The International Journal of Plant Reproductive Biology 11(2) July, 2019, pp.167-175

DOI 10.14787/ijprb.2019 11.2.

*e-mail: opalko_a@ukr.net

Received : 01.07.2019; Accepted and Published online: 02.07.2019

ABSTRACT

Keywords :

Non-morphogenetic post-trauma regeneration potential of Malus hallianaKoehne (Rosaceae Juss.)

Olga A. Opalko and Anatoly Iv. Opalko*

National dendrological park «Sofiyivka» of NAS of Ukraine, 12-а Kyivska Str., Uman, Cherkasy region, 20300 Ukraine

The dynamics of the display of a non-morphogenic post-traumatic regenerative potential of Malus halliana Koehne (Rosaceae Juss.) in the Malus spp. collection during the growing season had been studied. To do this, each decade the sections of peripheral tissues of 10–12 mm in the length and a width of 1.5 mm together with a cambial layer were cut by specially made cutter on the annual shoots (increments of the past season). As a result of the wounds healing from artificial injuries, the terms of the highest activity of non-morphogenic regeneration processes had been determined. The comparison of the pace and intensity of the wounds healing with the dates of artificial cuts made it possible to conditionally divide the growing season of Malus halliana by a regenerative potential display at the following stages: an increase in the rates of regeneration, their relative decline, the second wave of increase of the rate of regeneration, and a fairly rapid attenuation. The tendency of higher dependence of regenerative potential on temperature fluctuations than on precipitation or hydrothermal coefficient had been revealed. It was suggested that the index of regenerative ability indirectly confirmed the level of environmental adaptation of the investigated plants, and periods of the highest regenerative activity could be favorable for vegetative reproduction and other technological processes, in particular the formation of bonsai, etc., which had been followed by the plants damage.

adaptive modifications, bonsai, crabapple tree, evaluation, hydrothermal coefficient, juvenile, plant damage.

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The regeneration processes combine a wide range of published materials from other researchers devoted to the natural phenomena with different mechanisms of study of regeneration processes. Consequently, he came to the implementation. In living organisms, the phenomenon of conclusion that lower animals reproduced the lost parts renovation after a variety of natural trauma had been known much easier than highly organized ones. A tadpole (pollywog) from ancient times and described in the writings of Aristotle is the larval stage of an amphibian and it is capable of and Pliny the Elder. The first scientific and methodological reproducing lost organs much better than mature ones. In observation of regeneration had been made by the French higher vertebrates, such as birds and mammals, this ability is scientist Réaumur (René Antoine Ferchault de Réaumur), who extremely limited. At the same time Charles Darwin pointed to in 1712 described in detail the phenomenon of regeneration of the individual exceptions. For example, the ability to self-the limbs of crayfish. healing is almost absent in the rather low organized Nemertea,

Over the next half-century there has been a lot of new also known as “ribbon worms” or “proboscis worms” (Barrett information about regeneration in various organisms. In 2016, Carlson 2007, Trembley 1744).particular, the Swiss naturalist Abraham Trembley, trying to August Weismann analyzed the phenomenon of find out whether the Hydra (genus of small, fresh-water regeneration from evolutionary point of view (Barrett 2016, organisms) belongs to the Animal Kingdom or the Plant Carlson 2007, Weismann 2010). In 1892, he suggested that Kingdom, cut it and detected regeneration, continued to study regenerative ability had been formed in the process of this process and described it in 1744 (Carlson 2007, Galliot evolution due to adaptation to frequent damage, which 2013, Lenhoff and Lenhoff 2007, Trembley 1744). In 1745 generally coincided with the previously published materials of another Swiss scientist Charles Bonnet described the René Réaumur and Charles Darwin. But it was August regeneration of the ringed worms—annelids (Annelida), in Weismann who had formulated the integral concept of 1769 the Italian researcher Lazzaro Spallanzani described regeneration after generalization of the results of his own amphibians (Amphibia), and in 1770 the German scientist- observations of the works of predecessors (Weismann 2010).encyclopedist Peter Pellas (Peter Simon Pallas) described Unlike August Weismann, Thomas Hunt Morgan, an planarian (Planaria) (Dinsmore 2007, Elliott and Alvarado American biologist, denied the dependence of the 2018). regenerative capacity of the organism on the frequency of its

