CENH3 mediated haploid induction: application and future perspectives in crop plants

라이안자리,Dubey Kavita,한성수 한국원예학회 2023 Horticulture, Environment, and Biotechnology Vol.64 No.6

True homozygous pure lines are required for the development of new crop varieties. In conventional as well as molecu lar breeding strategies, it typically takes 7–9 generations to accomplish the appropriate level of homozygosity. On the contrary, haploids can have their chromosomes doubled in a single generation to create a true-breeding lineage. Over the period, researchers have developed several methods for haploids and doubled haploid induction, but these methods are only applicable to specifi c crop types. The discovery of the centromere-specifi c histone 3 variant (CENH3) and its manipulation is proving to be the most potent technique for haploid development. Recent advancements in this technol ogy have shown that non-transgenic changes to CENH3 can also induce haploids. Point mutations in CENH3 that can be induced by chemical agents may lead to haploid induction when crossed with wild-type CENH3 plants. These plants with the CENH3 mutation are fully fertile when selfed, develop properly, and can be found in already-existing collections of mutagenized plants. The current review encompasses the recent studies undertaken to utilize the CENH3 manipula tions strategy to develop various haploid plant crops with increased success rates. This review paper will provide a better insight into understanding the detailed mechanism of the CENH3-induced haploid induction process and help investigate the areas that need to be further explored.

Haploidy of somatic cells in mouse oocyte using somatic cell nuclear transfer

이연미,강은주 사단법인 한국동물생명공학회 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.4

Haploidization in somatic cells is the process of reducing the diploid somatic chromosomes to haploid. Several studies have attempted somatic haploidization using oocytes in mice and humans. Some researchers showed partial somatic haploidization, but none observed embryo development. Our study attempted somatic haploidization using the modified somatic nuclear transfer (SCNT) protocol with various combinations of chemicals or proteins in mice. This study induced the proper segregation of somatic homologous chromosomes and full embryo development in vitro. Furthermore, somatic haploid embryos established embryonic stem cells and produced live births. The current review summarizes this recent study on the success of somatic haploidization and provides an overview of other related studies on somatic haploidization.

Haploidy of somatic cells in mouse oocyte using somatic cell nuclear transfer

Yeonmi Lee,Eunju Kang The Korean Society of Animal Reproduction and Biot 2022 한국동물생명공학회지 Vol.37 No.4

Haploidization in somatic cells is the process of reducing the diploid somatic chromosomes to haploid. Several studies have attempted somatic haploidization using oocytes in mice and humans. Some researchers showed partial somatic haploidization, but none observed embryo development. Our study attempted somatic haploidization using the modified somatic nuclear transfer (SCNT) protocol with various combinations of chemicals or proteins in mice. This study induced the proper segregation of somatic homologous chromosomes and full embryo development in vitro. Furthermore, somatic haploid embryos established embryonic stem cells and produced live births. The current review summarizes this recent study on the success of somatic haploidization and provides an overview of other related studies on somatic haploidization.

High-purity Seed Production of Doubled Haploid Chinese Cabbage [Brassica rapa L. ssp. pekinensis (Lour.)] Through Microspore Culture

( Mi Hyun Lee ),( Chan Ju Lim ),( In Ho Lee ),( Jun Ho Song ) 한국육종학회 2014 Plant Breeding and Biotechnology Vol.2 No.2

The purpose of this study was to produce doubled haploid red color heading type Chinese cabbage using isolated microspore culture. Genotypic differences in embryogenic response and regenerative ability of microspore-derived embryos to plants were observed. A high rate of plant regeneration from microspore-derived embryos was achieved by an improved protocol involving replacement of culture media and adjustment of heat shock temperature. More than 60% of regenerated plants were spontaneous doubled haploids. Haploids were characterized by short and malformed stamen and few viable pollen grains as compared to spontaneous doubled haploids and dihaploids. The seeds harvested from the 142 spontaneous doubled haploid plants were designated as H1; 25 seeds in each population was grown and characterized. The H1 generation has been shown to display considerable phenotypic variation and high purity. These spontaneous doubled haploids may be directly exploited as new varieties in a red colored Chinese cabbage breeding program.

