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Введення в культуру in vitro тису ягідного (Taxus baccata L.) і отримання таксол-продукуючих калюсних ліній: Автореф. дис… канд. біол. наук / Л.Г. Ф

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Iaoe?iiaeueia Aeaaeai?y iaoe Oe?a?ie

?inoeooo ee?oeiii? a?ieia?? ? aaiaoe/ii? ?iaeaia???

O?EIIIAA Eaaea Aai???aia

OAeE 573.086.83:581.143.6

AAAAeAIIss A EOEUeOO?O IN VITRO OENO ssA?AeIIAI

(TAXUS BACCATA L.) ? IO?EIAIIss

OAENIE-I?IAeOEOTH*EO EAETHNIEO E?I?E

03.00.22 – ee?oeiia a?ieia?y

Aaoi?aoa?ao

aeena?oaoe?? ia caeiaoooy iaoeiaiai nooiaiy

eaiaeeaeaoa a?ieia?/ieo iaoe

Ee?a – 1999

Aeena?oaoe??th ? ?oeiien

?iaioa aeeiiaia a ?inoeooo? ee?oeiii? a?ieia?? ? aaiaoe/ii?

?iaeaia??? IAI Oe?a?ie

Iaoeiaee ea??aiee – /eai-ei?aniiiaeaio IAI Oe?a?ie,

aeieoi? a?ieia?/ieo iaoe,

Aethi ss?ineaa Ai?eniae/

?inoeooo ee?oeiii? a?ieia?? ? aaiaoe/ii? ?iaeaia??? IAI Oe?a?ie,

canooiiee aee?aeoi?a

Io?oe?ei? iiiiaioe – /eai-ei?aniiiaeaio IAI Oe?a?ie,

aeieoi? a?ieia?/ieo iaoe, i?ioani?, Eoiao
A?eoi? Aiaoie?eiae/,

?inoeooo iieaeoey?ii? a?ieia?? oa aaiaoeee IAI
Oe?a?ie,

caa?aeoth/ee a?aeae?e

eaiaeeaeao a?ieia?/ieo iaoe, annenoaio eaoaae?e
o?c?ieia?? oa

aeieia?? ?ineei Iaithoa Ieueaa Ieaenaiae??aia,

Ee?anueeee oi?aa?n?oao ?i. Oa?ana Oaa/aiea

I?ia?aeia onoaiiaa – Oeaio?aeueiee aioai?/iee naae ?iai? I. I. A?eoea
IAI Oe?a?ie.

Caoeno a?aeaoaeaoueny “22” ea?oiy 1999 ?ieo i 14 aiae. ia can?aeaii?
niaoe?ae?ciaaii? a/aii? ?aaee Ae.26.202.01 i?e ?inoeooo? ee?oeiii?
a?ieia?? ? aaiaoe/ii? ?iaeaia??? IAI Oe?a?ie ca aae?anith:

252143, Ee?a, aoe. aeaaeai?ea Caaieioiiai, 148

C aeena?oaoe??th iiaeia iciaeiieoenue o a??ae?ioaoe? ?inoeoooo
ee?oeiii? a?ieia?? ? aaiaoe/ii? ?iaeaia??? IAI Oe?a?ie, Ee?a, aoe.
aeaaeai?ea Caaieioiiai, 148.

Aaoi?aoa?ao ?ic?neaiee “17“ aa?aciy 1999 ?.

A/aiee nae?aoa?

niaoe?ae?ciaaii? a/aii? ?aaee E. A. Oa?anaiei

O?eiiiaa E. A. Aaaaeaiiy a eoeueoo?o in vitro oeno ya?aeiiai (Taxus
baccata L.) ? io?eiaiiy oaenie-i?iaeoeoth/eo eaethnieo e?i?e. — ?oeiien.

Aeena?oaoe?y ia caeiaoooy iaoeiaiai nooiaiy eaiaeeaeaoa a?ieia?/ieo iaoe
ca niaoe?aeuei?noth 03.00.22 — ee?oeiia a?ieia?y. — ?inoeooo ee?oeiii?
a?ieia?? oa aaiaoe/ii? ?iaeaia??? IAI Oe?a?ie, Ee?a, 1999.

Iaoa aeaii? ?iaioe — aea/aiiy caeiiii??iinoae eaethniooai?aiiy oa
i?iaeoeoaaiiy i?ioe?aeiaiai i?aia?aoo oaenieo a eoeueoo?ao ee?oei oeno
ya?aeiiai (Taxus baccata L.).

Aea/aii ?iciiae?e ai?noo oaenieo i?ae ie?aieie /anoeiaie ?ineei oeno a
15-oe eeiiao T. baccata ? 3-o eeiiao T. cuspidata. Anoaiiaeaii
i?aeeeiiaeueio aa??aaaeuei?noue ai?noo oaenieo a ei?? noiaao?a, ??/ieo
eeiiao oa oai? aeine?aeaeoaaieo aeae?a. Iaea?eueoee ai?no oaenieo oa
iaeiaioa eiai i?aeeeiiaeueia aa??aaaeuei?noue oa?aeoa?i? aeey ei?e
noiaao?a.

?ic?iaeaia iaoiaeeea io?eiaiiy eaethno c aaaaoaoeaieo a?oiueie, ieeye?a,
ian?iiy oa iaaii?aT. baccata. I?ae?a?aiee iioeiaeueiee neeaae iiaeeaieo
na?aaeiaeu oa oiiae eoeueoeaoaaiiy aeey eiaeiiai oeio aenieaio?a.
Aeyaeaii, ui aeey eoeueoo?e oeno aaeain ni?aa?aeiioaiiy aiii?th aei
i?o?aoo aaaeeea?oee, i?ae caaaeueiee ai?no iai?aai?/iiai acioo a
na?aaeiaeu?.

A?noieia?/iee aiae?c ee?oei oeno iieacaa, ui iaea?eueoa i?ie?oa?aoeaia
aeoeai?noue in vitro oa?aeoa?ia aeey eaia?th ? ia?aio?ie oeiaie.

