2. Tricholoma matsutake Ito et Imai(1ó­ ãËÓñ)

¢À ùÛÏп¡¼­ °¡Á®¿Â жñ»

ÛãûÜ

жñ»ÛãûÜ

ùÊÙ£

ÝÂ×îò¢æ´

ÝáÍÅ

1

FRI91001

T. matsutake

Ñõê«

92. 2. 17 (îïÝÁߣ×ùü»ÌÑæÚϼᶠÝÂåÓ)

2

FRI91009

T. matsutake

¾ç¾ç

94. 10. 15.

3

FRI91013

T. matsutake

°æµ¿½ÃÀå

94. 10. ?.

4

FRI91022

T. matsutake

ȫõ

95. 9. 21.

5

FRI91026

T. matsutake

¿ïÁø

96. 7. 25.

6

FRI91036

T. matsutake

Á¦Ãµ

97. 9. 19.

7

FRI91047

T. magnivelare

Ú¸ÏÐ

97. 10 (Oregon State Univ. ÝÂåÓ)

2.1. MYG medium¸¦ ì¦éÄÇÑ ùÛÏÐߧ жñ»ÀÇ ÐñÜâ ÛÆå×

2.1.1. Slant ¹× agar plate ñÞÝá (1998Ò´ 12êÅ 24ìí)

2.1.1.1. ÛÆò¢ ù±é©Õá

- plates : 3 plates/strain total 21 plates

- slants : 5 slants/strain total 35 slants

2.1.1.2. ÛÆò¢ðÚà÷

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

slantéÄ

plateéÄ

ÝáÍÅ

1

Distilled water

100§¢

250§¢

800§¢

1ó­ ñú׶â©

2

Malt extract

1g

2.5g

8g

ð¶ã¥Ñ¦Ò® ÜÁη

3

Yeast extract

0.4g

1.0g

3.2g

ð¶ã¥Ñ¦Ò® ÜÁη

4

Glucose

0.4g

1.0g

3.2g

5

agar

1.5g

4.25g

12g

¢Ñ 20§¢/plate & 10§¢/slant

2.1.2. ÛÆò¢ Øþж ¹× slant ñÞÝá

2.1.2.1. ÛÆò¢ ð²ðã ý­ plate pouring ¹× slant ñÞÝá

2.1.2.2. slant´Â ãùúÐãøÒ® table À§¿¡ À¯¸®°üÀ» õîÀ¸·Î ´µ¿©¼­ ñÞÝáÇÔ

2.1.3. ïÈðú ¹× ÛÆå× (1998Ò´ 12êÅ 25ìí)

2.1.3.1. Plates : 3 plates/strain

2.1.3.2. Slants : 3¡­5 slants/strain (#1Àº Ó£ñþ 3ËÁÀÇ slants¸¸ ¸¸µé ¼ö ÀÖ¾úÀ½)

2.1.3.3. ÛÆå× : 2öµÀÇ ùöè®Ðï(25¡É)¿¡ ³Ö¾î ÜÁη, ÛÆå×ÇÔ

¢Ñ ßæíþÀº ±×¸® ÁÁÁö ¸øÇÑ °ÍÀ¸·Î ¿©°ÜÁü

2.2. äûô÷ÛÆò¢(MYG & PDMP media)¸¦ ì¦éÄÇÑ ìíÜâжñ»ÀÇ ïÈðú

2.2.1. ØþжÐïÎý & ÛÆò¢ ñÞÝá (1999. 1. 12)

2.2.1.1. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

Komagome(ÏËçÊ) pipette

10§¢

2

autoclave

2

Silicon cap(pipetteéÄ)

10§¢éÄ

2

autoclave

3

Filtering mesh

120§­

4

autoclave on funnel

2.2.1.2. ÛÆò¢ - ÛÆò¢Ü¬ 2 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

2.2.1.2.1. MYG medium

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ(50§¢)

ÝáÍÅ

1

Distilled water

100§¢

50§¢

2

Malt extract

1g

0.5g

3

Yeast extract

0.4g

0.2g

4

Glucose

0.4g

0.2g

2.2.1.2.2. PDMP medium

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ(50§¢)

