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Journal of Fruit and Ornamental Plant Research Vol. 13, 2005: 109-122

SOMACLONAL VARIATION IN MICROPROPAGATEDTULIPS BASED ON PHENOTYPE OBSERVATION

M ałg o r z a t a P o d w y s z yńs k a

Research Institute of Pomology and FloriculturePomologiczna 18, 96-100 Skierniewice, POLAND

(Received November 28, 2005/Accepted December 8, 2005)

A B S T R A C T

A new protocol for tulip micropropagation was developed to produce virus-freestock plants, speed up breeding, and to introduce new cultivars on the market. Thismethod is based on cyclic multiplication of adventitious shoots in the presence ofthidiazuron. Before this method can be recommended for general use, it had to beverified that it could be used to produce high quality, true-to-type plants. Tulips of thecultivars ‘Blue Parrot’ and ‘Prominence’ were cultured in vitro from 1.5 to 6 yearsbefore being cultured ex vitro. Plants were observed for morphological changes whenthey began to flower. Juvenile plants were also examined for leaf variegation andother abnormalities. The first plants to flower did so in their third or fourth growingseason. Reliable information on somaclonal variation introduced during in vitropropagation can be obtained when more than 30% of the plants have flowered. Thiscan occur as early as the fourth or fifth growing season. The frequency of variationwas less than 3.3%, in progeny lines derived from cultures maintained in vitro for lessthan three years. But in the progeny lines derived from four-year-old cultures, allplants of ‘Blue Parrot’ and the half of plants of ‘Prominence’ were changed. In ‘BlueParrot’, all of the off-type plants had flowers which were red-purple instead of purple-violet. In ‘Prominence’, most of the off-type plants had minor changes, such as lily-like flowers, although some plants did have strongly malformed flowers. Variegationwas observed only in juvenile plants of progeny lines derived from shoot cultureswhich had been propagated in vitro for four years or longer. Micropropagation oftulips with this new method can increase the risk of mutation, especially when the invitro cultures are maintained for longer period. By limiting this period to three yearsor less, this risk can be significantly reduced. Systematic monitoring of the plantmaterial with DNA markers would further reduce the risk.

Key words: Tulipa gesneriana, somaclonal variation, in vitro propagation, flowering,variegated plants

M. Podwyszyńska

J. Fruit Ornam. Plant Res. vol. 13, 2005: 109-122110

INTRODUCTION

A new protocol for tulip micro-propagation was developed to producevirus-free stock plants, speed upbreeding, and to introduce newgenotypes on the market. The methodis based on the cyclic multiplication ofadventitious shoots in the presence ofthidiazuron (Podwyszyńska, 2001;Podwyszyńska and Marasek, 2003). Inthe tulip, the natural propagation rateis very low. Because each bulbproduces only two or three new bulbsa year, twenty or more years can passbetween the initial crossing andcommercial release of a new cultivar.The new method can shorten thisprocess by ten years.

Before this method can be recom-mended for general use, it had to bechecked to see if it could be used toproduce high quality, true-to-typeplants. It also had to be determinedwhether in vitro culture could inducegenetic or epigenetic changes dete-ctable in the plant phenotype.

Genetic and epigenetic variationwhich occurs during vegetative pro-pagation, including in vitro multi-plication, is called somaclonal varia-tion (Larkin and Scowcroft, 1981).The nature and causes of somaclonalvariation have been elucidated, andthe types of changes observed andtheir usefulness in plant breedinghave been reviewed by severalresearchers (Sabała and Orlikowska,1993; Jerzy et al., 1994; Borkowska,1995; Karp, 1995; Pląder, 1997; Jainand De Klerk, 1998).

Changes can be heritable ortransient, depending on whether the

changes occur in the germ line.Somaclonal variants can be detectedusing morphological, cytological,biochemical and molecular methods(Chen et al., 1998; Al-Zahim et al.,1999; Zhao et al., 2005).

Spontaneous mutations in a parti-cular gene occur once every 104 to107 cell divisions, depending on thegene in question. The mutation rateis considerable higher in tissuecultures, and, in extreme cases, canbe as high as several percent perlocus (Nadolska-Orczyk, 1991).

The mutation rate depends on thegenotype, the explant source, theregeneration system, the concentrationof growth regulators, the number ofmultiplication cycles, and the durationof the culture period.

Until now, little was knownabout the occurrence of somaclonalvariation during micropropagation oftulips because this method has onlybeen used on a very limited scale.

