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ISSN 0167-6857, Volume 102, Number 3

ORIGINAL PAPER

Production of in vitro haploid plants from in situ induced haploidembryos in winter squash (Cucurbita maxima Duchesne ex Lam.)via irradiated pollen

Ertan Sait Kurtar • Ahmet Balkaya

Received: 18 November 2009 / Accepted: 1 March 2010 / Published online: 23 March 2010

� Springer Science+Business Media B.V. 2010

Abstract The influence of pollen irradiation on the pro-

duction of in vitro haploid plants from in situ induced

haploid embryos was investigated in winter squash

(Cucurbita maxima Duchesne ex Lam.). Pollen were irra-

diated at different gamma-ray doses (50, 100, 200 and 300

Gray) and durations (9, 11, 15, 21, and 28 July). Production

of in vitro haploid plantlets was influenced by irradiation

dose, irradiation duration, genotype, and embryo type and

embryo stage. Embryos were only obtained from lower

irradiation doses (50 Gray and 100 Gray) and earlier irra-

diation durations (9, 11, and 15 July). The greatest embryo

number per fruit was procured from ‘‘G14’’ and ‘‘55SI06’’

genotypes at 50 Gray gamma-ray dose. Necrotic embryos

were higher than normal embryos at delayed harvest times

(5 and 6 weeks after the pollination). The convenient

harvest time for embryo rescue was observed about

4 weeks (between 25 and 30 days) after pollination. All

cotyledon and amorphous embryos had only diploid plants

while late-torpedo, arrow-tip, and pro-cotyledon embryos

produced 33.3, 50.0, and 66.7% haploid plant. The fre-

quency of haploid plantlets was 0.11, 1.17, 10.96 and 0.28

per 100 seeds, 100 embryos, 100 plantlets and a fruit at

50 Gray gamma-ray dose, respectively.

Keywords Irradiated pollen � In vitro haploidization �Winter squash (Cucurbita maxima Duchesne ex Lam.)

Introduction

In a conventional breeding programme, pure lines are

obtained after several generations of selfing and still may

not be 100% homozygous (Germana 2006). Haploidization

is the process of producing haploid plants in a single

generation. Following haploidization, the chromosome

number of haploid plants may be doubled (dihaploidiza-

tion) to obtain complete fertile homozygous lines. These

valuable lines are currently used for breeding programmes

and genetic research.

Haploid plants can be obtained spontaneously (andro-

genesis, gynogenesis, or parthenogenesis, semigamy and

polyembryony), but the frequency of haploid is very low

(Pochard and Dumas de Vaulx 1971; Lacadena 1974).

Recently, haploid plants are produced using in vitro

androgenesis (anther-microspore culture) and gynogenesis

(ovule-ovary culture), and in situ parthenogenesis (pollen

irradiation and treatment with chemicals, etc.).

Pollen irradiation (UV, gamma rays, and X-rays) is the

most widely used technique to induce in situ parthenoge-

netic haploid plants. Gamma rays are commonly used in

haploid programmes because of their simple application,

good penetration, reproducibility, high mutation frequency,

and low disposal (lethal) problems (Chahal and Gosal

2002). This technique was used firstly with embryo culture

on different species of Nicotiana (Pandey and Phung

1982).

Irradiated pollen can germinate on the stigma, grow

within the style and reach the embryo sac, but cannot

fertilize the egg-cell and the polar nuclei (Cuny 1992).

Genetically inactive but germinable pollen can be used to

stimulate the division of the egg cell, and thus induce

parthenogenesis or development of parthenocarpic fruit,

including gynogenic haploid production; overcoming

E. S. Kurtar (&)

High School of Profession of Bafra, Ondokuz Mayis University,

Bafra, Samsun, Turkey

e-mail: [email protected]

A. Balkaya

Horticulture Department of Agriculture Faculty, Ondokuz Mayis

University, Samsun, Turkey

123

Plant Cell Tiss Organ Cult (2010) 102:267–277

DOI 10.1007/s11240-010-9729-1 Author's personal copy

minor cross-incompatibilities, and for physiological studies

of incompatibility (Stairs and Mergen 1964; Savaskan and

Toker 1991; Todorova et al. 2004), gene transformation

(Pandey 1978) and nucleus substitution (Raquin et al.

