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콩() 하배축을 이용한 효율적인 원형질체 분리법

김재환1, 정영엽2,3,4, 박현우5, 서필준2,3,4, 김경도6,7,*

An Efficient Protoplast Isolation Method Using Hypocotyl in Soybean (Glycine max)

Jaehwan Kim1, Yeong Yeop Jeong2,3,4, Hyunwoo Park5, Pil Joon Seo2,3,4, Kyung Do Kim6,7,*
Korean Journal of Breeding Science 2025;57(1):1-11.
Published online: March 1, 2025

1명지대학교 생명과학정보학과

2서울대학교 식물유전체육종연구소

3서울대학교 화학부

4성균관대학교 생명과학과

5LG화학 차세대소재연구소 생분해성 SAP PJT

6세종대학교 스마트생명산업융합학과

7세종대학교 천연물소재융복합연구소

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4Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea

5Biodegradable SAP PJT, Advanced Materials Research Center, LG Chem, Seoul 07794, Republic of Korea

6Department of Integrative Biological Sciences and Industry, Sejong University, Seoul 05006, Republic of Korea

7Convergence Research Center for Natural Products, Sejong University, Seoul 05006, Republic of Korea

*Corresponding to Kyung Do KimTEL. +82-2-3408-1967E-mail. kdkim@sejong.ac.kr
• Received: September 11, 2024   • Revised: December 8, 2024   • Accepted: January 20, 2025

