Evaluation of total protein,peroxidase,and nutrients measured by pXRF for the determination of tissue rejuvenation/reinvigoration of Eucalyptus microcorys

Ontogenetic aging of tissues and the gradual decrease of adventitious rooting are known challenges for the clonal propagation of woody species,hampering clonal forestry programs.This study examined possible signatures of tissue rejuvenation/reinvigoration in different propagated materials of Eucalyptus microcorys by analyzing the total protein profile,peroxidase activity,macro-and micronutrient contents,and adventitious rooting of mini cuttings.The analyses were performed on E.microcorys shoots which were successfully obtained by seminal and grafting propagation,micropropagation with epicormic shoots,and indirect organogenesis.Among four mature trees used in the propagation,tissues from the one with the best propagation results were investigated for signs of tissue rejuvenation and/or reinvigoration.Five individuals from each technique were randomly selected and transferred to a semi-hydroponic"channel"system.After four weeks in the seedbed,the total protein,peroxidase activity,nutrient content and rooting of the mini cuttings were evaluated.SDS-PAGE enabled the differentiation of leaf samples obtained by grafting from the other propagation techniques,as revealed by two distinct bands.Materials obtained by micropropagation with epicormic shoots showed the highest peroxidase activity,while those obtained by seminal propagation and from the selected mature tree showed the lowest peroxidase activity.A portable X-ray fluorescence spectroscope(pXRF)identified adequate nutrient content in most of the nutrients tested in materials obtained by seminal and grafting propagation,and by indirect organogenesis.The analysis of adventitious rooting showed that the highest rooting percentage was observed in mini cuttings from seminal propagation(75%)followed by indirect organogenesis(35%).Based on principal component analysis,it was concluded that rooting of mini cuttings from both seminal propagation and indirect organogenesis was associated with phosphorous,sulphur,and potassium contents,which suggests a higher level of tissue rejuvenation/reinvigoration in these propagated plants.Further studies are recommended to search for other methods that present similarities with the responses to adventitious rooting in forest species and thus optimize the rescue and propagation of plants with distinct ontogenetic stages.

Low temperature,IBA concentrations and optimal time for adventitious rooting of Eucalyptus benthamii mini-cuttings

Eucalyptus benthamii is a forest species of economic interest that has difficulty with seed production and also is considered to have difficulty with adventitious rooting using propagation techniques, such as cutting or mini-cutting. We aimed to assess the adventitious rooting percentage under different storage times in low temperatures and at various IBA （indole-3-butyric acid） concentrations to determine the optimal time of permanence for rooting Eucalyptus benthamii minicuttings in a greenhouse. Shoots collected from mini-stumps cultivated in a semi-hydroponic system were used to obtain the mini-cuttings. For the first experiment, the mini-cuttings were stored at 4℃ for 0 （immediate planting）, 24, 48, 72, 96 and 120 h. The second experiment evaluated the rooting dynamic to determine the optimal time of permanence for minicuttings in a greenhouse. The basal region of the mini-cutting was treated with various IBA solutions： 0 （free of IBA）, 1,000, 2,000, 3,000 and 4,000 mg.L^-1. Every seven days （0 （immediate planting）, 7, 14, 21 and 28 days）, destructive sampling of the mini-cuttings was performed to evaluate the histology of the adventitious rooting. Eucalyptus benthamii minicuttings should be rooted immediately after the collection of the shoots. The 2,000 mg.L^-1 IBA concentration induced a greater speed and percentage of adventitious rooting, and an interval of 35 to 42 days was indicated for permanence of the mini-cuttings in the greenhouse. Expo- sure to low temperature induced adventitious root formation with diffuse vascular connections.

In vitro morphogenic response of leaf sheath of Phyllostachys bambusoides

Nodal segments from secondary branches of saplings of Phyllostachys bambusoides were inoculated in MS medium to assess the in vitro morphogenic response of leaf sheath through the induction to callogenesis by Picloram（4-amino-3,5,6-trichloropicolinic acid） at different concentrations of carbohydrate under the same conditions with presence or absence of luminosity.In our experiment,secondary explants were kept in MS medium containing 8.0 mg·L-1 of Picloram for the callus formation.Calluses were transferred in MS medium supplemented with sucrose,fructose and glucose（control,2%,4% and 6%）.Results show that Picloram induced the callogenesis in leaf sheath.The secondary embryogenesis was formed in yellow-globular callus.The sucrose as carbohydrate source in the absence of light was more efficient to induce rhizogenesis.Glucose was more efficiency in the presence of light.Callogenic induction and further embryogenesis evidenced the competence and determination of leaf sheath cells.