Later, Charles Robert Darwin, who studied on damage in past history. In 1901 he declared that regeneration is regeneration during his famous trip on board of HMS Beagle, the primary peculiarity of living organisms, which cannot be tried to analyze the results of his observations from the explained from the standpoint of Darwinism (Morgan 1901). evolution theory point of view, and compared them with After all, Thomas Morgan pointed out that if some useful

property, for example regenerative capacity, would occur it essential genotypic abolition of the ability of plants to self-would be leveled off with each generation due to the healing not only in ontogenetic dynamics (McMillan View interbreeding of an individual capable of regenerating with an 1999), but also in phylogenetic one (Jusufov 1978). It has been individual insolvent (Morgan 1901). In spite of certain shown that in all cases, young plants (in the juvenile age) skepticism of Thomas Morgan in relation to the concept of regenerate better (Radhakrishnan et al. 2018). Such regeneration, formulated by August Weismann, he information has not only general biological but also applied unequivocally spoke about the functional importance of significance, for example, with regard to the use of ornamental physiological regeneration, which is observed as a recovery plants in landscape compositions, in particular the planning of after natural deterioration (replacing mutilated root cork cells, the distance of plants from the pedestrian zone to the places of changing the tree cortex trunks, replacing old elements of mass visit (Opalko and Opalko 2006), as well as vegetative xylem with new ones, etc.). Thomas Morgan also pointed to propagation of the garden plants and planning of the need to find out how the damaged part of the organism is technological operations for garden care (Hartmann and regenerated. In some cases, there is proliferation, cell rapid Kester 1975, Opalko et al. 2014, Sinnott 2018).reproduction of the organism part damaged, from which the During the last decades, as a result of the spread of lost part is formed. Such a regeneration was called molecular genetic methods, in particular the regulation of “epimorphosis”. Otherwise, the cells of that part of the double-stranded RNA gene expression and transgenose, the remaining organism will not proliferate, but it itself study of classic regeneration models in animals and plants has transforms into a lost part without mitoses. Such a peculiar to intensified (King and Newmark 2012, Poss 2010, Ikeuchi et lower animals way of regeneration, Thomas Morgan called al. 2016 and Sugimoto et al. 2011).“morphallaxis” as regeneration by the transformation of It is well-known that plant cells are totipotent, that is, they existing body tissues (Maienschein 2007, Morgan 1901). are able to fully implement their genetic program and give a However, some modern authors (Agata et al. 2007) propose start to a new plant (almost as a zygote). Therefore, after the the abandonment of “epimorphosis” and “morphallaxis” injury of the stem, root, leaf, or any other part of the plant or the categories and their replacement by a new unifying principle, introduction of a separate lobe (explant) into the nutrient considering that epimorphosis is only versus morphallaxis. medium in vitro, an undifferentiated mass of callus cells are

All living organisms, during their growth and formed in a wounded place due to proliferation (rapid cell development, experience various damages and react to them division), and new shoots or roots can be regenerated; that is, according to their, genetically programmed regenerative cell dedifferentiation occurs on the way to the regeneration of potencies within the limits of a physiological reaction norm. a new plant (Krishnamurthy et al. 2015, Kunakh 2005, Pais At the same time, the quality of regeneration and the rate of 2019, Raven et al. 2013, Rost et al. 2006, Sugimoto et al. 2011, post-traumatic healing in plants are largely determined by Tvorogova and Lutova 2018). However, in contrast to this cambial activity (Barlow 2005, Larson 2012, Wilczek et al. established method, the recent plant regeneration studies show 2011). However, cambial activity, as well as the overall that plant cells can regenerate damaged tissues, apparently capacity for reparation and regeneration, affecting the without dedifferentiation (Sena et al. 2009). Consequently, potential productivity and ecological suitability of plants, are callus is an organized and differentiated tissue that is not controlled by the genotype (Zhuchenko 1988) and have the generated from all plant cells, but mainly from a specialized nature of adaptive modifications according to a stable population of primary cells (Atta et al. 2009, Sugimoto et al. genotype. The degree of regenerative potentials display is 2010, Klimešová and Klimes 2007).usually proportional to the strength and duration of the action Using the approaches proposed by Morgan (1901) to of a natural or artificial adverse factor, and the adaptability of interpret physiological regeneration in accordance with the the modification occurs only when the strength and duration of peculiarities of healing the wound surface of the injured organ, the traumatic factor does not exceed certain limits defined by compensatory regeneration can be recognized as an the past evolutionary history and are fixed by the genotype as intermediate phenomenon between Morgan’s “epimorphosis” reaction norm. and “morphallaxis”.