Copyright © 2025 by the Korean Society of Breeding Science

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  • Soybean is one of the most important crops because of its high protein and oil content. Previous studies have refined protoplast isolation methods for soybeans to enhance transfection efficiency. However, these methods have limitations due to the inconsistent number of viable protoplasts for various applications. In this study, we propose an optimized protoplast isolation method to overcome this challenge. Hypocotyls grown in the dark were selected to ensure rapid growth and a steady supply of plant materials. The hypocotyls were cut to 1–2 cm in length and halved longitudinally to achieve consistent protoplast yields. Our new hypocotyl cutting method demonstrated 1.5 times improved protoplast yield and improved protoplast viability compared to that of previous methods. The isolated protoplasts were purified using the sucrose density gradient purification method to remove residues while stacking viable protoplasts. Fluorescein diacetate (FDA) staining was performed to determine the proportion of healthy protoplasts throughout the process. Consequently, we propose a new protoplast isolation protocol that ensures a higher yield, better viability, and healthier conditions. This enhancement is expected to improve the efficiency of soybean transfection.
원형질체는 식물세포에서 세포벽을 제거한 상태의 세포로(Cocking 1960), 정상 세포와 유사한 특성과 활성을 가진다(Gresshoff 1980, Melchers & Labib 1974, Ozojiofor 2017). 최근까지 원형질체는 세포 내 작용 확인(Li et al. 2023, Marx 2016, Sheen 2001, Uhrig & Moorhead 2011), 식물체 재분화(Chupeau et al. 2013, Ikeuchi et al. 2019, Jeong et al. 2021, Sakamoto et al. 2022), CRISPR-Cas9을 이용한 유전자 교정 연구(Kim et al. 2015, Liang et al. 2017, Lin et al. 2018, Lin et al. 2020, Wang et al. 2017) 등에 활발하게 사용되고 있다.
일반적으로 원형질체는 배축, 잎 혹은 종자와 같은 식물 조직에서 추출된다(Davey et al. 2005). 원형질체 추출 효율은 식물 종, 식물 조직 및 방법에 따라 다르다. 애기장대, 담배, 옥수수의 경우 잎 조직에서 원형질체가 효율적으로 추출된다고 알려져 있으며(Kanai & Edwards 1973, Nagata & Takebe 1971, Yoo et al. 2007), 유채와 콩은 배축 조직에서 효율적으로 추출된다고 보고하고 있다(Hammatt & Davey 1988, Poulsen & Nielsen 1989). 원형질체를 추출할 때 사용되는 식물 조직의 성장 정도와 환경은 추출되는 원형질체의 수량과 상태에 큰 영향을 준다(Chen et al. 2023, Cho et al. 2021, Xu et al. 2021). 그러므로 원형질체를 추출할 때 사용하는 조직과 조직의 생장 정도를 선정하는 것은 중요하다. Wu & Hanzawa (2018)에서는 콩의 본엽을 이용하여 원형질체를 추출하였는데, 이 경우 잎이 성숙해짐에 따라 세포벽의 분해가 어려워져 초기 발달 단계의 잎만 사용해야 한다고 주장한다. 더 나아가, 원형질체 추출 시 사용하는 재료의 전처리 방법에 따라 달라지기도 한다. Moon et al. (2021)에서는 감자의 잎을 이용한 원형질체 추출 시 테이프를 이용하여 잎의 표피층을 제거한 방법, 잎을 2 mm의 두께로 얇게 자르는 방법, 잎맥을 따라 2-3 mm 크기로 상처를 내는 방법 총 세 가지 방법으로 원형질체를 추출하여 추출된 원형질체 수를 비교한 결과, 세 번째 방법이 첫 번째 방법보다 3배 이상 많은 원형질체 추출 효율을 보였다. 