Research Proposal:

Research Project : Improvement on the Efficiency of Double Haploid

Production in Minipaprika via Anther and Microspore
Culture

Responsible :Plant molecular and physiology laboratory, Department

organization of Horticulture, Kangwon National University (KNU)

Project Location :KNU and Mendel’s School Research Field, Hwacheon

Research Project :Prof. Kang Won Hee, PhD

/Academic Advisor

Researcher : Binod P. Luitel, PhD student, Department of

Horticulture, KNU

Project duration: 3 Year

Project commencement year: 2010/6/30

Expected termination date: 2013/06/30

Estimated total budget for project duration (3 years): Won (,000) ----------------

($ -------------------)
Research Proposal:

Improvement on the Efficiency of Double Haploid Production in Minipaprika

via Anther and Microspore Culture

1. Problem to be addressed
Sweet pepper (Capsicum annuum L.) is the most important vegetable crop in Korea both in terms
of cultivated area and economic value. However, yield of sweet pepper in Korea is relatively
lower than that of European and American countries (Shrestha, 2009). Breeding program of
sweet pepper in Korea is not advanced and cultivars grown so far in Korea are exotic F1 hybrids.
Sweet pepper cv. Minipaprika, is available in yellow, red or orange form, now gaining the
popularity in the market. It is consumed as either eaten as slice or dice and even in cooked
vegetable and it consists of small size, easy to handle, packaging and to transport as well. But
purchasing the hybrid seed every year from abroad is increased the production cost of the crop.
New and advanced breeding techniques for Minipaprika is needed to meet this challenge and
haploid technology i.e. production of (double) haploid plants from anther/or microspore culture
may one of the panacea to cope this problem.

Despite the first success of anther culture (George and Narayanaswamy 1973, Kuo et al., 1973,
Wang et al., 1973) in Capsicum annuum L., some factors have also restricted its widespread
application in pepper breeding. Species and genotype of donor plant, developmental stage of the
microspore, carbon source, stress treatment, light condition and culture medium affect on the
androgenesis. Manual work, low efficiency of haploid production and low frequency of
chromosome doubling have still hindered the application of anther culture in sweet pepper at
wider scale. Besides, rate of chromosome doubling depends on genotype and colchicines
application rate, method and time. High efficiency of double haploid production in optimized
condition through anther and microspore culture in sweet pepper would be useful tool for its
breeding program.

2. Rationale of the study

Sweet pepper (Capsicum annuum L.) is an important vegetable crop in the world. In Korea, it is
called paprika and widely grown under protected condition. Owing to good nutritional value of
paprika, its per capita consumption has been increased. In Korea, its cultivated area is 367 ha
with estimated total production 32,778 t (MFAFF, 2008). The cultivated area and total
production of paprika were increased by 14.7% and 13.5% than that of 2007. Existing production
of paprika cannot meet the galloping demand of domestic and international market. Development
of high yielding varieties with good quality through the advance breeding methods will cope the
farmers as well as market demand. Hence, it is imperative to search advance breeding techniques
in sweet pepper cv Minipaprika.

Development of sweet pepper varieties in accordance with the interests of consumers through
classical breeding is a long-term and labor consuming process. Due to uncontrolled foreign

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pollination, requirement for the maintenance of large space to isolation and lack of possibility of
vegetative propagation, breeding materials are now quickly degenerated. Difficulties in the
classical pedigree breeding may be overcome by the introduction of in vitro haploid production
from anther culture and genome diploidization. It is estimated that time to develop new cultivars
may be reduced by 50% less in spring grown crops when doubled haploid (DH) technology is
used following F1 crosses compared to classical pedigree breeding (Forster and Thomas 2004).
Time to market is becoming important for breeding industry. Thus, efficient DH production for
practical breeding provides a significant competitive advantage and potentially bigger market
shares. DH plant materials are the ideal material for genetic and breeding studies due to the
manifestation of genetic potency and mutations and these materials are extremely valuable for
heterosis breeding.

The first in vitro haploid pepper production via anther culture was obtained by Wang et al.
(1973). George and Narayanaswamy (1973) and Kuo et al (1973) studied the haploid
morphogenesis in Capsicum though the production of haploid individuals had been very low.
George and Narayanaswamy (1973) published the first report on pollen embryogenesis in the
anther culture of Capsicum annuum L. but Dumas de Vaulx et al (1981) was developed a
reproducible anther culture method. Several researchers (Abak et al. 1982; Pochard et al. 1983,
Hendy et al. 1985; Daubeze et al. 1990; Caranta et al. 1996) used anther culture to produce many
double haploid plants of bell pepper to use in breeding programs. However, information
regarding the efficient production of DH in sweet pepper cv. Minipaprika via anther culture at
optimized condition in Korea is lacking.

Nitsch (1974) first reported the success of microspore culture on Nicotiana tabacum and Datura
innoxia to regenerate haploids, then progress has been made in the production of haploids using
isolated microspore cultures. However, high frequencies of embryogenesis and plantlet
regeneration from isolated microspore culture have been obtained in only rapeseed, barley, wheat
and rice (Jahne and Lorz 1995; Palmer et al. 1996). Capsicum annuum L. is an economically
important crop in horticulture and several protocols have been reported to induce microspore
embryogenesis and plant regeneration in different varieties (Dumas de Vaulx et al 1981; Mityko
et al 1995, 1999; Dolcet-Sanjuan et al., 1997; Barany et al 2001). Anther culture is simple
method to produce haploid plant but it requires the manual work and low efficiency than
microspore culture. Furthermore, microspore culture avoids the formation of calli and embryos
from the somatic tissues of the anther. It also produces the higher number of embryos than does
anther culture. Therefore, this study has been proposed to explore the microspore culture in
sweet pepper cv. Minipaprika for increasing the efficiency of embryogenesis and plant
regeneration.

