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Researchproposal 110915022927 Phpapp01
Researchproposal 110915022927 Phpapp01
Researchproposal 110915022927 Phpapp01
Estimated total budget for project duration (3 years): Won (,000) ----------------
($ -------------------)
Research Proposal:
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.
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.
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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
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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).
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
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
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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
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.
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
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. 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
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.
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Under this scheme, following activities will be implemented;
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
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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.
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.
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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.
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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)
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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
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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
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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
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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
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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
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
24