In one study, no off-types werefound among flowering tulips whichhad been propagated in vitro, even ifthey had been produced with the helpof 2,4-D, an auxin with a relativelyhigh mutagenic activity (Langens,2001). In fact, flower quality in theseplants was higher than flower qualityin plants which had been propagatedby conventional methods.

When testing a new micro-propagation method, the tulips pro-duced should be morphologicallyevaluated for true-to-typeness whenthey are in the flowering phase. Thiscan be done as early as four yearsafter the bulblets were produced invitro (Le Nard et al., 1987). Some

Somaclonal variation in micropropagated tulips…

J. Fruit Ornam. Plant Res. vol. 13, 2005: 109-122 111

modifications which permit earliertesting have been investigated (Pod-wyszyńska and Nowak, 2004). Forexample, plants which had beenproduced in vitro were maintained ina growth chamber or greenhouse inorder to let them go through twogrowing cycles a year with minimumloss of bulbs due to winter freezing.However, the reproduction rate wasvery low. Better results wereobtained by growing the tulipsoutdoors under optimized conditions.In this way, two to five daughterbulbs per mother plant as well asa high rate of bulb weight increasecould be obtained every year.

The aim of this study was toevaluate the new method in terms oftrue-to-typeness and the rate ofmorphologically detectable somaclonalvariation.

MATERIAL AND METHODS

The tulip cultivars used in thisexperiment were ‘Blue Parrot’ and‘Prominence’. Shoots were multi-plied in vitro by the means ofadventitious regeneration for 1.5 to 6years before bulblets were generatedand cultivated ex vitro for two to sixyears (Podwyszyńska and Marasek,2003; Podwyszyńska, 2001; Podwy-szyńska and Nowak, 2004). Twoprogeny lines were produced for‘Blue Parrot’, BP-A and BP-B. Fiveprogeny lines were produced for‘Prominence’, Pr-A to Pr-E. Theconditions under which they werecultured and propagated are pre-sented in Table 1. Additionally, twoprogeny lines of ‘Blue Parrot’

derived from the four- and six-year-old cultures were planted in aninsect-proof tunnel in 2001 and 2003,respectively. These plants did notflower in 2005 (juvenile plants) andwere observed for leaf changes.

BP-A, Pr-A, Pr-B, Pr-D and Pr-Ewere grown in a growth chamber orgreenhouse for the first or secondgrowing cycle (GC) before beingtransferred outside to an insect-prooftunnel. BP-B and Pr-C were planteddirectly outdoors (Podwyszyńska andNowak, 2004).

Bulbs were transferred to theinsect-proof tunnels in mid-October,planted in plastic boxes filled withgrowth substrate, and mulched withbark to protect them from the frost.For each progeny line, bulbs wereplanted in order from largest tosmallest. In the spring, the plantswere fertilized, watered as needed,and treated to protect them frompests and diseases. At the beginningof July, the bulbs were lifted, dried,counted, weighed and stored at 20oCuntil they were planted again in thefall. In 2000, the first year plantswere transferred outdoors, no insect-proof tunnel was used and the plantswere accidentally exposed to aphids.

Bulbs of the progeny line Pr-Ewere grown in a greenhouse duringthe first GC. The next year, theywere rooted in a growth chamber at9oC from February until May, whenthey were transferred outdoors to aninsect-proof tunnel. In August, thebulbs were lifted and stored untilthey were replanted in October.

Morphology was evaluated whenthe plants were in full bloom, which

M. Podwyszyńska


occurred at the beginning of May for‘Prominence’, and in mid-May for‘Blue Parrot’.

The ratio of flowering plants tothe number of bulbs planted in thefirst GC was calculated. Morpho-logical evaluation was performed inaccordance with UPOV guidelineswith the help of published cultivardescriptions (Anonymous, 1988;Krause, 1986; Holitscher, 1968;1972; Van Scheepen, 1996).

The morphological traits recordedwere: plant height, leaf number perplant, leaf color, flower shape, flowerlength, flower color, base shape, basecolor, stamen color, and pollen color.Flower color was determined withthe help of the color chart developedby the Royal Horticultural Society ofLondon (Anonymous, 1996). Datawere also collected on juvenile plantswith altered leaf shape or withvariegated leaves.

Data on plant height and flowerlength were statistically elaboratedby analysis of variance, followed byStudent's t-test at P0.05.