For a significant excess of the usual strength and duration An effective way to estimate the dynamics of the post-of the traumatic factor, there may be non-adaptive changes traumatic regenerative potential of tree plants in vivo has where the plant can react inadequately and even die. However, already been developed. For the modeling of mechanical excessive stresses are weaker, which, on the one hand, are damage that may occur with plants, during the vegetation, a strong enough to cause hereditary changes and, on the other section of peripheral tissues along with a cambial layer was cut hand, not so strong to cause a lethal effect, can induce by a specially made cutter every decade on annual shoots mutations (Kosenko et al. 2008, Krenke 1950, Opalko and (increments of the past season). The regeneration potential Balabak 1999), including those that increase adaptability. was estimated at the pace and quality of healing (Opalko et al. Therefore, it is necessary to study and take into account the 2014). Such a technique contributed to the prediction of the

168 The International Journal of Plant Reproductive Biology 11(2) July, 2019, pp.167-175

periods of the highest activity of non-morphogenic (6–8 mm in diameter), spherical or slightly pear-shaped fruits regeneration processes, which is extremely important for the of red-purple color is a favorite for contained bonsai Trim. establishment of optimal terms of grafting and care The success of bonsai lies primarily in selection of plantoperations. However, due to the potential of non-morphogenic with high regeneration potential, regeneration it is also possible (with greater or lesser approximation) to judge the readiness for morphogenic .regeneration, which is equally important for the cutting by These considerations made it necessary to assess during classical methods, as well as using in vitro, where natural the growing season the peculiarities of the manifestation of the stress-induced stimuli (Opalko and Balabak 1999) similar to nonmorphogenous post-traumatic regeneration potential of irritation occurring in the process of propagation by cutting Malus halliana from the Malus spp. collection (Rosaceae had been modeling as a result of artificial injury to the plant. Juss.) of the National Dendrological park “Sofiyivka” of the An analysis of the dynamics of natural phytohormones National Academy of Sciences of Ukraine, depending on the content, the balance of inhibitors and stimulants will

date of the artificial injure.contribute to the selection of the hormonal composition of

MATERIAL AND METHODSartificial inductors of regeneration in vivo and in vitro (Opalko et al. 2015).

These studies were in the collections of the National An analysis of domestic and world literature reveals

dendrological park “Sofiyivka” of Ukraine’s NAS, situated significant gaps in the study of a number of important issues

in the Central-Dniepro elevated region of Podolsk-regarding to the connections between regenerative plant

Prydneprovsk area of the Forest-Steppe Zone of Ukraine. The potencies, in particular perennial woody plants, in vivo and in

area is characterized by temperate-continental climate with vitro, and their conjugacy with the genotype, physiological

unstable humidification and considerable temperature state, phytohormonal balance and environmental conditions.

fluctuations. Average many-year amount of precipitation per The last ones predetermine the need to deepen the theoretical

year is 633.0 mm, its amount being 300–310 mm at +10°С, and methodological fundamentals of improving the existing

which corresponds to the precipitation amount in dry southern propagation technologies, the production of planting material

areas of Ukraine. Average many-year air temperature is and the cultivation of horticultural plants.

