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  • 1. 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, KNUProject duration: 3 YearProject commencement year: 2010/6/30Expected termination date: 2013/06/30Estimated total budget for project duration (3 years): Won (,000) ----------------($ -------------------)
  • 2. Research Proposal: Improvement on the Efficiency of Double Haploid Production in Minipaprika via Anther and Microspore Culture1. Problem to be addressedSweet pepper (Capsicum annuum L.) is the most important vegetable crop in Korea both in termsof cultivated area and economic value. However, yield of sweet pepper in Korea is relativelylower than that of European and American countries (Shrestha, 2009). Breeding program ofsweet 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 thepopularity in the market. It is consumed as either eaten as slice or dice and even in cookedvegetable and it consists of small size, easy to handle, packaging and to transport as well. Butpurchasing 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 andhaploid technology i.e. production of (double) haploid plants from anther/or microspore culturemay 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 widespreadapplication in pepper breeding. Species and genotype of donor plant, developmental stage of themicrospore, carbon source, stress treatment, light condition and culture medium affect on theandrogenesis. Manual work, low efficiency of haploid production and low frequency ofchromosome doubling have still hindered the application of anther culture in sweet pepper atwider scale. Besides, rate of chromosome doubling depends on genotype and colchicinesapplication rate, method and time. High efficiency of double haploid production in optimizedcondition through anther and microspore culture in sweet pepper would be useful tool for itsbreeding program.2. Rationale of the studySweet pepper (Capsicum annuum L.) is an important vegetable crop in the world. In Korea, it iscalled paprika and widely grown under protected condition. Owing to good nutritional value ofpaprika, its per capita consumption has been increased. In Korea, its cultivated area is 367 hawith estimated total production 32,778 t (MFAFF, 2008). The cultivated area and totalproduction of paprika were increased by 14.7% and 13.5% than that of 2007. Existing productionof paprika cannot meet the galloping demand of domestic and international market. Developmentof high yielding varieties with good quality through the advance breeding methods will cope thefarmers as well as market demand. Hence, it is imperative to search advance breeding techniquesin sweet pepper cv Minipaprika.Development of sweet pepper varieties in accordance with the interests of consumers throughclassical breeding is a long-term and labor consuming process. Due to uncontrolled foreign 1
  • 3. pollination, requirement for the maintenance of large space to isolation and lack of possibility ofvegetative propagation, breeding materials are now quickly degenerated. Difficulties in theclassical pedigree breeding may be overcome by the introduction of in vitro haploid productionfrom anther culture and genome diploidization. It is estimated that time to develop new cultivarsmay be reduced by 50% less in spring grown crops when doubled haploid (DH) technology isused 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 forpractical breeding provides a significant competitive advantage and potentially bigger marketshares. DH plant materials are the ideal material for genetic and breeding studies due to themanifestation of genetic potency and mutations and these materials are extremely valuable forheterosis 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 haploidmorphogenesis in Capsicum though the production of haploid individuals had been very low.George and Narayanaswamy (1973) published the first report on pollen embryogenesis in theanther culture of Capsicum annuum L. but Dumas de Vaulx et al (1981) was developed areproducible 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 manydouble haploid plants of bell pepper to use in breeding programs. However, informationregarding the efficient production of DH in sweet pepper cv. Minipaprika via anther culture atoptimized condition in Korea is lacking.Nitsch (1974) first reported the success of microspore culture on Nicotiana tabacum and Daturainnoxia to regenerate haploids, then progress has been made in the production of haploids usingisolated microspore cultures. However, high frequencies of embryogenesis and plantletregeneration from isolated microspore culture have been obtained in only rapeseed, barley, wheatand rice (Jahne and Lorz 1995; Palmer et al. 1996). Capsicum annuum L. is an economicallyimportant crop in horticulture and several protocols have been reported to induce microsporeembryogenesis and plant regeneration in different varieties (Dumas de Vaulx et al 1981; Mitykoet al 1995, 1999; Dolcet-Sanjuan et al., 1997; Barany et al 2001). Anther culture is simplemethod to produce haploid plant but it requires the manual work and low efficiency thanmicrospore culture. Furthermore, microspore culture avoids the formation of calli and embryosfrom the somatic tissues of the anther. It also produces the higher number of embryos than doesanther culture. Therefore, this study has been proposed to explore the microspore culture insweet pepper cv. Minipaprika for increasing the efficiency of embryogenesis and plantregeneration.Production and frequency of haploids can be stimulated by different techniques and heat shocktreatment is one of them. Cold and heat-shock pre-treatments have positive effect on embryo orcallus formation from microspores in cultured anthers (Sangwan and Sangwan-Norreel, 1990).Likewise, carbon source affects the embryogenesis and embryo production. Chromosomedoubling of haploid pepper plants grown in a glasshouse or in the open air is generally known tobe inefficient and often troublesome. The application of colchicines during in vitro culture in theinduction medium (Barnabas et al. 1991) could be more efficient for doubling the chromosome 2