The main characteristics of ‘BlueParrot’ are: plant 55 to 60 cm inheight; flower purple-violet (CC80C); tepal goffering weaker than inother parrot tulips; flower 7.5 cmlong; base greenish blue edged withyellow; pollen grayish purple.

The main characteristics of‘Prominence’ are: plant 40 to 45 cmin height; flower dark red (46B);flower 7.0 cm long; base ivy greenedged with yellow; pollen bluishblack.

Plants of the original cultivarswhich had been propagated by

conventional means served as thereference.

In order to rule out morphologicalchanges due to viral infection, all off-type plants and randomly selected true-to-type plants were tested for virusesby ELISA in 2004 and 2005. Theviruses tested for were: Tulip breakingvirus (TBV), Lily symptomless virus(LSV), Cucumber mosaic cucumovirus(CMV), Tobacco rattle tobavirus(TRV), Tobacco necrosis virus (TNV),and the potyvirus group. Leaf samplesfor testing were collected from mid-April to mid-May. Testing was carriedout as previously described (Podwy-szyńska et al., 2005).

RESULTS

In 2003, the first micropro-pagated plants flowered after 2.5 to3 years of cultivation (Tab. 1). Theprogeny line BP-A was in its fourthGC and had a flowering rate of20.8%. Flower color was typical for‘Blue Parrot’, although the tepals didnot have any of the traits typical forparrot tulips. Pr-A was also in itsfourth GC and had flowers whichwere true-to-type, except for oneplant which had red and white tepals.This plant was infected by TBV andremoved from further study.

In 2004, BP-A, Pr-A and Pr-Bwere in their fifth GC. BP-A hada flowering rate of 37.5%, Pr-A hada flowering rate of 25.0%, and Pr-Bhad a flowering rate of 22.5%. Pr-Cwas in its fourth GC after a 0.5 yearsin the growth chamber and 2.5 yearsoutdoors, and had a flowering rate of23.5%. BP-B and Pr-D were in their



T a b l e 1 . Flowering and somaclonal variation in tulips derived frommicropropagation

Flowering plants[%]

Number of off-types

in 2005

Cultivar,progeny line,number of growingcycles (GCs)

until 2005,place of cultivation

c – growth chamberg – greenhouset – insect-proof tunnel

In vitromulti-plica-tion

period[years]

Year ofplantingex vitro

Number of

bulbsplantedin first

GC2003 2004 2005

minorchange

s

majorchanges

Somaclonalvariationin 2005

[%]

‘Blue Parrot’BP-A: 6 GCs: 1c, 5tBP-B: 4 GCs: 4t

‘Prominence’Pr-A: 6 GCs: 1c, 5tPr-B: 6 GCs: 2c, 4tPr-C: 5 GCs: 5tPr-D: 5 GCs: 1c, 4tPr-E: 5 GCs: 1g, 4t

24

1.52344

19992001

19991999200020012001

4891

324517

12068

20.80

3.1----

37.53.3

25.022.223.5

1.5-

72.95.8

78.1133.358.530.832.4

00

010

1710

06

01021

0.0100.0

0.03.30.0

51.450.0

third GC and flowered sporadically.BP-B had a flowering rate of 3.3%,and Pr-D had a flowering rate of1.5%. Some of the plants which hadbeen grown outdoors in 2000 withoutthe protection of an insect-prooftunnel showed symptoms of viralinfection. In ‘Blue Parrot’, the tepalshad dark purple and green streaks,and in ‘Prominence’, they had darkstreaks. In both cultivars, the leaveshad light green stripes and streaks.ELISA revealed the presence ofTulip breaking virus (TBV). Allsymptomatic plants were removed.

No distinct off-types were obser-ved among the healthy plants in 2004.‘Blue Parrot’ plants grown from bulbsover 10 grams generally were true-to-type, whereas plants grown from bulbsunder 10 grams often did not haveflowers of the characteristic parrotshape. Plant height ranged from 34 to50 cm, depending on bulb weight.

‘Prominence’ plants were pheno-typically true-to-type. Flowers deve-loped on plants grown from bulbsweighing at least 6 g. Plant heightranged from 37 to 46 cm.

