA and O.5 mg·L-1IAA.After hardening,90% of plants were successfully established under greenhouse conditions.Histological observation revealed that shoot primordium originated from subepidermal cells of hypocotyl explants and directly developed into adventitious shoots without caHus formation.     

Improvement of the tissue culture technique for Melaleuca alternifolia

Tea tree oil is extracted from the leaves and twigs of Melaleuca alternifolia(Maiden Betche) Cheel,and it is widely used in medicines, food preservatives,cosmetics and health care products. Traditional propagation of M. alternifolia from seeds does not necessarily transfer the desired characteristics from their mother trees,the seedlings are not uniform, and the multiplication rate from cuttings is relatively low. For these reasons, it is necessary to develop tissue culture techniques for this species. This study showed that an efficient explant initiation medium for M. alternifolia was MS 1/2 + BA0.6 mg L~(-1)+ NAA 0.1 mg L~(-1)+ sucrose 30 g L~(-1),which yielded a 75.9 % initiation rate. An efficient multiplication medium was MS + BA 0.3 mg L~(-1)+ NAA0.15 mg L~(-1)+ sucrose 30 g L~(-1), which yielded a 4.3multiplication rate and 3.2 cm shoot length. The rooting medium was MS 1/2 + IBA 0.1–0.25 mg L~(-1)+ sucrose15 g L~(-1), which yielded a 100 % rooting rate, 2.94–3.32 roots per individual and 1.36–1.44 cm root length. Local red-core soil was suitable as a transplant medium, and yielded 98 % survival. This study improved the tissue culture technique for mass-propagation of M. alternifolia,enabling the production of high quality plants for market.     

In Vitro propagation of juvenileNothofagus leoni Espinosa (Fagaceae)

Nothofagus leoni has a restricted distribution in Chilean forests. This work determines suitable culture conditions forin vitro multiplication and rooting through shoots obtained from seedlings. Broadleaved Tree Medium was suitable for shoot multiplication. A medium with a pulse of 0.55 μM BA in the first subculture and two subcultures on BA-free medium resulted in a multiplication rate at day 63 of × 5.7, without callus growth or shoot neoformation. Rooted shoots of good quality (number and length of roots without callus growth) were obtained with 1.23 μM IBA (91.4% of rooting). The first roots appeared at day 11, reaching a higher speed of rooting at day 15.     

Preservation of Juniperus thurifera L.:a rare endangered species in Algeria through in vitro regeneration

Juniperus thurifera L.is an endemic Cupres saceae from the Aure`s Mountains of north eastern Algeria and endangered,in part,due to the scarcity of viable seeds It is threatened by other abiotic factors and the lack of an effective management strategy will increase its risk o extinction.The dearth of information on its in vitro regeneration impedes its application in forest managemen programs.We therefore developed a micropropagation protocol using microcuttings with auxiliary buds.Cuttings were grown on different combinations of media supplemented with plant growth regulators at different concentrations.The highest number of shoots and branches regenerated from original shoots was obtained on Woody Plant Medium(WPM)supplemented with 6-benzylaminopurine(BAP)(0.5 mg L^-1)and 2,4-dichlorophe noxyacetic acid(2,4-D)(0.25 mg L^-1).The best elongation of shoots was achieved with WPM supplemented with0.5 mg L^-1of BAP and 0.25 or 1 mg L^-1 of 2,4-D.On the second subculture,shoots had a higher number of branches than those of the first.The highest rooting rate,38.8%,was obtained with shoots cultured in 1/2 Murashige and Skoog(MS)medium supplemented with 5.0 mg L^-1each of indol-3-butyric(IBA)and naphthalene acetic acid(NAA).Similarly,the highest root numbers and lengths were produced on 1/2 MS medium supplemented with IBA and NAA(5.0 mg L^-1each).During transfer to acclimatization,rates of plant losses of 50% occurred.The second part of the experiment showed that the best shoot callusing was on WPM supplemented with BAP and 2,4-D,with either the combination 0.5+0.25 or 0.25+0.25 mg L^-1.The results of this research provide a starting point for further studies on in vitro regeneration of J.thurifera for the sustainable management of its unique ecosystem in the Mediterranean basin.     

Embryo culture and rapid propagation of Syringa

Embryo of lilacs (Syringa L) culture in vitro and the rapid propagation were studied. The orthogonal experiments, including the selection of basal medium, embryo age and other factors such as sugar, benzyladenine (BA), naphthalene acetic acid (NAA) and glutamine (Gln), were carried out. The results indicated that the optimal medium for embryo culture was Monnier medium supplemented with NAA (0.001 mg.L^-1), BA (0.1 mg.L^-1), sugar (50 g.L^-1), and Gin (400 mg.L^-1), with a germination rate of 91.7% at least; the optimal embryo age was 50 d; and Gln had significant effects on the germination rate of embryo.Moreover, the optimal medium for subculture was MS BA (2 mg.L^-1) NAA (0.001 mg.L^-1) Gln (0.5 mg.L^-1), with the propagation coefficient of 3.6 at least.     

Establishment of Efficient Regeneration System of Populus×euramericana cl.‘Bofeng’

Compared with the species of Leuce section,the difficulty in plant regeneration during tissue culture for the species of Aigeiros section is a key limiting factor for their application in genetic engineering of forest trees.In this study,the plant growth regulators combination,copper concentration,light intensity,and the selection pressures of kanamycin were investigated using leaves of Populus×euramericana cl.’Bofeng’ as explants,and a stable and high efficient regeneration system was established.Using this system,both the shoot regeneration rate and rooting rate were up to 100%,and the average number of differentiated shoots in each leaf explant was up to 20,and the survival rate of seedlings reached 98%.The optimal selection pressure of kanamycin in shoot inducement for leaf-explant was 40 mg·L^-1,and the optimal selection pressure of kanamycin in root inducement of adventitious bud was 20 mg·L^-1.The authors also found that differentiation rate of adventitious buds of P.×euramericana cl.’Bofeng’ could be significantly promoted by increased copper concentration,and this is the first report about the key role of copper（Cu） in explant regeneration of Poplars.This regeneration system with high frequency which was established by the authors was as good as that of species of Leuce section,and it provides a good technological platform for genetic engineering of forest tree which uses the species of Aigeiros section as model materials.