In vitro propagation of conifers using mature shoots

Mostafa K.Sarmast

Introduction

Plant tissue culture ideally allows the rapid proliferation of selected trees in a limited time frame,although it is desirable to select individual plants with greater growth rates or individuals that are more resistant to diseases.These could basically provide more options for breeding purposes(Thorpe 1985).In long-lived trees such as conifers,the genetic fidelity of plantlets derived by indirect organogenesis should be tested through several techniques before establishing clonally propagated offspring in the field.Despite considerable amounts of research,most gymnosperm species are still regarded as extremely dif ficult to regenerate.For clonal production of recalcitrant trees,not only the explant position of the mother trees is important,but also the seasonal effects should be taken into consideration because such factors play decisive roles in the regeneration response(Bonga et al.2010).Apart from a few successful examples with optimized procedures of micropropagation,mostclonalpropagation attempts encounter dif ficulties,either in the culture media or thereafter.Dif ficulties may include the inability to continue elongation,necrosis,low rooting ef ficiency,morphological changes,vitri fication,low regeneration,and excessive phenolic exudation,especially in older tissues.In somatic embryogenesis(SE),somatic cells undergo a developmental process similar to the development of zygotic embryos.However,somatic embryos must be tested for genetic stability before being established in the field.The SE technology for many conifers has yet to be evaluated in detail.In contrast to SE,the in vitro establishment of conifers through axillary and adventitious shoots—as they generally originate from mature differentiated stems—have a much lower chance of in vitro variation(Sarmast 2016).However,information on explant disinfestation and shoot multiplication through axillary and adventitious buds in conifers is sparse.Therefore,here we review the available literature on surface sterilization and in vitro propagation of conifers,exclusively from axillary and adventitious shoots.

Explant disinfestation

Success in plant tissue culture depends on obtaining aseptic mother plants;thus,controlling microbial contamination is the first and most important step for in vitro plant propagation and genetic transformation.Pathogen-free plants are also required for germplasm storage and moving living materials across international borders.Tissue culture regeneration of plants without viruses,bacteria,and fungi via shoot tip culture has been widely used since the 1960s(Thorpe 2007).However,meristem culture is now commonly used to remove viruses from infected plants.Epiphytic and endophytic microorganisms may cause severe problems for in vitro cultures(Cassells 1991;Debergh and vanderschaeghe 1988;Leifert et al.1991).In some cases,no signs or symptoms of infection are visible,yet infection can stop the growth of explants(Leifert et al.1989;1992).Bacterial contamination is especially hard to treat and dif ficult to detect in old woody plant species and conifers,because bacterial infections are mostly untraceable inside the plant tissues(Viss et al.1991;Buckley et al.1995).In contrast,however,fungal contamination is,in most cases,easily removable by ethanol and sodium hypochlorite.Internal contamination is often present in many in vitro propagation systems(Chanway 1998;Ewald and Suss 1993;Ewald et al.1997,2000;Laukkanen et al.2000;Traore et al.2005,Sarmast et al.2011).Generally,the surface sterilization of seeds is easier than other approaches such as sterilizing shoots or roots.Explants from fieldgrown perennial plants,especially explants obtained during the fall and winter or from tissues close to or inside the soil,are particularly dif ficult to disinfest due to the possible presence of epiphytic and endophytic microbes(Buckley et al.1995).It is common knowledge that the highest number of decontaminant explants,mostly reported in percentages,must be achieved with the least possible injury during establishment.Even a minor injury caused by a chemical is likely to act as a barrier for future growth and developmentofthe explants.Ethanoland sodium hypochlorite are two common chemicals used to decontaminate woody species,but explants from mature conifers are dif ficult to sterilize without injury(Keathley 1984;Timmis and Ritchie 1984).Gupta and Durzan(1985)obtained 90%aseptic explants from shoots of mature trees ofPinus lambertianaDougl.andPseudotsuga menziesii(Mirb.)Franco by successively applying H2O2(30%for 15 min),bleach(10%for 10 min),then HgCl2(0.05%for 10 min).Vegetative buds ofP.menziesiiwere surfacesterilized by bleach(20%and 100%)and flaming(for 3,5 s,or until the flame self-extinguished).According to this study,spring buds have higher contamination rates than winter buds.However,3–5 s of flaming,in combination with bud dissection,was preferred to bleach treatments for sterilizing buds in a highly contaminated environment before the in vitro culture is initiated.Increasing the duration of flaming apparently causes a decrease in the percentage of healthy,actively growing buds obtained(Traore et al.2005).An improved method of sterilization was reported,but it involved the use of toxic agents such as mercuric chloride(HgCl2),which should be avoided.Previous reports suggest that ethanol and sodium hypochlorite cannot completely controlErwinia herbicolacontamination when treating in vitro cultures ofAraucaria excelsaR.Br.,and the use of common antibiotics(i.e.,submersing in 200 mg L-1antibiotic solutions of cefotaxime,gentamicin,amoxicillin,ampicillin or tetracycline for 60 and 120 min after surface sterilization)can have dramatic side effects on the explants ofA.excelsa(Sarmast et al.2011).However,adding silver nanoparticles(AgNPs)is quite effective in controllingP. fluorescens,as the treatment drastically decreasedE.herbicolacontamination.The authors suggested that AgNPs,which induce physiochemical changes,have several modes of action and a higher surface area per mass,enabling them to prevent bacterial infections and actively block ethylene through the release of silver ions(Sarmast et al.2015).The presence of elevated levels of ethylene may also reduce explant growth and development,depending on the species(Sarmast et al.2010,2011,2015).

