Anther Culture of Transgenic Pepper (Capsicum annuum L.)

김지연,Young Soon Kim,이기범,Kyung-Moon Kim 한국육종학회 2005 한국육종학회지 Vol.37 No.5

Pepper (Capsicum annum L.) is an important vegetable crop that can surely benefit from plant biotechnology. Using transgenic pepper lines resistant to fungal disease, anther culture system was applied to show the possibility to use in molecular breeding program. We compared some conditions for enhancing anther culture efficiency. We did not find the beneficial effect of low pretreatment of anthers in both wild type and transgenic lines. By comparing medium compositions, anthers produced more microspore-derived embryos or embryogenic callus in combination of 0.1 mg/L of 2,4-D and 0.1 mg/L of kinetin. In anther culture response, wild type had better androgenesis than transgenic lines. PCR results showed that plants regenerated from anthers of transgenic pepper lines had transgenes, HPT/PepEST. By hygromycin tests, plants developed from anthers of transgenic lines showed resistance to hygromycin. A total of 48 plants were produced from anther culture of wild type and transgenic lines, among them, 43 were developed from wild type and 5 plants from transgenic lines. Six plants and one plant spontaneously set pepper fruits in wild type anther-derived plants and anther derived plants from transgenic lines, respectively. Regenerated plants showed various phenotypes and some of plants grew normally. These results showed the possibility to use anther culture system for accelerating breeding programs by the combination with plant biotechnology.

당근의 약배양으로부터 직접 및 간접 배형성을 통한 식물체 생산 체계 확립

김미영,형남인 한국육종학회 2014 한국육종학회지 Vol.46 No.4

당근의 약배양을 통한 효율적 식물체 재생 시스템을 확립하고자 실험을 수행하였다. 당근 유전형 ‘S&P2342’의 약을 2,4-D와 NAA를 각각 0.1 mg/L 또는 1.0 mg/L 단독 또는 조합 처리한 배지에 치상하여 암조건에서 18주간 배양하였을 때, 2,4-D 1.0 mg/L + NAA 0.1 mg/L 조합처리에서 캘러스 형성율 22.0%, 배형성율 2.0%로 반응이 가장 좋았다. 약으로부터 직접적으로 유도된 1차배를 분리하여 명조건에서 2주간 배양 후 재생배지로 옮겨 배양하였을 때 배양 4주 후부터 발아하다가 생장이 멈추어진 1차배로부터 다수의 2차배가 형성되기 시작하였다. 배양 8주 후 62.5%가 식물체로 전환되었으며, 12주 후 본엽이 전개된 소식물체로 발달하였다. 또한 약으로부터 유도된 캘러스를 분리하여 재생배지에서 배양하였는데, 8주 후 93.9%의 캘러스로부터 1차배가 형성되었으며, 38.8%에서 1차배로부터 2차배 형성을 거쳐 식물체로 전환되었다. 약배양 유래 소식물체(본엽 2매 이상, 초장 2-4 cm)의 건전한 기내 생장을 유도하기 위해 실시한 광도(10, 30, 100, 150 μmol·m-2·s-1) 처리에서 100 μmol·m-2·s-1이 소식물체의 생장에 가장 효과적이었다. 소식물체의 효율적인 기외 활착을 위하여 본엽 4매 이상, 초장 5 cm 이상의 소식물체를 플러그 트레이에 이식 후 플라스틱 재배용기 안에 넣은 다음 온도 (15, 20, 25℃)와 광도(μmol·m-2·s-1) 처리를 각각 2주간에 걸쳐 실시하였는데, 온도 처리에서는 15℃가 기외 활착에 가장 적합하였으며, 광도 처리에서는 100 μmol·m-2·s-1 처리구만 기외 활착이 원활하였다. 결론적으로 당근의 약배양으로부터 직접 배형성 또는 캘러스 단계를 거치는 간접 배형성이 이루어졌으며, 1차배로부터 2차배가 형성된 후 발아를 거쳐 소식물체가 재생되었으며 이를 성공적으로 활착시켜 정상적인 식물체를 얻을 수 있었다. 본 연구를 통하여 당근에서의 약배양으로부터 직접 및 간접 배형성을 통한 식물체 재생 시스템을 구축하였으며, 향후 당근의 F1 잡종 생산을 위한 순수 동형접 합성 유지계통 및 화분친 모본계통의 획득에 활용할 수 있을 것으로 생각된다. We tried to develop the protocol for embryogenesis and plant regeneration from anther culture of carrot (Daucus carotaL.) genotype ‘S&P2342’. Anthers were cultured on MS medium with B5 vitamins containing different combinations of 2,4-D and NAA for 18 weeks in the dark. The best induction of callus and embryo was obtained in the medium containing 0.1 mg/L 2,4-D and 0.1 mg/L NAA, on which 22.0% callus and 2.0% embryo were induced. When primary embryos induced directly from anther culture were transferred to the regeneration medium, secondary embryos were initiated from primary embryos after 4 weeks of culture and 62.5% converted into plantlets after 8 weeks of culture. The plantlets with true leaves were obtained after 12 weeks of culture. When the calli derived from anther culture were transferred to the regeneration medium, 38.8% of the calli produced primary embryos and plantlets after 8 weeks of culture. The plantlets with 2 or more leaves cultured on the regeneration under the different light intensity for the growth of in vitro plantlets. The plantlets cultured at 100 μmol・m-2・s-1 showed the highest growth rate. For the acclimatization, the in vitro plantlets with 4 or more leaves cultivated under the different light intensity and temperature, respectively. The survival rate and growth of plantlets was best at 15℃ and 100 μmol・m-2・s-1, respectively. The plants were successfully acclimatized and had a normal phenotype. The anther culture system could be used to prepare the doubled haploid lines as an appropriate breeding material for F1 hybrid breeding program.

