Lev P.Trofimuk·Pavel S.Kirillov,2·Aleksandr A.Egorov,2

Abstract Abies gracilis Kom.(Pinaceae)is one of the rarest and endangered conifers in the Russian flora,which must be cultivated ex situ to ensure its survival.Cuttings of A.gracilis do not take root without biostimulants.We used a selection of biostimulants,concentrations,and conditions of their use to significantly increase Abies gracilis rooting,and to accelerate the production of planting material,and to reduce rooting time to one season.We tested 4 rooting systems:IBA,IBA with glucose and glycine,and original biostimulants(S-try and S-5).The original S-5 biostimulating system had the most balanced ratio of components.The number of rooted samples increased 2.7 times and the length of roots increased 1.8 time when using S-5 as compared to IBA.S-try and S-5-original biostimulant systems were synthesized and collected in 2011 and 2014,respectively, and were tested in St. Petersburg Forest Technical University.

Keywords Root formation·Abies gracilis·Biostimulant·Planting·Vegetative reproduction

Introduction

Abies gracilis Kom.(Pinaceae)is one of the rarest conifers in the Russian flora.It was described in 1901 by Komarov(1912).The taxonomic independence of this species is not recognized by all scholars.Most researchers(Turkov and Shamshin 1963;Naumenko et al.1986;Neshataeva and Firsov 2006;and others)accept species independence of the taxon and consider it a relict species of broad-leaved and coniferous mixed forests of the Early Pleistocene epoch.It was widely distributed on Kamchatka Peninsula prior to the beginning of the Pleistocene glaciation.

Abies gracilis is listed in the Kamchatka Red Book and is contained in the supplementary list in the annex of the Red Data Book of the Russian Federation:‘‘The list of the plant and mushroom taxa standing in need of special attention to their status in the environment and monitoring’’.A.gracilis is also included in the first and second editions of the Red Data Book of the USSR as a relict endemic species.Regrettably,it has erroneously not been included in the Red Data Book of the Russian SFSR,since it was inadvertently‘‘blended’’in the synonyms of Abies sachalinensis(Firsov et al.2008,2010,2015).

IUNC Red List of Threatened Species(Katsuki et al.2013)shows the taxonomic status of this taxon in the variety rank: Abies sachalinensis var. gracilis (Kom.)Farjon.Since the taxon population is isolated,it is assumed that it is here for one reason out of two:either an introduction or a Tertiary relict.Semerikova and Berkutenko(2009)determined species independence of A.gracilis and its mainland origin in Kamchatka by genomic-based research of allele frequency, and mitochondrial and chloroplast DNA analyses.An isolated population of A.gracilis is of scientific and practical interest,being the most cold resistant among East Asian Firs and ornamental(Firsov et al.2008,2015).At the Komarov Botanical Institute of the Russian Academy of Sciences(BIN RAS)since 1986 A.gracilis has been grown from seeds collected from the wild(Firsov et al.2008).The first fructification was observed in 2007.The seeds were brought from the Kamchatka Kronotsky Reserve by S.M. Shevchuk,research fellow of the BIN RAS,they were taken to the BIN RAS and to St.Petersburg State Forest Technical University(SPbSFTU)and sown in 1992.In the Botanical Garden of SPbSFTU only one sample out of three survived and was transplanted to its permanent site in 2000(Firsov et al.2010).There the sample is kept away from cold and is characterized by a slow growth rate in a vegetative state.

In its natural environments A.gracilis propagate both from seeds and by offshoots from the lower sides of branches. Root development requires up to 80 days(Dokuchaeva 1967;Den Ouden and Boom 2012).In tree nurseries most A.gracilis cannot be propagated by vegetative means.When propagating by cuttings,calluses form and becomes large,and then grow out from the cutting and disturb the root development.In autumn the callus is subject to decay which causes a cutting failure.

There is little information on distribution of A.gracilis in botanical gardens.In the Information-Searching System?Botanical collections of Russia and the adjacent states?(Firsov et al.2010)only A.sachalinensis is listed and,in some cases,it can be A.gracilis.Besides the botanical gardens of BIN RAS and SPbSFTU A.gracilis is also found in the Main Moscow Botanical Garden of Academy of Sciences(Demidov 2005).