Production and frequency of haploids can be stimulated by different techniques and heat shock
treatment is one of them. Cold and heat-shock pre-treatments have positive effect on embryo or
callus formation from microspores in cultured anthers (Sangwan and Sangwan-Norreel, 1990).
Likewise, carbon source affects the embryogenesis and embryo production. Chromosome
doubling of haploid pepper plants grown in a glasshouse or in the open air is generally known to
be inefficient and often troublesome. The application of colchicines during in vitro culture in the
induction medium (Barnabas et al. 1991) could be more efficient for doubling the chromosome
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number rather than applied to young plants. But application of colchicines for the first hours of
application in the induction medium in anther and microspore culture has not studied in sweet
pepper. Thus, heat shock, carbon source and colchicines application in anther and microspore
culture will be studied to investigate the embryogenesis and double haploid production in
Minipaprika.

Foreign ovary co-culture (wheat ovary ‘CY-45’) method has found effective in embryoid
production than pepper ovaries in Hungarian and Spanish pepper cultivars. Microspore culture
with wheat ovary co-culture may improve the embryoid production and development on sweet
pepper genotypes and therefore, this would be effective approach to get the more number of
haploid plants. Anther and microspore derived spontaneous DHs, and colchicines treated DHs
may exhibit the agronomic variation and so far, studies on this aspect are lacking. Furthermore,
double haploids produced either from anther and microspore culture, may also show the genetic
difference and genetic diversity through molecular markers has not studied yet. Hence, this study
is proposed in sweet pepper cv. Minipaprika to explore agronomic variation as well as genetic
variation among the DH populations.

3. Objectives of the study

3.1 General objective

To improve the efficiency of double haploid production via anther and microspore culture
in Minipaprika for its breeding program

3.2 Specific objectives

To improve the embryogenesis and plant regeneration through isolated microspore
culture of Minipaprika
To achieve the high frequency of DHs using colchicines treatment in isolated microspore
culture of Minipaprika
To obtain the high efficiency of DHs using anther culture of Minipaprika
To achieve the high frequency of DHs using colchicines treatment in anther culture of
Minipaprika
To assess the agronomic variation among the microspore and anther derived spontaneous
double haploids (S-DHs) and colchicines induced double haploids (C-DHs) populations
of Minipaprika
To examine the genetic diversity anther and microspore derived double haploids (DH-R2)
of Minipaprika using RAPD analysis
To demonstrate/learn the technical know-how about the haploid breeding method to
graduate and undergraduate students, commercial paprika growers and technician of
breeding and agriculture research institute

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4. Literature review
Kim et al. (2008) had found that the heat shock treatment in sucrose-starvation medium was
more effective than in B5 medium. They demonstrated the superiority of sucrose over maltose
with achieving the highest frequency of embryo production in 9% (w/v) sucrose in hot pepper cv.
Milyang-Jare. They had obtained over 54 embryos and an average of 5.5 cotyledonary embryos
when 10 x 104 microspores were grown on an individual plate but optimized the plating density
of 8x104-10x104/ml. Shrestha and Kang (2009) had obtained the highest percentage of
regenerated plantlets in cv. Phenlene (2.67%) followed by Bossanova (2.41%) at Dumas de
Vaulx R (CP medium) followed by MS (Murashige and Skoog, 1962) medium. They were found
the highest (40%) haploids plants in Minipaprika and 36.1% haploids in cv. Bossanova. The
frequency of spontaneous DH plants in anther culture of bell pepper i.e. 35.6% by Dumas de
Vaulx et al. (1981) or 32.6% by Gyulai et al. (2000).

Microspores are haploid, unicellular and the generation of haploid/doubled plants from isolated
microspores offers the opportunities for genetic transformation, gene mapping and selection for
desired dominant and recessive traits which let the production of homozygous doubled haploid
plants (Graner 1996; Polsoni et al. 1988; Stoger et al., 1995). Use of isolated microspores is now
becoming a realistic approach for haploid induction because production of embryos from isolated
microspores of several plant species has been shown to be very efficient and reproducible (Davis
and Morton, 1998).

Isolated microspore culture might be an alternative method to obtain androgenic response in poor
and non-responsive pepper varieties. Isolated microspore culture has been successful in many
species such as rapeseed (Coventry et al. 1998; Custers et al. 1994), tobacco (Touraev et al.
1996), barley (Davis and Mortan 1998; Kasha et al. 2001) and maize (Nageli et al. 1999).
Successful establishment of isolated microspore cultures of 3 Hungarian and 3 Spanish pepper
genotypes are reported using a modified cereal microspore culture protocol (Pauk et al., 2003;
Lantos et al., 2005). Juhasz et al. (2009) reported the application of 3% maltose in the induction
phase for six days at 350C, resulted the increase ratio of responding anthers and in plant
regeneration in sweet and spice pepper. They found that heat pre-treatment onto 0.3 M mannitol
had an important effect on the microspores development.

In some monocots including wheat (Mejza et al. 1993), durum wheat (Cistue et al. 2006),
triticale (Eudes and Amundsen 2005) and barley (Li and Deavaux, 2001), ovary co-culture has a
significant on the efficiency of microspore-derived plant production. Lantos et al. (2009) found
that co-cultures with wheat line ‘CY-45’ ovaries exhibited enhanced frequency of embryoid
production than those with pepper ovaries. They had observed the differences in efficiency of
isolated pepper microspore culture among different pepper genotypes.