  • 4. number rather than applied to young plants. But application of colchicines for the first hours ofapplication in the induction medium in anther and microspore culture has not studied in sweetpepper. Thus, heat shock, carbon source and colchicines application in anther and microsporeculture will be studied to investigate the embryogenesis and double haploid production inMinipaprika.Foreign ovary co-culture (wheat ovary ‘CY-45’) method has found effective in embryoidproduction than pepper ovaries in Hungarian and Spanish pepper cultivars. Microspore culturewith wheat ovary co-culture may improve the embryoid production and development on sweetpepper genotypes and therefore, this would be effective approach to get the more number ofhaploid plants. Anther and microspore derived spontaneous DHs, and colchicines treated DHsmay 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 geneticdifference and genetic diversity through molecular markers has not studied yet. Hence, this studyis proposed in sweet pepper cv. Minipaprika to explore agronomic variation as well as geneticvariation among the DH populations.3. Objectives of the study3.1 General objective To improve the efficiency of double haploid production via anther and microspore culture in Minipaprika for its breeding program3.2 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 3
  • 5. 4. Literature reviewKim et al. (2008) had found that the heat shock treatment in sucrose-starvation medium wasmore effective than in B5 medium. They demonstrated the superiority of sucrose over maltosewith 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 embryoswhen 10 x 104 microspores were grown on an individual plate but optimized the plating densityof 8x104-10x104/ml. Shrestha and Kang (2009) had obtained the highest percentage ofregenerated plantlets in cv. Phenlene (2.67%) followed by Bossanova (2.41%) at Dumas deVaulx R (CP medium) followed by MS (Murashige and Skoog, 1962) medium. They were foundthe highest (40%) haploids plants in Minipaprika and 36.1% haploids in cv. Bossanova. Thefrequency of spontaneous DH plants in anther culture of bell pepper i.e. 35.6% by Dumas deVaulx et al. (1981) or 32.6% by Gyulai et al. (2000).Microspores are haploid, unicellular and the generation of haploid/doubled plants from isolatedmicrospores offers the opportunities for genetic transformation, gene mapping and selection fordesired dominant and recessive traits which let the production of homozygous doubled haploidplants (Graner 1996; Polsoni et al. 1988; Stoger et al., 1995). Use of isolated microspores is nowbecoming a realistic approach for haploid induction because production of embryos from isolatedmicrospores of several plant species has been shown to be very efficient and reproducible (Davisand Morton, 1998).Isolated microspore culture might be an alternative method to obtain androgenic response in poorand non-responsive pepper varieties. Isolated microspore culture has been successful in manyspecies 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 peppergenotypes 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 inductionphase for six days at 350C, resulted the increase ratio of responding anthers and in plantregeneration in sweet and spice pepper. They found that heat pre-treatment onto 0.3 M mannitolhad 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 asignificant on the efficiency of microspore-derived plant production. Lantos et al. (2009) foundthat co-cultures with wheat line ‘CY-45’ ovaries exhibited enhanced frequency of embryoidproduction than those with pepper ovaries. They had observed the differences in efficiency ofisolated pepper microspore culture among different pepper genotypes.A significant increase in doubling was observed with 300 mg l-1 in the low androgenicresponding wheat cv. Caramba. Colchicines incorporation during the first hours of culture 4
  • 6. 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 respondinggenotypes and upto 75% doubling in cv. Caramba in microspore and anther culture (Soriano etal., 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 doubledhaploid plants. In vitro colchicines treatment in pepper applied to regenerated haploid explantsresulted in 75% diploidization success rate (Mityko and Fari, 1997). The application ofcolchicines during the earlier periods of in vitro culture has been reported as an efficient methodto increase the production of DH plants in anther/ or microspore culture of some species such asBrassica 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 foundthat development of total number of embryos and the number of cotyledonary embryos werehighest 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 atCapsicum frutscens L. and also observed the equal number of haploids and diploids inregenerants. Incubating treatment in heat conditions at 35 0C in darkness for 8 days, the next 4days to light conditions (12 –h photoperiod at 25 0C ) on Dumas De Vaulx (CP) medium andthen transferring the explants to R1 medium for 4 weeks, anthers produced embryos (Koleva-Gudeva et al, 2007).5. Methodologies of the studyActivity 5.1. Field survey of the major commercial sweet pepper (Capsicum annuum L) farm inKangwon Do Province and assessment of popular cultivars of sweet pepper for domestic andexport 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 cultureActivity 5.2.1. Effect of heat pretreatment duration on embryogenesis and regeneration ofisolated 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. 5