ÝáÍÅ

1

Distilled water

100§¢

50§¢

2

Potato dextrose broth

2g

1g

3

Malt extract

0.3g

0.15g

4

Peptone

0.1g

0.05g

pH = 5.5

2.2.1.3. ñÞÝá : Øþжô¥×âµÈ ÐïÎý¿Í ÛÆò¢¸¦ ÙíжßÉ¿¡ ³Ö°í í¹èâàÊ lamp¸¦ ÄÑ ³õ¾ÆµÒ

2.2.2. äûô÷ÛÆò¢ ïÈðú - ÙíжßÉ¿¡¼­ íÂåö (1999. 1. 13 çíîñ)

2.2.2.1. ÕÒíúÜÁη Çß´ø slant media ß¾ÀÇ Tricholoma matsutake culture #51, #70 ÞÅéÄ

2.2.2.2. ïÈðúê« handling

¢Ñ ØþжÇÏ¿© ñÞÝáµÈ MYG ÛÆò¢ÀÇ ÇÑ ¡â-flask¿¡¼­ ÛÆå×äû 10§¢¸¦ komagome pipetteÀ» ÞÅéÄÇÏ¿© õÎõóÇÏ¿© #51 slant media¿¡ ¿Å°Ü ³ÖÀ½

¢Ñ `¤¡`ÀÚ·Î ³¡ÀÌ ÈÖ¾îÁø ÛÜÐÝ켸¦ ì¦éÄÇÏ¿© slant mediaÀÇ Ð¶ÞêöµÀ» ºÎµå·´°Ô ±Ü¾î¼­ ËíˬÇÑ Ð¶Þ곪 øàí­ ÔõÀÌ ºÎ¾î³ÖÀº äûô÷ÛÆò¢¿¡ ³ì¾Æµé ¼ö ÀÖµµ·Ï ÇÔ

¢Ñ õöÝÂÈ÷ жÞêô÷°¡ ¶³¾îÁ® ³ª¿Â °ÍÀ¸·Î ¿©°ÜÁö¸é, ãËúÐηҮÀÇ îïô÷ éÁäûÀ» filtering mesh¸¦ ÷×Φ½ÃÅ°¸é¼­ äûô÷ÛÆò¢¿¡ ¿Å°Ü ïÈðú(mesh size : 120§­)

¢Ñ °°Àº Û°ÛöÀ¸·Î ´Ù¸¥ жñ»(#70)¿¡ ÛÆå×äû 10§¢¸¦ komagome pipetteÀ» ÞÅéÄÇÏ¿© õÎõó, ¿Å±ä ý­ жÞêô÷¸¦ ÝÂ×î, ïÈðú½ÃÅ´(mesh size : 120§­)

¢Ñ ÀÌ ý­, PDMP ÛÆò¢¸¦ ì¦éÄÇÏ¿© ÔÒìéÇÏ°Ô ñÞÝá, ïÈðúÇÔ

2.2.2.3. äûô÷ÛÆò¢ ß¾¿¡¼­ÀÇ ïÚô÷ÛÆå×(ÛÆå×è®Óø : ßÈè®, ãùúÐãø)

¢Ñ ßæíþÀÌ ³Ê¹« ´ÊÀº °ÍÀ¸·Î ¿©°ÜÁ® ùöè®ãø·Î 1êÅ 20ìí ì¹ÔÑ, ÛÆå×

¢Ñ 2ñÎìí ÛÆå× ý­¿¡µµ жÞêÕáÀÌ ò¸Ð¿È÷ Àû¾î¼­ ´Ù¸¥ Û°ãÒÀÇ ãËÓñ°¡ ù±é©ÇÔ

2.3. ùÛÏÐ ¹× ìíÜâߧ áæéÇ Ð¶ñ»ÀÇ î¢ÛÆå×

2.3.1. áæéÇ Ð¶ñ»(5жñ» - ùÛÏÐߧ #2, 3; Ú¸ÏÐߧ #7, ìíÜâߧ #51, 71)ÀÇ ÛÆå× ñÞÝá

2.3.1.1. slant - ùÛÏÐߧ ¹× Ú¸ÏÐߧ жñ» Í©ÓÛÛÆå×éÄ (1999. 1. 27)