A significant increase in doubling was observed with 300 mg l-1 in the low androgenic
responding wheat cv. Caramba. Colchicines incorporation during the first hours of culture
4
improved percentage of doubling in wheat genotypes (Chris, Pavon, Caramba and DH24033).
Application of 300 mg l-1 colchicine improved the percentage of doubling in 2 low responding
genotypes and upto 75% doubling in cv. Caramba in microspore and anther culture (Soriano et
al., 2007). Supena et al. (2006) had reported that the in vitro application of colchicines (100 µM)
during the first week of culture was highly effective in increasing the percentage of doubled
haploid plants. In vitro colchicines treatment in pepper applied to regenerated haploid explants
resulted in 75% diploidization success rate (Mityko and Fari, 1997). The application of
colchicines during the earlier periods of in vitro culture has been reported as an efficient method
to increase the production of DH plants in anther/ or microspore culture of some species such as
Brassica napus (Moller et al., 1994 and Zhao et al., 1996) and maize (Saisingtong et al. 1996).

Lee et al. (2007) reported MN medium as the most efficient for embryo induction and they found
that development of total number of embryos and the number of cotyledonary embryos were
highest when microspores were cultured in dark for 4 weeks and then in light for one week.
Nowaczyk et al. (2006) obtained the low efficiency of androgenesis in red and yellow forms at
Capsicum frutscens L. and also observed the equal number of haploids and diploids in
regenerants. Incubating treatment in heat conditions at 35 0C in darkness for 8 days, the next 4
days to light conditions (12 –h photoperiod at 25 0C ) on Dumas De Vaulx (CP) medium and
then transferring the explants to R1 medium for 4 weeks, anthers produced embryos (Koleva-
Gudeva et al, 2007).

5. Methodologies of the study

Activity 5.1. Field survey of the major commercial sweet pepper (Capsicum annuum L) farm in
Kangwon Do Province and assessment of popular cultivars of sweet pepper for domestic and
export market

Field survey will be carried out in major paprika producers of Kangwon Do Province to
assess the popular cultivars for domestic and export market. At least 10 commercial
farms will be visited and information will be collected via discussion with producers.
From the discussion, commercially important paprika varieties will be identified.

Activity 5.2. Double haploid production in Minipaprika via isolated microspore culture

Activity 5.2.1. Effect of heat pretreatment duration on embryogenesis and regeneration of

isolated microspore culture in Minipaprika
Sweet pepper grown in farmers field with well managed condition will be the source of
anther donor plants and flower buds will collected in the morning (8-9 a.m.). Anther with
1/4 purple color (20-75% late uninucleate stage) and anthers with 3/4 purple color ( >75%
early binucleate pollen) will be taken and surface sterilize with 50 ml, 2% (w/v) sodium
hypochlorite (or 5% Calcium hypochlorite) + one/two drop of Tween-20 for 10 or 20
minutes.
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 Anthers (30 to 35 flower buds) will be isolated, collected, mixted with 10 ml of sucrose
starvation medium (0.37 M mannitol, 10 mM CaCl2, 1 mM MgSO4.7H2O, 1 mM KNO3,
200 µM KH2PO4, 1 µM KI, and 100 nM CuSO4.5H2O, and the pH adjust to 5.8
Anthers will be placed for heat shock pretreatment at 32 0C in the dark for 0, 1, 3, 5, 7
days
Microspore isolation
Isolated anthers macerate in a mortar containing 10 ml distilled water or sucrose
starvation medium or mannitol solution (70 g/l).
Remove anther debris by filtering through sieves with mesh sizes of 75 and 38 µm. The
resulting suspension centrifuged at 500 rpm for 5 min, and the microspore pellet wash
twice with sucrose-starvation medium
About 30 ml of suspension at a microspore density of 8 x 104 – 10 x 104/ml, obtain from
30 to 35 flower buds.
Place 1 ml microspore per plate (35 x 10 mm) in culture medium, seal with parafilm, then
will be incubated the culture to 25 0C in dark condition until 4 weeks for further
development
Culture media
NLN medium (Swanson, 1990) Appendix 1, modified by Kim et al. (2008),
supplemented with 0.83 mg/l of KI with no hormones, containing 10% (w/v) sucrose,
culture 25 0C at dark till 4 weeks
After 4 weeks, cotyledonary embryos transfer to basal B5 medium (Gamborg et al., 1968)
Appendix 2, supplemented with 2% (w/v) sucrose and 0.35% (w/v) Phytagel.
Transfer culture into growth chamber, maintain at 25 0C with 16/8 h light-dark
photoperiod
Experimental set up/Design
Completely Randomized Design (CRD), Minipaprika (3 form), and heat shock treatment
(5 level), each treatment replication 6 times (6 plates per treatment, repeat if necessary)

Observation and analysis: No of embryos per plate (globular and heart, cotyledonary or embryo
like structures), using stereomicroscope, total embryos and no of green plants

Activity 5.2.2. Effect of different carbohydrate source and concentration on embryogenesis and
regeneration of isolated microspore culture in Minipaprika

Experimental set up/design

Microspore isolation method is same as above mentioned. NLN medium, supplemented
with different concentration of sucrose, 6%, 9%, 12%, 15% and maltose, 6%, 9%, 12%,
15%
Other procedure (Same as above mentioned)

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 Completely randomized design (CRD), genotypes (2 level), carbohydrate source (2 level)
and concentration (4 level), replicate 6 times (6 plates, repeat if necessary)
Observation and analysis: same as above

Activity 5.2.3 Study the effect of ovary co-culture on the embryoid production and plant
regeneration of isolated microspore culture in Minipaprika

Experimental set up/Design.