?ic?iaeaia i?ioeaaeo?a eoeueoeaoaaiiy i?ioiieano?a T. baccata.
I?e?ieieii?? i?iyaeyee i?ie?oa?aoeaio aeoeai?noue i?ioyaii ia?oeo 7-14
ae?a. Iio?i ee?oeiiee iiae?e i?eieiyany, aea i?e?ieieii?? caa??aaee
aeeoo?caeaoi?noue i?ioyaii 2-o oeaei?a.

Aiae?c ai?noo oaenieo a io?eiaieo eaethnieo e?i?yo T. baccata ca
aeiiiiiaith aenieiaoaeoeaii? ??aeeiii? o?iiaoia?ao?? — iaa?iaiee aa??aio
— iieacaa iayai?noue o ieo ?a/iaeie a e?eueeinoyo, ui oa?aeoa?i? aeey
?ineei-aeiii??a.

Io?eiai? eaethni? e?i?? T. baccata, ui noaa?eueii ?inoooue ?
caa??aathoue a?ineioaoe/io aeoeai?noue i?e o?eaaeiio eoeueoeaoaaii?.

Eeth/ia? neiaa: oen ya?aeiee (Taxus baccata L.), eaethnia eoeueoo?a,
i?ioiieanoe, a?noieia?/iee aiae?c, a?ineioac oaenieo.

Oeeiiiaa E. A. Aaaaeaiea a eoeueoo?o in vitro oenna yaiaeiiai (Taxus
baccata L.) e iieo/aiea oaenie-i?iaeooee?othueo eaeeoniuo eeiee. —
?oeiienue.

Aeenna?oaoeey ia nieneaiea o/aiie noaiaie eaiaeeaeaoa aeieiae/aneeo iaoe
ii niaoeeaeueiinoe 03.00.22 — eeaoi/iay aeieiaey. — Einoeooo eeaoi/iie
aeieiaee e aaiaoe/aneie eiaeaia?ee IAI Oe?aeiu, Eeaa, 1999.

Oeaeue aeaiiie ?aaiou — eco/aiea caeiiiia?iinoae eaeeoniia?aciaaiey e
i?iaeooee?iaaiey i?ioeai?aeiaiai i?aia?aoa — oaeniea a eoeueoo?a eeaoie
oenna yaiaeiiai (T. baccata L.).

Eco/aii ?ani?aaeaeaiea niaea?aeaiey oaeniea iaaeaeo ioaeaeueiuie /anoyie
?anoaiee a 15-oe eeiiao T. baccata e 3-o eeiiao T. cuspidata.
Onoaiiaeaia iaaeeeiiaeueiay aa?eaaaeueiinoue niaea?aeaiey oaeniea a ei?a
noaiea, aiaee/iuo iiaaaao e oaie yoeo aeaeia. Iaeaieueoaa niaea?aeaiea
oaeniea e iaeiaiueoay aai iaaeeeiiaeueiay aa?eaaaeueiinoue oa?aeoa?iu
aeey ei?u noaiea.

?ac?aaioaia iaoiaeeea iieo/aiey eaeeona ec aaaaoaoeaiuo ii/ae,
iueueieeia e iiaaaia T. baccata. Iiaeia?ai iioeiaeueiue ninoaa
ieoaoaeueiuo n?aae e oneiaey eoeueoeae?iaaiey aeey eaaeaeiai oeia
yenieaioia. Onoaiiaeaii, /oi aeey eoeueoo?u oenna aaeain niioiioaiey
aiiiiey e ieo?aoo aieaa aaaeai, /ai iauaa niaea?aeaiea iai?aaie/aneiai
acioa a n?aaea.

Aenoieiae/aneee aiaeec eeaoie oenna iieacae, /oi iaeaieueoay
i?ieeoa?aoeaiay aeoeaiinoue in vitro oa?aeoa?ia aeey eaiaey e ia?aioeiu
oeiyiu.

?ac?aaioaia i?ioeaaeo?a eoeueoeae?iaaiey i?ioiieanoia T. baccata.
Iee?ieieiiee i?iyaeyee i?ieeoa?aoeaioth aeoeaiinoue a oa/aiea 7-14
aeiae. Caoai eeaoi/iia aeaeaiea i?ae?auaeinue, ii iee?ieieiiee nio?aiyee
aeecianiiniaiinoue a oa/aiea 2-o iaaeaeue.

Aiaeec niaea?aeaiey oaeniea a iieo/aiiuo eaeeoniuo eeieyo T. baccata i?e
iiiiue aunieiyooaeoeaiie aeeaeeinoiie o?iiaoia?aoee iieacae iaee/ea a
ieo aauanoaa a eiee/anoaao, oa?aeoa?iuo aeey ?anoaiee-aeiii?ia.

Iieo/aiu eaeeoniua eeiee T. baccata, eioi?ua noaaeeueii ?anooo e
nio?aiytho aeineioaoe/aneoth aeoeaiinoue i?e aeeeoaeueiii
eoeueoeae?iaaiee.

Eeth/aaua neiaa: oenn yaiaeiue (Taxus baccata L.), eaeeoniay eoeueoo?a,
i?ioiieanou, aenoieiae/aneee aiaeec, aeineioac oaeniea.

Filonova L. G. Introduction in vitro culture of Taxus baccata L. and
obtaining of taxol produced callus lines. — Manuscript.

Thesis for gaining a scientific degree of Candidate of Biological
Sciences on speciality 03.00.22 — Cell Biology, Institute of Cell
Biology and Genetic Engineering, National Academy Science of Ukraine,
Kiev, 1999.

The diterpenoid taxol belongs to the class of efficient antineoplastics
drugs, taxoids. Taxol promotes abnormal microtubule assembly and
stabilizes the previously formed microtubules. Further spindle formation
is arrested. Due to this unique antimitotic mechanism, this natural
compound has become a well known anticancer drug.