  • 7. 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 daysMicrospore 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 developmentCulture 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 photoperiodExperimental 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 embryolike structures), using stereomicroscope, total embryos and no of green plantsActivity 5.2.2. Effect of different carbohydrate source and concentration on embryogenesis andregeneration of isolated microspore culture in MinipaprikaExperimental 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) 6
  • 8. 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 aboveActivity 5.2.3 Study the effect of ovary co-culture on the embryoid production and plantregeneration of isolated microspore culture in MinipaprikaExperimental 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 andtorpedo, ELS embryo, no of shoot per petri dish, no of rooted plants per petri dish and no ofacclimatized plants per petri dish etc.Activity 5.2.4. Effect of colchicines treatment on double haploid production of isolatedmicrospore culture in MinipaprikaExperimental 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 embryolike structures), total embryos and no of green plants, doubling (%) (no. of double haploids per100 analyzed plants), regeneration percentage (number of regenerated plants per 100 embryos) 7
  • 9. Activity 5.3. Double haploid production in Minipaprika through anther cultureActivity 5.3.1. Effect of abiotic (cold and heat) stress pretreatment of flower bud on androgenesisin 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 minutesStress 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 plantsActivity 5.3.2. Effect of colchicines treatment on the double haploid production of anther culturein MinipaprikaExperimental 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 regeneratedplants per 100 embryos) 8
  • 10. Activity 5.4. Cytological studies of anther and microspore derived plants of MinipaprikaActivity 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 doublehaploids (DH1) of minipaprikaActivity 5.1 Study the agronomic variation among the microspore and anther derivedspontaneous DHs and colchicines induced DHs of MinipaprikaExperimental 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. 9
  • 11. 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 analysisExperimental 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 andundergraduate), commercial paprika growers, members of agriculture research station andbreeding institution at Mendel School’s Research FieldActivity 6.1 Providing the practical skills (learning by doing) on haploid breeding method atMinipaprika 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
  • 12. 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
  • 13. Activity 6.2 Training, seminar and field demonstration of anther/microspore derivedhomozyogous lines to commercial Minipaprika growers, technician of breeding and agricultureresearch 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 person1. 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 Hwacheon2. Minipaprika Breeding: Anther culture and Slides, figures and Mendel School’s ’’ advance method of variety development flow charts Research Field, Hwacheon3. Production of double haploids/in vitro Photos, videos, Mendel School’s ’’ haploid production factsheet with data, Research Field, booklets etc Hwacheon4. Field demonstration of homozygous plants Field observation Mendel School’s ’’ and observation on morphological Research Field, characters Hwacheon5. Maintenance of inbred/homozygous line for Field observation Mendel School’s ’’ breeding (self pollination) Research Field, Hwacheon6. 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 HwacheonActivity 7. Statistical analysis (SAS Institute, 1999), preparation manuscripts (PhD thesis) andbooklets, 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
  • 14. 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
  • 15. 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
  • 16. 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 III1. Field survey of paprika farm andassessment of major paprika varieties fordomestic and export market2. DH production of sweet pepper(Capsicum annuum L) cv.Minipaprika viaisolated microspore culture3. DH production of sweet pepper(Capsicum annuum L) cv. Minipaprikathrough anther culture4. Ploidy analysis and chromosomalobservation of anther and microsporederived plants of sweet pepper (Capsicumannuum L.) cv.Minipaprika5. Assessment of genetic variabilitybetween anther and microspore deriveddouble haploids (DH1) of sweet pepper(Capsicum annuum L.) cv.Minipaprika6. Training, seminar, and fielddemonstration to horticulture studentscommercial paprika growers, techniciansof breeding and agriculture researchinstitute at Mendel School’s ResearchField7. Statistical analysis, preparationmanuscripts (PhD thesis), booklets, reportsand paper publication in SCIE (ScienceCitation Index Expanded) and SCI(Science Citation Index) journals Trim I- (Jan.-Apri.), Trim II- (May-Aug.) and Trim III - (Sept.-Dec.)