Microspore isolation same as above. Place 1 ml microspore per plate (35 x 10 mm) in
culture medium, seal with parafilm, then will be incubated the culture to 25 0C in dark
condition until 4 weeks for further development
For ovary co-culture (OCC), spikes of wheat (any variety) ovaries and pepper (same
species) will be collected 2 days before pollination, add the isolated wheat ovary and
pepper ovary (no of ovary vary upto 7) to each petri dish containing freshly isolated
microspore cultures
Incubate the petri dishes at 28 0C and 80% humidity and maintain in the dark for 2
months.
Other procedure (Same as above mentioned)
Completely Randomized Design (CRD), with 4 replications, Minipaprika (3 form) and
ovary co-culture (3 level, without ovary, wheat ovary and pepper ovary) at least 4 plates
for each treatment (repeat if necessary)

Observation and analysis : No of embryoids/petri dish, no of globular and hear, cotyledonary and
torpedo, ELS embryo, no of shoot per petri dish, no of rooted plants per petri dish and no of
acclimatized plants per petri dish etc.

Activity 5.2.4. Effect of colchicines treatment on double haploid production of isolated

microspore culture in Minipaprika

Experimental set up/design

Isolated microspore placed in induction medium containing different concentration of
colchicines; 0, 100, 200, 300 mg/L (1.5ml induction medium), for 48 hrs (2 days) and 72
h (3 days) at 250C
Completely randomized design (CRD), colchicines treatment duration (2 level), and
concentration (4 level), 5 replication (5 plates per treatment, repeat if necessary)

Observation and analysis: No of embryos per plate (globular and heart, cotyledonary or embryo
like structures), total embryos and no of green plants, doubling (%) (no. of double haploids per
100 analyzed plants), regeneration percentage (number of regenerated plants per 100 embryos)

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Activity 5.3. Double haploid production in Minipaprika through anther culture

Activity 5.3.1. Effect of abiotic (cold and heat) stress pretreatment of flower bud on androgenesis
in anther culture in Minipaprika

Sweet pepper grown in farmers field with well managed condition will be the source of
anther donor plants and flower buds will collected in the morning (8-9 a.m.). Anther with
1/4 purple color (20-75% late uninucleate stage) and anthers with 3/4 purple color ( >75%
early binucleate pollen) will be taken and surface sterilize with 50 ml, 2% (w/v) sodium
hypochlorite (or 5% Calcium hypochlorite) + one/two drop of Tween-20 for 10 or 20
minutes
Stress pretreatment
Cold and heat stress pretreatment: Flower buds will be subjected to different temperature
40C, 10 0C (cold treatment) and 32 0C and 35 0C ( heat treatment) in the dark for 3 and 7
days (incubation)
Culture media: As mentioned by Dumas de Vaulx et al. 1981(Appendix 4).
Experimental set up/Design
CRD, Minipaprika (3 form), stress pretreatment (4 level), and duration (2 level) each
treatment replication 6 times (10 plates per treatment, repeat if necessary)

Observation and analysis: No of embryos per 100 anthers and type of embryo (globular and heart,
cotyledonary or embryo like structures), total embryos and no of green plants

Activity 5.3.2. Effect of colchicines treatment on the double haploid production of anther culture
in Minipaprika

Experimental set up/design

Minipaprika form (3 form) and colchicine will be treated in anther culture medium
Isolated anther will be placed at colchicines containing 0, 100, 200, 300 mg/L CP
medium (containing 1.5 ml medium for 48 and 72 hrs at 25 0C)
Treated anther will be washed with 2 ml culture medium (C medium) and then 10-15
anther per petri dish (depend on size) and 60-75 anthers per treatment, culture at 25 0C 4
weeks in dark and following culture in regeneration medium
Completely randomized design (CRD), colchicines treatment duration (2 level), and
concentration (4 level), 5 replication (5 plates per treatment, repeat if necessary)

Observation and analysis: No of embryos per 100 anthers and type of embryo (globular and heart,
cotyledonary or embryo like structures), total embryos and no of green plants, doubling (%) (no.
of double haploids per 100 analyzed plants), regeneration percentage (number of regenerated
plants per 100 embryos)

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Activity 5.4. Cytological studies of anther and microspore derived plants of Minipaprika

Activity 5.4.1 Ploidy analysis of anther and microspore derived regenerants

Transfer the regenerated plantlets (1-2 leaves) into glass tube or plastic plug (6cm x 6 cm,
mixture of sterilized vermiculite), containing growth regulator free MS (Murashige and
Skoog, 1962) medium (Appendix 3) with 2% sucrose and then acclimatize under 16 h
photoperiod at 25 0C µmol.m-2.S-1 of fluorescent light
Ploidy of well-rooted plantlets with 3-4 leaves will be determined by flow cytometry
using a ‘PARTEC I’ flow cytometer (Partec GmbH, Munster, Germany)

Activity 5.4.2 Observation on stomatal length, chloroplast and chromosome number

Procedures of observing stomatal length, chloroplast and chromosome number will be
followed as mentioned by Qin and Rotino, 1995.
The first three fully expanded leaflets from the top of well developed plantlets, almost
touching the vessel lid, will be used to determine the stomatal length and the number of
chloroplasts per guard cell pair.
Peel the lower epidermis, place onto a glass microscope slide, and stained with 1-2 drops
of a 1% silver nitrate solution.
After 5 min, a cover slip will be mounted and observations in oil immersion will be made
using a phase contrast microscope at 1000x magnification.
The stomatal length will be measured with an ocular micrometer. Measurements from ten
stomata of three leaflets (stomata length and chloroplast number) sampled from each
androgenic plant will be recorded.
Chromosome counts will be carried out on the same plantlets subjected to chloroplast
counts and stomatal length.
Root-tips of greenhouse-grown plantlets were pretreated with a saturated solution of α-
bromonaphthalene for 24 h or with a 1% colchicine solution for 1 h, then hydrolyzed in
1N HCl at 60°C for 20 min, stained following the Feulgen method and squashed in 45%
acetic acid.
Chromosomes will be counted in 2 to 5 well-spread cells of three root tips per androgenic
plant.