A.gracilis is rare in nature,poorly represented in the collections of the botanical gardens and has reduced reproductive capacity.Having studied reports on the rooting success of true firs (Abies spp.) (Rosier et al.2004,2005)and phytohormone levels in A.nordmanniana(Rasmussen et al.2009),we experimentally attempted to root cuttings of A.gracilis,primarily for the purpose for preservation and increasing the number of this rare species in the Botanical gardens of St.Petersburg and using it for urban greening.Because cuttings seldom develop roots,a prerequisite for our experiments was to identify a growth stimulant that would increase the frequency and quality of roots developed from cuttings.

Materials and methods

We studied the effects of 4 growth stimulants on A.gracilis survival and callus formation,viz:A:well water,the control;B:IBA(3-indolilbutyric acid)(67 mg/L);C:IBA(67 mg/L)+glucose (5 g/L)+glycine (Gly) (1.3 g/L);D:biostimulant S-try(3-(1H-indole-3)-2-(4-tiokso-1,3,5-triazinil-1) propanoic acid) (67 mg/L)+glucose (5 g/L)+glycine(Gly)(1.3 g/L);S-try-original biostimulant synthesized from tryptophan(Try)in 2011 and tested in SPbSFTU;E:original S-5 biostimulating system contained in 1-L of solution:IBA-50 mg;D,L-glutamic acid(Glu)-167 mg; D,L-aspartic acid (Asp)-233 mg; L-α-alanine(Ala)-167 mg; L-α-aminobutyric acid-67 mg; γaminobutyric acid-67 mg; L-leucine(Leu)-67 mg; Ltyrosine(Tyr)-67 mg;Gly-0,4 g;glucose-3 g.S-5-new system collected from known components and tested in SPbSFTU in 2014.

Concentration were: IBA-0.005% ; amino acids-0.15% ;glucose-0.5% .The selected concentration of IBA was recommended in previous reports(Tarasenko 1959;Gamburg et al.1979;Basra 2000).The original biostimulants listed as D and E have also been tested for A.gracilis rooting.

The experiment was performed in 2013-2016, in February the cuttings were pruned from 5 A.gracilis grafters planted in 1980s in BIN RAS.Before rooting,the cuttings were stored in snow.In April,2014 the cutting was pruned from the sixth grafter at age 10 years from a private nursery at the settlement of Koloskovo in Leningrad Region.The branches were pruned from the lower part of the canopy.The cuttings were taken with 2-3 internodes of 10-18 cm length.We collected 400 cuttings,80 of which were used as controls.The rooting was carried out during the first 10 days of May.

Before planting,the cuttings were stored in a container with fresh solutions of stimulants.The cuttings were stored there for 20 h at a temperature of 18-20°C in broad daylight.

After treatment,the cuttings were planted in a small hotbed(outdoor bed in a wooden case)separated from soil by geotextile.The hotbed was shaded by dull,white,polyethylene film at a height of 30 cm.The shading protected the plants on east and south exposures.Hotbeds were filled to a depth of 12 cm with a mixture of sand and highmoor peat(pH 3.5-5.0)at a ratio 4:1.Transplant furrows were arranged on a north-south axis.

Cuttings were planted using methods described by Ermakov(1975).Cuttings were inserted into the slightly compacted and watered substrate to a depth of 4-7 cm with 3-4 cm between rows to provide aeration and to avoid decreasing the rate of photosynthesis. Cuttings were watered at 5-7 day intervals.

The rooting process can be divided into two stages:callus formation and root formation.Notably,callus formation is not always accompanied by root formation and can lead to further callus growth which in turn can affect root formation.This effect was called the formation of‘‘excessive’’callus.

Before being transplanted from hotbeds to open-ground beds,the rooted cuttings were measured when grafting in late August.The numbers of rooted cuttings and of cuttings that formed‘‘excessive’’callus were counted and percentages were calculated.

Statistical analysis consists of two stages:rooting and root progression.At the first stage we report rooting rate and 95% exact confidence intervals in natural conditions and for each of 4 kinds of stimulants,and perform comparative analysis using logistic regression GLM.Rooted cuttings only are involved into the root progression analysis. Root progression is analyzed by using one-way ANOVA model.We report mean value of root length for each group with 95% confidence interval,and check significant difference of the mean values between groups using F test.In order to investigate main contrasts and report more precise statistical findings we use Sheffe multiple comparison method(S-method).Statistical analysis performed using R statistical software(R Core Team 2018).