- MYG medium, Hamada ÊÀÊÀ 15ËÁ¾¿(3 жñ»; жñ»Ü¬ 5ËÁ¾¿)

¡Ø MYG(¢Ñ 10§¢/ãËúÐη)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

200§¢

2

Malt extract

1g

2g

3

Yeast extract

0.4g

0.8g

4

Glucose

0.4g

0.8g

5

Agar

1.5g

3.0g

¡Ø Hamada medium(¢Ñ 10§¢/ãËúÐη)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Tap water

100§¢

200§¢

2

Glucose

2g

4g

3

Yeast extract

0.5g

1.0g

4

Agar

1.5g

3.0g

pH = 5.5

- Øþж ý­ table À§¿¡ ´µ¿©¼­ ½ÄÈû

2.3.1.2. äûô÷ÛÆò¢ - ÛÆò¢Ü¬·Î ùÛÏÐߧ ¹× Ú¸ÏÐߧ жñ» 3ËÁ¾¿, ìíÜâжñ» ÊÀ 1ËÁ¾¿

- MYG medium, PDMP ÊÀÊÀ 11ËÁ

¡Ø MYG(¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

250§¢

2

Malt extract

1g

2.5g

3

Yeast extract

0.4g

1.0g

4

Glucose

0.4g

1.0g

¡Ø PDMP medium(¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

250§¢

2

Potato dextrose broth

2g

5g

3

Malt extract

0.3g

0.75g

4

Peptone

0.1g

0.25g

pH = 5.5

2.3.1.3. жÞêï·Ó¨ ñÞÝá

- À¯¸® »þ¾Ë·¹, Pasteur pipette : (жñ»Ü¬) ÊÀÊÀ 5ËÁ

- Blade, pipette holder

2.3.2. ìíÜâ áæéÇ Ð¶ñ»(2жñ» - #51, 70)ÀÇ Ð¶Þêô÷ ï·Ó¨ ¹× äûô÷ÛÆå× (1999. 1. 27)

2.3.2.1. source ¹× ÓßßÚ

2.3.2.1.1. source : slant ß¾ÀÇ Tricholoma matsutake strains

- Áö³­ 1êÅ 13ìí¿¡ liquid culture ïÈðúê«À¸·Î ÞÅéÄÇÏ¿´´ø slants

2.3.2.1.2. ÓßßÚ : MYG ¹× PDMP liquid media

2.3.2.2. ô¥×â Û°Ûö

2.3.2.2.1. slantß¾ÀÇ Ð¶ñ»¸¦ ±Ü¾î³»¾î ØþжÇÑ À¯¸® »þ¾Ë·¹¿¡ ³õÀ½

2.3.2.2.2. ¾ç³¯ ØüÓﳯÀ» 100§¢ ºñÀÌÄ¿ÀÇ ¾ËÄڿÿ¡ ´ã¾Æ µÎ°í handlingÀ» À§ÇÑ Çɼ ñÞÝá

2.3.2.2.3. ¾ËÄڿÿ¡ ´ã¾Æ µÎ¾ú´ø ¾ç³¯ ØüÓﳯÀ» ÇɼÂÀ¸·Î ²¨³»¾î áÀûý½ÃÅ´

2.3.2.2.4. À¯¸® »þ¾Ë·¹ À§ÀÇ Ð¶Þêô÷¸¦ ØüÓﳯÀ» ì¦éÄÇÏ¿© Àß°Ô ÀÚ¸§(å³ 3¡­5ÝÂÊà ô¥×â)