1989).

The irradiated pollen technique is an effective method

for the induction of haploid embryos in Cucurbit. Induction

of in situ haploid embryos and obtaining in vitro haploid

plants have been achieved using an irradiated pollen

technique in watermelon (Gursoz et al. 1991; Sari 1994),

melon (Sauton and Dumas de Vaulx 1987; Cuny 1992;

Maestro-Tejada 1992; Sari et al. 1992; Abak et al. 1996;

Lotfi et al. 2003), cucumber (Truong-Andre 1988;

Niemirowicz-Szczytt and Dumas de Vaulx 1989; Sauton

1989; Caglar and Abak 1999; Faris et al. 1999; Lotfi et al.

1999; Dolcet-Sanjuan et al. 2006), snake cucumber (Taner

et al. 2000), summer squash (Kurtar et al. 2002), and

pumpkin (Kurtar et al. 2009).

Winter squash is an annual cultivated species of

Cucurbit (Whitaker and Bemis 1964) and genetically dif-

ferent from Cucurbita pepo and Cucurbita moschata

(Athanasios et al. 2009). Cucurbita pepo shares a common

ancestor with C. moschata and C. argyrosperma, but not

with C. maxima (Decker-Walters et al. 1990). Isozyme

study showed high allelic diversity in C. pepo, C. mosch-

ata, and C. maxima (Heikal et al. 2008). To the authors’

knowledge, this is the first report on in vitro haploid pro-

duction in winter squash.

The objective of the present study was to determine the

effects of irradiation dose (Co60), irradiation duration,

genotype, embryo type and embryo stage on production of

in vitro haploid plants from in situ induced haploid

embryos in winter squash.

Materials and methods

Plant material

The experiment was carried out with six winter squash

genotypes (57SI06, 57SI21, 55BA02, 55BA03, 55CA06,

and G14) selected from four provinces (Samsun, Amasya,

Sinop and Bolu) of the Black Sea region of Turkey (genetic

material used in the project was funded by TUBITAK-

TOVAG -104O144), except ‘‘G14’’ genotype which was

provided by the Turkish Seed Gene Bank, Menemen, Izmir

(Fig. 1). The seeds were sown in plastic flats (cell volume

150 cm3 and 28 cells per flat) containing mixture of peat-

moss: perlite (2:1 v/v) on 20 April 2008. Seedlings were

raised in unheated glasshouse, and 15 seedlings from each

genotype were planted at 3–4 leaf stage with spacing of

3 9 3 m on 11 May.

Irradiation and pollination

Female flowers were isolated with white cloth bags

(15 9 10 cm) and male flowers were collected around

noon on the day before anthesis. Anthers without filaments

were excised and mixed equally for each genotype, and

placed into small cardboard boxes (5 9 7 9 2 cm). Sam-

ples were irradiated at 50, 100, 200 and 300 Gray doses of

gamma rays by a Co60 source of Theratron 780-C equip-

ment on the same day at 1,482 Ci source activity and

11.96 Gymin-1 dose rate. Irradiation experiments were

performed at different durations (9, 11, 15, 21, and 28 July)

to evaluate the effects of irradiation duration on embryo

induction. Irradiated anthers were incubated at room tem-

perature overnight. Female flowers were pollinated by

irradiated pollen in the morning of the following day at

0700–0900 hours. Pollen of flowers 0 days old (the day

following irradiation) and 1 day old were used for polli-

nation. Female flowers were then isolated with cloth bags

again to avoid undesired pollen contamination. Cloth bags

were removed at 3rd or 4th days of pollination.

Embryo culture

Immature fruits were harvested from 3 to 6 weeks after

pollination to determine the convenient harvest time.