  • 17. 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 fordomestic and export market2. DH production of sweet pepper (Capsicum annuum L) cv. Minipaprika viaisolated microspore culture2.1 Effect of heat pretreatment duration2.2 Effect of different carbohydrate source and concentration2.3 Study the effect of ovary co-culture on the embryoid production and plantregeneration2.4 Effect of colchicines treatment on DH production3. DH production of sweet pepper (Capsicum annuum L) cv.Minipaprika throughanther culture3.1. Effect of abiotic (cold and heat) stress pretreatment3.2 Effect of colchicines treatment on the DH production of anther culture4. Ploidy analysis and chromosomal observation of anther and microsporederived plants of sweet pepper (Capsicum annuum L.) cv.Minipaprika4.1 Ploidy analysis of anther and microspore derived regenerants4.2 Observation on stomatal length, chloroplast and chromosome number5. Assessment of genetic variability between anther and microspore deriveddouble haploids (DH1) of sweet pepper (Capsicum annuum L.) cv.Minipaprika5.1 Study the agronomic variation among the microspore and anther derivedspontaneous DHs and colchicines induced DHs5.2 Examine the genetic diversity in anther and microspore derived doublehaploids (DH-R2) lines using RAPD analysis6. Training, seminar, and field demonstration to horticulture students commercialpaprika growers, technicians of breeding and agriculture research institute atMendel School’s Research Field6.1 Providing the practical skills (learning by doing) on haploid breeding methodat Minipaprika to graduate and undergraduate students of horticulture science6.2 Training, seminar and field demonstration of anther/microspore derivedhomozyogous lines to commercial Minipaprika growers, technician of breedingand agriculture research institute.7. Statistical analysis, preparation manuscripts (PhD thesis), booklets, reports andpaper publication in SCIE (Science Citation Index Expanded) and SCI (ScienceCitation Index) journals8. Salary, travel, field expenses, gasoline, lab (equipments, chemicals ) andstationaryTotal (000’ Won)
  • 18. 11. Literature citedAbak K, Pochard E and Dumas de Vaulx R. 1982. Transmission of resistance to Phytophthora capsici on roots and stems of pepper plants; study of doubled haploid lines issued from the cross ‘PM217’ x ‘Yolo Wonder’ through anther culture. Capsicum Nwsl 1:62-63.Barany I, Testillano PS, Mityko J and Risueno. 2001. The switch of the microspore developmental programme in Capsicum involves HSP70 expression and leads to the production of haploid plants. Int. J. Dev. Biol. 45, S39-S40.Barnabas B, Pfahler PL and Kovacs. 1991. Direct effect of colchicines on the microspore embryogenesis to produce dihaploid plants in wheat (Triticum aestivum L.). Theor Appl Genet 81:675-678.Caranta C, Palloix A, Gebre-Selassie K, Lefevre V, Moury B and Daubeze AM. 1996. A complementation of two genes from susceptible Capsicum annuum lines confers a new and complete resistance to pepper veinal mottle virus. Phyto pathology 86:739-743.Cistue L, Soriano M, Castillo AM, Valles MP, Sanz JM and Echavarri B. 2006. Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant Cell Rep 25:257-264.Coventry J, Kott L and Beversdorf WD. 1988. Manual for microspore culture technique for Brassica napus. University of Guelph, Guelph, Guelph, pp1-35.Custers JBM, Cordewener JHG, Nollen Y, Dons HJM and Campagne MMV. 1994. Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Rep 13:267-271. doi:10.1007/BF00233317Daubeze AM, Palloix A and Pochard E. 1990. Resistance of androgenetic autodiploid lines of pepper to Phytopthora capsici and tobacco mosaic virus under high temperature. Capsicum Nwsl 8-9:47-48.Davis PA and Morton S. 1998. A comparison of barley isolated microspore and anther culture and the influence of cell culture density. Plant Cell Rep 17(3):206-210. Doi:10.1007/s002990050379Dolcet-Sanjuan R, Claveria E and Huerta A. 1997. Androgenesis in Capsicum annuum L. –Effects of Carbohydrate and Carbon Dioxide Enrichment. J. Amer. Soc. Hort. Sci. 122 (4):468-475.Dumas de Vaulx R, Chambonnet D and Pochard E. 1981. Culture in vitro d’antheres du piment (Capsicum annuum L.): amelioration des taux d’obtention de plantes chez different genotypes par des traitements a +35 0C. Agronomie 1, 859-864.Eudes F and Amundsen E. 2005. Isolated microspore culture of Canadian 6x triticale cultivars. Plant Cell Tissue Org 82:233-241. doi :10.1007/s11240-005-0867-9Forster BP and Thomas WTB. 2004. Doubled haploids in genetics and plant breeding. Plant Breed Rev 25:57–88Gamborg OL, Miller RA and Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151-158.George L and Narayanaswamy S. 1973. Haploid Capsicum through experimental androgenesis. Protoplasma 78:467-470. doi:10.1007/BF01275781 17
  • 19. Graner A. 1996. RFLP-mapping the haploid genome of barley (Hordeum vulgare L.) In: Jain SM, Sopory SK, Veilleux RE (eds). In vitro haploid production in higher plants. Vol 3. Kluwer, Dordrecht, pp. 127-150.Gyulai G, Gemesne JA, Sagi ZS, Venczel G, Pinter P, Kristof Z, Torjek O, Heszky L, Bottka S, Kriss J and Zatyko L. 2000. Doubled haploid development and PCR-analysis of F1 hybrid derived DH-R2 paprika (Capsicum annuum L.) lines. J. Plant Physiol. 156:168-174.Hendy H, Pochard E and Dalmasso A. 1985. Transmission de la resistance aux nematodes Meloidogyne chitwood (Tylenchida portee par 2 lignees de Capsicum annuum L. Etude de descendances homozygotes issues d’androgenese. Agronomie 5:93-100.Jahne A and Lorz H. 1995. Cereal microspore culture. Plant Sci, 109:1-12.Juhasz AG, Kristof Z, Vagi P, Lantos C and Pauk J. 2009. In vitro anther and isolated microspore culture as tools in sweet and spice pepper breeding. Acta Hort. 829, ISHS.Kasha KJ, Simion E, Oro R, Yao QA, Hu TC and Carlson AR. 2001. An improved in vitro technique for isolated microspore culture of barley. Euphytica 120:379-385. doi:10.1023/A:1017564100823Kim M, Jang IC and Kim JA. 2008. Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep, 27:425-434Koleva-Gudeva LR, Spasenoski M and Trajkova F. 2007. Somatic embryogenesis in pepper anther culture: The effect of incubation treatments and different media. Scientia Horticulturae 111:114-119.Kuo JS, Wang ZZ, Chien NF, Ku SJ., Kung ML and Hsu HC. 1973: Investigations on the anther culture in vitro of Nicotiana tabacum L. and Capsicum annuum L. Acta Botanica Sinica 15: 43-47.Lantos C, Juhasz AG and Somogyi G. 2009. Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tiss Organ Cult, 97:285-293.Lee JS, Park EJ and Kim M. 2007. Influence of donor plant growth condition, microspore isolation method, culture medium and light culture on the production of embryos in microspore culture of hot pepper (Capsicum annuum L.). J Plant Biotechnol 34 (4):363-373.Li H and Devaux P. 2001. Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep, 20:475-481. Doi:10.1007/s002990100368Mejza SJ, Morgant V, DiBona DE and Wong JR. 1993. Plant regeneration from isolated microspores of Triticum aestivum. Plant Cell Rep 12:149-153. Doi:10.1007/BF00239096MFAFF. 2008. Ministry of Food, Agriculture, Forestry and Fisheries. Statistic of protected cultivated vegetables production. Khyoungi Province. p7.Mityko J and Fari M. 1997. Problems and results of doubled haploid plant production in pepper (Capsicum annuum L.) via anther- and microspore culture. Acta Hortic. 447:281-287.Mityko J, Andrasfalvy A, Csillery G and Fari M. 1995. Anther culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.). Plant Breeding 114, 78-80.Mityko J, Szabo L and Barnabas B. 1999. Colchicine induced ultrastructural changes in barley and pepper microspores. J. Slovak Acad. Sci. 54, 24-25. 18
  • 20. Moller C, Iqbal MCM and Robbelen G. 1994. Efficient production of doubled haploid Brassica napus plants by colchicines treatment of microspores. Euphytica 75, 95-104.Murashige T and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.Nageli M, Schmid JE, Stamp P and Butter B. 1999. Improved formation of regenerable callus in isolated microspore culture of maize: impact of carbohydrates, plating density and time of transfer. Plant Cell Rep 19:177-184. Doi:10.1007/s002990050730Nitsch C. 1974. Pollen culture-a new technique for mass production of haploid and homozygous plants. In: Kasha KJ (ed) Haploids in higher plants: advance and potential. University of Guelph Press, Guelph, pp 123-135.Nowaczyk P, Kisiala A and Olszewska. 2006. Induced androgenesis of Capsicum frutescens L. Acta Physiologiae Plantarum, 28 (1):35-39.Palmer CE, Keller WA and Arnison PG. 1996. Experimental haploidy in Brassica species. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 2. Kluwer. The Netherlands, pp 143-172.Pauk J, Mihaly R, Monostori T and Puolimatka M. 2003. Protocol of triticale (Triticosecale Wittmack) microspore culture. P. 129-Pochard E, Selassie KG and Marchoux G. 1983. Oligogenic resistance to potato virus Y pathogype 1-2 in the line ‘Perennial’. Capsicum Nwsl 2: 137-138.Polsoni SK, Kott LS, Beversdorf WD. 1988. Large-scale microspore culture technique for mutation selection studies in Brassica napus. Can J Bot 66:1681-1685.Qin X and Rotino GL. 1995. Chloroplast number in guard cells as ploidy indicator of in vitro- grown androgenic pepper plantlets. Plant Cell Tissue Org. Cult. 41, 153-160.Saisingtong S, Schmind JE, Stamp P and Butter B. 1996. Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.) Theor. Appl. Genet. 92, 1017-1023Sangwan RS and Sangwan-Norreel BS. 1990. Anther and pollen culture. In: Bhojwani, SS (Ed.) Plant tissue culture: Application and Limitation. Development in Crop Science, Vol 19. Elsevier, Amsterdam, pp. 220-241.Shrestha SL and Kang WH. 2009. Effect of genotype of donor plants on the success of anther culture in sweet pepper (Capsicum annuum L.). Korean J. Plant Res. 22 (6): 506-512.Shrestha, S.L.2009. Establishment of hybrid breeding system in sweet pepper (Capsicum annuum L.), PhD Thesis, Kangwon National University, Chuncheon, Republic of Korea.Soriano M, Cistue L, Valles MP and Castillo AM. 2007. Effects of colchicines on anther and microspore culture of bread wheat (Triticum aestivum L.). Plant Cell Tiss Organ Cult. 91:225-234. Doi 10.1007/s11240-007-9288-2Stoger E, Fink C, Pfosser M and Heberle-Bors E. 1995. Plant transformation by particle bombardment of embryogenic pollen. Plant Cell Rep 14:273-278.Supena EDJ, Muswita W, Suharsono S and Custers. 