2.3.2.2.5. Àß°Ô À߶óÁø жÞêô÷¸¦ ØþжÇÑ Pasteur pipetteÀ» ì¦éÄÇÏ¿© ¿øÇÏ´Â äûô÷ÛÆò¢·Î ¿Å°Ü ³Ö¾î ïÈðúÇÔ - ÀÌ ¶§, ñÞÝáµÈ ÛÆò¢¿¡¼­ ÛÆå×äûÀ» ìéÝ» ²¨³»¾î »þ¾Ë·¹¿¡ ºÎÀº ý­ ´Ù½Ã жÞêô÷¿Í ´õºÒ¾î êüìý½ÃÄѼ­ ïÈðúÅä·Ï ÇÔ

2.3.2.3. ж ÛÆå×(MYG #51, PDMP #51, MYG #70, PDMP #70)

- 2öµÀÇ ùöè®ãø(25¡É, 12ãÁÊà ÎÃñÎÑ¢)¿¡ ³Ö¾î ÛÆå×

2.3.3. ùÛÏÐ ¹× Ú¸ÏÐߧ жñ»(3жñ» - #2, 3, 7) Í©ÓÛÛÆå× (1999. 1. 28)

2.3.3.1. Í©ÓÛÛÆå× source ¹× ÓßßÚ

2.3.3.1.1. source : øÁ÷ùÛÆå× ñéÀÎ Tricholoma matsutake strains (#2, 3, 7)

2.3.3.1.2. ÓßßÚ : MYG ¹× Hamada slants (ÊÀ жñ»Ü¬·Î 6ËÁ¾¿)

2.3.3.2. ïÈðú ¹× ÛÆå×

2.3.3.2.1. 1ËÁÀÇ øÁ÷ùÛÆò¢ ß¾¿¡¼­ 24ËÁÀÇ ÀÛÀº(1¡­2§® øëÀÇ ïáÞÌÊÇû¡) ïÈðúê«À» ¸¸µê

2.3.3.2.2. ÊÀ slants¿¡ 2ËÁ¾¿ ïÈðúê«À» ß¾öǽÃÄÑ ÛÆò¢Ü¬·Î ÊÀÊÀ 6ËÁÀÇ slant ñÞÝá

- slantÀÇ ¨÷ ¹× ¨ø¿¡ ú±Ó×ÇÏ´Â ò¢ïÇ¿¡ ÊÀÊÀ ïÈðúê«À» ß¾öǽÃÅ´ (40ÝÂ/жñ»)

2.3.3.2.3. slants¿¡ ïÈðúÇÑ ý­ жÞêô÷ ï·Ó¨ ¹× äûô÷ÛÆå× ãùã¿

2.3.3.3. ж ÛÆå× : 2öµ ùöè®ãø(25¡É, 12ãÁÊà ÎÃñÎÑ¢)

2.3.4. ùÛÏÐ ¹× Ú¸ÏÐߧ жñ» жÞêô÷ ï·Ó¨ ¹× äûô÷ÛÆå× (1999. 1. 28)

2.3.4.1. source ¹× ÓßßÚ

2.3.4.1.1. source : øÁ÷ùÛÆå×ñéÀÎ Tricholoma matsutake strains (#2, 3, 7)

2.3.4.1.2. ÓßßÚ : MYG ¹× PDMP liquid media (ÊÀ жñ»Ü¬·Î 3ËÁ¾¿)

2.3.4.2. ô¥×â Û°Ûö

2.3.4.2.1. 1ËÁÀÇ øÁ÷ùÛÆò¢ жñ»·Î 3ËÁÀÇ äûô÷ÛÆò¢¿¡ ïÈðú

2.3.4.2.2. ¾ç³¯ ØüÓﳯÀ» 100§¢ ºñÀÌÄ¿ÀÇ ¾ËÄڿÿ¡ ´ã¾Æ µÎ°í handlingÀ» À§ÇÑ Çɼ ñÞÝá

2.3.4.2.3. slantß¾ÀÇ Ð¶ñ»¸¦ ±Ü¾î³»¾î ØþжÇÑ À¯¸® »þ¾Ë·¹¿¡ ³õÀ½

2.3.4.2.4. ¾ËÄڿÿ¡ ´ã¾Æ µÎ¾ú´ø ¾ç³¯ ØüÓﳯÀ» ÇɼÂÀ¸·Î ²¨³»¾î áÀûý½ÃÅ´

2.3.4.2.5. À¯¸® »þ¾Ë·¹ À§ÀÇ Ð¶Þêô÷¸¦ ØüÓﳯÀ» ì¦éÄÇÏ¿© Àß°Ô ÀÚ¸§(å³ 3¡­5ÝÂÊà ô¥×â)