Washed fruits were surface-sterilized in 2% sodium hypo-

chlorite solution for 30 min and then flame-sterilised using

ethanol. Prior to extraction, the laminar-flow hood was

disinfected with UV light for 15 min following 70% etha-

nol. Thereafter, seeds were extracted under axenic condi-

tions in a laminar-flow hood. Liquid medium culture (Lotfi

et al. 2003) and direct extraction methods were investigated

to embryo rescue. Embryos were rescued from 21- to 42-

day-old immature fruits and cultured in magenta boxes and

culture tubes containing solid E20A medium (Sauton and

Dumas de Vaulx 1987) (Table 1). Embryos were classified

according to embryo type and stage of embryo development

(Raghavan 1986; Kurtar et al. 2002), and cultured at

28 ± 1�C with 16-h photoperiod (3,000 lux) thereafter.

Transplantation and acclimatization

After 5–15 days of culture, mini-plantlets (having root and

shoot) were transferred onto fresh E20A medium for fur-

ther development, individually. Well-generated complete

plantlets (27–41 days old) were undergone acclimatization

process. First, the covers of magenta boxes and culture

tubes were gradually opened for 8–10 days. Plantlets were

removed and the roots were washed carefully in running

tap-water. The roots were then soaked for 10 min in 0.2%

solution of fungicide (Maxim XL035FS) to prevent pos-

sible contamination at the beginning of the acclimatization.

268 Plant Cell Tiss Organ Cult (2010) 102:267–277

123

Author's personal copy

The plantlets were transplanted into plastic cups (150 cm3)

containing sterile peat-moss. Each cup was closed a

transparent cup and the plantlets were acclimatized in a

growth cabinet (Nuve TK120) at 28 ± 1�C with 16-h

photoperiod (6,000 lux) and 95% humidity. The transpar-

ent cups were opened gradually and completely removed at

6 days. The humidity of the growth cabinet was regulated

at intervals of 5% over a period of 2 days, until the

greenhouse or open field condition was reached.

Ploidy determination

The ploidy level of plantlets was determined by direct

(chromosome counting in root tip) and indirect (stomata

size, stomata density, and chloroplast number of the guard

cells) methods.

Chromosome counting in root tip was realised by

‘‘Feulgen technique’’ (Darlington and La Cour 1963). In

chromosome counting, root tips of plantlets were excised at

the beginning of acclimatization process. The 4th or 5th

leaves from shoot apex were used to measure stomata size

(width and length), stomata density (number of stoma per

mm2) and chloroplast number (each side of the guard cells)

(Sari 1994). In the measurements, lower epidermal strips of

leaves was placed onto a microscope slide after the addi-

tion of one drop of tap-water, and the cover glass was then

closed (Dore 1986). The sizes of 8 stomata per leaf were

measured for stomata size. 1% AgNO3 solution was used

for chloroplast counting (Rouselle 1992). Stomata density

and chloroplast number were counted in 6 visual areas.

Chromosomes and stomata were observed (40 910 mag-

nification) and photographed (100 910 magnification) by a

light microscope (Nikon, Alphapot, YS-2 model).

Statistical analysis

Responses to induction of in situ haploid embryos and

production of in vitro haploid plants are expressed as

Fig. 1 Representative fruits of investigated genotypes

Plant Cell Tiss Organ Cult (2010) 102:267–277 269

123


percentage (%) due to unequal test materials. Statistical

analysis were only carried out in stomata measurements as

completely randomized experimental design including four

replications and the differences between means were

evaluated by using ANOVA test.

Results

Embryo induction

Only 50 Gray and 100 Gray gamma-ray doses and polli-

nation with 0-day-old pollen stimulated embryo induction.

Overall, 50 fruits, 7,071 seeds and 686 embryos were

obtained from the investigated genotypes, and mean

embryo number per fruit was 13.7. The highest fruit (29),

seed (4,981) and embryo number (673) were procured from

50 Gray gamma-ray dose (Table 2). Relatively higher

gamma-ray doses (200 and 300 Gray) were not effective on

embryo induction and gave fruits more or less but fruits

were seedless.