이처럼 원형질체 추출의 효율은 다양한 요소에 의해 영향을 받기 때문에 땅콩(Biswas et al. 2022), 배추(Sivanandhan et al. 2021), 옥수수(Hu et al. 2020)와 같은 식물에서 원형질체 추출법에 대한 최적화를 위한 연구가 진행되었다. 원형질체를 이용한 실험 진행 시 효소에 의해 분해되고 남은 세포벽의 잔해와 죽은 원형질체의 잔해는 원형질체의 사멸을 유발하여 실험 결과에 부정적인 영향을 줄 수 있다(Castelblanque et al. 2010). 이를 방지하기 위해 실험의 목적에 따라 추출한 원형질체를 sucrose density gradient 기반의 정제방법을 사용해 세포벽과 죽은 원형질체의 잔해를 제거하여 살아있는 원형질체만 정제하여 사용한다(Brandt et al. 2020, Panda et al. 2024).
콩은 식물성 단백질과 기름의 원료가 되는 중요한 작물 중 하나이며(Park et al. 2016, Sedivy et al. 2017, Song et al. 2013), 최근까지 콩에서도 다양한 연구를 위해 원형질체가 사용되어 왔다. 콩 원형질체 추출에는 잎(Lin 1983, Sun et al. 2015, Wu & Hanzawa 2018), 미성숙 자엽(Demorest et al. 2016), 미성숙 종자(Patil et al. 2022)와 같은 조직을 사용하고 있는데, 사용하는 조직에 따라 조직을 전처리하는 방법이나 사용하는 효소 용액의 조성이 조금씩 다르다. Sun et al. (2015)에서는 잎을 얇게 잘라 0.5% cellulose R10, 0.5% macerozyme R10, 0.1% pectolase Y23, 0.6 M mannitol, 10 mM MES pH 5.7, 20 mM KCl, 10 mM CaCl2 and 0.1% BSA 용액으로 원형질체를 추출하였으며, Demorest et al. (2016)에서는 미성숙 자엽을 활용해 0.5 M D-Mannitol 과 20 mM MES, 2% cellulose, 0.5% macerozyme 용액으로 원형질체를 추출하였다. Patil et al. (2022)에서는 미성숙 종자를 0.5 mm 크기로 자른 후 0.45 M D-Mannitol, 20mM MES (pH 5.7), 2% Cellulase R-10, 0.5% Macerozyme R-10 용액을 사용하여 원형질체를 추출하였다. Cho et al. (2021)에서는 여러 콩 조직을 비교하여 원형질체 추출 효율이 가장 높은 조직과 효소 용액 조합을 제시하였다. 콩의 하배축, 본엽, 삼복엽에서 원형질체를 추출하여 비교하였을 때 암 조건으로 기내 배양한 하배축 조직이 원형질체 분리의 효율이 가장 높음을 확인하였고, 다양한 효소 용액의 조합을 사용한 비교 실험을 통해 0.5% Cellulase, 0.5% Pectinase, 1% Viscozyme 조합의 효소 용액에서 효율이 가장 높은 것을 밝혔다.
본 연구에서는 이전 연구에서 원형질체 추출 효율이 가장 높은 하배축 조직을 이용하되, 조직 전처리 방법을 최적화하여 기존 방법보다 콩 원형질체 추출 효율을 향상시키고자 하였다. 원형질체 추출에는 암 조건에서 7일간 기내 배양한 콩 하배축을 사용하였으며, 하배축을 0.5~1 mm 크기로 자르는 기존 절단 방법(Cho et al. 2021)과 1~2 cm 크기로 자른 후 수직으로 이등분하는 개선된 절단 방법으로 원형질체를 추출하여 결과를 비교하였다. Fluorescein diacetate (FDA) 염색법을 통해 살아있는 원형질체의 비율을 확인하였으며, sucrose density gradient를 이용해 원형질체를 정제한 결과를 확인하였다.
식물재료
콩 실험 재료는 Williams 82를 사용하였다. 암 조건에서 기내 배양한 하배축을 원형질체 추출을 위한 조직으로 사용하였으며, 동일 조직을 사용하는 선행 연구(Cho et al. 2021)의 하배축 절단 방법을 참고하여 비교 실험을 진행하였다.
건실한 상태의 종자를 선별하여 소독을 위해 70% ethanol에 2분간 교반하고, 락스 수용액 (25% 락스, 0.1% Tween-20)에 20분간 교반하였다. 소독이 끝난 종자는 멸균한 증류수로 3~4번 세척하여 잔여 락스 수용액을 제거하고, 멸균된 filter paper 위에 종자를 올려 물기를 제거하였다. 물기를 제거한 종자를 1/2 MS 배지(2% sucrose, 0.7% plant agar, pH 5.8) plate (150 mm×20 mm)에 9립씩 치상하여 랩 테이프로 밀봉 후 25℃에서 암 조건으로 7일간 배양하였다(Fig. 1A).
원형질체 추출