2.3.4.2.6. Àß°Ô À߶óÁø жÞêô÷¸¦ ØþжÇÑ Pasteur pipetteÀ» ì¦éÄÇÏ¿© ¿øÇÏ´Â äûô÷ÛÆò¢·Î ¿Å°Ü ³Ö¾î ïÈðúÇÔ - ÀÌ ¶§, ñÞÝáµÈ ÛÆò¢¿¡¼­ ÛÆå×äûÀ» ìéÝ» ²¨³»¾î »þ¾Ë·¹¿¡ ºÎÀº ý­ ´Ù½Ã жÞêô÷¿Í ´õºÒ¾î êüìý½ÃÄѼ­ ïÈðúÅä·Ï ÇÔ

2.4. ÊÀ áæéÇ Ð¶ñ»ÀÇ protoplast preparationÀ» êÓÇÑ î¢ÛÆå×

2.4.1. ÛÆå×ÓßßÚ Ð¶ñ» õÅÕá

ÛÆò¢

MYG

PDMP

жñ»

#51

#70

#2

#3

#7

#51

#70

#2

#3

#7

ÛÆå×ìí

1/13

1/27

1/13

1/27

1/28

1/28

1/28

1/13

1/27

1/13

1/27

1/28

1/28

1/28

Õá

1

1

1

1

3

3

3

1(0)

1

1

1

3

3

3

жñ»Ü¬

2

2

3

3

3

2(1)

2

3

3

3

ÛÆò¢Ü¬

13

13(12)

- ÛÆò¢Ü¬·Î ÊÀÊÀ 13ËÁ¾¿ õÅ 26ËÁ ß²ÊÇÇöó½ºÅ© ñÞÝá

- жñ»Ü¬·Î ÊÀÊÀ 5ËÁ¾¿ õÅ 10ËÁÀÇ filtering mesh(190§­) ñÞÝá

2.4.2. ÐïÎý Øþж ¹× ÛÆò¢ ñÞÝá

2.4.2.1. ØþжÐïÎý (1999. 2. 7)

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

Filtering mesh

190§­

11

autoclave on funnel

2

Glass petri-dish

¥õ: 6cm

11

autoclave

3

Pasteur pipette

3¡­5§¢éÄ

11

autoclave

4

Silicon cap(pipetteéÄ)

PasteuréÄ

5

autoclave

5

Small spatula

жô÷ïÚ×âéÄ

11

autoclave

2.4.2.2. ÛÆò¢ - ÛÆò¢Ü¬ 12¡­13 flasks

2.4.2.2.1. MYG medium : 13 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

300§¢

2

Malt extract

1g

3.0g

3

Yeast extract

0.4g

1.2g

4

Glucose

0.4g

1.2g

2.4.2.2.2. PDMP medium : 12 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

250§¢

2

Potato dextrose broth

2g

2.5g

3

Malt extract

0.3g

0.75g

4

Peptone

0.1g

0.25g

pH = 5.5

2.4.2.3. ÐïÎý´Â ¸ðµÎ ÐÝèøìí¿¡ ñÞÝáÇÏ°í, ÛÆò¢´Â ìíèøìí¿¡ ñÞÝáÇÏ¿© ìíèøìí¿¡ ±â±¸¿Í PDMP ÛÆò¢´Â ØþжÇÏ°í, MYGÛÆò¢´Â êÅèøìí ¾Æħ¿¡ Øþжô¥×â

2.4.2.4. ñÞÝá : Øþжô¥×âµÈ ÐïÎý¿Í ÛÆò¢¸¦ ÙíжßÉ¿¡ ³Ö°í í¹èâàÊ lamp¸¦ ÄÑ ³õ¾ÆµÒ

2.4.3. äûô÷¹è¾ç жñ»ÀÇ Ð¶Þêô÷ ï·Ó¨ ¹× î¢ÛÆå× (1999. 2. 8)