The total seed and embryo number showed varied

responses to different irradiation durations. At earlier

irradiation durations (9, 11, and 15 July) produced the

highest total seed (2,903, 2,979, and 855) and embryo

number (223, 318, and 122) whereas seed (216 and 118)

and embryo number (12 and 11) were the lowest at later

irradiation durations (21 and 28 July), sequentially. The

highest mean embryo number per fruit was recorded as

19.9 on 11 July. Mean seed and embryo number were

varied from genotypes, and the highest mean seed and

embryo number were 381 and 21.2 in ‘‘G14’’ while

55CA06 had the lowest mean seed and embryo number

(133 and 8.2), respectively.

Regeneration efficiency

Both normal (white) and necrotic embryos were rescued

and classified according to stages of embryo development

(Raghavan 1986; Kurtar et al. 2002) (Fig. 2). Liquid

medium culture (Lotfi et al. 2003) was found to be inef-

fective on embryo excising, and embryos were not visu-

alized and identified. Moreover, contamination was

observed and caused embryo loss.

Overall, 103 plantlets were regenerated from 686

embryos, and regeneration rate was 15.0%. The number of

necrotic embryos (379) was higher than normal embryos

(307), and regeneration rate were found 31.9% (95 plant-

lets) in normal and 2.1% (8 plantlets) in necrotic embryos.

Well-developed embryos (cotyledon) had higher regener-

ation rate than amorphous and at early developmental

stages embryos (pro-cotyledon, late-torpedo, and arrow-

tip). Despite globular and heart embryos did not regenerate

cotyledon embryos had the highest regeneration rate

(58.9%). Arrow-tip (31.3%) and late-torpedo (28.6%)

embryos also gave noteworthy results compared to pro-

cotyledon embryos (16.0%) (Table 3).

Frequency of haploid plantlets

Frequency of haploid plantlets was influenced from embryo

type and embryo stage, and all haploid plantlets were

obtained from normal embryos. Overall, 8 haploid plantlets

were regenerated from investigated genotypes (3 of

57SI06, 2 of G14, and 3 of 57SI21) at 50 Gray gamma-ray

dose. All cotyledon and amorphous embryos gave only

diploid plantlets, while late-torpedo, arrow-tip, and pro-

cotyledon embryos produced haploid plantlets with values

33.3, 50.0, and 66.7%, respectively (Table 4).

The frequency of haploid in per 100 plantlets was 8.00,

12.2, and 13.3 on 9, 11, and 15 July, respectively. Later

irradiation durations (21 and 28 July) had no effect on

embryo induction. Overall, the frequency of haploid

plantlets in per 100 seeds, 100 embryos, 100 plantlets, and

fruit was determined as 0.11, 1.17, 10.96, and 0.28 at 50

Gray dose, respectively (Table 5).

Ploidy determination

Chromosome counting

The results of chromosome counting on root tips of 73 plant-

lets (8 of arrow-tips, 6 of late-torpedo, 3 of pro-cotyledon,

Table 1 Composition of ‘‘E20A’’ medium

Macro and micro elements (mg l-1)

KNO3 1,075 MnSO4�7H2O 11.065

NH4 NO3 619 ZnSO4�7H2O 1.812

MgSO4�7H2O 206 H3 BO3 1.575

CaCl2�2H2O 156.5 KI 0.345

KH2PO4 71 Na2MgO4�2H2O 0.094

Ca(NO3)2�4H2O 25 CuSO4�5H2O 0.008

NaH2PO4�4H2O 19 CoCl2�6H2O 0.008

(NH4)2SO4 17 FeSO4�7H2O 27.8

KCl 3.5 Na2EDTA 37.3

Vitamins and amino acids (mg l-1) Others

Myo-inositol 50.300 Sucrose 20.00 g l-1

Pyridoxine–HCl 5.500 Agar 7.00 g l-1

Nicotinic acid 0.700 IAA 0.01 mg l-1

Thiamine–HCl 0.600 pH 5.90

Calcium pantothenate 0.500

Glycine 0.100

Biotine 0.005


123


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52 of cotyledon, and 4 of amorphous) indicated that ploidy

level changed with embryo stages. Haploid plantlets

(n = 20) were achieved from arrow-tip (4), late-torpedo

(2), and pro-cotyledon (2) embryos. All cotyledon and

amorphous embryos produced only diploid plantlets

(2n = 40).