배양한 plate에서 오염이 없는 식물체를 선별해 의료용 메스로 뿌리와 자엽을 제거하여 하배축만 추출하였다. 하배축을 자르는 방법에 따른 결과 비교를 위해 참조한 논문에서 제시한 0.5-1 mm 크기로 자른 대조군과 1-2 cm 크기로 자른 후 단면에 수직으로 자른 실험군으로 나누어 비교 실험을 진행하였다. 자른 하배축을 petri dish (90 mm×20 mm)에 옮겨 0.5 M D-mannitol 용액(2 mM MES.H2O, 0.5 M D-mannitol, pH 5.8) 20 mL에 하배축의 단면적이 용액에 완전히 잠기도록 하여 1시간 동안 상온에서 반응시켰다(Fig. 1B). 1시간 동안 반응시킨 후 petri dish의 0.5 M mannitol 용액을 최대한 제거하고 MMC solution (10 mM MES.H2O, 0.467 M D-mannitol, 10 mM CaCl2, pH 5.8)에 2% Viscozyme (Viscozyme®, Novozymes, Denmark), 1% Celluclast (Celluclast®, Novozymes, Denmark), 1% Pectinex (Pectinex® ultra SP-L, Novozymes, Denmark)을 혼합한 VCP 효소 용액을 20 mL 넣어주었다. VCP 효소 용액을 넣어준 후 상온과 암조건에서 40 rpm으로 12-16시간 동안 진탕 반응시켰다. 반응시킨 VCP 효소 용액의 상등액을 40 µm 세포 여과망(SPL Cell Strainer, SPL, Republic of Korea)으로 여과하여 원형질체를 제외한 잔해물을 걸러주었다. 여과한 상등액은 50 mL centrifuge tube에 10 mL씩 옮기고 W5 용액(2 mM MES.H2O, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM Glucose, pH 5.8)을 5 mL을 추가하였다. 스윙형 로터를 사용해 150×g에서 10분간 원심분리하고, 상등액을 제거하였다.
원형질체를 세척하기 위해 튜브에 W5 용액 10 mL을 넣어준 후 조심스럽게 튜브를 기울여주며 침강된 원형질체를 완전히 풀어주었다. 세척한 원형질체는 스윙형 로터를 사용해 150×g에서 10분간 원심분리하고, 상등액을 제거하였다. 위 작업을 같은 방식으로 한 번 더 세척하여 총 2번 W5 용액으로 세척해주었다.
Sucrose density gradient 정제
W5 용액으로 세척한 원형질체는 sucrose density gradient 정제 방법에 따른 결과를 비교하기 위해 정제를 진행한 실험군과 그렇지 않은 대조군의 원형질체의 개수와 상태를 비교하였다. 대조군은 침강된 원형질체에 W5 용액 5 mL를 넣고 풀어준 후 혈구계수기를 사용하여 원형질체의 개수를 측정하였다. 실험군은 침강된 원형질체에 W5 용액 5 mL를 넣고 풀어주었다. 풀어준 원형질체를 0.6 M sucrose 용액(0.6 M sucrose, 2 mM MES.H2O, pH 5.8) 10 mL가 들어있는 50 mL centrifuge tube에 벽을 따라 조심스럽게 넣고, W5 용액층과 0.6 M sucrose 용액층이 분리된 상태(Fig. 1C)에서 40×g에서 5분간 원심분리 하였다. 원심분리 후 W5 용액과 0.6 M Sucrose 용액의 경계면에 생성된 원형질체층(Fig. 1D)을 새로운 50 mL centrifuge tube로 옮겨주었다. 이후 원형질체 세척을 위해 튜브에 W5 용액 10 mL를 넣고 100×g에서 10분간 원심분리 후 상등액을 제거하는 작업을 2회 반복하였다. 침강된 원형질체에 W5 용액 5 mL를 넣어준 후 혈구계수기를 사용하여 원형질체의 개수를 측정하였다.
FDA 염색
원형질체 계수 후 세포 상태를 비교하기 위해 fluorescein diacetate (FDA) 형광 염색법(Jones & Senft 1985)을 다음과 같이 진행하였다. 원형질체가 들어있는 W5 용액 5 mL에 FDA 용액(5 mg of FDA in 1 mL acetone) 10 µL를 넣고 4℃ 암 조건에서 5분간 반응시킨 후 150×g에서 10분간 원심분리 하였다. 상등액을 제거하고 침강된 원형질체에 W5 용액 5 mL을 넣고 튜브를 부드럽게 흔들어 세포를 풀어주었다. 공초점 현미경(C1 confocal microscope, Nikon, Japan)을 이용해 살아있는 원형질체 내 esterase가 FDA를 fluorescein으로 분해하여 녹색 형광을 발현하는 원형질체의 수를 계수하였다(Jones & Senft 1985). 모든 원형질체는 20배율로 관찰하였고 살아있는 상태의 원형질체 비율은 (녹색 형광 발현 원형질체 수/전체 원형질체 수)×100%로 계산하였다.
데이터 통계적 분석
원형질체 추출 효율과 viability를 확인한 모든 실험은 3반복하였으며 결과값은 단측 t-검정(one-tailed Student’s t-test)을 수행하여 통계적 검증을 하였다. t-검정 결과 p-value가 0.05 이하인 경우에만 유의한 차이가 있는 것으로 판단하였다.
절단 방법에 따른 결과 비교
본 연구에서는 원형질체 추출에 사용하는 하배축 조직을 0.5 M mannitol 용액에서 1시간 상온 반응을 시키기 전 해당 조직을 전처리하는 방법에 따른 원형질체 추출 효율을 비교하였다. Cho et al. (2021)에서 사용한 전처리 방법으로 하배축을 0.5-1 mm로 자르고 수직으로 이등분하지 않는 기존의 절단 방법(Fig. 2A)과 하배축을 1-2 cm 간격으로 절단한 후 수직으로 이등분하는 본 연구의 개선된 절단 방법(Fig. 2B)으로 원형질체를 추출하였으며, 이를 3반복 하여 그 결과를 비교하였다(Fig. 2C-2F). 기존 절단 방법의 경우 평균 8.6×105 protoplasts/g의 원형질체가 추출되지만, 개선된 절단 방법의 경우 기존 방법보다 약 1.5배 더 많은 평균 13.23×105 protoplasts/g의 원형질체가 추출되었다(Fig. 3A, p-value=0.012). 두 가지 방법으로 추출한 원형질체를 FDA 염색법(Jones & Senft 1985)을 사용해 살아있는 원형질체의 비율을 비교하였다. 기존 방법의 경우 전체 원형질체 중 살아있는 원형질체의 비율이 평균 56% (49%, 52%, 67%)이었으며, 개선된 방법의 경우 살아있는 원형질체의 비율이 평균 79% (70%, 75%, 92%)로 기존 방법보다 약 1.4배 더 높은 비율을 보였다(Fig. 3B, p-value=0.029). 결과적으로 기존 절단 방법의 경우 4.8×105 protoplasts/g의 효율로 살아있는 원형질체를 추출하였고 개선된 절단 방법의 경우 2배 이상 많은 10.5×105 protoplasts/g의 효율로 살아있는 원형질체를 추출하였다.