+7.4°С.Representatives of Malus Mill. genus (Rosaceae Juss.), in

particular Malus halliana Koehne are among the undervalued in Ukraine ornamental plants promising for horticulture. In contrast, in many countries, Malus halliana cultivars and other ornamental crabapple have been taken a good place in

The wound, where the cut was made, was covered with garden design for many years (Cornille et al. 2014, Dirr 2002,

transparent scotch-tape to avoid infection and withering (Fig. Fiala 1994, Jefferson 1966, Marosz 2007, Opalko et al. 2016,

2 b). The equation to calculate regeneration coefficient was Tucker et al. 2000 and Volk et al. 2015).

adapted to a 9-point scale of the evaluation of regeneration efficiency (Opalko et al. 2014). The course of the healing process of the wound was observed every ten days with help of a magnifying glass, and the intensity of callus genesis was estimated at 1 point, if callus formation did not occur or its surface was less than 5% of the wound (Fig. 2 c). Objects with callus areas equal to 85.5–100% were estimated at 9 points (Fig. 2 d).

Regeneration coefficient was calculated in units of Common name of that species is Hall

regeneration coefficient (urc) using the approach of Olga A. crabapple tree. Malus halliana is woody plant species that

Opalko (Opalko et al. 2014) :natived to the North-Central, South-Central and Southeast China, with secondary habitats in Japan and North Carolina of USA (Malus halliana Koehne ... 2019). The Chinese name of Hall crabapple is “Chui Si Hai Tang”. Hall crabapple tree due to its small size and very beautiful flowers, reddish leaves with small teethed edges and a tapered tip (Fig. 1 a, b) and small

as well as in determination of

the pruning season when the tree can adequately heal the wound created during pruning

To evaluate regeneration ability on one-year-old shoots of the previous year of the plants studied the notchings (10–12 mm long, 1.5 mm wide) were being performed (Fig. 2 a). It is important to notch into the cambium.

Although ornamental crabapples (Malus spp.) did appear in gardens of Europe

thbefore the 18 century, with the introduction of Malus coronaria, Malus prunifolia, and Malus baccata, but the profile of crabapples was raised considerably owing to introduction of Malus floribunda, Malus halliana, Malus sieboldii, and Malus tschonoskii, and the very large hybrid populations those all now well established in cultivation in the

The notchings were being made every ten days from thlate 19 century (Barnes 2012).

third decade of March till first decade of October with a Malus halliana is one of the more widely used Malus spp.

special cutter (Fig. 2 f).in classical gardens in the Yangtze River basin in China (Zhang et al. 2019).

where :R—regeneration coefficient, urc;S—intensity of callus genesis, points;n —number of days after cuts were made to the 1

appearance of the first signs of callus;

,21

2

nn

SR

+=

2019 169Non-morphogenetic post-trauma regeneration potential of Malus halliana Koehne (Rosaceae Juss.)

Fig.1– Malus halliana blossom tree: (a–general view of blossoming Malus halliana; (b)–flowers in inflorescence.

Fig. 2–The technique of notchings and estimation of the artificial wounds overgrowing (according to A.I. Opalko et al. 2014):(a)–wound made with manual cutter (notching 10–12 mm long and 15.5 mm wide); (b)–wound covered with transparent scotch-tape to avoid infection and withering; (c)–surface of callus occupies less than 5% of the wound (1 point); (d)–surface of callus occupies 85.5–100% of the wound (9 points); (e)–manual notch cutter.

A B C D

E

A B

170 The International Journal of Plant Reproductive Biology 11(2) July, 2019, pp.167-175

n —number of days after cuts were made to the 2

completion or termination of callus development.

where : em–HTС– hydrothermal coefficient;SQ– precipitation amount for period of time with of temperatures higher than +10°С;SТ– sum of active temperatures (higher than +10°С) for some period of time.

Variations of Malus halliana average-seasonal regeneration coefficients indices, and precipitation amount, and air temperature in 2012–2015: (a)–average-seasonal regeneration coefficients, urc; (b)– mean air temperature, °С; (c)–precipitation sum, mm

the average seasonal coefficients of regeneration over the years from 2.59 to 4.04 urc (Fig. 4).