The only known source of taxol is from Taxus species. Family Taxaceae
is represented by ancient slowly growing species with narrow natural
habitats, and the levels of taxol production by intact plants do not
exceed 0.1% on the dry weight basis. Large-scale exploitation of natural
stands of Taxus spp. for taxol production will eventially lead to
complete disappearance of these species. Development of alternative ways
of taxol production is required to meet current demands of medicine.
Cell culture of Taxus spp. attracts much attention as a promising
alternative tool to produce sufficient amounts of taxol.

In Ukraine and other European countries, the only indigenously growing
Taxus spp. is Taxus baccata L. Cell culture of T. baccata was studied
less deeply, than other species. The aim of the present study is to
describe peculiarities of callusogenesis and taxol production in vitro
cultured cells of Common yew (Taxus baccata L.).

Distribution of taxol content in different parts of the plant has been
analysed for 15 genotypes of T. baccata and 3 genotypes of T. cuspidata.
The highest levels of taxol have been found in the bark, what is 10-fold
higher than in the needles and one-year-old twigs. Concentration of
taxol varies significantly depending on the genotype. The least
variation has been demonstrated for the bark. Also, both T. baccata and
T. cuspidata exhibited high interclonal variation of taxol content in
three types of samples analysed. A veraged values of taxol content in
two Taxus spp. differed significantly for needle samples only.

The culture protocols have been developed to establish callus cultures
from different types of explants. The temporal pattern and frequency of
callus formation from shoot segments, vegetative buds and pollen have
been shown to be independent on the time of collecting plant material.
In contrast, degree of seed maturity determines both parameters with the
highest callusogenic activity occuring in September. Primary
seed-originated callus was less susceptible to necrotic process than
shoot-originated callus which, however, grew faster.

Five basal nutrient compositions routinely used for culturing
coniferous species have been tested. The best medium was SH (Shenk,
Hilderbrandt, 1972) showing 2 to 2.5 times higher frequency of callus
formation (91%) compared to other media. Experiments have shown that
ammonium/nitrate molar ratio is more important factor than the total
concentration of inorganic nitrogen.

Optimization of the balance between exogenous auxin and cytokinin has
shown that the use of NAA with or without cytokinin has no effect on
induction of callusogenesis whereas combined application of cytokinin
and 2.4-D exerted stimulatory effect.

The pattern of influence of plant growth regulators on cell
proliferation was ambiguous, and depended on basal medium composition
and plant genotype. Biomass increase, averaged over six growth regulator
treatments and cell lines, originated from two genotypes of T. baccata,
was higher on SH medium. Interestingly, cytokinins promoted growth of
all long-term cell lines. Under the best culture conditions, biomass
increase of some cell lines was as high as 350%, that corresponded to
production of 3 g of fresh weight callus from a single stem segment
within 3-week sybculture interval.

The callus started to develop after approximately 20 days for stem
explants and after 50 days for seeds. Histological annalysis has
revealed that the cambium and phloem of shoot explants are the primary
tissues participating in callus formation. All the cell types of seed
including even multinucleate gametophytic cells can dedifferentiate in
vitro to give rise callus. Callus, originated from these cells was
homogenous, whereas embryo-originated callus revealed zonal structure.
The clear indications of xylogenesis in zygotic embryo-originated callus
were found in areas of hypocotil and cotyledons. These early cell
differentation events underlyng callusogenesis in yew are similar to
with those found during adventitious bud formation in other coniferous
species (e.g., spruce).

The procedure for T. baccata protoplast culture has been elaborated.
Colonics of protoplasts held proliferative activity for the first 7 to
14 days of culture, followed by ceasing cell divisions without the loss
of cell viability for the next 7 days.

The established culture procedure enabled to obtain stable-growing
callus lines retaining biosynthetic activity under long-term
subculturing. The level of taxol accumulation in callus lines of T.
baccata checked well with the content of drug found in donor plants, and
varied in the range of 0.0003-0.001%. When studying dynamics of the
biosynthesis of taxol in peak at 28th day of culture. A strong positive
correlation found between biomass increase and taxol accumulation level
in callus cultures of T. baccata (R=0.9; p0.01) appears an adequate
evidence of the need for preselection of fast growing cell lines for
biotechnological production of taxol.

Key words: Taxus baccata L., callus culture, protoplasts, histological
analysis, biosynthesis of taxol.

CAAAEUeIA OA?AEOA?ENOEEA ?IAIOE

Aeooaeuei?noue i?iaeaie. I?aaenoaaieee ?iaeeie oeniaeo (Taxaceae)
i?eaa?oathoue aei naaa i?aeaeuaio oaaao a/aieo /a?ac ?o caeaoi?noue
neioacoaaoe oaenie — oi?eaeueiee aeeoa?iaiiaee aeeaei?ae, ui
oa?aeoa?eco?oueny iniaeeaei aioei?oioe/iei iaoai?ciii ae??.

Ia a?aei?io a?ae aioei?oioe/ieo aaaio?a oeio a?iaeano?io oa eieo?oeeio,
ye? ?oeiothoue aa?aoaii iiae?eo, oaenie ?iaeoeo? ooai?aiiy
i?e?io?oai/ie, aeeeeeath/e iacai?ioio iie?ia?ecaoe?th iieaeoe ooaoe?io,
ui aeieo? iiae?e ee?oeie i?e ia?aoiae? a i?oic (Shiff et al., 1979).
O?i?/i? aiaeiae ? iio?aei? oaenieo, oaenaie, iiaeooue i?iyaeyoe iiae?aio
aeoeai?noue (Blechert et al., 1991).

Oe? aeanoeaino? coiiaeththoue i?aeaeuaiee ?ioa?an aei aea/aiiy oaenieo
ye i?aaenoaaieea i?ioe?aeiaeo i?aia?ao?a iiaiai iieie?iiy (Nragg et al.,
1993). Aea? eiai e?eoaaeuei? oi?ie — oaenie ? aeioeaoaenae — oaea
nueiaiaei? canoiniaothoueny a i?aeoe/i?e iieieia?? a a?eueo i?ae 50
e?a?iao na?oo (Goldspiel, Kohler, 1994).