Stomata observations

Diploid plants had 10 or 12 chloroplast in guard cells

whereas haploid plants had 6 or 8 chloroplast. Average

chloroplast number was counted 11.17 in diploids and 7.21

in haploids. Besides, average stomata length and width

Fig. 2 Some of the different stages of embryos: a,b globular; c,d heart; e arrow-tip; f,g late-torpedo; h,i pro-cotyledon; j,k cotyledon; l,mamorphous; n,o necrotic


123


were 30.51 and 21.11 lm in diploids, and 21.82 and

17.37 lm in haploids, respectively. While diploid plants

had larger stomata than haploids, haploid plants had higher

stomata density (428.6) than diploids (311.4) (Table 6;

Fig. 3).

Discussion

Embryo induction was achieved only with 50 and 100 Gray

gamma-ray doses and pollination with 0-day-old pollen.

The best result was obtained from 50 Gray gamma-ray

Table 3 Effects of embryo type (ET) and embryo stage (ES) on regeneration rate (R) (%)

ET ES ID Genotypes

57SI06 57SI21 55BA02 55BA03 55CA06 G14 R

E P E P E P E P E P E P E P R

N G 50 6 0 11 0 4 0 6 0 1 0 11 0 39 0 0

100 1 0 0 0 1 0 0 0 0 0 2 0 4 0 0

H 50 10 0 7 0 4 0 7 0 5 0 4 0 37 0 0

100 1 0 0 0 0 0 0 0 0 0 1 0 2 0 0

A 50 7 3 8 2 7 1 4 2 2 1 4 0 32 10 31.3

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

LT 50 3 0 8 1 4 1 8 2 3 1 2 1 28 8 28.6

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PC 50 7 2 5 1 2 0 6 1 1 0 4 0 25 4 16

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C 50 20 13 33 21 13 7 23 13 7 4 16 9 112 66 58.9

100 2 1 1 0 0 0 0 0 0 0 2 1 5 2 40

AM 50 2 0 8 1 6 1 3 1 1 1 3 1 23 5 21.7

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NE G 50 13 0 13 0 7 0 4 0 5 0 6 0 48 0 0

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

H 50 11 0 14 0 4 0 8 0 1 0 11 0 49 0 0

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

A 50 11 0 13 0 11 0 8 0 5 0 5 0 53 0 0

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

LT 50 5 0 9 0 7 0 5 0 4 0 4 0 34 0 0

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PC 50 5 0 7 0 3 0 8 0 2 0 5 0 30 0 0

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C 50 20 2 32 1 17 1 26 1 8 0 22 2 122 7 5.7

100 1 0 0 0 0 0 1 0 0 0 0 0 2 0 0

AM 50 8 0 12 1 6 0 6 0 2 0 4 0 38 1 2.6

100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

RN 50 55 20 80 26 40 10 57 19 20 7 44 11 296 93 31.4

100 4 1 1 0 1 0 0 0 0 0 5 1 11 2 18.2

RNE 50 73 2 100 2 55 1 65 1 27 0 57 2 377 8 2.1

100 1 0 0 0 0 0 1 0 0 0 0 0 2 0 0

R 50 128 22 180 28 95 11 122 20 47 7 101 13 673 101 15

100 5 1 1 0 1 0 1 0 0 0 5 1 13 2 15.4

Overall 133 2 181 28 96 11 123 20 47 7 106 14 686 103 15

ID Irradiation dose (Gray), E embryo number, P plantlet number, G globular, H heart, A arrow-tip, LT late-torpedo, PC pro-cotyledon, Ccotyledon, AM Amorphous, N normal, NE necrotic


123


dose, and embryo induction was not performed at 200 and

300 Gray doses. Embryos and haploid plants were also

obtained from lower irradiation doses (25 and 50 Gray) in

summer squash (Kurtar et al. 2002) and in pumpkin (50 and

100 Gray) (Kurtar et al. 2009). On the other hand, haploid

embryo induction was obtained at relatively higher doses

(200–300 Gray) in watermelon (Gursoz et al. 1991; Sari

et al. 1994), melon (Sauton and Dumas de Vaulx 1987;

Cuny 1992; Maestro-Tejada 1992; Sari et al. 1992; Abak

et al. 1996; Lotfi et al. 2003), cucumber (Niemirowicz-

Szczytt and Dumas de Vaulx 1989; Sauton 1989; Caglar

and Abak 1999), and snake cucumber (Taner et al. 2000).