DNA marker를 이용한 벼의 조직배양 효율 재선

김경민,Kim Kyung-Min 한국생명과학회 2006 생명과학회지 Vol.16 No.3

The purpose of this study is to improve the culturability of a indica type rice cultivar, IR 36, using DNA marker associated with the ability of plant regeneration in anther and seed culture. The varietal difference of ability of callus formation and plant regeneration was investigated in anther and seed culture of 8 rice cultivars. Three japonica rice cultivars showed to have better culturability than those of tongil and indica type genotypes. But two indica/japonica lines, 'MGRI 079' and 'MGRI 036', which were selected to have good culturability in previous study showed the highest regenerability (20%) in anther culture of 8 rice cultivars. Thirty four $BC_2F_4$ lines were selected by marker screening using RZ400 for 100 $BC_2F_4$ lines derived from a cross $'MGRI\;079/IR\;36^{^*3}'$. The frequency of callus formation of 30 $BC_2F_4$ lines was higher than those of 'IR 36' in anther culture of the selected $BC_2F_4$ lines. The ability of plant regeneration of 15 lines was higher than that of 'IR 36' in the seed culture of 34 $BC_2F_4$ lines. A promising line, $BC_2F_4-28$, was selected to have better culturability in the anther and seed culture of the $BC_2F_4$ lines. The heading date and grain shape of the $BC_2F_4-28$ was similar to 'IR 36'. In seed culture of 50 $BC_4F_3$ lines derived from a rice cross $'MGRI\;079/IR\;36^{^*5}'$, 11 lines including $BC_4F_3-3$ showed to have higher regenerability compared with 'IR 36'. The highest frequency of plant regeneration (11%) was obtained from $BC_4F_3-46$ in seed culture of the $BC_4F_3$ lines. 벼의 약 및 현미 배양효율과 관련된 DNA marker를 이용하여 인디카형 벼 품종인 'IR 36'의 조직배양 효율을 개선하기 위하여 실험한 결과를 요약하면 다음과 같다. MGRI집단의 약배양에서 식물체분화율이 높은 계통으로 선발된 'MGRI 079'와 'MGRI 036'의 약배양 효율은 각각 19.8%, 19.9%로 가장 높게 나타났다. 'MGRI 079'에 'IR 36'이 여교배되어 양성된 $BC_2F_4$ 100 계통에 대한 marker 검정을 실시하여 선발된 34 계통중 약배양에서 캘러스 형성률이 'IR 36'보다 높은 계통은 30계통이었고, 현미배양에서 'IR 36'보다 식물체 분화율이 높은 계통은 $BC_2F_4-28$ 외 14 계통이었다. $BC_2F_4$ 34계통 중에서 식물체분화능력이 높은 계통으로 선발된 $BC_2F_4-28$은 간장이 'IR 36'보다 큰 편이었으나 출수기와 미립특성은 'IR 36'과 비슷하였다. 'MGRI 036'에 'IR 36'을 반복친으로 여교배하여 양성된 $BC_4F_3$ 50계통의 현미배양을 실시한 결과 'IR 36'보다 식물체 재분화율이 높은 계통은 $BC_4F_3-3$ 외 10 계통이었고, 그중 $BC_4F_3-46$의 식물체 재분화율이 11%로 가장 높게 나타났다.