Aia?oa oaenie aoa aeae?eaiee ?c ei?e oeno oeoiieaainueeiai Taxus
brevifolia (Wani, Taylor, 1971). Ai?no oaenieo neeaaea? eeoa
0,0001-0,00001% a?ae nooi? iane iaoa??aeo (Vidensek et al., 1971).
Caaaeueia ae iio?aaa a oaenie? o?eueee aeey e?eoaaiiy oai?eo, ye?
no?aaeaeathoue ?aeii y?/iee?a, aaea iaeaeeae/ei /anii neeaaea aeecueei
200 ea/??e (Stokigt, 1995), a aeey iaea?aeaiiy 1 ea ?a/iaeie iaiao?aeii
7200 ea ei?e 50-60-e?oi?o aea?aa (oiiaii — 2-3 aei?ineeo aea?aaa ia
iaoe??ioa (Donovan, 1995).

Oen iaeaaeeoue aei ae?aai?o aeae?a, ui iia?eueii ?inoooue, ?c aocueeeie
a?aaeaie iioe?aiiy (Eicoaia, Io?aoiaa, 1986). Ianiaa i?iieneiaa
aenieoaoaoe?y i?e?iaeieo ianaaeaeaiue noaaeoue i?ae caa?ico ?nioaaiiy
oe??? oe?iii? ?ineeie, caianaii? a nienee aeae?a, ui ioi?iiythoueny.
Iaea?eueo ?aioaaaeueiei nueiaiaei? ? aeae?eaiiy iiia?aaeiee?a oaenieo —
aaeaoeio-III ? aeeaoeaoeeaaeaoeio-3 ?c oai? ? ia?inoe?a oeno ya?aeiiai c
iiaeaeueoei neioacoaaiiyi c ieo oaenieo (Donovan, 1995). A?ioaoiieia?y
i?iiiio? 2 aeueoa?iaoeaieo oeyoe iaea?aeaiiy oaenieo: c eoeueoo? a?oie
oaenie-neioacoth/eo a?ea?a oa c ee?oeiii? eoeueoo?e naiiai oeno
(Heinstein et al., 1994). I?ioa a eoeueoo?? a?eaa oaenie iaeiie/o?oueny
a iacia/ieo e?eueeinoyo ? eiai a?ineioacii ea?oaaoe neeaaeii (Jaziri et
al., 1996). Eoeueoo?a ee?oei ? oeaiei oeno in vitro a?aee?eaa?
iiaeeea?noue ?ic?iaee a?ioaoiieia?/ii? nenoaie, ui ii?aeio? a nia?
i?eioeeie aoaeoeaiino? ? eiio?ieth a?ineioaco oaenieo (Shuler et al.,
1992). ?ineeii? ee?oeie iiaeooue aooe aeaea?aeii oe?eiai niaeo?o
oaenai?a, ui oaeiae iathoue aioei?oioe/io aeoeai?noue.

Ca inoaii? ?iee io?eiaii eaethni? oa noniaic?ei? eoeueoo?e aeayeeo
aeae?a oeno, ianaiia?aae T. brevifolia ? T. cuspidata, ? iieacaii, ui
ie?ai? eaethni? e?i?? i?iaeoeothoue oaenie o e?eueeinoyo, oa?aeoa?ieo
aeey ?ioaeoieo ?ineei (Fett-Neto et al., 1992; Wickremesinhe, Arteca,
1993), a a aeayeeo aeiaaeeao ai?no oaenieo a eaethnieo e?i?yo ia?aaaaea?
oaeee o aeo?aeiiio iaoa??ae? (Winkremesinhe et al., 1992). Neeaaeiith
caeeoa?oueny i?iaeaia noaa?e?caoe?? ?inoo ciai?oi?o ? ?icoaoiaaieo
ana?aaeei? eaethnii? eoeueoo?e ee?oei oeno, ine?eueee i?ia?anoth/a
noa??iiy in vitro, ui aeanoeaa aeey an?o oaieieo (Oe?y?aa oa ?i., 1992),
iniaeeai ae?aaeaia aeey i?aaenoaaiee?a ?iaeo Taxus (Gibson et al., 1993;
Fett-Neto et al., 1992).

A Oe?a?i? o i?e?iaeieo oiiaao ?inoa o?eueee oen ya?aeiee (Taxus baccata
L.), i?eeiie eoeueoeaoaaiiy aeey yeiai ia ?ic?iaeai?. Oea e iaoiiaeei
aea?? iaie T. baccata ye aieiaiiai aeine?aeaeoaaiiai ia’?eoa.

Iaoa ?iaioe — aaaaeaiiy a eoeueoo?o in vitro T. baccata ? aea/aiiy
aeeiai?ee iaeiie/aiiy oaenieo a eaethn?. A?aeiia?aeii aei iaoe aoee
iinoaaeai? eiie?aoi? caaaeaiiy:

– aecia/eoe nooi?iue aa??aaaeueiino? ai?noo oaenieo a ??cieo /anoeiao
?ineei i?ae ie?aieie eeiiaie o iiioeyoe?yo T. baccata ? T. cuspidata,
?io?iaeoeiaaieo o Oeaio?aeueiiio aioai?/iiio naaeo ?i. I.I. A?eoea
IAI Oe?a?ie;

– aecia/eoe oiiae aeey oni?oiiai eoeueoeaoaaiiy in vitro aaaaoaoeaieo ?
?ai?iaeoeoeaieo oeaiei ? i?aai?a T. baccata;

– i?iaanoe a?noieia?/ia aeine?aeaeaiiy iniaeeainoae aoaeiae iaaiia oa
ian?iiy oeno ya?aeiiai, a oaeiae ci?i no?oeoo?e a i?ioean?
aeaaeeoa?aioe?thaaiiy oa i?ie?oa?aoe?? ee?oei T. baccata in vitro;

– aea/eoe iiaeeeaino? iaea?aeaiiy aeeoo?caeaoieo i?ioiieano?a
T. baccata, ui ae?eyoueny;

– ii??aiyoe ai?no oaenieo a eaethn? e o oeaieiao ?ineei-aeiii??a.