Hordeum bulbosum 을 利用한 半數性 보리의 同定

Dong Hee Chung(鄭東熙),Kyung Soo Min(閔庚洙),Jong Un Chun(千鍾殷) 한국육종학회 1994 한국육종학회지 Vol.26 No.3

This study was conducted to authenticate barley haploids by cytological, electrophoretical and morphological observations in order to establish an effective bulbosum method. Somatic chromosome numbers were 7 in the suspected barley hybrids compared to 14 in both H. bulbosum and H. vulgare, even though the chromosomes of the two species were not exactly distinguished. Barley haploid was clearly shown because of the different isozyme patterns of the two species, and suspected barley haploids. The colchicine-induced diploids with H. bulbosum chromosomes always displayed several characteristics of H. bulbosum, especially prostrate habit of growth. Progeny of the diploids was morphologically identical to their seed parents, which was the additional proof that they were diploids of genetically complete barley haploids. Genotypic variation for haploid embryo formation in eleven selected Korean barley cultivars ranged from 15 to 52%, and the variation for plant development from embryo culture ranged from 18 to 55%. H. bulbosum pollinator, GBC gave a little better results for embryo formation and plant development than Sp. D. did.

Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

김영천,Thu May Phyo,Rahman Falguni Maliha,윤영재,Jang Jin Hoon,이옥란,이정환 한국식물생명공학회 2024 Plant biotechnology reports Vol.18 No.2

Pak-choi (Brassica rapa ssp. chinensis) is a popular vegetative crop in southern China, East Asia, and Southeast Asia. Owing to the threat of climate change, rapid breeding strategies for vegetable cultivars that are tolerant to abiotic and biotic stresses are required. Thus, the rapid fixation of useful agronomic traits using doubled haploid technology is urgent. The haploid- inducer gene is key to doubled haploidization. Two known CENH3 and pPLAIIγ genes, in which altered or partially deleted forms lead to haploid induction, were selected, and direct editing of Pak-choi CENH3 and pPLAIIγ genes (BcCENH3 and BcpPLAIIγ) was conducted using an Agrobacterium-mediated CRISPR/Cas9 system. First, BcCENH3 and BcpPLAIIγ genes were characterized by analyzing the spatial expression patterns and subcellular localization. The CENH3 expression levels in carpels and pPLAIIγ in various parts of Pak-choi flowers were higher than those of other parts. BcCENH3 and BcpPLAIIγ proteins targeted in the nucleus and plasma membrane, respectively. Whole plants were successfully regenerated from the shoot apical meristem (SAM) regions of Pak-choi seedlings using the optimized procedure and culture conditions. The regeneration results of SAM explants after Agrobacterium-mediated transformation of constructs expressing CRISPR/Cas9 and BcCENH3 or BcpPLAIIγ sgRNAs confirmed four independent BcCENH3-targeted transgenic lines with 2.1%, 1.8%, 1.8%, and 1.7% INDEL frequencies, and three independent BcpPLAIIγ-targeted transgenic lines with 24.5%, 33.7%, and 33.0% INDEL frequencies. Thus, our results suggested the possibility of developing transgenic Pak-choi lines by applying the CRISPR/Cas9 genome editing technology to BcCENH3 and BcpPLAIIγ as two haploid-inducer genes.