원형질체를 사용하는 실험에서는 살아있는 원형질체를 높은 효율로 일관되게 추출하는 것이 매우 중요하다. 이를 위해 효소용액과 식물조직의 반응시간을 비교하거나(Jones et al. 2012), 추출된 원형질체의 생존율을 높이는 버퍼 조성을 사용하는 등(Matchett-Oates et al. 2021, Savviou et al. 2021) 다양한 연구가 선행되었다. 특히, 원형질체 추출 시 식물세포에서부터 세포벽을 분리하기 위해 효소용액를 처리하는데, 이 과정에 사용되는 식물조직시료를 준비하는 전처리 과정에 따라 원형질체의 분리효율이 달라지기도 한다. Li et al. (2022) 에서는 동백(Camellia oleifera)의 잎을 진공 처리하여 원형질체를 최대 3.5×107 protoplast/g 의 높은 효율로 추출하였으며, Deng et al. (2023)에서는 효소용액 처리 전 제비쑥(Artemisia japonica)의 잎을 원형질 분리 처리(pre-plasmolysis treatment)하여 원형질체 추출 효율(1.01× 106 protoplast/g)과 생존력을 모두 향상 시키기도 하였다.
식물조직의 전처리 과정이 중요한 이유는 원형질체를 추출할 때 효소용액과 반응하는 단면적의 넓이와 조직의 손상되는 정도가 추출 효율에 영향을 주기 때문이다. 효소용액에 반응하는 단면적을 넓이기 위해 조직에 손상을 과하게 주게 된다면 추출하고자 하는 세포도 손상되는 상충 관계가 발생할 수 있다. Wu et al. (2009)에서는 테이프를 사용하여 애기장대 잎의 표피를 제거하는 방식을 새롭게 제시하여 잎을 자르는 기존 방식과 원형질체 추출 효율을 비교하였으며, 그 결과 잎을 자른 방식이 더 높은 원형질체 추출 효율을 보였으나 잎 표피를 제거하는 방식에서 손상되지 않은 원형질체가 더 균일하게 추출되었다. 이에 비해 Moon et al. (2021)에서는 감자 잎에서 두 방식의 원형질체 추출 효율을 비교하였는데, 감자 잎의 경우 잎을 자르는 방식이 조직의 손상도가 적어 잎 표피를 제거하는 방법에 비해 4배 이상의 원형질체 추출 효율을 보였으며, 배양 및 캘러스 유도에도 적합하였다. 이와 같이 동일한 방법이라도 사용하는 식물 종과 조직에 따라 전처리 효과가 다를 수 있기에 살아있는 원형질체를 높은 효율로 일관되게 추출하기 위해서는 사용하고자 하는 종과 조직에 최적화된 전처리 방법이 정립되어야 한다.
본 연구에서는 콩 원형질체 추출 효율과 생존력을 높이기 위해 전처리 과정에서 하배축을 절단하는 방식을 변경하여 조직의 손상을 최대한 줄이고자 하였다. Cho et al. (2021)에서는 하배축을 0.5-1 mm 간격으로 자르고 세로로 이등분하지 않는 방식을 사용하였으나 살아있는 세포를 정량한 결과를 제시하지 않아 해당 방법에서 추출된 살아있는 세포의 비율을 정확히 알 수 없었다. 본 연구에서는 이를 동일한 실험조건에서 비교하기 위해 FDA 염색법을 사용하여 두 가지 방법을 반복실험을 통해 비교하였다. 결과적으로 같은 양의 하배축을 사용했을 때 1-2 cm 간격으로 자른 후 세로로 이등분 하는 방식으로 원형질체 추출 시 살아있는 원형질체 수가 2배 이상 많았다. 콩 하배축은 세포 신장을 통해 성장하는데(Shen & Chen 2022, Wang et al. 2018), 기존 방법과 같이 신장하는 방향과 수직으로 절단 시에는 세포에 손상이 가해질 가능성이 높다. 이에 비해 신장하는 방향과 수평으로 절단하고 수직으로 절단하는 간격을 0.5-1 mm에서 1-2 cm로 늘려 절단 횟수를 1/20로 줄이게 되면 기존 방식에 비해 효소용액과 반응하는 단면적은 큰 차이가 없으나 하배축 조직 내 세포의 손상을 줄여 원형질체 추출 효율을 높이는 것으로 사료된다. 본 연구에서 제시하는 방법을 사용하여 원형질체를 추출한다면 살아있는 원형질체의 수를 늘림으로써 일시적 형질전환이나 재분화 실험에 긍정적인 영향을 줄 수 있을 것이다.
Sucrose density gradient 정제에 따른 결과 비교