Iaoeiaa iiaecia. Aia?oa i?iaaaeaii aiae?c ai?noo oaenieo a iiioeyoe??
T. baccata, iieacaii aenieee nooi?iue aa??aaaeueiino? ai?noo ?a/iaeie
i?ae eeiiaie ? /anoeiaie ie?aii? ?ineeie.

Anoaiiaeaii i?yio ei?aeyoe?th i?ae ?ioaineai?noth i?ie?oa?aoe?? ee?oei ?
ai?noii oaenieo a aeiaaio?eaaei ianiaaieo eaethnieo e?i?yo. Aia?oa
aea/aia iiaeeea?noue iaea?aeaiiy ? eoeueoeaoaaiiy i?ioiieano?a, iieacai?
ni?euei? c ?ioeie oaieieie caeiiii??iino? ?o iiaaae?iee a eoeueoo?? in
vitro.

Ia i?aenoaa? a?noieia?/iiai aiae?co oi?ioaaiiy eaethno c iaaii?a ?
ian?iiy T. baccata io?eiaia aeeiai?ea no?oeoo?ieo ci?i ??cieo oeaiei o
i?ioean? aeaaeeoa?aioe?thaaiiy ee?oei i?e ?iaeoeoe?? eaethniaaiaco.

I?iaaaeaia ?iaioa — ia?oee a Oe?a?i? aeina?ae oni?oiiai io?eiaiiy
noaa?eueieo eaethnieo eoeueoo? T. baccata, ui i?noyoue oaenie ? iathoue
no?eeo a?ineioaoe/io aeoeai?noue.

I?aeoe/ia oe?ii?noue ?iaioe iieyaa? a ?ic?iaoe? iaoiaeeee aaaaeaiiy a
eoeueoo?o in vitro T. baccata. Aoaeoeai?noue iaoiaeeee aecia/a?oueny
iiaeeea?noth oe?ieiai aeai?o oei?a aenieaio?a, iecueeith /anoioith ?o
?io?eoaaiiy oa aenieith /anoioith oi?ioaaiiy eaethno. Iioei?caoe?y oiia
eoeueoeaoaaiiy eaethno aeicaieeea io?eiaoe aeiaaio?eaaei ianiaai?
eaethni? e?i??, ui i?noyoue oaenie. ?ic?iaeaia iaoiaeeea aeae?eaiiy ?
eoeueoeaoaaiiy i?ioiieano?a T. baccata aeicaiey? iaea?aeaoe
aeeoo?caeaoi? i?ioiieanoe, ui ooai?ththoue eieii??.

Ai?iaaoe?y ?acoeueoao?a aeena?oaoe??. Iaoa??aee aeena?oaoe??
i?aaenoaaeai? o 14 ioae?eaoe?yo. Iaoa??aee iaaiai?thaaeeny ia: II
c’?cae? Oe?. Oiaa?enoaa oiciieiaia ?ineei, Ee?a, 1993; VIII
International Congress of International Association Plant Tissue
Culture, Firenze, Italy, 1994; Simposium Plant. Cell, Tissus and Organ
Culture in Liquid Media, Prague, 1994; 44th Annual Congress on Medicinal
Plant Research, Prague, 1996; Simposium Principles Regulating
Biosynthesis and Storage of Secondary Products, halle, 1996; XVIII
Congress of SPPS the Scandinavian Society for Plant Physiology, Uppsala,
Sweden, 1997; International Conference Problems and Perspectives of
Biotechnology Development in ornamental Gardening and Horticnlture,
Yalta, Ukraine, 1997; II International Symposium on Natural Drugs,
Maratea, Italy, 1997; VII Iaaeaeoia?iaeiie eiioa?aioeee “Aeieiaey eeaoie
?anoaiee in vitro, aeioaoiieiaey e nio?aiaiea aaiioiiaea”, Iineaa,
?inney, 1997.

No?oeoo?a aeena?oaoe??. Aeena?oaoe?y aeeeaaeaia ia 109 noi??ieao,
aeeth/a? 7 oaaeeoeue, 15 iaethie?a, i?noeoue anooi, o?e ?icae?ee (iaeyae
e?oa?aoo?e, aenia?eiaioaeueio /anoeio, ?acoeueoaoe oa iaaiai?aiiy),
caeeth/aiiy, aeniiaee oa nienie e?oa?aoo?e, yeee aeeth/a? 218 aeaea?ae.

IAOA??AEE ? IAOIAeE

?ineeiiee iaoa??ae

Aeey aaaaeaiiy a eoeueoo?o in vitro aeei?enoiaoaaee ??ci? oeaieie e
i?aaie T. baccata ?c eieaeoe?? aeaiae?a??th Oeaio?aeueiiai aioai?/iiai
naaeo ?i. I.I. A?eoea IAI Oe?a?ie. Ca?? ??/ieo iaaii?a i?iaiaeeee
uii?nyoey, aaaaoaoeaieo a?oiueie ? ieeye?a — o ea?oi?-o?aai?, ian?iiy ?
a?eeon?a — uii?nyoey c /a?aiy ii aeiaoaiue. A iaeiiio c aenia?eiaio?a
aeei?enoiaoaaee ??/i? iaaiie aeaio /?oei oaiioeiiai a?aei?iieo aaiioei?a
T. baccata — oei?io?e-aeaoaeoiiai (CD) ? ii?iaeueiiai uiaei neioaco
oei?io?eo (N). Iaoa??ae caa??aaany i?e +4N ia a?eueoa, i?ae o?e oeaei?.

Aenieaioe noa?ee?coaaee 9%-iei ?ic/eiii a?iioei?eoo iao??th (NaClI).
O?eaae?noue ii?aoethaaiiy ?ic/eiii NaClI neeaea 60 oa. aeey naaiaio?a
iaaii?a aeiaaeeiith 1,5-2 ni; 15 oa. — aeey aaaaoaoeaieo a?oiueie oa
i/euaieo a?ae ian?iii? iaieiiee iae?iaaiaoio?o?a; 4-5 oa. — aeey ieeye?a
? a?eeon?a. ?ineeiiee iaoa??ae iii?uaee a /aoee Iao?? ae?aiao?ii 120 ii
ia aaa?eciaai? iiaeeai? na?aaeiaeua. O eiaeiiio aeine?ae?
aeei?enoiaoaaee ia iaioa, i?ae 50 aenieaio?a eiaeiiai oeio.