2.4.3.1. ô¥×â ê«öÎ

2.4.3.1.1. 1ËÁÀÇ ß²ÊÇ Çöó½ºÅ© ÛÆå× Ð¶ñ»´Â ´Ù½Ã 1ËÁÀÇ ß²ÊÇÇöó½ºÅ©¿¡ ïÈðú

2.4.3.1.2. ÔÒìéÇÑ Ð¶ñ»¸¦ ¸ÕÀú ì¹ãÕ, ïÈðúÇϹǷΠµÎ °¡Áö ÛÆò¢°¡ ÔÒãÁ¿¡ ñÞÝáµÇ¾î¾ß ÇÔ

2.4.3.1.3. ô¥×ââ÷ßí

- #51 : PDMP(1), MYG(2), knife change, pipette tip change

- #70 : PDMP(2), MYG(2), knife change, pipette tip change

- #2 : PDMP(3), MYG(3), knife change, pipette tip change

- #3 : PDMP(3), MYG(3), knife change, pipette tip change

- #7 : PDMP(3), MYG(3)

2.4.3.2. ô¥×â Û°Ûö

2.4.3.2.1. ¾ç³¯ ØüÓﳯÀ» 100§¢ ºñÀÌÄ¿ÀÇ ¾ËÄڿÿ¡ ´ã¾Æ µÎ°í handlingÀ» À§ÇÑ Çɼ ñÞÝá

- ØüÓﳯÀ» ÚâÀ¸·Î À߶ó¼­, ÇɼÂÀ» å´ÊÎ ´¯Èù û¡÷¾·Î Áã¾î ÞÅéÄ

2.4.3.2.2. Small spatula¸¦ ì¦éÄÇÏ¿© Ðþðí ÛÆå×жñ»ÀÇ Ð¶Þ길À» °ñ¶ó À¯¸® »þ¾Ë·¹¿¡ ¿Ã·Á³õÀ½(¶Ñ²±ÀÌ ÞÅéÄÇϱ⠴õ ÁÁÀ½)

2.4.3.2.3. ¾ËÄڿÿ¡ ´ã¾Æ µÎ¾ú´ø ¾ç³¯ ØüÓﳯÀ» ÇɼÂÀ¸·Î ²¨³»¾î áÀûý½ÃÅ´

2.4.3.2.4. À¯¸® »þ¾Ë·¹ À§ÀÇ Ð¶Þêô÷¸¦ ØüÓﳯÀ» ì¦éÄÇÏ¿© Àß°Ô ÀÚ¸§(å³ 5ÝÂÊà ô¥×â)

2.4.3.2.5. Àß°Ô À߶óÁø жÞêô÷¸¦ ØþжÇÑ Pasteur pipetteÀ» ì¦éÄÇÏ¿© 190§­ÀÇ filter¸¦ Åë°ú½ÃÅ°¸é¼­ ÁغñµÈ »õ äûô÷ÛÆò¢·Î ¿Å°Ü ³Ö¾î ïÈðúÇÔ - ÀÌ ¶§, ñÞÝáµÈ ÛÆò¢¿¡¼­ ÛÆå×äûÀ» ìéÝ» ²¨³»¾î »þ¾Ë·¹¿¡ ºÎÀº ý­ ´Ù½Ã жÞêô÷¿Í ´õºÒ¾î êüìý½ÃÄѼ­ ïÈðúÅä·Ï ÇÔ

¡Ø 1ËÁ ãËÖù ô¥×â¿¡ å³ 10Ý á¶é©

¡Ø çíîñ ñé¿¡ ìíÜâжñ» ô¥×â, çíý­¿¡ #2, #7 жñ», Àú³á¿¡ #3 жñ» ì¹ãÕ, ïÈðúÇÔ

2.5. Protoplast preparation ñÞÝá

2.5.1. ØþжÐïÎý ñÞÝá : 5 жñ» ô¥×âéÄ (1999. 2. 15 çíîñ)