원형질체의 손상을 최소화하기 위해서는 원형질체의 정제방법이 중요하다(Moon et al. 2021). 추출한 원형질체에서 sucrose density gradient 정제 효과를 확인하기 위해 정제를 진행하지 않은 대조군과 정제를 진행한 실험군으로 나누어 비교 실험을 진행했다(Fig. 4). 그 결과, 대조군의 평균 12.8×105 protoplasts/g의 원형질체가 추출되었으며 실험군의 경우 평균 6.7×105 protoplasts/g의 원형질체를 추출할 수 있었다(Fig. 5A, p-value=0.013). 이후, FDA 염색법을 이용하여 대조군과 실험군의 원형질체에서 건강한 원형질체의 비율이 비교되었다. 대조군에서는 평균 67%의 건강한 원형질체가 관찰되었으며, 실험군에서는 평균 90%의 건강한 원형질체가 확인되었다(Fig. 5B, p-value=0.017). 결과적으로, 대조군에서는 7.68×105 protoplasts/g, 실험군에서는 6.03×105 protoplasts/g의 건강한 원형질체를 정제할 수 있었다. 또한 실험군에서는 sucrose density gradient 정제를 통해 세포의 사멸을 유발하는 세포벽과 죽은 세포의 잔해(Zhang et al. 2022)가 대부분 제거되어 관찰하기 어려웠다(Fig. 4). 이러한 결과로부터, sucrose density gradient 정제 방법은 건강한 원형질체의 비율을 높이고 세포벽 및 죽은 세포의 잔해를 제거하여 이후 원형질체의 생존율을 높일 수 있을 것으로 사료된다.
콩은 경제적, 산업적으로 중요한 곡물 중 하나이다. 식물의 원형질체는 재분화, 일시적인 형질전환, 유전자 교정 등에서 사용된다. 본 연구에서는 콩에서의 원형질체 추출법을 최적화 하기 위해 생장이 빠르고 환경요소의 변수가 적은 암 조건에서 기내 배양한 콩의 하배축을 재료로 사용하였다. 기존의 하배축을 사용한 원형질체 추출법을 개선하기 위해 하배축을 자르는 방법을 변경하여 1.5배 이상 많은 원형질체를 추출하였으며 살아있는 상태의 원형질체만을 비교하였을 때 2배 이상 많은 원형질체를 추출하였다. 또한 sucrose density gradient 정제 방법을 통해 건강한 원형질체의 비율을 1.3배 이상 올릴 수 있었으며, 세포의 사멸을 유발하는 세포벽과 죽은 세포의 잔해를 제거할 수 있음을 확인하였다. 이를 통해 콩 원형질체를 이용한 연구 시 긍정적인 영향을 줄 수 있을 것으로 사료된다.
본 논문은 농촌진흥청 연구사업(과제번호: RS-2023-00220176)의 지원에 의해 이루어졌습니다.
Fig. 1
Process of sucrose density gradient purification method. (A) 7 days-old seedlings grown at 25℃ under dark condition. (B) Preplasmolysis of hypocotyls in 0.5 M mannitol solution. (C) Protoplast resuspension solution with 0.6 M sucrose solution. Protoplasts are resuspended in W5 solution (upper, cloudy solution) and 0.6 M sucrose solution (lower, transparent solution). (D) After centrifugation of protoplast resuspension solution. The red circle indicates protoplast layer between W5 solution (upper) and 0.6 M sucrose solution (lower).
kjbs-57-1-1-f1.jpg
Fig. 2
Comparison of protoplast viability by using different methods of hypocotyl sample preparation. (A) Hypocotyls cut by 0.5-1 mm in length. (B) Hypocotyls cut by 1-2 cm in length and longitudinally in half. Bright field image (C) and fluorescence field image (E) of protoplast isolated from hypocotyls cut by 0.5-1 mm in length. Bright field image (D) and fluorescence field image (F) of protoplast isolated from hypocotyls cut by 1-2 cm in length and longitudinally in half. All images are observed under Confocal Laser Scanning Microscopy (CLSM) at 20X magnification. Viable protoplasts are stained by FDA. Fluorescence of FDA is shown in green. The scale bar is 40 µm.
kjbs-57-1-1-f2.jpg
Fig. 3
Protoplast isolation efficiency and viability by using different methods of hypocotyl sample preparation. (A) Quantification of protoplast counts per fresh tissue weight (FW) (g). (B) Viability of protoplast (%). Previous method: hypocotyls cut by 0.5-1 mm in length, improved method: hypocotyls cut by 1-2 cm in length and longitudinally in half. The error bars indicate the standard deviation of three replicates. One-tailed Student’s t-tests were conducted to test the differences between the previous and improved methods statistically (*p-value<0.05).
