The use of microclonal propagation to create a genetic pool of Quercus robur L. for breeding and seed production

. In the present study seed progeny of a number of plus and normally the best trees of floodplain and forest-steppe forms of pedunculate oak has been introduced and propagated in vitro. To do this a full cycle of microclonal propagation of juvenile pedunculate oak plants grown from mature zygotic embryos has been carried out. This approach makes it possible to obtain mother juvenile plants with true and scaly leaves in two weeks and use them to get explants and increase the reproduction rate of plus trees in the shortest possible time.


Introduction
In Transdnistria, the State Program for the period from 2021 to 2041 "Restoration of hightrunk oak trees on the lands of the State Forestry Fund of Pridnestrovie" was adopted.To implement the program, it is necessary to develop appropriate methods for the study, use, breeding and conservation of natural populations of oak forests in varying growing conditions.First of all oak is characterized by the frequency of fruiting and acorns belong to the seeds of the recalcitrant type and are not stored for a long time.Pedunculate oak trees older than 10 years are difficult to graft objects and at last their growths are slow with long ontogenesis.The use of microclonal propagation technologies makes it possible to accumulate material for creating a genetic pool of the breed in the form of seed production objects.However, the available literature data indicate that not all stages of microclonal propagation for hard rock's have been worked out [1][2][3][4][5][6].Of great importance is the regenerative ability of the genotype, the ontogenetic state of the explant, the time of its removal and the correctly selected composition of the media used.Since oak is a slowgrowing species in vitro, ways to accelerate its growth rate are also important.
The goal of our research is to clarify a method of microclonal propagation of pedunculate oak on the basis of the obtained juvenile plants growing from mature zygotic embryos in vitro.Research objectives: to clarify the mode of sterilization of acorns for the removal of mature pedunculate oak embryos and their cultivation in vitro; to define the composition of the nutrient medium that is optimal for accelerated growth of embryos and obtaining plantlets for subsequent cuttings; to determine composition of media for induction of shoots, their growth, multiplication and rooting.

Plant material
Forest-steppe and seed material of floodplain forms of plus and normally better trees of Q. robur was used for in vitro multiplication.The model genotype for the refinement of the microclonal propagation technique was the plus tree No. 8.It is the floodplain form of Q. robur from Transdnistrian Kitskany forestry.Acorns taken at the end of October till the first decade of November without prior stratification from the 2022 harvest were used.Sterilization of acorns was carried out according to the method [3] in our modification.The modification included the use of the commercial sterilizing agent Domestos, cotyledon's pruning, and change composition of the media and calendar age of the used explant.

Growing plants from mature zygotic embryos
Pruned cotyledons with embryos were placed in vials on a nutrient medium (BTM+0.25mT+0.25K+0.25+IAA+0.1-2.4D) for growing ten or two week-old seedlings.The vials were placed in a cultural cabinet, working mode: photoperiod 16/8, t=25°C, humidity 60%, light 4000 lux.All parts of the maternal plantlets in the juvenile state were used: apex, nodal segments with a true and scaly leaves.The obtained explants were later used to multiply the shoots, which were rooted and grown to new juvenile plants.

Multiplication and rooting
The obtained zygotic seedlings of mother trees in the juvenile stage were multiplied on the medium of TMV with hormonal additives according to [1,2], but with our some modification [Table1].If necessary, the shoots were grown for 2-3 weeks on TMV medium with hormonal additives of 0.25 mT + 0.25 IAA+ 0.2 G. Shoots with a height of more than 1.5 cm with real leaves were transplanted to a rooting.Well-developed shoots were rooted on TMV medium with the addition of IBA according to [1,2] or without hormonal additives.

Pot culture
Peat was used as a substrate for pot culture.Plants with a root size of 2.5-6.5 cm were washed from the nutrient medium in a weak solution of potassium permanganate, soaked with filter paper and planted in prepared pots in a damp substrate, lightly watered and covered with a plastic cup to create a wet chamber.
The data obtained were processed by statistically accepted methods.