Agro-morphological Characterization of Anther Derived Plants in Sweet Pepper (Capsicum annuum L. cv. Boogie)

Surendra Lal Shrestha,Binod Prasad Luitel,강원희 한국원예학회 2011 Horticulture, Environment, and Biotechnology Vol.52 No.2

This study was conducted in sweet pepper ‘Boogie’ to produce the haploids and diploids via anther culture, and to characterize their plant and fruit characters. Anthers were cultured on Dumas de Vaulx medium, (C-medium) supplemented with 0.1 mg・L-1 kinetin and 0.1 mg・L-1 2, 4-D, and 3% sucrose and 0.32% phytagel. Cultures were first incubated in dark at 35℃ for one week and then transferred at 25℃ under 16 h photoperiod for 40 days. Regenerated plantlets including haploids and diploids were evaluated for their plant and fruit characters at Hwacheon in spring, 2007. Results revealed that 39.6 % callus was formed in total anthers (6100) whereas only 1.78% plants were regenerated among them. Ploidy analysis showed that 78.1% regenerants were diploids. Haploid plants were characterized by short plant height, narrow leaf size, short internode length, small flower bud size, and small fruit with low volume than diploid plants. Average fruit volume measured in diploids was 62.7% higher than haploids. Variation in plant and fruit characters was observed within the diploid population and pepper breeder could utilize such variation in breeding program. Based on this characterization and compared to original cultivar, SP94, SP117, SP148, SP171 and SP174 are found superior diploid lines and recommended them to evaluate for variety improvement in sweet pepper.

야생보리(Hordeum bulbosum)의 주요 특성 및 이를 이용한 보리 반수체육성

鄭東熙,閔庚洙,千鍾殷 韓國作物學會 1993 Korean journal of crop science Vol.38 No.6

Hordeum bulbosum에 의한 보리 반수체 이용육종은 단기간에 목적 형질을 고정하는 일반적인 이점 외에 세포학적으로 안정된 많은 반수체를 용이하게 얻을 수 있는 기능성이 있기 때문에 이 방법의 실용화을 제고하기 위한 방법을 체계화하기 위해 일년의 실험을 실시하였으며 우선, H. bulbosum의 여러 형질의 특성 및 재배종과의 교배시 유배형성율등을 조사하였다. H. bulbosum은 H. vulgare에 비하여 간이 가늘고 간장이 길며 세엽이나, 수장이 길고 수당영화수가 2배이상 많으며 수장개화일수가 3배이상 길었으며, H bulbosumrks에는 차이가 적었다. H. bulbosum중 4배체는 2배체에 비하여 유배형성율은 8%, 식물체 발생율은 2배이상 높으나 염색체의 일부가 완전히 소실되지 않으므로 보리의 반수체를 이용한 육종에는 활용할 수 없는 것으로 인정되었다. Bulbosum method, which haploids are developed by pollinating with H. bulbosum have been productive to be recommended as an effective method for the production of genetically stable normal barley hybrids. The purpose of this study is to investigate several characters of H. bulbosum, seed set and embryo development rate pollinated with H. bulbosum in order to establish a method for improvement of embryo development and to find conditions favoring plant development from the embryos cultured in vitro. Three lines of H. bulbosum used as pollinators: GBC(2~times ), Spenish diploid(2~times ) and var. Jaaska (4~times ) were morphologically similar, having characteristically narrow leaves, narrow and long culms, long spikes and anthesis duration in comparison with H. vulgare. H. bulbosum, var. Jaaska(4~times ) on being pollinated to barley cultivars, increased embryo formation by 8% and plant development in vitro by three times compared to the other diploid lines. the plants developed were not normal barley hybrids but had some H. bulbosum chromosomes uneliminated, indicating that the line was unstable as a pollinator to induce barley haploids.