To calculate precipitation amount and air temperature Such fluctuations of the average seasonal coefficients sums, the data of Uman meteorological station were used. The of regeneration have led to the assumption that it is hydrothermal coefficient of G.G. Selyaninov (Shein and fluctuations in meteorological conditions, especially the Goncharov 2006) was calculated using the formula : amount of precipitation and air temperature could affect the

regeneration capacity of Malus halliana plants. Comparison of the regeneration coefficient indices on average over the season showed that the highest rate over the years of research was obtained in 2012 at 4.04 urc (Fig. 4 a). The year 2012 was characterized by the highest average air temperature (Fig. 4 b) and the lowest rainfall during the vegetation period (Fig. 4 c) of Malus halliana (March–October). By contrast, 2014 was the coldest and most voluptuous, which apparently affected

The statistical analysis of the experimental data was the seasonal average rate of the regenerative coefficient, carried out by the methods of R.A. Fisher (2006). which in 2014 was the smallest in the years of research and

was only 2.59 urc.RESULTS AND DISCUSSION

The comparison of the pace and intensity of the wound growing with the dates of an artificial injury gave rise to the conditional classification of the vegetative period by the regenerative potential for the following stages–regeneration rise, relative decrease, a second rise wave, and rather fast damping (Fig. 3).

Fig. 3–Seasonal variations of regeneration coefficients indices Malus

halliana in 2012–2015

In the spring, Malus halliana showed an increase in the regenerative coefficient from 1.28 in the third decade of March to 5.47 urc in the first decade of May. By the end of May, the regeneration coefficient remained rather high (5.43-5.21 urc). In the first decade of June, it dropped to 3.43 urc, followed by an increase to the maximum during the season of 5.61 urc in the third decade of June, after which it remained almost on the same level (about 5 urc) until the first decade of August. From the second decade of August, the plants of Malus halliana began gradually decay regeneration processes until complete extinction by the end of the first decade of

Fig. 4–October. At the same time, in years with different meteorological conditions, differences in the overall regenerative capacity have been observed, with fluctuations of

,1.0 åå=

Т

QНТС

annual (January-December) growing season (March-October)spring-summer (May-August)

C

annual (January-December) growing season (March-October)spring-summer (May-August)

B

A

2019 171Non-morphogenetic post-trauma regeneration potential of Malus halliana Koehne (Rosaceae Juss.)

In order to find out the links between the display of us for agricultural climate assessment of the region for the regenerative activity and the seasonal variations of the level of moisture provision for certain cultivated plants, in vegetation conditions, the correlation coefficients between the particular Malus halliana, is calculated as the ratio of the average seasonal regeneration coefficients indices Malus amount of precipitation for the period with average daily air halliana and the meteorological parameters (Fig. 5) have been temperatures above 10° C to the 10 times reduced amount of calculated, in particular, the average annual air temperature active temperatures (ΣT) for the same time (Shein and and the amount of precipitation per year, the hydrothermal Goncharov 2006). Therefore, the results obtained by us coefficient value, as well as the amount of precipitation and concerning the strong reciprocal dependence of regenerative average air temperature for March–October (research period) potentials on the hydrothermal coefficient (r=-0.93) confirm and May–August (the period of the highest regenerative the already established regularities regarding the connection activity). between the indicators of regeneration coefficient and the

amount of precipitation.The variation of the display of non-morphogenic callus

genesis over the years, as well as during the season, is objective and the analysis of the boundaries of this variability may make it possible to better understand the essence of the phenomenon under study. To measure the variation, several methods are used in statistics, among which evaluation by coefficient of variation (CV) is considered rather informative (Reed et al., 2003).

The data obtained in our experiments on the estimation of the intra-seasonal variation of the Malus halliana regeneration coefficients indices in some years of research have shown an annual significant volatility of the regenerative ability (Fig. 6).