Iiaeeai? na?aaeiaeua ? iaoiaee eoeueoeaoaaiiy

Aeey aecia/aiiy iioeiaeueiiai i?ia?aeueiiai neeaaeo iiaeeaiiai
na?aaeiaeua iaaiie eoeueoeaoaaee ia na?aaeiaeuao SH (Schenk,
Hilderbrandt, 1972); IS (Murashige, Skoog, 1962); LP (von Arnold,
Eriksson, 1982); B5 (Gamborg et al., 1968); DBM2 — ?? (Gresshoff, Doy,
1972) c aeiaeaaaiiyi ?iiceoo (50 ieI), i?eioeiiai? eeneioe (0,8 ieI),
i??eaeieneio-HCl — A6 (0,5 ieI), o?ai?io-HCl — A1 (3 ieI),
2,4-aeeoei?oaiieneioeoiai? eeneioe (2,4-Ae; 10 ieI), e?iaoeio (5 ieI),
3% naoa?ice ? 0,7% aaa?o. Aeey eoeueoeaoaaiiy ?ioeo oei?a aenieaio?a
iniiaiei aoei na?aaeiaeua SH. Eoeueoo?e ?ieoaoaaee a oaiiio? i?e 22-26N,
ianoaaee ia na?ae? na?aaeiaeua /a?ac eiaei? 2-3 oeaei?.

Aieea ??cieo eiia?iaoe?e aoenei?a (2,4-Ae ? -iaooeeioeoiai? eeneioe —
IIE) ? oeeoie?i?i?a (6-aaiceeai?iiio?eio — AAI ? e?iaoeio) ia
?iaeoeoe?th oa ?ioaineai?noue ?inoo eaethno aea/aany ia eaethn?,
io?eiaiiio c iaaii?a ? aaaaoaoeaieo a?oiueie. sse aaciaa aeei?enoiaoaaee
na?aaeiaeua SH.

I?e aea/aii? aieeao iniiaiiai neeaaeo iiaeeaiiai na?aaeiaeua ? eiai
o?oiai?iiiaeueiiai aaeaino ia ??no aeiaaio?eaaei ianiaaiiai eaethno c
iaaii?a aeaio aaiioei?a T. baccata ye aaciaa aeei?enoiaoaaee na?aaeiaeua
INM10 (Bozhkov et al., 1993) ? SH. Iaeaeaa na?aaeiaeua aoee aeiiiaiai?
7,4 ieI A1, 8,1 ieI ??, 2,4 ieI A6, 0,55 iI ?iiceoo, 3 iI aeooai?io,
1 iI ania?aa?io ? 2% naoa?ice. Eaethn o aeaiiio aenia?eiaio? aoa
io?eiaiee ia na?aaeiaeu? SH ?c aeiaeaaaiiyi 5 ieI 2,4-Ae. *a?ac 6
oeaei?a eoeueoeaoaaiiy eaethn ia?aiineee ia na?aaeiaeua SH ? INM10 c
aeiaeaaaiiyi 1% IAI, 10 ieI 2,4-Ae, 1 ieI IIE, 1 ieI AAI, 1 ieI e?iaoeio
a 12 ??cieo eiia?iaoe?yo.

On? na?aaeiaeua noa?ee?coaaee aaoieeaaoaaiiyi. ?ic/eie ania?aa?io oa
aeooai?io noa?ee?coaaee ca aeiiiiiaith o?eueo?oaaiiy /a?ac iaia?aii?
o?eueo?e “Millipore” (NOA) c ae?aiao?ii ii? 0,45 iei.

A?noieia?/iee aiae?c oi?ioaaiiy eaethno

A aenia?eiaio? aeei?enoiaoaaee /anoeie iaaii?a oa i/euaia ian?iiy i?ney
0, 7, 14 ? 35 ae?a eoeueoeaoaaiiy ia na?aaeiaeu? SH. Iaoa??ae o?enoaaee
o?ii-aoeaoi-oi?iieii (10:1:4) ca Iaaaoeiei (1985), aeaeueoo ia?iaeo
i?iaiaeeee ca iaoiaeeeith Iaooaai? (1970). I?aia?aoe oa?aoaaee ?aaeoeaii
Oeooa ca Oaeueaaiii c i?aeoa?aiaoaaiiyi na?oeei caeaiei.

Aeae?eaiiy ? eoeueoeaoaaiiy i?ioiieano?a

I?ioiieanoe aeae?eyee c ia?aeiiiai (a?e 3 oeaei?) ? aeiaaio?eaaei
ianiaaiiai eaethno (a?e 27 oeaei?a). Oa?iaioiee ?ic/ei i?noea 0,4 I
naoa?ice, 0,2 I aetheice,5 iI CaCl2, 0,5% ne?iaaoeiaiai aeueaoi?io
(ANA), 0,5% iaeoeiace ? 1% oeaetheace “Onozuka”. I?ioiieanoe a?aeieaaee
noi?ooth oaeiai ae neeaaeo ?c aeiaeaaaiiyi 0,7 iI NaH2PO4 aac ANA. An?
?ic/eie aiooaaee ia 3 iI ?ic/ei? oinoaoiiai aooa?o aai
2-N-ii?oie?iiaoainoeueoiiiai? eeneioe (IAN), ?I 5,9. Oa?iaioiee neeaae ?
aaciaee ?ic/ei aoee iiaeeo?eiaai? ia iniia? cai?iiiiiaaieo aeey
aeae?eaiiy i?ioiieano?a Pinus oocarpa (Laine et al., 1988), P. banksiana
(Tautorus et al., 1990) ? P. contorta (Hakman, von Arnold, 1983).