Activity 5. Assessment of genetic variability between anther and microspore derived double
haploids (DH1) of minipaprika

Activity 5.1 Study the agronomic variation among the microspore and anther derived
spontaneous DHs and colchicines induced DHs of Minipaprika

Experimental set up/Design

Spontaneous double haploids, colchicine induced double haploid plants produced from
microspore and anther culture will be grown in plastic green house, until maturity. All
plants (S-DH, C-DH) will be self pollinated to produce a double haploid line, harvest the
fruit and seed separately in each line.
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 Group Balanced Block Design: Population 4 groups, A (A-SDH), B (M-SDH) and C (C-
DH) (each group consist of no of populations, each group consist of 1 block (block may
vary according to entry or population no.), with replication 3 times. All microspore
derived SDH will be treated in the same block and other DH will be same as like this.
Statistical analysis: Analyses of variance (ANOVA) and orthogonal contrast for mean
separation.

Activity 5.2 Examine the genetic diversity in anther and microspore derived double haploids
(DH-R2) lines of Minipaprika using RAPD analysis

Experimental set up
DH-R1 seeds harvested previous season (regenerated plants self-pollinate and harvest
fruit seed) will be germinated, separately in petri dishes for molecular analysis.
For genomic DNA extraction, 2 young fresh leaves will be collected from each DH-
haploid lines ( approx. 30 individual lines)
For control, a bulk of DNAs of 5 individual of anther donor plants will be used.
PCR-analysis: Amplification reaction will be run by GeneAMP PCR Thermal Cycler,
Reaction mixture: 4 deoxynucleotides (dATP, dCTP, dGTP, dTTP); Taq-polymerase,
RAPD primers, 10 x PCR buffer, dH2O and template DNA.
PCR-Primers : OP/A, I-20 for RAPD analysis.
After PCR amplification- followed by the agarose gel electrophoresis of PCR amplified
samples, then bands verification under UV illumination will be observed.

Activity 6. Training, demonstration and field visit to horticulture students (graduate and
undergraduate), commercial paprika growers, members of agriculture research station and
breeding institution at Mendel School’s Research Field

Activity 6.1 Providing the practical skills (learning by doing) on haploid breeding method at
Minipaprika to graduate and undergraduate students of horticulture science
Anther culture/microspore culture is one of the best approach for the production of
double haploid. Classical breeding requires six to eight generation to develop,
homozygous lines but through the anther/microspore culture, genetically stable,
homozygous lines can be produced even in single generation.
In addition to theoretical knowledge, practical know-how will be very important for
graduate/undergraduate students of horticulture and they will gain in-depth knowledge
from learning by doing approach.
Dissemination of research findings and innovative ideas to the students through the
practical experiment will broaden their knowledge and thinking.
International and national students will get the exposure of training and higher education
leading to MS and PhD degree and undergraduate students will get the opportunities to
commence the academic research in breeding field.

10
Under this scheme, following activities will be implemented;

S.N. Activities Location Responsible Person

1. Sweet pepper: Overview (introduction, varieties, KNU & Mendel’ School Prof. Kang Won
nutrition and export potential in Korea ) Research Field, Hwacheon Hee
2. Flower morphology, flower bud, stage of flower bud KNU & Mendel’ School ’’
and development stage of microspore Research Field, Hwacheon
3. Isolation of anther/microspore from flower bud, KNU & Mendel’ School ’’
Principles of anther culture and microspore culture Research Field, Hwacheon
4. Haploid breeding in crop species and culture medium KNU, Plant molecular and ’’
– an overview physiology Lab
5. Factors affecting the androgenesis (/embryogenesis & KNU, Plant molecular and ’’
organogenesis) physiology Lab
6. Practical demonstration of callus/embryoid production KNU, Plant molecular and ’’
physiology Lab
7. Colchicine: Role of colchicines in chromosome KNU, Plant molecular and ’’
doubling, method and time of application physiology Lab
8. Plant regeneration and acclimatization of regenerated KNU, Plant molecular and ’’
plantlets physiology Lab & Mendel’
School Research Field,
Hwacheon
9. Ploidy analysis and observation of chromosome KNU, Plant molecular and ’’
number physiology Lab
10. DNA isolation and PCR (Polymerase chain reaction) KNU, Plant molecular and ’’
based markers for molecular breeding physiology Lab
11. Field visit of the students for plant Mendel’ School Research ’’
phenological/morphological observation Field, Hwacheon
12. Selection criteria for homozygous lines for better Mendel School’s Research ’’
variety development/ or heterosis breeding Field, Hwacheon
13. Heterosis breeding, heritability, estimation of SCA Mendel School’s Research ’’
(specific combining ability) and GCA (General Field, Hwacheon
combining ability) of inbred lines
14. Hybridization, variety testing (genotype x Mendel School’s Research ’’
environment) and stability analysis Field, Hwacheon

11
Activity 6.2 Training, seminar and field demonstration of anther/microspore derived
homozyogous lines to commercial Minipaprika growers, technician of breeding and agriculture
research institute
Commercial paprika growers, technician of breeding and agriculture research institutes
are also the beneficiary of this research. Hence, research findings as well as information
of this crop will be disseminated among stakeholders through the effective teaching and
learning methods which are given here.