Optimal cytokinin/auxin balance for indirect shoot organogenesis of Eucalyptus cloeziana and production of ex vitro rooted micro-cuttings

Shoot organogenesis is critical for the shortening of long breeding cycles and circumvent the barrier of cloning mature Eucalyptus cloeziana trees.It enables large-scale production of plants from transformed tissues.This study evaluates the effect ofα-naphthaleneacetic acid(NAA),thidiazuron(TDZ)and benzylaminopurine(BAP)on the organogenesis of E.cloeziana from hypocotyls and cotyledonary leaves.In the induction stage,hypocotyls and cotyledonary leaves were established in a Murashige and Skoog(MS)culture medium supplemented with NAA or TDZ.Callus tissues were cultivated in a MS culture medium containing only BAP or different concentrations of BAP/NAA in the differentiation stage.Adventitious buds were multiplied in vitro and elongated in a WPM culture medium supplemented with 0.89μM BAP and 0.05μM NAA.Cotyledonary leaves exhibited the best in vitro regeneration.The induction of adventitious buds occurred only in calluses induced from TDZ.In the differentiation stage,4.4μM BAP treatment promoted an increase of adventitious bud regeneration.Micro-cuttings from regenerated shoots were acclimatized and rooted ex vitro in mini-incubators.The results confirm the establishment of an efficient protocol for the in vitro regeneration of E.cloeziana by indirect organogenesis,providing new insights regarding cloning of this species.

Effects of chemical sterilization of the culture media,porous membranes and luminosity on in vitro culture of Eucalyptus grandis×Eucalyptus urophylla

Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro cultivation.Active chlorine and sealing systems are often used to reduce contamination and increase gas exchange.The aim of the present study is to evaluate the establishment,multiplication,elongation and adventitious rooting of E.grandis × E.urophylla.Two clones(C1 and C2) and four active chlorine concentrations(0.000%,0.001%,0.003%,and 0.005%) were tested in the establishment and multiplication phases.Three sealing forms(W/M,1/M and 3/M) and the same four active chlorine concentrations were applied to the elongation phase.Two luminosities(dark and light)and three sealings(W/M,1/M and 3/M) were tested during adventitious rooting.Active chlorine concentration of0.005% led to the lowest fungal contamination rate and to the highest in vitro establishment.Active chlorine concentration of 0.003% resulted in the greatest length and highest number of shoots per explant in the multiplication phase.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was maintained in comparison with those grown in an autoclave.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.

Decreased morphogenetic potential in peach palm stem-like cells in long-term in vitro conditions

Peach palm(Bactris gasipaes Kunth)has been micropropagated from pre-procambial cells that provide stem-like cell niches,(i.e.,pre-procambial cells),multipotent,pluripotent and totipotent for direct vascularization,adventitious buds and somatic embryogenesis,respectively.The direct induction of adventitious buds and somatic embryogenesis reduces the frequency of mutations when compared to indirect morphogenesis.Long-term in vitro cultivation of perennial species such as peach palm cause the clones to age and deteriorate;however,the consequences for morphogenesis potential are not fully clear.The morphogenic potential of peach palm clones established and in vitro cultivated for 8 years(regeneration of adventitious buds without callus formation)was investigated in leaves,roots and stem bases using histological and histochemical analyses.Data from long-term cultures(8-years-old)was compared to data from short-term cultures(1-year-old).Morphogenic pathways monitoring for direct induction of somatic embryos and adventitious buds revealed a strong morphogenic reduction potential in the pre-procambial cells,parenchyma cells in the proximal region of stem bases,and external cells of leaf sheaths.Initial cells of shoot apical meristems and pre-procambial cells commit cell reprogramming to the undifferentiated state and subsequent acquisition of cellular competence.These results are applicable in the micropropagation of peach palm,with consideration to obtaining clones and their long-term in vitro culture.

Mini-cutting technique for Khaya anthotheca: selection of suitable IBA concentration and nutrient solution for its vegetative propagation

Khaya anthotheca is a hardwood species from Africa and recently introduced to Brazil. This species yields high-quality wood for diverse applications, but little has been done toward an effective propagation method for large-scale production in nurseries. We evaluated the effects of different concentrations of macro and micronutrients（i.e., 100, 50 and 25% of the concentration of a referenced solution） combined with indole-3-butyric acid（IBA） at 0 and 2 g L-1 on the survival rate of mini-stumps and the survival and adventitious rooting of K. anthotheca mini-cuttings. The mini-stumps were grown from a source of seeds imported from Ghana, West Africa. The ministumps survived at a high rate（97%）. Consistently high shoot yields were obtained from the mini-stumps（average of 1.8 per mini-stump）. High survival percentage of minicuttings and adventitious rooting were observed when solutions with 100 and 50% of the nutrients concentration were combined with 2 g L-1 IBA. Histological sections analyzed through optical microscopy indicated the tissues from mini-cutting stems were juvenile, which might have stimulated adventitious rooting. These results have important implications for further work aimed at establishing propagation strategies for K. anthotheca, which are of prime importance for assisting breeding programs of this species.

Correction to:Effects of chemical sterilization of the culture media,porous membranes and luminosity on in vitro culture of Eucalyptus grandis×Eucalyptus urophylla

Correction to:J.For.Res.https://doi.org/10.1007/s11676-020-01240-5 In the original publication of the article,there were errors in the fig 3 and fig 6.The corrected Figs.3 and 6 are given below:The original article has been corrected.