I?ney 12-15 aiae. ?ieoaaoe?? eaethno a ?ic/ei? oa?iaio?a i?e oaiia?aoo??
25N a ?ieoaaoe?eio noi?o, ui neeaaeaeany c i?ioiieano?a ? o?aaiaio?a
eaethno, aeiaaaeyee oaeee aea ia’?i ?ic/eiieea oa?iaioo. Io?eiaiee
?ic/ei i?aeaeaaaany a?oaiio i/euaiith /a?ac eai?iiiaee o?eueo?
(300 iei). O?eueo?ao ia?aiineee o i?ia??eo, iiaa?o iaoa?iaoaaee 1 ie
0,5%-iiai ?ic/eio naoa?ice ? oeaio?eooaoaaee 4-5 oa. I?ioa?ie
i?ioiieano?a a?aeae?aee noa?eeueiith i?iaoeith, iii?uaee a ?ioo i?ia??eo
? aeiaaaeyee noi?o aeey a?aeieaaiiy. Aiiiaaiao oeaio?eooaoaaee i?ioyaii
5 oa. Inaae iii?uaee a ??aeea iiaeeaia na?aaeiaeua SH, iiaeeo?eiaaia
oeyoii ca?eueoaiiy ai?noo CaCl2 aei 5 iI ? aeiaeaaaiiyi 0,4 I iai?oo oa
0,2 I ni?a?oo (?I 5,9).

I?ioiieanoe eoeueoeaoaaee a oaiiio? a /aoeao Iao?? (ae?ai. 40 ii) i?e
26N. Eoeueoo?o ?icaaaeyee /a?ac eiaei? 10-14 aei?a na?aeei iiaeeaiei
na?aaeiaeuai oiai ae neeaaeo, aea ?c ciaioaiith aaea?/? eiioeaio?aoe??th
iniioee?a, o i?iii?oe?? 1:1. I?eaeeoo?aa oa?aoaaiiy cae?enithaaee
o?aiaiiaei nei?i. I?ae?aooiie io?eiaieo i?ioiieano?a i?iaiaeeee a eaia??
Ai?y?aa.

Aiae?c ?inoo ? ?icaeoee eoeueoo?

O ca’yceo c ??cieie oa?i?iaie ?iaeoeoe?? eaethno c ??cieo oei?a
aenieaio?a /anoioo eaethniaaiaco aecia/aee /a?ac 4 oeaei? eoeueoeaoaaiiy
aeey a?ae??ce?a iaaii?a ? /a?ac 6 oeaei?a — aeey aaaaoaoeaieo a?oiueie ?
iae?iaaiaoio?o?a (oiaoi /a?ac 2 oeaei? i?ney ?i?oe?aoe?? eaethno), ye
i?ioeaioia ni?aa?aeiioaiiy e?eueeino? aenieaio?a ?c eaethnii aei
caaaeueii? e?eueeino? aenieaio?a. O aeiaaeeo aaiioei?a N ? CD i?e??no
eaethno aecia/aany ca 3-oeaeiaaee ianaae i?ney 24 oeaei?a
eoeueoeaoaaiiy, ye [(e?ioeaaa ne?a iana) — (ii/aoeiaa ne?a iana)/
ii/aoeiaa ne?a iana] x100%.

Aiae?c ai?noo oaenieo ca aeiiiiiaith aenieiaoaeoeaii? ??aeeiii?
o?iiaoia?ao?? (AA?O)

Aeey aecia/aiiy ai?noo oaenieo an? c?acee i?aeaeaaaee eiiaaeoe?eiiio
aenoooaaiith i?e 35N (ianoithaaiiy 2 aeiae). Iio?i c?acee o?e/?
aeno?aaoaaee i?e e?iiaoi?e oaiia?aoo?? a 10 ie nooiai aoeaoiio,
aeno?aeoe ii?aeioaaee e aoeaoii aeia?iaoaaee a iioioe? acioo. Io?eiaiee
nooee caeeoie o?e/? ia?iaeyee ii 2 ie aaenaio. Aaenaiia? aeoyaeee
a?aeeeaeaee, inaae ia?a?ic/eiyee a 2 ie 90%-iiai iaoaiieo. Iio?i
i?aia?ao o?e/? aeno?aaoaaee ii 2 ie nooiai oei?ioi?io. Oei?ioi?ii?
aeoyaeee ia’?aeioaaee. I?ney aeia?iaoaaiiy ?ic/eiieea a iioioe? acioo
caeeoie ?ic/eiyee a 2 ie aoeaoii?o?eeo. ?ic/ei o?eueo?oaaee ?
aeei?enoiaoaaee aeey aiae?co ai?noo oaenieo ca aeiiiiiaith AA?O.

Ai?no oaenieo a io?eiaieo aeno?aeoao aecia/aee iaoiaeii aenieiaoaeoeaii?
??aeeiii? o?iiaoia?ao?? (iaa?iaii-oaciaee aa??aio) ia ??aeeiiiio
o?iiaoia?ao? “I?e?o?ii-4” (I?ae, ?in?y), aeei?enoiaoth/e eieiieo
2×80 ii, caiiaiaio SEPARON-SGX-RP/S, 5 iei. Oaeaee?noue aeththaaiiy —
50 iee/oa., ia’?i i?iae — 2 iee.

Aeey aeththaaiiy aeei?enoiaoaaee nooi?i/aoee a?aae??io aoeaoii?o?eeo
(oaca A) o 20 iI aiaeiiai ?ic/eio i?oioinoi?ii? eeneioe (oaca A): 2 oa.
— 0% A a A; 2 oa. — 17% A a A; 4 oa. — 33%; 6 oa. — 50%; 6 oa. — 67%;
4 oa. — 83%; 20 oa. — 100% A.