S.N. Activities Methods Location Responsible

person
1. Minipaprika : Overview (Statistics, market Survey and statistic Mendel School’s Prof. Kang
demand and export potentiality ) report, figure, Research Field, Won Hee
graph, slides etc Hwacheon
2. Minipaprika Breeding: Anther culture and Slides, figures and Mendel School’s ’’
advance method of variety development flow charts Research Field,
Hwacheon
3. Production of double haploids/in vitro Photos, videos, Mendel School’s ’’
haploid production factsheet with data, Research Field,
booklets etc Hwacheon
4. Field demonstration of homozygous plants Field observation Mendel School’s ’’
and observation on morphological Research Field,
characters Hwacheon
5. Maintenance of inbred/homozygous line for Field observation Mendel School’s ’’
breeding (self pollination) Research Field,
Hwacheon
6. Selection parameters of better variety for Field observation Mendel School’s ’’
commercial cultivation/high yielding on fruit quality Research Field,
variety with better quality characters Hwacheon

Activity 7. Statistical analysis (SAS Institute, 1999), preparation manuscripts (PhD thesis) and
booklets, paper presentation, publication in SCIE (Science Citation Index Expanded) and SCI
(Science Citation Index) journals.

Data will be entered in MS excel sheet of all the experiments. Frequencies, percentage,
histogram, graphs, mean, standard deviation and, one way and two way ANOVA will be
used to interpret the result using SAS software (Version 7; SAS, Cary, NC). Correlation
and regression will be carried out wherever necessary. Manuscripts (PhD) thesis will be
prepared from the effective and significant data and paper will be published in respective
SCIE (Science Citation Index Expanded) and SCI (Science Citation Index) journals.

12
6. Expected Output
Method of obtaining high efficiency of embryogenesis and plant regeneration through
isolated microspore and anther culture in Minipaprika will be achieved
High frequency of DHs obtaining by colchicines treatment method and rate in isolated
microspore and anther culture in Minipaprika will be obtained.
Agronomic variation among the microspore and anther derived spontaneous double
haploids (S-DHs) and colchicines induced double haploids (C-DHs) populations of
Minipaprika will be identified
Genetic diversity anther and microspore derived double haploids (DH-R2) of Minipaprika
using RAPD analysis will be assessed.
Knowledge of practical skills for developing the haploid and its uses in breeding new
cultivar will be learned by students, growers and agriculture technicians.

7. Beneficiary of the research

University graduate students and undergraduate students will be benefitted by obtaining
the exposure of new methodological skills in haploid breeding and they will achieve the
graduate studies by giving the significant contribution to paprika breeding. In addition,
academician, research institution, private breeding and agriculture research institution
will be benefited by obtaining the elite breeding material. Furthermore, government
research institution, development organization like NGO (Non-government organization)
and INGO (International Non-Government Organization) and as whole commercial
paprika growers in Kangwon Do Province will be the beneficiary of this research.

13
8. Description of the activities:
S. List of activities Begin End Location Responsible
N. person
1. Field survey of paprika farm and assessment of major May 010 Dec. 010 Kangwon Binod P
paprika varieties for domestic and export market Do Luitel/Taek J.
Province Lee
2. DH production of sweet pepper (Capsicum annuum L) cv. Jun. 010 Jun. 011 KNU Binod P. Luitel
Minipaprika via isolated microspore culture
2.1. Effect of heat pretreatment duration Jun. 010 Jun. 011
2.2.Effect of different carbohydrate source and Jun. 010 Jun 011
concentration
2.3 Study the effect of ovary co-culture on the embryoid Jun. 010 Jun.010
production and plant regeneration
2.4 Effect of colchicines treatment on DH production Jun. 010 Jun.010
3. DH production of sweet pepper (Capsicum annuum L) cv. May 011 Aug. 012 KNU Binod P. Luitel
Minipaprika through anther culture
3.1 Effect of abiotic (cold and heat) stress pretreatment May 011 Aug. 012
3.2 Effect of colchicines treatment on the DH production May 011 Aug. 012
4. Ploidy analysis and chromosomal observation of anther April 011 Aug. 012 KNU Binod P. Luitel
and microspore derived plants of Minipaprika
4.1 Ploidy analysis of regenerants April 011 Aug. 012
4.2 Observation on stomatal length, chloroplast and April 011 Aug. 012
chromosome number
5. Assessment of genetic variability between anther and May 011 May 013 KNU and Binod P. Luitel
microspore derived double haploids (DH1) of sweet Mendel’s
pepper (Capsicum annuum L.) cv. Minipaprika School
5.1 Study the agronomic variation among the microspore April 011 Dec 012
and anther derived spontaneous DHs and colchicines
induced DHs
5.2 Examine the genetic diversity in anther and April 011 May 013
microspore derived double haploids (DH-R2) lines using
RAPD analysis
6. Training, seminar, and field demonstration to horticulture Aug. 010 May 013 Mendel’s Prof. Kang Won
students, commercial paprika growers, technicians of School, Hee/ /Binod P.
breeding and agriculture research institute at Mendel Hwacheon Luitel
School’s Research Field
6.1 Providing the practical skills (learning by doing) on Aug. 010 Aug. 012 Prof. Kang Won
haploid breeding method at Minipaprika to graduate and Hee
undergraduate students of horticulture science
6.2 Training, seminar and field demonstration of May 012 May 013 Prof. Kang Won
anther/microspore derived homozyogous lines to Hee
commercial Minipaprika growers, technician of breeding
and agriculture research institute.
7. Statistical analysis, preparation manuscripts (PhD thesis), Jan. 012 May 013 KNU Binod P
booklets, reports and paper presentation and publication in Luitel/Prof Kang
SCIE (Science Citation Index Expanded) and SCI Won Hee
(Science Citation Index)