Endogenous bacterial infections are apparently inevitable during the culture conifers in vitro and may persist even after several subcultures(Ewald et al.2000).Ideal antibiotics to eliminate these bacteria are soluble in water,heat and light stable,unaffected by pH and the media used and are deleterious only on bacteria and fungi,not to the plant cells.Preferably they are active against a broad range of bacteria,and bactericidal rather than bacteriostatic.They ought to be optimally effective,even when used in combination with other antibiotics.Ideal antibiotics are expected to be non-resistance inducing,inexpensive,nontoxic to humans and other nontarget organisms and should have at least two modes of action(Falkiner 1990).However,common antibiotics are phytotoxic or may disrupt explant growth by inhibiting metabolic processes(Dodds and Roberts 1981;Falkiner 1990;Leifert et al.1992).For the genetic transformation ofPinus pineaL.,the application of cefotaxime,vancomycin,and ticarcillin is frequently advised to eliminate excessive growth ofAgrobacterium tumefaciensafter co-cultivation.These antibiotics are essentially nontoxic to stone pine cotyledons and enable significantly more buds to form on cotyledons(Humara and Ordas 1999).However,carbenicillin has inhibitory effects during somatic embryogenesis of Sitka spruce[Picea sitchensis(Bong.)Carr.;Sarma et al.(1995)]and Norway spruce[Picea abies(L.)Karst.;Mala´et al.(2009)].For conifers,the results are inconsistent.Cefotaxime appears to be detrimental to general health following the regeneration ofPinus radiatacotyledons(Holland et al.1997),whereas it had little negative effect on explant growth and development ofPicea glauca(Moench)Voss(Ellis et al.1989),Picea abies(Hood et al.1990),Pinus nigraJ.F.Arnold(Lo´pez 2000),Larix kaempferi×L.decidua(Levee et al.1997)andPinus pineaL.(Humara and Ordas 1999).These reports are in contrast with many other reports on nonconiferous plant species(Holford and Newbury 1992;Yepes and Aldwinckle 1994;Nauerby et al.1997;Ogawa and Mii 2005;Mendes et al.2009)in which the negative effect of cefotaxime on shoot regeneration was con firmed.Mala´et al.(2009)recommended amoxicillin and meropenem to eliminate infections byAgrobacteriumin explants ofPicea abiesduring their somatic embryogenesis because it did not adversely affect growth.When Le-Feuvre et al.(2013)was usingAgrobacteriumto transformP.radiata,even 400 mg L-1timentin did not inhibitAgrobacteriumgrowth after cocultivation ofP.radiataembryonal masses withA.tumefacienswhen cultured on modi fied Murashige and Skoog(MSG)medium(Becwar et al.1990).

The procedures for in vitro sterilization of many conifers are summarized in Table 1.The decontamination procedure is now mostly aimed at mature tissues of conifers.Recent reports have focused on the micropropagation of conifers via somatic embryogenesis through seed-based materials,which are not dif ficult to sterilize.Yet,new approaches have not been developed,and we still have little information on the decontamination of mature tissues in conifers other than several procedures that can reduce bacterial contamination in plant tissue culture systems.Such measures include sanitation of growth rooms,use of laminar flow hoods,and proper autoclaving and training of operators to reduce contamination of samples from mother plants from the field.Proper prewashing of explants with different antifungal compounds,mechanical shaking,and applying multiple decontaminants via vacuum in filtration can improve the effectiveness of the decontamination.Heat treatment of whole plants or plant parts(for up to 4 h at 43.5–57 °C),thermotherapy (exposure forweeksat 37–38 °C)or heat combined with meristem excision(0.10–0.15 mm in length)can most likely be effective for excluding bacteria and viruses.Visual inspection and specialized media(indexing cultures)should also be used to detect slow-growing bacteria,viruses,endophytes,or bacteria that do not grow on plant tissue culture media.Media supplemented with ribavirin or tribavirin can partially be successful in producing virus-free plants.Finally,a widespread range of antibiotics at different concentrations should be tested to determine the most effective concentrations with minimal phytotoxicity(Reed and Tanprasert 1995).