Contrary to these reports, the best irradiation dose was

found 100 Gray (Faris et al. 1999; Lotfi et al. 1999), 150

Gray (Xie et al. 2005), and 500 Gray (Dolcet-Sanjuan et al.

2006) in cucumber, and 750 Gray in melon (Sun et al.

2006).

These results may be attributed to the radio-resistance of

pollen and also to biologic efficiency of irradiation. A

linear relationship between radio-resistance and pollen

size, which is also a function of the amount of DNA in the

nucleus has been reported (Brewbaker and Emery 1962;

Alison and Casareft 1968; Shridhar 1992; Jain et al. 1996).

Pollen of winter squash are one of the largest pollen (as in

squash and pumpkin) in vegetables (average width

180 lm). Melon, watermelon, and cucumber pollen are

smaller than winter squash (average 50, 60 and 65 lm,

respectively) (Sensoy et al. 2003). Moreover, melon,

watermelon and cucumber have 3 apertures, whereas

winter squash has 12 apertures. Hence, winter squash

pollen are more sensitive to dehydration and rapid loss of

their viability as reported in squash (Nepi and Pacini 1993).

Pollen viability, germination ability and fruit and seed-

set also decreased along with increasing of irradiation dose,

irradiation duration and pollen age in pumpkin and winter

squash (Kurtar 2009).

Biologic efficiency of irradiation relates to the source

activity (Ci) and dose rate (Gymin-1) (Ozalpan 2001).

Irradiation efficiency influences from oxygen and water

contents of tissue (Bernstein et al. 1993), division volume

of cell (Tokarek et al. 1994) and also type of radiation

(Goldschmidt et al. 1994). Although the same irradiation

procedure was followed in our previous work, the best

result was obtained from 25 to 50 Gray gamma ray doses in

summer squash (Kurtar et al. 2002). Irradiation treatments

were realised at 6,313 Ci source activity and 44.64

Gymin-1 dose rate in summer squash while these values

were 1,482 Ci and 11.96 Gymin-1 in the present study,

respectively. Based on this concept, source activity (Ci),

dose rate (Gymin-1), pollen characteristics (size, sensitiv-

ity condition of dehydration) and irradiation and

Table 4 Effects of embryo type (ET) and embryo stage (ES) on

haploid plantlet number (HN) and haploid plantlet rate (HR) (%)

ET ES E P IP HN HR (%)