14
 9. Time Frame
2010 2011 2012 2013
Activities Trim I Trim II Trim III Trim I Trim II Trim III Trim I Trim II Trim III Trim I Trim I Trim III

1. Field survey of paprika farm and

assessment of major paprika varieties for
domestic and export market
2. DH production of sweet pepper
(Capsicum annuum L) cv.Minipaprika via
isolated microspore culture
3. DH production of sweet pepper
(Capsicum annuum L) cv. Minipaprika
through anther culture
4. Ploidy analysis and chromosomal
observation of anther and microspore
derived plants of sweet pepper (Capsicum
annuum L.) cv.Minipaprika
5. Assessment of genetic variability
between anther and microspore derived
double haploids (DH1) of sweet pepper
(Capsicum annuum L.) cv.Minipaprika
6. Training, seminar, and field
demonstration to horticulture students
commercial paprika growers, technicians
of breeding and agriculture research
institute at Mendel School’s Research
Field
7. Statistical analysis, preparation
manuscripts (PhD thesis), booklets, reports
and paper publication in SCIE (Science
Citation Index Expanded) and SCI
(Science Citation Index) journals
Trim I- (Jan.-Apri.), Trim II- (May-Aug.) and Trim III - (Sept.-Dec.)
10. Budget summary
Activities 2010 2011 2012 2013 Total
(000’Won) (000’Won) (000’Won) (000’Won) (000’Won)
1. Field survey of paprika farm and assessment of major paprika varieties for
domestic and export market
2. DH production of sweet pepper (Capsicum annuum L) cv. Minipaprika via
isolated microspore culture
2.1 Effect of heat pretreatment duration
2.2 Effect of different carbohydrate source and concentration
2.3 Study the effect of ovary co-culture on the embryoid production and plant
regeneration
2.4 Effect of colchicines treatment on DH production
3. DH production of sweet pepper (Capsicum annuum L) cv.Minipaprika through
anther culture
3.1. Effect of abiotic (cold and heat) stress pretreatment
3.2 Effect of colchicines treatment on the DH production of anther culture
4. Ploidy analysis and chromosomal observation of anther and microspore
derived plants of sweet pepper (Capsicum annuum L.) cv.Minipaprika
4.1 Ploidy analysis of anther and microspore derived regenerants
4.2 Observation on stomatal length, chloroplast and chromosome number
5. Assessment of genetic variability between anther and microspore derived
double haploids (DH1) of sweet pepper (Capsicum annuum L.) cv.Minipaprika
5.1 Study the agronomic variation among the microspore and anther derived
spontaneous DHs and colchicines induced DHs
5.2 Examine the genetic diversity in anther and microspore derived double
haploids (DH-R2) lines using RAPD analysis
6. Training, seminar, and field demonstration to horticulture students commercial
paprika growers, technicians of breeding and agriculture research institute at
Mendel School’s Research Field
6.1 Providing the practical skills (learning by doing) on haploid breeding method
at Minipaprika to graduate and undergraduate students of horticulture science
6.2 Training, seminar and field demonstration of anther/microspore derived
homozyogous lines to commercial Minipaprika growers, technician of breeding
and agriculture research institute.
7. Statistical analysis, preparation manuscripts (PhD thesis), booklets, reports and
paper publication in SCIE (Science Citation Index Expanded) and SCI (Science
Citation Index) journals
8. Salary, travel, field expenses, gasoline, lab (equipments, chemicals ) and
stationary
Total (000’ Won)
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Appendix 1. Composition of the NLN medium

(Nitsch, C and Nitsch, J.P. 1967) - (Lichter R. 1981)- (Lichter, R. 1982). (Swanson, 1990),
Lionneton et al (2001) -Kim et al (2008),
Macro-elements mgL-1 mg (500 ml) gL-1
KH2PO4 125 62.5 0.125
KNO3 125 62.5 0.125
MgSO4.H2O 125 62.5 0.125
Ca(NO3)2.4H2O 500 250 0.500
NaFeEDTA 40 20 0.040
Minor and micro-element
CuSO4.5H2O 0 0 0.025
CoCl2.6H2O 0.025 0.0125 0.025
H3BO3 6.2 3.1 6.2
KI 0.83 0.415 0.83
MnSO4.4H2O 22.3 11.15 22.3
Na2MoO4.2H2O 0.025 0.0125 0.250
ZnSO4.7H2O 8.6 4.3 8.6
Organic substance
D+ Biotine 0.05 0.025 0.005
Folic acid 0.5 0.25 0.05
L-Glutamine 800 400 0.800
L-Glutathione 30 15 0.030
L-Serine 100 50 0.100
Glycine 2 1 0.20
Myo-inositol 10 5 10.00
Nicotinic acid 5 2.5 0.50
Pyridoxine HCL 0.5 0.25 0.05
Thiamine HCl 0.5 0.25 0.05
Sucrose (10%) 100000 50000 100
pH 6.0 6.0 6.0