kjbs-57-1-1-f3.jpg
Fig. 4
Comparison of protoplast viability by using different methods of protoplast purification. Bright field image (A) and fluorescence field image (C) of protoplast isolated without sucrose density gradients purification. Bright field image (B) and fluorescence field image (D) of protoplast isolated with sucrose density gradients purification. Red points indicate cell wall and dead cell debris. All images are observed under Confocal Laser Scanning Microscopy (CLSM) at 20X magnification. Viable protoplasts are stained by FDA. Fluorescence of FDA is shown in green. The scale bar is 40 µm.
kjbs-57-1-1-f4.jpg
Fig. 5
Protoplast isolation efficiency and viability by using different methods of protoplast purification. (A) Quantification of protoplast counts per fresh tissue weight (FW) (g). (B) Viability of protoplast (%). Control group: sample without sucrose density gradient purification, Experimental group: sample with sucrose density gradient purification. The error bars indicate the standard deviation of three replicates. One-tailed Student’s t-tests were conducted to test the differences between the previous and improved methods statistically (*p-value<0.05).
kjbs-57-1-1-f5.jpg
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An Efficient Protoplast Isolation Method Using Hypocotyl in Soybean (Glycine max)
Korean. J. Breed. Sci.. 2025;57(1):1-11.   Published online March 1, 2025
DOI: https://doi.org/10.9787/KJBS.2025.57.1.1
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An Efficient Protoplast Isolation Method Using Hypocotyl in Soybean (Glycine max)
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DOI: https://doi.org/10.9787/KJBS.2025.57.1.1
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An Efficient Protoplast Isolation Method Using Hypocotyl in Soybean (Glycine max)
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Fig. 1 Process of sucrose density gradient purification method. (A) 7 days-old seedlings grown at 25℃ under dark condition. (B) Preplasmolysis of hypocotyls in 0.5 M mannitol solution. (C) Protoplast resuspension solution with 0.6 M sucrose solution. Protoplasts are resuspended in W5 solution (upper, cloudy solution) and 0.6 M sucrose solution (lower, transparent solution). (D) After centrifugation of protoplast resuspension solution. The red circle indicates protoplast layer between W5 solution (upper) and 0.6 M sucrose solution (lower).