Clonal multiplication

The three common methods for clonal propagation include the production of plantlets via axillary shoots with the lowest somaclonal variation,direct and indirect adventitious bud induction,and somatic embryogenesis(Murashige 1974).An efficient in vitro regeneration procedure is a prerequisite forcommercialmicropropagation and genetic transformation.Immature or mature embryos of conifers are a popular explant for clonal production because regenerations from other types of conifer explants such as leaf and mature shoots are too laborious.Tang et al.(2006)reported cotyledon-derived embryos ofPinus elliottiion severalbasalmedia;higherfrequencies(34–46%)of callus induction were obtained on B5,SH,and TE media than on DCR,LP,MSG and MS media.On shoot formation media,adventitious shoots were regenerated from callus on B5,SH and TE within 6–12 weeks,with higher frequencies of callus induction(26–35%)on SH and TE,but with low frequencies(6–9%)on B5.They induced the formation of roots on adventitious shoots in media supplemented with 0.01 mg L-1indole-3-acetic acid(IAA)and 0.01 mg L-1indole-3-butyric acid(IBA),but the frequency of rooting was not mentioned.Tang and Newton(2005)also indued calli ofPinus strobuson PS medium(Tang and Newton 2005)supplemented with high concentrations of 2,4-Dichlorophenoxyacetic acid(2,4-D),naphthaleneacetic acid(NAA)or IAA(15 μM)within 6 weeks.They reported that the 6 μM thidiazuron(TDZ)was more effective than 6-benzyladenine(BA)and 6(Dimethylallylamino)purine(2iP)regarding the formation of adventitious shoots.They also concluded that treating the callus cultures at 4°C increases shoot formation,the average number of shoots per gram of callus and the rooting frequency in the PS medium.In addition,putrescine(0.01–1 μM)had positive effects on callus induction and shoot formation,most probably by decreasing lipid peroxidation in callus cultures.However,in protocols where the callus is the basal source of regeneration,the genetic fidelity of regenerated plantlets is more questionable than those that are directly derived from axillary shoots,which are used for many woody species.Burrows et al.(1988)reported that explants had a negative response to high concentrations of plant growth regulators(PGRs),

especially BA in the culture media.These results agree with observations by Traore et al.(2005),Selby et al.(2005)and Sarmast et al.(2012a).Furthermore,Stojicic et al.(1999)reported that the average number of buds per explant,average shoot length,and the explant survival percentage ofPinus heldrichiiChrist.grown on GD medium(Gresshoff and Doy 1972)was better than growth on LP or MS.In their experiment,BA was the best PGR,and 1 mM IBA pulse treatments of shoots propagated in vitro was ideal for inducing roots.Stojicic and Budimir(2004)later reported that a 1 h pulse treatment with 222 μM BA of explants from 30-day-oldP.heldreichiiseedlings on GD medium resulted in an average of 4.6 shoots per explant,but only 10%of the shoots formed roots.Adventitious shoot formation(caulogenesis)by cotyledons excised from mature embryos ofP.pineacan take place directly in the presence of BA.Nonetheless,adventitious shoot formation not only depends on the applied concentration of BA in addition to the genotype,but also on the extent of differentiation within the cotyledon after BA treatment(Valde´s et al.2001).According to Moncalea´n et al.(2005)and Alonso et al.(2007),BA-mediated caulogenic gene expression depends on BA concentration.Loureiro et al.(2007)evaluatedJuniperus phoeniceaL.on five media with different PGRs and found that the best elongation was achieved on Driver and Kuniyuki(DKW)medium supplemented with kinetin,either alone or with NAA.When well-developed internodes ofJuniperus phoeniceaL.were exposed to 2.4 μM IBA for 5 min,40%rooting was obtained after they were transferred to OM-free PGR medium(Rugini 1984).Combining BA and NAA can increase the percentage of responsive explants,and the mean number of buds per explant(Zhu et al.2010).Furthermore,the explants were superior to those on media without NAA;this situation can be of signi ficance compared to when media is supplemented with BA alone(Zhu et al.2010).Cortizo et al.(2009a)reported the first successful procedure for micropropagation ofPinus pineaadult trees via axillary buds using TDZ in LP medium(Quoirin and Lepoivre 1977).De Diego et al.(2010)