N G 43 0 0 0 0.0

H 39 0 0 0 0.0

A 32 10 8 4 50.0

LT 28 8 6 2 33.3

PC 25 4 3 2 66.7

C 117 68 49 0 0.0

AM 23 5 4 0 0.0

NE G 48 0 0 0 0.0

H 49 0 0 0 0.0

A 53 0 0 0 0.0

LT 34 0 0 0 0.0

PC 30 0 0 0 0.0

C 124 7 3 0 0.0

AM 38 1 0 0 0.0

RN 307 95 73 8 10.96

RNE 379 8 0 0 0.0

E Embryo number, P plant number, IP investigated plant number,

G globular, H heart, A arrow-tip, LT late-torpedo, PC pro-cotyledon,

C cotyledon, AM amorphous, N normal, NE necrotic

Table 5 Frequency of haploid production

IT ID HP S E IP F 100

seeds

100

embryos

100

plantlets

Per

fruit

9 50 2 2,136 216 25 9 0.09 0.93 8 0.22

100 0 767 7 0 7 0 0 0 0

11 50 4 2,185 312 33 9 0.18 1.28 12.2 0.44

100 0 794 6 0 7 0 0 0 0

15 50 2 430 122 15 5 0.47 1.64 13.3 0.4

100 0 425 0 0 4 0 0 0 0

21 50 0 124 12 0 3 0 0 0 0

100 0 92 0 0 2 0 0 0 0

28 50 0 106 11 0 3 0 0 0 0

100 0 12 0 0 1 0 0 0 0

R 50 8 4,981 673 73 29 0.16 1.19 10.96 0.28

100 0 2,090 13 0 21 0 0 0 0

Overall 8 7,071 686 73 50 0.11 1.17 10.96 0.28

IT Irradiation time in July, ID irradiation dose (Gray), HP haploid

plant number, S seed number, E embryo number, IP investigated plant

number, F fruit number

Table 6 Stomata dimension and density of haploid and diploid plants

SL SW SD CN

Haploid 21.82 b 17.37 b 428.6 a 7.21 b

Diploid 30.51 a 21.11 a 311.4 b 11.17 a

Means followed by the same letter in the same column are not sig-

nificantly different

SL (lm) Stomata length, SW (lm) stomata width, SD (number/per

mm2) stomata density, CN chloroplast number


123


pollination details (time from collection to irradiation and

incubation and storage conditions of anthers) must be

clarified in irradiation experiments like this.

Embryo induction (number/per fruit) was achieved from

all investigated genotypes and varied in the range of 3.5–

52.0. Genotype specificity was observed, but the general

reaction of the genotypes was low. When comparing the

average values, ‘‘G14’’ (21.2) demonstrated good response.

Data presented in this paper confirm the relationship

between embryo induction and genotype that has been

previously demonstrated for cucumber (Sauton 1989; Faris

et al. 1999), summer squash (Kurtar et al. 2002) and

pumpkin (Kurtar et al. 2009). Genotypic effect can be

explained by the responsiveness of parthenogenesis

(Pandey and Phung 1982) or lower parthenocarpic response

(Sauton 1989; Faris et al. 1999). Moreover, embryo yield

was highly influenced by different factors such as irradia-

tion dose, irradiation duration, and genotype (Brewbaker

and Emery 1962; Niemirowicz-Szczytt and Dumas de

Vaulx 1989; Sauton 1989; Sari et al. 1992, 1994;

Ficcadenti et al. 1995; Abak et al. 1996; Caglar and Abak

1999; Kurtar et al. 2002, 2009).

Overall, 686 embryos were rescued and the number of

necrotic embryos (379) was higher than normal embryos

(307) due to delayed harvest times (5 and 6 weeks). A total

of 103 plantlets were regenerated from 686 embryos, and

the regeneration rate was 15.0%. The greatest number of

plantlets (95 plantlets) were regenerated from normal

embryos. Regeneration rate changed with embryo type and

embryo stage. Globular and heart embryos were not

regenerated, and the greatest regeneration rate was

observed in cotyledon embryos (58.9%). Regeneration rate

was determined 31.3% in arrow-tip, 28.6% in late-torpedo,

and 16.0% in pro-cotyledon embryos. Regeneration rate

was reported 12.0% in arrow-tip, 31.3% in torpedo, 44.4%

in heart, and 31.3% in cotyledon embryos in summer

squash (Kurtar et al. 2002). On the other hand, cotyledon

embryos had the highest regeneration rate (66.7%), and

torpedo (45.5%), heart (44.0%) and arrow-tip (41.7%)

embryos also gave better results in pumpkin (Kurtar et al.

2009). Regeneration rate was 80% in heart embryos and

20% in globular embryos in melon (Sari et al. 1999b).

28.1% of globular, 55.0% of heart, 80% of torpedo, and

81.8% of cotyledon type embryos were regenerated in

cucumber (Caglar and Abak 1999). However, Faris et al.

(1999) found relatively lower regeneration efficiency (3.3–

7.7%) in cucumber. The success of embryo rescue depends

on the stage of embryo development and composition of

culture media (Jaskani et al. 2005). Different culture media

and several subcultures may be used for increasing

regeneration efficiency (Ondrej et al. 2002).

Embryo rescue is a very laborious and tedious process in

winter squash. Opening 100 seeds within a fruit takes

approximately 70–80 min. Therefore, liquid medium cul-

ture (Lotfi et al. 2003) was investigated to facilitate embryo

rescue. This procedure appeared simple and convenient,

but it was found to be ineffective in winter squash.