C iaoith iaeneiaeueiiai a?aene?aeeiaoaaiiy OO-iiaeeiaiiy oaenieo
aeaoaeooaaiiy i?iaiaeeeiny ii 5 eaiaeao ia aeiaaeeiao oaeeue 202, 212,
228, 270, 300 ii. Aeey ioe?iee ai?noo oaenieo a c?aceao aeei?enoiaoaaee
iaoiae aaniethoiiai eae?a?oaaiiy aeey eiioeaio?aoe?e 0,01, 0,05 ? 1 ia/e
noaiaea?oo. Ii?aoethaaiiy o?iiaoia?ai ? ?ic?aooiie ieiu? i?e?a, ui
a?aeiia?aeathoue oaenieo, i?iaiaeeee c aeei?enoaiiyi i?ia?aie
Ioeueoeo?ii 2,6 (“Ioeueoeo?ii”, Iineaa, ?in?y) ia aeiaaeei? oaee? 228 ii
(ae?oaee iaeneioi iiaeeiaiiy oaenieo).

Noaoenoe/ia ii?aoethaaiiy ?acoeueoao?a

Aenia?eiaioe i?iaiaeeee a 2-3-e?aoi?e iiaoi?iino?, o eiaeiiio
aeei?enoiaoaaeiny ia iaioa 50 aenieaio?a eiaeiiai oeio. An? oeeo?ia?
aeai? iiaeai? o aeaeyae?: na?aaei? (x) na?aaei? eaaae?aoe/ia
a?aeoeeaiiy (Sx).

Iioeaea aiae?oe/iiai aecia/aiiy oaenieo ia ia?aaeuoaaea 20%.

?ACOEUeOAOE ? IAAIAI?AIIss

Aiae?c ai?noo oaenieo a ?ioaeoieo ?ineeiao

A aenia?eiaio? aeei?enoiaoaaeenue aaniyia oaiy, ??/i? iaaiie ? ei?a
ieaeiuei? /anoeie noiaao?a 15-oe eeii?a T. baccata ? 3-o eeii?a
T. cuspidata ?c eieaeoe?? aeaiae?a??th OeAN ?i. I.I. A?eoea IAI Oe?a?ie,
c?a?ai? iaeii/anii (oaae. 1).

Oaaeeoey 1. Oa?aeoa?enoeea ?ineeiieo c?ace?a

AeaeT. baccataT. cuspidataeeii123456789101112131415161718e-oue aea?aa

o eeii?613446681691245191

Ca ai?noii oaenieo i?iaiae?ciaai? iaie eeiie iiaeia oiiaii ?icae?eeoe ia
/ioe?e a?oie (iae. 1):

1) eeiie, aeey yeeo oa?aeoa?iee i?eaeecii iaeiaeiaee ai?no oaenieo a
noaaeao ? oai? (2, 4, 8, 12, 15, 17);

2) eeiie c i?i?iaeueiei ai?noii oaenieo a iaaiiao (5, 6, 9, 10, 11, 13,
18);

3) eeiie c i?i?iaeueiei ai?noii oaenieo a oai? (1, 14);

4) eeiie, aeey yeeo oa?aeoa?ia iayai?noue oaenieo o?eueee a ei?? (3).

Iayai? a iaoiio ?icii?yaeaeaii? eeiie T. baccata, iiae?aii aei
T. brevifolia (Videncek et al., 1990; Fett-Neto et al., 1992), i?noeee
iaeneiaeueio e?euee?noue oaenieo a ei?? noiaao?a, yea aoea ia
ii?yaeie aeuith, i?ae o iaaiiao ? oai?. Aeey eeii?a T. cuspidata
aeyaeaia iiae?aia oaiaeaioe?y, ui noia?a/eoue aeniiaeai a?eueoino?
aaoi??a, ye? a?aecia/athoue iaeneiaeueia iaeiie/aiiy ?a/iaeie o oai?
(Witherup et al., 1990; Kwak et al., 1995). Iaeiae, an? iayai? ?iaioe
i?enay/ai? aea/aiith ai?noo oaenoeo a ?ineeiao, a?ae?a?aieo a a?aaeao ?o
i?e?iaeiiai c?inoaiiy. C iaeyaeo ia cia/iee aieea oiia c?inoaiiy ia
i?ioeane neioaco ? iaeiie/aiiy oaenoeo (Wheeler et al., 1992; Kwak et
al., 1995), iiaeia i?eionoeoe, ui ia?aaaaeia eiai iaeiie/aiiy a ei??
T. cuspidata o iaoiio aeiaaeeo iia’ycaia c ?io?iaeoeoe??th ?ineeie a
Oe?a?io. Aeyaeaia cia/ia aa??aaaeuei?noue ai?noo oaenieo i?ae ??cieie
oeaieiaie a iaaeao iaeiiai eeiio (iae. 1). Oea ia aea? iiaeeeaino?
aeae?eeoe ie?aiee eeii-noia?i?iaeooeaio oaenieo, ui a?aecia/aeiny ?
?ai?oa i?e aea/aii? iiioeyoe?e T. brevifolia ? T. cuspidata (Wheeler et
al., 1992; Choi et al., 1995).

Iaeiaioa aa??aaaeuei?noue oa?aeoa?ia aeey ei?e noiaao?a. A?aecia/aia
oaeiae aeniea i?aeeeiiaeueia aa??aaaeuei?noue ai?noo oaenieo a iaio
aeae?a o an?o o?ueio oeiao c?ace?a (iae. 1).

I?e ii??aiyii? na?aaei?o cia/aiue ai?noo oaenieo a ei??, oai? oa
iaeii??/ieo iaaiiao aeaio aeae?a oeno noaoenoe/ii cia/oua ??cieoey
aeyaeaia eeoa o aeiaaeeo oai? (oaae. 2).

Oaaeeoey 2

Na?aaeiy ioe?iea ai?noo oaenieo a ??cieo /anoeiao ?ineei

T. baccata ? T. cuspidada

AeaeAi?no oaenieo, % nooi?
iane*ei?aiaaiieoaiyT. baccata0,0114+0,0015a0,0005+0,0001a0,001+0,0002aT.
 cuspidata0,0149+0,0024a0,0007+ 0. 0002a0,0027+0,0002a

* — na?aaei? cia/aiiy a noiai/eeao, ui noi?iaiaeaeothoueny iaeiaeiaeie
aoeaaie, a?ae??ciythoueny ianooo?ai i?e

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