DNA marker를 이용한 벼의 조직배양 효율개선

김홍집,김태헌,손재근 경북대학교 농업과학기술연구소 2009 慶北大農學誌 Vol.27 No.-

벼의 약 및 현미 배양효율과 관련된 DNA marker를 이용하여 인디카형 벼 품종인 'IR 36'의 조직배양 효율을 개선하기 위하여 실험한 결과를 요약하면 다음과 같다. 벼품종 간에 약 및 현미배양 효율을 비교한 결과 자포니카 > 통일형 > 인디카 형의 순으로 나타났다. 그러나 MGRI집단의 약배양에서 식물체분화율이 높은 계통으로 선발된 'MGRI 079'와 'MGRI 036'의 약배양 효율은 각각 19.8%, 19.9%로 가장 높게 나타났다. 'MGRI 079'에 'IR 36'이 여교배되어 양성된 90 계통에 대한 marker검정을 실시하여 positive band를 나타내는 34계통을 선발할 수 있었다. 선발된 34계통 중 10 계통의 약배양에서 캘러스 형성률은 'IR 36' 보다 현저히 높았다. 선발된 10 계통의 현미배양에서도 캘러스형성 능력과 식물체재분화율이 'IR 36' 보다 높게 나타났다. 계통 중에서 식물체분화능력이 높은 계통으로 선발된 -28은 간장이 'IR 36'보다 큰 편이었으나 출수기와 미립특성은 'IR 36'과 비슷하였다. The purpose of this study was to improve the culturability of 'IR 36', a indica type rice cultivar using DNA marker associated with the ability of plant regeneration in anther and seed culture. The culturability of 6 rice cultivars and 2 indica/japonica lines ('MGRI 036', 'MGRI 079') were investigated in anther and seed culture. The culturability of 3 japonica rice cultivars were much higher than tongil and indica rice cultivars, and 'MGRI 036' and 'MGRI 079' has high culturability with 20% regenerability, also. 34 4 lines were selected by marker screening using RZ400 among 90 lines derived from a cross between 'MGRI 079' and 'IR 36'. The frequency of callus formation of 10 lines were higher than 'IR 36' in anther culture among selected 34 lines. The ability of plant regeneration of 10 lines were higher than 'IR 36' in the seed culture among selected 34 lines. A promising line, -28, was selected to have better culturability in the anther and seed culture among selected 34 lines. The heading date and grain shape of the -28 was similar to 'IR 36'. Using the RZ400 DNA marker associated with the culturability will be useful method for improving of indica rice culticvar's culturability in rice breeding program.

SNP-based analysis of genetic diversity in anther-derived rice by whole genome sequencing

Jeong, In-Seon,Yoon, Ung-Han,Lee, Gang-Seob,Ji, Hyeon-So,Lee, Hyun-Ju,Han, Chang-Deok,Hahn, Jang-Ho,An, Gynheung,Kim, Tae-Ho Springer New York 2013 Rice Vol.6 No.-