Fig. 2 Comparison of protoplast viability by using different methods of hypocotyl sample preparation. (A) Hypocotyls cut by 0.5-1 mm in length. (B) Hypocotyls cut by 1-2 cm in length and longitudinally in half. Bright field image (C) and fluorescence field image (E) of protoplast isolated from hypocotyls cut by 0.5-1 mm in length. Bright field image (D) and fluorescence field image (F) of protoplast isolated from hypocotyls cut by 1-2 cm in length and longitudinally in half. All images are observed under Confocal Laser Scanning Microscopy (CLSM) at 20X magnification. Viable protoplasts are stained by FDA. Fluorescence of FDA is shown in green. The scale bar is 40 µm.
Fig. 3 Protoplast isolation efficiency and viability by using different methods of hypocotyl sample preparation. (A) Quantification of protoplast counts per fresh tissue weight (FW) (g). (B) Viability of protoplast (%). Previous method: hypocotyls cut by 0.5-1 mm in length, improved method: hypocotyls cut by 1-2 cm in length and longitudinally in half. The error bars indicate the standard deviation of three replicates. One-tailed Student’s t-tests were conducted to test the differences between the previous and improved methods statistically (*p-value<0.05).
Fig. 4 Comparison of protoplast viability by using different methods of protoplast purification. Bright field image (A) and fluorescence field image (C) of protoplast isolated without sucrose density gradients purification. Bright field image (B) and fluorescence field image (D) of protoplast isolated with sucrose density gradients purification. Red points indicate cell wall and dead cell debris. All images are observed under Confocal Laser Scanning Microscopy (CLSM) at 20X magnification. Viable protoplasts are stained by FDA. Fluorescence of FDA is shown in green. The scale bar is 40 µm.
Fig. 5 Protoplast isolation efficiency and viability by using different methods of protoplast purification. (A) Quantification of protoplast counts per fresh tissue weight (FW) (g). (B) Viability of protoplast (%). Control group: sample without sucrose density gradient purification, Experimental group: sample with sucrose density gradient purification. The error bars indicate the standard deviation of three replicates. One-tailed Student’s t-tests were conducted to test the differences between the previous and improved methods statistically (*p-value<0.05).

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An Efficient Protoplast Isolation Method Using Hypocotyl in Soybean (Glycine max)

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