Fig. 5–Correlation coefficients between the indices of regeneration

ability of Malus halliana and precipitation amount, and temperature, and HTC when notchings are made

Calculations of the correlation coefficients of the average regeneration coefficient for the season and individual meteorological indicators over the years of research have

Fig. 6–Coefficients of variation of Malus halliana average – seasonal revealed its direct dependence on the average air temperature regeneration coefficient indices, CV.

and strong reciprocal—from the amount of precipitation and the value of the hydrothermal coefficient. Such a reciprocal dependence of regenerative potentials on the amount of precipitation seems somewhat weird. At the same time, the intensity of non-morphogenic callus genesis depended more on the average temperature from May to August, that is, in the period with the highest regenerative activity, reaching r = 0.71, than the average annual air temperature (r = 0.55) and for March-October r = 0.41, while the strong reciprocal dependence on the amount of precipitation was maintained in all periods ranging from r = -0.78 to r = -0.97. As already noted, the hydrothermal coefficient of G. Selianiniva used by

Comparison results of Fig. 6 and Fig. 4a indicate that in the less favorable conditions for regeneration in 2014, variation of Malus halliana average-seasonal regeneration coefficient indices was the highest (CV = 70.66%). In the more favorable for regeneration years, the variation of average-seasonal regeneration coefficient indices was significantly lower with CV = 57.96% (in 2012) and CV = 43.95% (in 2015). However, in 2013, when the average-seasonal regeneration coefficient index was 3.83 urc, the variation of the average-seasonal regeneration coefficient indices was rather high and reached CV = 70.41%, which requires additional reflection.

172 The International Journal of Plant Reproductive Biology 11(2) July, 2019, pp.167-175

The results of the quadrennial monitoring of the healing characteristics of artificial wounds indicate significant differences in the rates and quality of healing wounds, depending on the notching date and the concrete weather conditions of each year.

Notchings had been made in the third decade of April 2014, formed callus in 11 days, and estimated at 1 point (Fig. 7 c), and in the second decade of June 2015, in 12 days, healed with a score of 7 points (Fig. 7 d), indicating a significantly more favorable conditions for regeneration in June. Also callus formed quickly on the notchings made in the third At the same time, in the third decade of March, nochings decade of June 2012 (Fig. 7 e), when the highest score of 9 callus was formed slowly and artificial wounds healed for a points was reached in 18 days. Almost at that pace (in 17 days long time (Figure 7), which was estimated at 3 points in 29 after injury) the 9 point score (Fig. 7 f) was achieved when days after injury in 2012 (Figure 7 a), and in unfavorable healing of the artificial wounds from the notchings made in the conditions in 2014, the score itself was obtained only in 42 first decade of August 2015.days after the injury (Fig. 7 b).

At the same time, all injuries, which had been made in March–August, but in some years in September every year, had varying intensity, but healed completely to 9 points. Only the last one to four injuries made in September–October did not fully heal until the end of the vegetation, stopping the regeneration processes, when the degree of healing of the wound was estimated at 1–8 points. In 2014, no signs of regenerative activity had been noted in wounds made in the third decade of September and in the first decade of October,

CONCLUSION

The variation in the Malus halliana regeneration coefficient indices during vegetation generally corresponded to the seasonal development rhythms with appropriate fluctuations connected with the conditions of the research year. The average daily air temperature during the regeneration period had greater influence on the dynamics of regenerative capacity during the season, than the amount of precipitation or the value of the hydrothermal coefficient. Regarding the average regeneration coefficient for the season, a direct correlation dependence of its parameters on the average air temperature and a strong reverse dependence on the amount of precipitation and the value of the hydrothermal coefficient had been found.Acknowledgements—This material is partly based on the work supported by the National Dendrological park “Sofiyivka” of NAS of Ukraine (№ 0112U002032) in compliance with the thematic plan of the research work. The authors are grateful to the corresponding member of NAS of Ukraine DSc on Biology Ivan Kosenko and Ph.D. Liudmyla Zagoruiko for consultations and discussion, as well as to the Head, of the Genetics, Plant Breeding and Reproductive Biology Ph.D. Oleksandr Balabak, for providing necessary facilities.

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