In 2005, BP-A, Pr-A and Pr-Bwere in their sixth GC. BP-A hada flowering rate of 72.9%, Pr-A hada flowering rate of 78.1%, and Pr-Bhad a flowering rate of 133.3%. Thereason why the percentage washigher than 100% for Pr-B was thatflowers developed not only from theprimary bulbs, but from many of thedaughter bulbs as well. For ‘Promi-nence’ progeny lines in their fifthseason, those which had been grownoutdoors in all growing seasons hadflowering rates higher than lineswhich had been grown in a green-house or growth chamber beforebeing transfered outdoors. All plantsof BP-A and Pr-A were true-to-type.These were the progeny lines which

M. Podwyszyńska


a b

c d

e f



Figure 1. In vitro-produced tulip plants observed at full flowering in 2005a) True-to-type plants of progeny line BP-A.b) Off-type plant of BP-B with red purple flower.c, d, e) Variegated plants of ‘Blue Parrot’ derived from six-year-old in vitro culture.f) True-to-type plants of Pr-A.g) Off-type plant of Pr-D with narrow tepals.h) Off-type plant of Pr-D with abnormal flower.i) Off-type plant of Pr-D with lily-like flower.

had been cultivated ex vitro for thelongest time (Fig. 1a,f). In theseprogeny lines, flower length was thesame as in the conventionallypropagated reference plants, but plantheight and bulb weight were lower

(Tab. 2). In Pr-D and Pr-E, bothflower length and plant height weresignificantly lower than in thereference plants. Plant height wasgenerally positively correlated withbulb weight.

g h

i

M. Podwyszyńska


T a b l e 2 . Quality parameters of true-to-type tulips derived from micropropagation.Bulb weights from 2004. Plant height and flower length from 2005

Cultivar,progeny

Number ofplants

observed

Mean bulbweight

[g]

Weight oflargest bulb

[g]

Plantheight

[cm]

Flower length[cm]

‘Blue Parrot’BP-AReferenceFemp

2120

7.318.3

15.540.1

57.6 a60.9 b5.98**

6.4 a6.3 a< 1

‘Prominence’Pr-APr-BPr-CPr-DPr-EReferenceFemp

21539

191220

12.011.914.5

8.37.3

21.0

26.128.726.515.813.338.6

42.2 b42.2 b40.1 bc40.9 b36.6 c45.1 a7.02**

7.8 a7.7 a7.6 a7.2 b7.0 b7.7 a

6.85**

Means marked with the same letter do not differ significantly at P 0.05 according to Student t-test** level of significance 0.01 according to F-test

In 2005, some off-types wereobserved, especially in those progenylines, which had been cultured invitro for four years (Tab. 1). In BP-B, all of the flowers were red-purple(CC 66A) instead of purple-violet.Tepal goffering was also atypical forthis cultivar (Fig.1b). In Pr-D and Pr-E, about half of the plants were off-types, although only three plantswere extremely atypical. One ofthese plants had very narrow tepals(Fig.1g). The other two plants wereonly 25 cm high and had abnormalflowers with yellow bases, mal-formed stamens and anthers, andfringed, narrow tepals with whitestreaks (Fig. 1h). The other off-typeshad normal colored flowers and onlyminor changes: tepals with acute tipsor tepals which were curved back asin lily-flowered tulips (Fig.1i).

In Pr-B, which had been culturedin vitro for two years, two off-typeswere found. One had lily-like flowers.The other was strongly changed. It wasonly 30 cm high and had abnormalflower like those observed in Pr-D.

Variegation was observed only injuvenile plants of ‘Blue Parrot’ pro-geny lines derived from shoot cultureswhich had been propagated in vitro forlong periods. For example, thepercentage of variegated plants was1.0% in one progeny line which wasplanted ex vitro after four years of invitro culture , and 1.7% in anotherline which was planted ex vitro aftersix years of in vitro culture (Tab. 3,Fig. 1c,d,e). These juvenile plantsalways had only one leaf with variouspatterns of white or yellow stripesconsisting of cells which did notcontain chlorophyll. These patterscould be either symmetrical or asym-



metrical, regular or irregular, wide ornarrow, marginal or central.T a b l e 3 . Number of variegatedplants in ‘Blue Parrot’ progeny fromlong-term in vitro cultures in 2005

In vitrocultureperiod[years]

Firstseason

ofex vitroculture

Totalplants

Variegatedplants

46

20012003

1545469

15 (1.0%)8 (1.7%)

Sporadic juvenile plants withvery narrow leaves or longitudinallyfolded leaves were also observed.ELISA did not reveal the presence ofany of the viruses tested for in theseplants. We are currently studyingvariegation and other morphologicalchanges observed in these juvenileplants.