reported an efficient biotechnological approach for micropropagating adultPinus sylvestris;the highest organogenesis was obtained by culturing dormant shoot buds on DCR(Gupta and Durzan 1985)and WPM(McCown and Lloyd 1980).Media supplemented with 25 μM meta-topolin[6-(3-hydroxybenzylamino)purine]was also effective for organogenesis.De Diego et al.(2008)revealed thatPinus pinastershoot buds generated the most axillary shoots when cultured on DCR medium supplemented with 25 μM zeatin and meta-topolin,and in combination with 25 or 50 μM 6-benzyladenine.When Cortizo et al.(2009b)investigated the cause of reductions in the cytokinin-induced formation of adventitious shoots from embryos ofP.pinea,they postulated that differing concentrations of BA/[9R]BA inside the explants is the most probable cause of this germination-related loss in the cotyledon capacity to produce buds.The germination process not only decreases the endogenous concentration of BA/[9R]BA,but also hinders the occurrence of the peak where free bases and ribosids become evident(the active forms of BA metabolites).The procedures for in vitro shoot induction pertaining to the majority of conifers are summarized in Table 2.

Table 1 Treatments for surface sterilization of some conifer species

Table 1 continued

Table 2 Summary of treatments used to induce tissue production by various explants from conifers species

Renau-Morata et al.(2005)and Piola et al.(1999)achieved the highest proliferation rate(nearly 4.5 buds per explants)inCedrus libaniafter pulse treatment of microcuttings with 2.2 μM BA and 0.1 mM BA,respectively.In one report,adventitious shoots were regenerated fromC.atlanticaembryos,achieving 4.2 buds per embryo after 60 days.They were cultured on MSBN/2 media(halfstrength MS salt and half-strength Bourgin and Nitsch[1967]vitamins and Skoog[1944]amino acids)when preliminarily exposed to 9.0 μM zeatin for the induction stage,and 4.6 μM for further development.The adventitious shoots then continued to elongate on cytokinin-free media.However,Tamta and Palni(2004)reported a mean of 33.75 shoots per explant forC.deodaraon LP medium after exposure to 0.1 μM BA and kinetin for 28 days before transfer to PGR-free LP.But these shoots turned yellow during proliferation and elongation and,in most cases,they did not survive.For overcoming dehydration of shoots produced in subsequent subcultures,explants should be inverted on LP supplemented with 20.0 μM BA for 10 days,then shifted back to their typical position on PGR-free medium.Montalba´n et al.2011 established an efficient procedure whereby 100 mg embryonic tissue can produce 1700 rooted shoots via a combined pathway of SE and organogenesis inPinus radiata.The researchers reported significantly more shoots per explant in freshly collected somatic embryos that were exposed to 4.4 μM BA for 3 or 4 weeks,when compared to somatic embryos that were a week overdue for germination.The evaluation of two variables together,i.e.,the percentage of embryo formingshoots(EFS)and the number of shoots per embryo,called the shoot elongation capacity(SEC),provides a realistic assessment of the ef ficiency of the culture conditions(Pulido et al.1992).Freshly collected embryos ofPinus radiatatreated with suitable levels of BA for 3–4 weeks,resulted in a high ef ficiency of induction(Montalba´n et al.2011).Stump sprouts were produced from the basal zone of the juvenile crown of old trees and were successfully used as explant sources to micropropagateSequoia sempervirens(Boulay 1979)andCunninghamia lanceolataLamb.(Bigot and Engelmann 1987).Epicormic shoots have also been as an explant source for micropropagation(Burrows et al.1988;Vidal et al.2003).

Table 2 continued

Conclusion

Proper elimination of unwanted microbial contamination concerning in vitro conditions could contribute to a better establishment of mature shoots in conifers.Before surface sterilization,elements such as selection of explant source,physiological activity of explants are pivotal,and proper exposure and combination of decontaminants during surface sterilization are critical for removing surface contaminants.SE protocols for many conifer species are not adequate;therefore,mature shoots of conifers offer a good alternative for micropropagation.Although there are dif ficulties in the clonal multiplication of conifers,such as higher levels of contamination on mature shoots,low rates of regeneration,and excessive phenolic exudation,plantlets derived from axillary shoots and adventitious shoots of conifers generally have a much lower incidence of somaclonal variation,making this procedure more reliable than callus-derived techniques.

AcknowledgementsI thank Dr.Jenna E.Gallegos(Department of Molecular and Cellular Biology,University of California,Davis)for critically reading the manuscript.Mohsen Hamedpour-Darabi is thanked for editing the English.

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