Assessment of genetic diversity in androgenic-based doubled haploid-derived improved restorer lines of indica rice

Bhuyan Sudhansu Sekhar,Barik Durga Prasad,Dash Byomkesh,Rout Prachitara,Pattnaik Snigdha Samir,Verma Ramlakhan,Katara Jawahar Lal,Parameswaran C.,Devanna B. N.,Sahoo Raj Kishore,Mishra Anindita,Sabari 한국작물학회 2024 Journal of crop science and biotechnology Vol.27 No.2

Hybrid rice has a considerable yield advantage over inbred lines, but the adoption rate in India is quite slow. In a three-line hybrid rice breeding programme, the development of fertility restorer lines that have enhanced grain quality is crucial. Employment of doubled haploid (DH) technology will accelerate the development of improved R lines in a short period of time, thus hastening the pace of hybrid rice breeding. This investigation was carried out for the evaluation of genetic diversity and assessment of population differentiation in 113 DHs derived from BC1F1s of IMP. IR 42266-29-3R and Nagina 22. A total of 46 SSR (Simple Sequence Repeat) markers resulted in an average of 2.00 alleles per locus. Polymorphism information content (PIC) value ranged from 0.31 to 0.37 with an average of 0.36. RM496, RM25520, RM483 and RM553 were found to be the best markers for the identification of genetic diversity. Structure analysis classified 113 DHs into four groups, which matched the Neighbour-Joining method using UPGMA cluster analysis. The AMOVA results demonstrated substantial genetic variations within subpopulation than among sub-population. The SSR marker-based molecular fingerprinting could serve as a sound basis in the identification of genetically distant as well as in the duplicate sorting of the morphologically close population in future breeding programmes

고추의 소포자 배양 시 배지 첨가와 진탕이 배의 생산에 미치는 영향

안동주,박은준,김문자 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.1

본 연구에서는 고추의 나출 소포자 배양 시 배양 중 새배지의 첨가와 진탕이 배의 생산에 미치는 영향을 조사하였다. 액체-2층 배양에서 초기 액체배양 시에 새 배지를 첨가하면 배의 발생과 발달 모두 크게 증가하였다. 가장 효과적인 첨가 시기는 초기액체 배양을 시작한 5일후 이었다. 한편 액체-2층 배양에서 후기 2층배양 시의새 배지 첨가는 초기 액체배양 시 첨가 때에 비해 그 효과가 적었다. 액체-2층 배양에서 후기 2층배양 시의 1주간 진탕은 정상 자엽배 생산에 효과적이었다. 액체배양시에도 배양 1주 후의 1주간 진탕은 배의 발달에 효과적이었다. 그러나 액체-2층배양 시와 액체배양 시 모두 진탕기간이 2 ~ 3 주간으로 길어질 때에는 배의 발달이 비정상적이었다. 본 실험 결과 얻어진 정상 자엽배들은 재분화 배지에 이식 시 용이하게 유식물체로 발달하였다. 재분화 식물체들 중에는 반수체와 배가반수체가 혼재하였으며, 이들 간에는 공변세포 내 엽록체 수의 차이가 뚜렷하였다. 이와 같은 결과들은 고추에서 다수의 정상자엽배를 생산할 수 있는 소포자 배양 시스템을 확립하는데 중요한 기초자료가 될 것이다. The influences of the agitation as well as the addition of medium during culture on the production of embryos were invested in isolated microspore culture of hot pepper (Capsicum annuum L.). When the culture medium was added during initial liquid culture step of liquid-double layer culture, the embryo yield and quality greatly increased. The most effective time point for medium addition was 5 days after the culture commenced. On the other hand, the effect of medium addition at later double layer culture step in liquid-double layer culture on the embryo production was less compared to that of medium addition during the initial liquid culture step. Agitating the culture for 1 week during later double layer culture step in liquid-double layer culture effectively increased the production of normal cotyledonary embryos. In the case of liquid culture, agitating the culture for 1 week from 7 days after the culture commenced was also effective for embryo development. However, when the total agitation time was longer (2 to 3 weeks) during liquid-double layer culture or liquid culture, the embryos developed abnormally in both cases. The normal cotyledonary embryos obtained in this study successfully developed to plants when transferred to regeneration media. These regenerated plants were either diploid or haploid, and there was a difference in the number of chloroplasts between guard cells of diploid and haploid. These results can be used as an important data for developing an efficient microspore culture system with high quality embryo production in hot pepper.