DISCUSSION

Plants which had been micro-propagated in vitro began floweringin the fourth growing season. Theonly atypical trait observed at thisstage was the lack of parrot flowersin the ‘Blue Parrot’ progeny line BP-A. These flowers were very similarto those of ‘Bleu Aimable’, the non-parrot cultivar from which ‘BlueParrot’ originated as a mutant. It hasbeen supposed that ‘Blue Parrot’ isa chimera with elements of twogenotypes, one with parrot flowersand the other without. The lack ofparrot flowers in some of themicropropagated plants might be dueto segregation of the genotypesduring adventitious shoot regene-ration. The plants which failed to

produce typical parrot flowers in2003 did produce them in 2004,however. The ability to produceparrot flowers was thus related tobulb size. Bulbs over a criticalweight produced parrot flowers,whereas smaller bulbs did not.Further study is needed to moreaccurately determine the critical bulbweight, but it is about 10 grams.

In Pr-D, which had been culturedin vitro for four years, 1.5% of theplants flowered in fourth season.None of them were phenotypicallyabnormal. The next season, 30.8% ofthe plants flowered, and about half ofthem were phenotypically abnormal.Based on the results of this study, wecan conclude that reliable informa-tion on somaclonal variation intro-duced during in vitro propagationcan be obtained when more than 30%of the plants have flowered. This canoccur as early as in the fourth or fifthgrowing season.

Very few off-types if any wereobserved in progeny lines derivedfrom shoot cultures which had beenpropagated in vitro for three years orless. Micropropagated plants wereshorter than the reference plants, butthat is because of smaller bulb size.In outdoor cultivation, the annualrate of weight increase in micropro-pagated bulbs was between two andfive (Podwyszyńska and Nowak,2004). Thus most of the bulbs shouldreach the size of the reference bulbsin the next season.

Somaclonal variation was obser-ved far more often in progeny linesderived from shoot cultures whichhad been propagated in vitro for four

M. Podwyszyńska


years or more. Because thesechanges were widespread, distinctand repeated, the source of thevariation may be a mutation.

There have been several reportsof somaclonal changes in variousplants which had been propagated invitro, such as bushy growth inrhubarb, and malformed flowers inPhalenopsis and the oil palm (Zhaoet al., 2005; Chen et al., 1998; Rivalet al., 2000).

Whether the changes induced byin vitro propagation are stable can beconfirmed by growing the resultingprogeny line from seed. This hasbeen done with Capsicum annuum(Anu et al., 2004). However, this isnot possible with plants which takea long time to mature or in plantswith highly heterogenous genomes,as in fruit trees and ornamentalperennials, including tulips. Therefore,it has been suggested that somaclonalvariation in tulips would be studiedwith the help of DNA markers, ashas been done in the date palm(Gurevich et al., 2005). Work iscurrently underway to developmethods of detecting somaclonalchanges using molecular techniquessuch as randomly amplified poly-morphic DNA (RAPD) and inter-simple sequence repeats (ISSR).

Some of the changes observed inthis study may be due to epigeneticchanges resulting from geneexpression or repression. One of thebest known mechanisms of regulatinggene expression is DNA methylation,which can be strongly influenced byvarious factors of in vitro culture, suchas growth regulators. In the oil palm

trees produced in vitro, DNAhypomethylation was found to causeflower malformation in 50% of theplants (Rival et al., 2000). Thisphenomenon vanished after nine yearsof cultivation. Variation due to thechanges in the rate of DNA methyl-ation has been also detected inmicropropagated plants of cucumber,potato and almond (Pląder et al., 1998;Cassells et al., 1999; Channuntapipat etal., 2003).

Our results show that the occur-rence of somaclonal changes andvariegation increases with the timethe progeny lines are maintained invitro. Marked changes could beobserved after four, five or six years.This confirms earlier reports thatsomaclonal variation increases withthe duration of in vitro culture, andespecially with the number ofmultiplication cycles (Gavidia et al.,1996; Yang et al., 1999; Al-Zahim etal., 1999; Devarumath et al., 2002).A statistical model to predict thetheoretical mutation rate in in vitroplant culture has been developedusing the number of multiplicationcycles as the primary parameter(Côte et al., 2001).