2006. Evaluation of crucial factors for implementing shed-microspore culture of Indonesia hot pepper (Capsicum annuum L.) cultivars. Scientia horticulturae, 107 : 226-232Swanson EB. 1990. Microspore culture in Brassica : In : Pollard JW, Walker JM (eds) Methods in molecular biology, vol 6. Plant cell and tissue culture. Humana Press, New Jersey, pp. 159- 170. 19
  • 21. Touraev A, Ilham A, Cicente O and HeberleBors E. 1996. Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep 15:561-565. Doi:10.1007/BF00232453Wang YY., Sun CS., Wang CC. and Chen NF. 1973: The induction of pollen plantlets of Triticale and Capsicum annum from anther culture. Scientia Sinica 16: 147-151.Zhao JP, Simmonds DH and Newcomb W. 1996. Induction of embryogenesis with colchicines instead of heat in microspores of Brassica napus L. cv. Topas. Planta 198, 433-439. 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
  • 22. Appendix 2. Composition of the B5 mediumMacro-elements mgL-1 mg (500 ml) gL-1CaCl2.2H2O 150 75.0 0.150KNO3 3000 1500 3.0MgSO4.7H2O 500 250 0.500NaH2PO4.2H2O 150 75 0.150(NH4)2SO4 134 67.0 0.134Na2EDTA* 37.3 18.65 0.0373Minor and micro-elementCoCl2.6H2O 0.025 0.0125CuSO4.5H2O 0.025 0.0125FeSO4.7H2O* 27.8 13.9H3BO3 3 1.5KI 0.75 0.375MnSO4.4H2O 13.2 6.6Na2MoO4.2H2O 0.25 0.125ZnSO4.7H2O 2 1Organic substanceMyo-inositol 100 50Folic acid 0 0Nicotinic acid 1 0.5Thiamine HCl 10 5Pyridoxine HCl 1 0.5Sucrose (2%) 20000 10000Phytagel (0.35%) 3500 1750pH 5.8 5.8 21
  • 23. Appendix 3. Composition of MS mediumMacro-elements mgL-1 mg (500 ml) gL-1NH4NO3 1650 825 1.650KNO3 1990 995 1.990CaCl2.2H2O 440 220 0.440MgSO4.7H2O 370 185 0.370KH2PO4 170 85 0.170Minor and micro-elementH3BO3 6.2 3.1MnSO4.4H2O 22.3 11.15ZnSO4.7H2O 8.6 4.3KI 0.83 0.415Na2MoO4.2H2O 0.25 0.125CuSO4.5H2O 0.025 0.0125CoCl2.6H2O 0.025 0.0125Na2EDTA.2H2O 37.3 18.65FeSO4.7H2O 27.8 13.9Organic substanceGlycine 2 1Mesoinositol 100 50Nicotinic acid 0.5 0.25Pyridoxine (HCl) 0.5 0.25Thiamine (HCl) 0.1 0.05Carrot extract 200 ml 100 mlCharcoal (1%) 10000 5000Sucrose (3%) 30000 15000Agar (0.8%) 8000 4000pH of the media 5.5 5.5 22
  • 24. Appendix 4. Composition of C medium and R mediumMacro-element C Medium R1 medium C Medium R1 medium mgL-1 mgL-1 mg (500 ml) mg (500 ml)NH4NO3 1238 1238 619.0 619.0KNO3 2150 2150 1075.0 1075.0KCl 7 7 3.5 3.5CaCl2.2H2O 313 313 156.5 156.5Ca(NO3)2.4H2O 50 50 25 25MgSO4.7H2O 444 444 222 222(NH4)2SO4 34 34 17 17KH2PO4 142 142 71 71Minor and micro-elementNaH2PO4.H2O 38 38 19 19H3BO3 3.15 1.55 1.575 0.775MnSO4.H2O 22.13 20.13 11.065 10.065ZnSO4.7H2O 3.625 3.225 1.8125 1.6125KI 0.695 0.33 0.3475 0.165Na2MoO4.2H2O 0.188 0.138 0.094 0.069CuSO4.5H2O 0.016 0.011 0.008 0.0055CoCl2.6H2O 0.016 0.011 0.008 0.0055Na2EDTA.2H2O 18.65 18.65 9.325 9.325FeSO4.7H2O 13.9 13.9 6.95 6.95Organic substancesGlycine 0.1 0.1 0.05 0.05Mesoinositol 50.3 50.3 25.15 25.15Ca-pantothenate 0.5 0.5 0.25 0.25Nicotinic acid 0.7 0.7 0.35 0.35Pyridoxine (HCl) 5.5 5.5 2.75 2.75Vitamin B12 0.03 0 0.015 0Thiamine (HCl) 0.6 0.6 0.3 0.3Biotine 0.005 0.005 0.0025 0.0025Sucrose (3%) 30000 30000 15000 15000Agar (0.8%) 8000 8000 4000 4000pH 5.9 5.9 5.9 5.9Growth regulatorsKinetin 0.01 or 2 0.1 0.005 or 1 0.052,4-D 0.01 or 1 0.005 or 0.5 23
  • 25. 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