Aseptic preparation of sufficient amounts of fast-growing juvenile plant material
Two-stage sterilization of acorns in our modification has shown high efficiency.When planting explants on the medium, the contamination of in vitro cultures did not exceed 2%, and corresponded to the standard method.2% were acorns with cotyledons hidden under the pericarp, so their pruning contributed to the removal of the affected areas (Fig. 1, 2).Planting embryos with cotyledons remains on media was accompanied by active growth of shoots.The formation of scaly and true leaves in the shoots with simultaneous growth of the root was noted on the 10 th -15 th day, which were suitable for cutting explants (Table 1).This method gives a significant gain in the time (from 4 to10 times) to obtain stock material of explants compared to literature data [1,2,3,4,5].Yet, the embryos germinate on all tested media in almost the same way till the end of the second week and the height of the formed shoot is not significantly differentiated by variants probably due to the supply of nutrients of the cotyledon fragment.However, the best option is marked on medium No. 2 with gibberellin A3, and this medium can be used to grow shoots.On the medium No. 3, the most effective was the number of pledged nodes, with a high level of probability (P = 0.01) this option exceeds all others.This medium is optimal for multiplication.The resulting mother plants were used to refine the composition of the nutrient medium for the proliferation of shoots.The obtained mother plants were cut and the isolated explants were used to refine the composition of the nutrient medium for forcing new shoots (Table 2).A total of 143 explants of model genotype were examined.Most of the work on oak propagation was carried out using nodal segments in the juvenile phase with high efficiency [1][2][3][4][5].
Our data also confirm the high shoot-forming capacity of juvenile explants, as well as the influence of the physiological state of the maternal explant, determined by the composition of the nutrient medium.Analysis of the obtained data revealed the best variants (No. 3 and No. 4), in which the intensity of shoot formation varied from 130 to 156%, and the height of the average shoots are maximum and the best shoots reached up to 1.68-1.78cm.

Clarification of the conditions and composition of the nutrient medium optimal for the rooting of the obtained shoots
According to the literature, various hormonal additives are used for active rhizogenesis [1][2][3]5], but the most optimal medium for rooting is considered to be BTM medium with the addition of indolylbutyric acid (IBA 0.3 mg/l).At the same time, rhizogenesis is also observed with the addition of other auxins and even on hormone-free medium.Our results confirmed the high efficiency of the medium with IBA.Options of hormone-free medium are also interesting, but taking into account the physiological state of the shoots [Table3, Fig. 3].In addition, the effect of short-term (within a week) shading and a decrease in temperature to 12°C on the rooting of the resulting shoots was analyzed, as additional factors possibly increasing the production of endogenous growth hormones.The best option (87%) was on the medium with IBA and short-term shading.However, the difference between the variants was not statistically sufficient.The results of pot growing and the survival rate is not yet high enough and varies from 40% up to 55% (Fig. 4).Survival rate in pot culture varied depending on the timing of cultivation and increased in the summer, possibly with optimization of substrate temperature.At the same time, the survival rate decreased with direct intense insolation of plantlets, causing leaf burns.Therefore, shading favored the survival of regenerants in the summer.

Conclusions
The mode of sterilization of pedunculate oak planting material for introducing in vitro has been worked out.The composition of the nutrient medium for the accelerated development of mature zygotic embryos has been optimized.A full cycle of microclonal propagation of juvenile pedunculate oak plants grown from mature zygotic embryos has been carried out.Seed progeny of a number of plus and normally the best trees of floodplain and forest-steppe forms of pedunculate oak has been introduced and propagated in vitro.

Fig. 1 .
Fig. 1.Removal of woody pericarps (A, B) and pruning of cotyledons (C) in aseptically prepared acorns before placing explants in vitro.

Fig. 2 .
Fig. 2. Growth of shoots and formation of juvenile plantlets with scaly and true leaves.

Table 1 .
Growth dynamic of Q. robur plantlets developed from mature embryos of the plus tree №8

Table 2 .
The shoot formation efficiency of maternal explants depending on the physiological state of the mother plant and the composition of the medium on which explants were planted.
Note: The composition of medium and it's numbers are similar to those in Table1

Table 3 .
Shoot rooting efficiency depending on the composition of the nutrient medium, cultivation conditions, and physiological state of the shoots