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7

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2

á³û¡ ãËúÐη(morton îû)

íþ : 10cm

65

ËëæðØþж, ýüà· íÂåöéÄ

3

Glass petri-dish

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6

ËëæðØþж,

4

Pasteur pipette

6

ËëæðØþж,

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Spreader

11

ËëæðØþж,

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Mini spatula

6

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7

Pipetman tip(á³, üÜßä)

0.1§¢

11

autoclave, ýüà· ¹× ïÈðúéÄ

8

Pipetman tip(ñé, ôìßä)

1.0§¢

3

autoclave, ýüà·äû ñÞÝáéÄ

9

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

3

autoclave, plate ñÞÝáéÄ

10

Silicon cap(pipetteéÄ)

5.0§¢

6

autoclave

11

Filtering mesh

59¡­60§­

6

autoclave on funnel

12

Membrane filter set

25§®, 0.4§­

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autoclave in cap

¢Ñ membrane filterÀÇ ¹Ý¦ÀÌ´Â ºÎºÐÀÌ À§·Î(¶Ñ²± ºÎºÐÀ») ÇâÇϵµ·Ï ³ÖÀ½

2.5.2. Regeneration medium ñÞÝá (1999. 2. 16 çíîñ)

2.5.2.1. ÛÆò¢ - MYG medium + sucrose for osmotic buffering

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1

Distilled water

100§¢

100§¢

200§¢

100§¢

200§¢

1ó­ ñú׶â©

2

Malt extract

1g

1.0g

2.0g

1.0g

2.0g

3

Yeast extract

0.4g

0.4g

0.8g

0.4g

0.8g

4

Glucose

0.4g

0.4g

0.8g

0.4g

0.8g

5

Sucrose

17.1g

17.1

34.2g

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6

Agar

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¢Ñ + øöãÆ : sucrose, agar øÐùß 200§¢/300§¢ ¡â-flask ¡æ regeneration medium

sucrose øÐùß 100§¢/300§¢ ¡â-flask ¡æ dilutionéÄ

¢Ñ - øöãÆ : agar øÐùß 200§¢/300§¢ ¡â-flask ¡æ regeneration medium for control

sucrose Ú±øÐùß 100§¢/300§¢ ¡â-flask ¡æ dilutionéÄ

2.5.2.2. Regeneration medium ñÞÝá(pouring)

¢Ñ èÐõúäû øÐùß êóÙí¿¡ µû¶ó 60¡¿15§® sterilized petri-dish ÞÅéÄ 50ËÁ¾¿ ñÞÝá

¢Ñ Pipetman°ú ÓÞúþ tip(5§¢éÄ)À» ì¦éÄÇÏ¿© 4§¢¾¿ petri-dish¿¡ ÝÂñ»

¢Ñ ÝÂñ»°¡ ³¡³­ ý­ ½Ä¾î¼­ ͳïÒÀÌ µÇ¸é ¶Ñ²±¿¡ ýüà·ÛÃ×Ë°ú buffer ôÕÊ¥æ¨Üú øöãÆ

¡Ø + øöãÆ : sucrose, agar øÐùß ¡æ regeneration medium

- øöãÆ : agar øÐùß, sucrose Ú±øÐùß ¡æ ÓßðÎÏ¡

2.5.3. Enzyme ñÞÝá

2.5.3.1. CCZ- øöñÞ ý£áÈ ûèùêéÁäû for Coprinus cinereus

¢Ñ 0.1% (4 units/§¢) of Chitinase (Sigma)

¢Ñ 2% Cellulase Onozuka R-10

¢Ñ 0.1% Zymolyase 60,000

¢Ñ 0.5% mannitol

¢Ñ 50mM maleate buffer

¢Ñ pH 5.5

¡Ø ÓÞÝ»ÝÂÀÇ ÐñÜâ ý£áÈ´Â ÕÒíúÍ·(ÕÒÔÐãø)¿¡ ÜÁηÇÔ

2.5.3.2. ÇÑ ¹ø¿¡ 30¡­40§¢ ïïÓøÀÇ ý£áÈäûÀ» ¸¸µé¾î¼­ ÕÒÔÐÇØ µÎ¾ú´Ù°¡, ÞÅéÄÇÒ ¶§ ßÈ访¡¼­ ³ì¿© membrane filter·Î ØþжÇÑ ý­ ÞÅéÄ