20
Appendix 2. Composition of the B5 medium
Macro-elements mgL-1 mg (500 ml) gL-1
CaCl2.2H2O 150 75.0 0.150
KNO3 3000 1500 3.0
MgSO4.7H2O 500 250 0.500
NaH2PO4.2H2O 150 75 0.150
(NH4)2SO4 134 67.0 0.134
Na2EDTA* 37.3 18.65 0.0373
Minor and micro-element
CoCl2.6H2O 0.025 0.0125
CuSO4.5H2O 0.025 0.0125
FeSO4.7H2O* 27.8 13.9
H3BO3 3 1.5
KI 0.75 0.375
MnSO4.4H2O 13.2 6.6
Na2MoO4.2H2O 0.25 0.125
ZnSO4.7H2O 2 1
Organic substance
Myo-inositol 100 50
Folic acid 0 0
Nicotinic acid 1 0.5
Thiamine HCl 10 5
Pyridoxine HCl 1 0.5
Sucrose (2%) 20000 10000
Phytagel (0.35%) 3500 1750
pH 5.8 5.8

21
Appendix 3. Composition of MS medium
Macro-elements mgL-1 mg (500 ml) gL-1
NH4NO3 1650 825 1.650
KNO3 1990 995 1.990
CaCl2.2H2O 440 220 0.440
MgSO4.7H2O 370 185 0.370
KH2PO4 170 85 0.170
Minor and micro-element
H3BO3 6.2 3.1
MnSO4.4H2O 22.3 11.15
ZnSO4.7H2O 8.6 4.3
KI 0.83 0.415
Na2MoO4.2H2O 0.25 0.125
CuSO4.5H2O 0.025 0.0125
CoCl2.6H2O 0.025 0.0125
Na2EDTA.2H2O 37.3 18.65
FeSO4.7H2O 27.8 13.9
Organic substance
Glycine 2 1
Mesoinositol 100 50
Nicotinic acid 0.5 0.25
Pyridoxine (HCl) 0.5 0.25
Thiamine (HCl) 0.1 0.05
Carrot extract 200 ml 100 ml
Charcoal (1%) 10000 5000
Sucrose (3%) 30000 15000
Agar (0.8%) 8000 4000
pH of the media 5.5 5.5

22
Appendix 4. Composition of C medium and R medium
Macro-element C Medium R1 medium C Medium R1 medium
mgL-1 mgL-1 mg (500 ml) mg (500 ml)
NH4NO3 1238 1238 619.0 619.0
KNO3 2150 2150 1075.0 1075.0
KCl 7 7 3.5 3.5
CaCl2.2H2O 313 313 156.5 156.5
Ca(NO3)2.4H2O 50 50 25 25
MgSO4.7H2O 444 444 222 222
(NH4)2SO4 34 34 17 17
KH2PO4 142 142 71 71
Minor and micro-element
NaH2PO4.H2O 38 38 19 19
H3BO3 3.15 1.55 1.575 0.775
MnSO4.H2O 22.13 20.13 11.065 10.065
ZnSO4.7H2O 3.625 3.225 1.8125 1.6125
KI 0.695 0.33 0.3475 0.165
Na2MoO4.2H2O 0.188 0.138 0.094 0.069
CuSO4.5H2O 0.016 0.011 0.008 0.0055
CoCl2.6H2O 0.016 0.011 0.008 0.0055
Na2EDTA.2H2O 18.65 18.65 9.325 9.325
FeSO4.7H2O 13.9 13.9 6.95 6.95
Organic substances
Glycine 0.1 0.1 0.05 0.05
Mesoinositol 50.3 50.3 25.15 25.15
Ca-pantothenate 0.5 0.5 0.25 0.25
Nicotinic acid 0.7 0.7 0.35 0.35
Pyridoxine (HCl) 5.5 5.5 2.75 2.75
Vitamin B12 0.03 0 0.015 0
Thiamine (HCl) 0.6 0.6 0.3 0.3
Biotine 0.005 0.005 0.0025 0.0025
Sucrose (3%) 30000 30000 15000 15000
Agar (0.8%) 8000 8000 4000 4000
pH 5.9 5.9 5.9 5.9
Growth regulators
Kinetin 0.01 or 2 0.1 0.005 or 1 0.05
2,4-D 0.01 or 1 0.005 or 0.5

23
 Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum
L.)

1. J. Mitykó1 A. Andrásfalvy1,

2. G. Csilléry2,
3. M. Fári1

Plant Breeding
Volume 114, Issue 1, pages 78–80, February 1995

Get PDF (416K)FIND IT @ KANGWON NAT’L UNIV

Keywords:

Capsicum annuum;

anther culture;

doubled haploids;
flow cytometry

Abstract
An in vitro anther-culture method has been improved by using young mother plants and by using frequent subcultures,

thus increasing the androgenic yield in different Capsicum annuum L. genotypes. An assortment of peppers was

used, composed of 15 genotypes (four breeding lines, seven cultivars and four F1 hybrids). A new system for

qualifying the androgenic response was established. For use in practical breeding, a minimum of 5 % of plant

regeneration was proposed as the criterion for a fair response. Accordingly, one excellent, one good and eight fair

responses were identified among the genotypes investigated. As compared to the standard cultivar. 2 genotypes
gave a significantly better response, i.e. ‘Fehérözön’ (75.8%) and ‘Szechuan 90716’ (21.0%). In comparative

investigations, F1 hybrids, produced from crosses between poor/non-responsive and responsive genotypes, showed a

fair level of response, even the case of a poor response in donor parent. The ploidy level of the resulting plants was

determined by flow-cytometric analysis.

24