<P><B>Background</B></P><P>Anther culture has advantage to obtain a homozygous progeny by induced doubling of haploid chromosomes and to improve selection efficiency for invaluable agronomical traits. Therefore, anther culturing is widely utilized to breed new varieties and to induce genetic variations in several crops including rice. Genome sequencing technologies allow the detection of a massive number of DNA polymorphism such as SNPs and Indels between closely related cultivars. These DNA polymorphisms permit the rapid identification of genetic diversity among cultivars and genomic locations of heritable traits. To estimate sequence diversity derived from anther culturing, we performed whole-genome resequencing of five Korean rice accessions, including three anther culture lines (BLB, HY-04 and HY-08), their progenitor cultivar (Hwayeong), and an additional japonica cultivar (Dongjin).</P><P><B>Results</B></P><P>A total of 1,165 × 10<SUP>6</SUP> raw reads were generated with over 58× coverage that detected 1,154,063 DNA polymorphisms between the Korean rice accessions and <I>Nipponbare</I>. We observed that in Hwayeong and its progenies, 0.64 SNP was found per one kb of <I>Nipponbare</I> genome, while Dongjin, bred by a conventional breeding method, had a lower number of SNPs (0.45 SNP/kb). Among 1,154,063 DNA polymorphisms, 29,269 non-synonymous SNPs located on 30,013 genes and these genes were functionally classified based on gene ontology (GO). We also analyzed line-specific SNPs which were estimated 1 ~ 3% of the total SNPs. The frequency of non-synonymous SNPs in each accession ranged from 26 SNPs in Hwayeong to 214 SNPs in HY-04.</P><P><B>Conclusions</B></P><P>The genetic difference we detected between the progenies derived from anther culture and their mother cultivar is due to somaclonal variation during tissue culture process, such as karyotype change, chromosome rearrangement, gene amplification and deletion, transposable element, and DNA methylation. Detection of genome-wide DNA polymorphisms by high-throughput sequencer enabled to identify sequence diversity derived from anther culturing and genomic locations of heritable traits. Furthermore, it will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/1939-8433-6-6) contains supplementary material, which is available to authorized users.</P>

옥수수 약배양 연구 동향 및 전망

김경희(Kyung-Hee Kim),문준철(Jun-Cheol Moon),김재윤(Jae Yoon Kim),이병무(Byung-Moo Lee) 한국육종학회 2016 한국육종학회지 Vol.48 No.2

Anther culture is useful and significant tool for producing haploid or doubled haploid (DH) plants in crop breeding system. Androgenesis is the way of inducing haploid and DH plants from anther (immature pollen) or microspore culture. In vitro androgenesis is efficient technique for introducing complete homozygous lines in one generation, thus less time and expense could be necessary than conventional plant breeding. In maize, anther culture is important system for shortening the breeding cycle and enhancing selection efficiency. Anther culture technique is also applicable to various researches such as molecular genetics, genetic engineering, genomics, and plant biotechnology. We review the past and present studies on anther culture and provide useful information for future researches on androgenesis in maize. The combination of androgenesis with other techniques such as molecular breeding and biotechnology is producing a variety of variety of maize species. In addition, we suggest strategy to develop androgenesis technique adapted to Korean research environment.

Petunia hybrida의 약배양(約培養)으로부터 callus 형성(形成)에 미치는 배양조건(培養條件)의 영향(影響)

정재동,이정희,지선옥,Chung, Jae Dong,Lee, Jung Hee,Jee, Sun Ok 경북대학교 농업과학기술연구소 1992 慶北大農學誌 Vol.10 No.-

본 연구는 Petunia hybrida $F_1$ hybrid를 약배양(葯培養)하여 반수체(半數體)를 얻을 목적으로 약(葯)의 치상시기(置床時期)와 배지(培地)의 종류(種類), 생장조절물질(生長調節物質)의 종류와 농도, 약(葯)의 전처리(前處理)와 품종(品種)에 따른 캘러스 형성률을 조사하였던 바 다음과 같은 결과를 얻었다. 약배양하기 전 0.8-1.2cm의 길이의 화뢰(花雷)가 달린 모본(母本)의 줄기를 $4^{\circ}C$에서 15일간 저온처리(低溫處理)하였다. 약을 채취하여 1/2 MS배지에 NAA 5.0mg/${\ell}$, BAP 0.5mg/${\ell}$를 첨가한 배지(sucrose 30g/${\ell}$, 2g/${\ell}$, pH 5.8)에서 배양 4주후 연녹색의 캘러스가 형성되었으며, 이들 캘러스로부터 배양 3주후 2ip 2.0mg/${\ell}$가 함유된 MS배지에서 식물체가 분화되었다. The study was conducted to get basic information for haploid production of Petunia hybrida through anther culture by investigating several factors, namely anther stage, culture medium, cold treatment, and genotypes. The results are summarized as follows; Four genotypes of Petunia hybrida were used in a study of anther culture. Plants of each genotype were grown in controlled environments at $20-30^{\circ}C$ with a 16h photoperiod. Equal numbers were harvested from each genotype. The anthers were taken from buds which had received the 14 days' cold treatment at $4^{\circ}C$. Anthers were dissected out aseptically and plated on 1/2 strength MS agar medium containing 5.0mg/${\ell}$ 6-Benzylaminopurine(BAP). Four weeks after culture, light green callus was formed. From these calli, plantlets were regenerated on MS medium containing 2.0mg/${\ell}$ 2-isopentenyl adenine(2ip) after 3 weeks.