Embryos were not visualized and identified because of

having the thick seed-coat. Moreover, contamination was

occurred in magenta boxes excessively and led to embryo

loss. In a solution, X-ray technique can also be used for

identification of embryos (Savin et al. 1988; Sauton 1989).

But this technique requires specific equipment which is not

available in many laboratories.

The frequency of haploid plantlets was the highest at

earlier harvest times (3rd and 4th week), because of later

harvest times (5th and 6th week) led to a greater number of

necrotic embryos (Renata and Visser 1987; De Witte 2000;

Taner et al. 2000). Overall, 8 haploid plants were regen-

erated from arrow-tip (4), late-torpedo (2) and pro-cotyle-

don (2) embryos. The frequency of haploid plantlets was

0.11, 1.17, 10.96 and 0.28 per 100 seeds, 100 embryos, 100

plantlets and fruit, respectively. Frequency of haploid

plantlets was found per 100 seeds, 100 embryos and fruit

1.2, 10.4 and 0.7 in summer squash (Kurtar 1999), and

0.24, 0.94 and 0.33 in pumpkin (Kurtar et al. 2009),

respectively. Low frequency of haploid (range 0.11–0.36)

was also reported in cucumber (Caglar and Abak 1999).

Fig. 3 Stomata dimension and chloroplast number in guard cells: haploid (a), diploid (b)


123


Stomata size, stomata density, chloroplast number and

chromosome number in root tip were different from hap-

loid to diploid plants. Measurement of stoma and chloro-

plast counting was simple and more practical than

chromosome counting. These results indicated that stomata

observations can be used successfully to determine the

ploidy in winter squash. Abak et al. (1996) in melon, Sari

et al. (1999a) in watermelon, Kurtar et al. (2002) in sum-

mer squash and Kurtar et al. (2009) in pumpkin reported

similar results. Flow-cytometry can be used a potential

method to determine the ploidy level, but it is expensive,

labour-intensive and requires specific equipment (Sari et al.

1999a). Besides, no convenient method for flow-cytometry

has been found by many researchers working on haploid/

dihaploid production (Lim and Earle 2009) because many

plants scored as diploid by flow-cytometry do not produce

pollen or fruit-set in vivo (Lim and Earle 2008). This may

be explained by genetic modification of plants (especially

polyploidy, aneuploidy, euploidy and mixopoidy) in tissue

culture (Berlyn et al. 1986). Aneuploid plants were also

reported in cucumber (Truong-Andre 1988; Niemirowicz-

Szczytt and Dumas de Vaulx 1989) and in melon (Ezura

and Oosawa 1994). Considering these findings, in addition

to chromosome counting and stomata observations, mor-

phological observation (checking of flowers and pollen,

fruit-set and growth characteristics, etc.) seems to be nec-

essary to determine the ploidy level in winter squash.

However, morphological observations are time consuming,

as it requires plant development to reach an appropriate

stage.

Although our results indicated low frequency of haploid

plantlets, this is the first report on induction of partheno-

genetic haploid embryo via irradiated pollen technique in

winter squash. Low frequency of haploid may be explained

by the greatest number of necrotic embryos (due to delayed

harvest times), absence of regeneration (globular and heart

embryos were not regenerate) and spontaneous diploidi-

zation. Spontaneous diploidization has also been reported

for root meristems of cucumber (Sauton 1989) and melon

(Sauton 1988; Lotfi et al. 2003).

In conclusion, the irradiated pollen technique is pro-

posed to induce embryos and obtain haploid plantlets in

winter squash. However, in order to evaluate the irradiated

pollen approach for the recovery of haploid plants in winter

squash, further research should be based on appropriate

irradiation doses, irradiation durations, genotypes, culture

media and culture conditions, and also acclimatization

processes.

Acknowledgments We gratefully acknowledge the support of the

Scientific and Technical Research Council of Turkey (TUB_ITAK

Project No: TOVAG 108 O 390) and partial funding by the Voca-

tional School of Bafra, Ondokuz Mayis University in Turkey.

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