Results and discussion

Experimental data of 2013-2016 showed that growth stimulants varied in their effects on survival and formation‘‘excessive’’callus.Mean first-order root lengths are listed in Table 1 and depicted graphically in Fig.1.

Fig.1 Percentage of cuttings of Abies gracilis under the action of root biostimulants:with roots,‘excessive’’callus and died

The cuttings of A.gracilis did not take root without treatment using growth stimulants. The best results,67.50% rooting,resulted from use of the original S-5(E) biostimulating system. Using IBA with additives(C)and S-try with additives(D)the percentages of surviving cuttings were 42.50% and 41.25% ,respectively.The lowest rooting percentage resulted from use of IBA(B)-25% rooting.This was consistent with the results obtained at the Main Moscow Botanical Garden of Academy of Sciences-27% rooting(Demidov 2005).The best biostimulator E showed its effectiveness in the survival of cuttings by 2.7 times compared to the B biostimulator and 1.6 times compared to C and D biostimulators.

Figure 2 shows photos of the cuttings 1 year after treatment with stimulants the formation of roots.

Figure 3 shows a general view of the Abies gracilis rooted cuttings plantation aged 4 years in 2018.

The surviving cuttings measured the length of the root in accordance with the stimulator group(Table 2).

The efficiency of the E biostimulator display the root length(Table 2),which increased the root length compared to the B biostimulator in 1.8 times,C-1.5 times,D-1.2 times.

Classical ANOVA approach displays significance of stimulator type for the expected root length(F test statistic:13.82 under 3 and 137 df,p value 6.28×10-8).Furthercontrasts analysis(Table 3)indicates that the expected root length under the action of the E biostimulator is significantly larger then under the action of the standard widely used biostimulator IBA(B)and its improved version(C).Statistical non-significance of advantage in stimulating the root length of the biostimulantor E over the biostimulantor D is probably due to the small samples sizes.A comparison of the previous version E biostimulator-S-try(D)with the standard(B)indicates its significant advantage in stimulating the root length.

Table 1 Growth stimulant effects on rooting of Abies gracilis cuttings,2013-2016(indices shown the 95% exact confidence intervals)

Fig.2 Photos of root development on cuttings of Abies gracilis 1 year after treatment using stimulants:a-B stimulant,b-C,c-D,d-E(decoding of reductions of stimulants is given above in the text)

Fig.3 Photographs of a transplanted 3-year seedlings and b transplanted rooted 4-year seedling of Abies gracilis in 2018

Table 2 Growth stimulant effects on root progression of Abies gracilis cuttings,2013-2016(indices shown 95% confidence intervals)

Table 3 Advanced analysis of growth stimulant effects on root progression of Abies gracilis cuttings, 2013-2016 (simultaneous confidence intervals and p values)

Conclusions

Rooting of A.gracilis cuttings without biostimulators is impossible.The results of the statistical analysis revealed a significant influence of biostimulators on survival(up to 67.5% for original S-5 biostimulating system)and root size of Abies gracilis cuttings.A comparison with the action of a standard stimulant(IBA,survival 25.0% )showed a significant increase in survival up to 2.7 time and the length of rootsin 1.8 time.The use of other biostimulating systems,a variant of the standard(IBA+glucose+glycine)and early original system(S-try+glucose+glycine),led to an increase in survival up to about 1.6 and the length of roots up to about 1.5 time(reliable)and 1.2 time(nonreliable)respectively.

Appropriate selection of biostimulants,concentrations,and conditions of their use can significantly increase A.gracilis rooting, accelerate the production of planting material, and reduce rooting time to one season. For effective A.gracilis rooting it is necessary to use the stimulants containing growth regulators and bioactive substances developed to maximize their effect and to serve as a nutrient medium at the same time.

The simplest stimulating system with such composition is the IBA with glucose and glycine(C)and the biostimulant S-try(D).The original S-5 biostimulating system has the most balanced ratio of components.

AcknowledgementsThe authors express their deep gratitude to Dr.S.Malov(Leading researcher,Theodosius Dobzhansky Center for Genome Bioinformatics,St.Petersburg State University,Russia)for consultations and mathematical processing of the research data.