Inheritance of Herbicide (glufosinate) Resistance in Transgenic Rice Plant through Anther Culture

Hyeon-Jung Kang,Hyun-Soon Kim,Jeong-Kwon Nam,Young-Tae Lee,Seung-Yeob Lee,Chung-Kon Kim 한국육종학회 2008 한국육종학회지 Vol.40 No.3

Haploid system by anther culture allows the development of homozygous lines when doubled. The response of anther culture to Basta (glufosinate) resistance was investigated on transgenic plants (cv. Anjungbyeo) in order to identify inheritance of bar gene associated with Basta. Most of the regenerated transgenic plants were sterile, and only a few plants produced viable seeds (A1) in the greenhouse. The bar gene was analysis by PCR in basta resistant transgenic plant (TA0). The transgenic seeds (A1) were significantly germinated in Basta solution compared with non-transformed seeds. As a result of anther culture, in regenerated haploid plants, segregation ratio was 1:1 in five of eight cross combinations. In diploid plants, segregation ratio was 1:1 in seven of eight cross combinations. Although there was some differences in the cross combinations, most of the combinations had 1:1 segregation ratio which supports the theory. The difference may be a result of the small sample size or the difference of anther culture response caused by genotypic difference. Hence, when many cross combinations were anther-cultured the results would support the theory.

The Effect of Cultural Factors on Anther Culture in Spring Wheat (Triticum aestivum)

Kang,Tae-Jin,Yang,Deok-Chun 한국자원식물학회 2003 Plant Resources Vol.6 No.2

Effects of shaking, medium consistency and anther density on polyhaploid production in two wheat cultivars, Pavon and Chris, were studied using a modified 85D12 medium. Pavon produced more calli in shaking and more albino plants tban Chris. However, Chris produced threefold more green plants than Pavon in non-shaking treatment. More calli and green plants were derived from non-shaking treatment than those from shaking treatment. Anthers were cultured on both liquid and semi-solid 85D12 media, using two anther densities, 48 and 96 anthers per plate. Although Pavon generally produced more calli and albino plants than Chris, Chris produced more green plants than Pavon. More green plants were derived from semi-solid medium than those from liquid medium. A factor that may affect plant regeneration from anthers is the length of time on initiation medium. Most of the calli for both genotypes were transferred during the first two time periods. Fertility, as measured by seed set, was determined for all surviving regenerated plants. About 24% of Chris and Pavon anther-derived green plants in the experiment of medium consistency and anther density produced seed.

BREEDING OF RESISTANT PEPPER LINES (CAPSICUM ANNUUM L.) TO BACTERIAL SPOT (XANTHOMONAS CAMPESTRIS PV. VESICATORIA) THROUGH ANTHER CULTURE

Hwang, J.K.,Cho, D.H.,Paek, K.Y. 충북대학교 한국과학재단 지정 첨단원예기술개발 연구센터 1997 연구보고서 Vol.2 No.-

The experiments were conducted to obtain pepper lines resistant to bacterial spot with superior horticultural characteristics through anther culture techniques. Thirty three breeding lines and plants derived from anthers of 3 F₁combinations. Nine lines including PI 163184 were shown to be resistant to the disease and PI 183439 and PI 244669 were moderately resistant, but others were susceptible. Pretreatment of anthers at 35℃ for 8 days under the darkness was more effective than 4℃ treatment for plant regeneration. The anthers at late uninucleate to binucleate stage responded better than those at other stages in inbred line-1×PI 163292 and inbred line -5×PI 183481 combinations, but in MS line-3×PI 183431 anthers at early uninucleate stage were more responsive. Large variations in the length of main stem and floral bud were observed with plants derived from anthers, and 2 chimeral plants with partial albino leaves were also observed. Most of the anther-derived plants were moderately resistant to the bacterial spot, and only few plants were susceptible.