The frequency of somaclonalvariation also depends on the regene-ration system and growth regulatorsused. Variation is more often observedin multiplication by adventitious shootregeneration than in micropropagationby lateral bud development (Karp,1995; Pląder et al., 1998). The newmethod employed in this study isbased on adventitious shoot regene-ration stimulated by thidiazuron,a growth regulator with a high



cytokinin activity. In this method,thidiazuron was the main factorwhich made successful cyclic shootmultiplication possible. So far, nomethods based on micropropagationof lateral shoots have been developedfor use in tulip. This is not sur-prising, considering how difficult itis to induce lateral bud formation inmonocotyledonous plants like thetulip.

Micropropagation of tulips withour new method can therefore increasethe risk of mutation, especially whenthe progeny lines are maintained invitro for four years or more. Bylimiting the period of in vitro culture tothree years or less, this risk can besignificantly reduced. Systematicmonitoring of the plant material withDNA markers would further reducethe risk. Molecular techniques havebeen used to assess the genotypestability during in vitro culture, andhave been recommended for routineuse (Rani et al., 1995; Al-Zahim et al.,1999; Cassells et al.,1999; Devarumathet al., 2002; Martins et al., 2004;Gurevich et al., 2005).

The aim of micropropagation is torapidly produce a large number of true-to-type plants. Somaclonal variation istherefore undesirable. However, inplant breeding, somaclonal variationcan be a valuable source of newgenetic material. In this study, severalinteresting variants were seen,including ‘Blue Parrot’ progeny withred-purple flowers or variegatedleaves, and ‘Prominence’ progeny withlily-like flowers. These variants mayprovide interesting material forbreeding.

This study was supported byMinistry of Science and InformationSociety Technologies, Grant No.PBZ-MIN-007/P04/2003.

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M. Podwyszyńska


ZMIENNOŚĆSOMAKLONALNA TULIPANÓWROZMNOŻONYCH IN VITRO NA PODSTAWIE OCENY

FENOTYPU

M ałg o r z a t a P o d w y s z yńs k a

S T R E S Z C Z E N I E

Celem badańbyła weryfikacja nowej technologii mikrorozmnażania tulipanaprzez ocenętożsamości odmianowej rozmnożonych in vitro roślin. Metoda ta opierasięna cyklicznym namnażaniu pędów w obecności tidiazuronu. W badaniachwykorzystano rośliny tulipanów ‘Blue Parrot’ i ‘Prominence’ uzyskane z kulturpędów rozmnażanych in vitro przez okres od 1,5 do 6 lat. Obserwacje fenotypudotyczyły roślin uprawianych w tunelu owadoszczelnym przez 2-5 lat. Wykonywanoje w okresie pełni kwitnienia. W przypadku roślin juwenilnych obserwowanopojawianie sięchimer typu variegata. Wykazano,że u tulipana rozmnażanego in vitropierwsze rośliny zakwitły w 3 lub 4 cyklu uprawy (1,5-23,1%). W kolejnym sezoniezakwitło od 58,5 do 133,3% roślin. Wiarygodne obserwacje można byłoprzeprowadzićpo 4 latach uprawy, gdy zakwitło ponad 30% roślin. U tulipanówuzyskanych z kultur pędów in vitro rozmnażanych przez 1,5-3 lata zmiennośćniewystępowała lub była sporadyczna. Wysoki poziom zmienności – 100% u ‘BlueParrot’ i około 50% u ‘Prominence’, stwierdzono wśród roślin pochodzących z 4-letnich kultur in vitro. U ‘Blue Parrot’ zmany dotyczyły barwy kwiatów,a u ‘Prominence’ ich zmienionego kształtu, np. na „liliokształtny” (większośćsomaklonów), czy kwiatów silnie zdeformowanych (kilka somaklonów). Wśródroślin ‘Blue Parrot’ otrzymanych z długoterminowych kultur in vitro pojawiły sięchimery typu variegata (1,0-1,7%).

Uzyskane wyniki wskazują, że nowoopracowana metoda może byćstosowanaprzy zachowaniu zasady, iżcykliczne namnażanie pędów nie powinno trwaćdłużejniż 2-3 lata, a materiałroślinny powinien byćsystematycznie kontrolowanyz użyciem markerów molekularnych (obecnie prowadzone sątakie badania).

Słowa kluczowe: Tulipa gesneriana, zmiennośćsomaklonalna, rozmnażanie in vitro,rośliny typu variegata

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