2.5.3.3. Abe Ôõ(1982)ÀÇ ÌÑéç¿¡´Â Òýá´ ´Ù¸¥ ý£áÈéÁäûÀ» ÞÅéÄÇÏ¿´À½

¢Ñ 60mM MgSO4¡¤7H2O

¢Ñ 0.5% Cellulase Onozuka R-10 (Kinki Yakult Mfg. Co.)

¢Ñ 0.5% Zymolyase 5,000 (Kirin Brewery Co.)

¢Ñ 1.5% ¥â-glucuronidase (Sigma Co.)

¢Ñ 50mM maleic acid - NaOH buffer

¢Ñ pH 5.6

2.6. Protoplast preparation (1999. 2. 16 çíý­)

2.6.1. Protoplast extraction

2.6.1.1. ý£áÈ ¹× жÞêô÷ ñÞÝá

¢Ñ Water bath¿¡ ¹°À» ä¿ì°í ï³ê¹À» ÄѼ­ è®Óø¸¦ 30¡É·Î settingÇÔ

¢Ñ ÕÒÔÐÜÁηÇÏ´ø enzyme solutionÀ» ²¨³»¾î ãùúÐãø¿¡ ³õ¾ÆµÎ¾î ßÈ访¡¼­ ³ìÀÓ

¡Ø ãùòõîÜÀ¸·Î ý£áÈ°¡ ³ì´Âµ¥ á¶é©µÇ´Â ãÁÊàÀº 1ãÁÊà ïïÓøÀ̾úÀ½

¢Ñ Clean-bench ¿·ÀÇ table ¼­¶ø¿¡¼­ ñ¼ÞÒÐï ¼¼Æ®¸¦ ²¨³»¾î clean-bench¿¡ ³Ö¾îµÒ

¢Ñ ñ¼ÞÒÐï ¼¼Æ®¸¦ membrane filterÀÇ À­ ݻݿ¡ ֧̿ÇÏ°í ØþжÇÑ ÐÞîû ãËúÐηÀÇ À§¿¡ membrane filter kitÀÌ Ö§Ì¿µÇµµ·Ï ÇÑ ý­, ñ¼ÞÒÐï¿¡ ³ìÀº enzyme solutionÀ» ³Ö°í Á¶½É½º·´°Ô ñ¼ÞÒÐï ÇÚµéÀ» ¹Ð¾î ý£áÈéÁäûÀÌ ØþжµÇµµ·Ï ô¥×â

¢Ñ 2êÅ 8ìí¿¡ ïÈðúÇÏ¿© ÛÆå×ÇÏ´ø ¼ÛÀÌ(T. matsutake) äûô÷ÛÆå×äûÀ» °¡Á®¿Í ñÞÝá

¡Ø ùÛÏРжñ» #2, #3

¡Ø Ðìöâ жñ»´Â ßæíþÕá(жô÷Õá)ÀÌ ³Ê¹« À۾Ƽ­ ãùúп¡ ÞÅéÄÇÒ ¼ö ¾ø¾úÀ½

2.6.1.2. жÞê µ¢¾î¸® ñÞÝá ¹× ïÒÕá

¢Ñ ÐÞîûÀ¸·Î ¸·Èù ãËúÐη 2°³ÀÇ ñìÕá ö´ïÒ ¹× ãËúÐη¿¡ ¸ÅÁ÷ÆæÀ¸·Î ÑÀÖâ

¢Ñ 3¡­5ËÁÀÇ ß²ÊÇÇöó½ºÅ©¿¡ ÛÆå×µÈ Ð¶Þ긦 ØþжµÈ mini spatula¸¦ ì¦éÄÇÏ¿© жÞ길À» °É·¯³»¾î spatula¸¦ ì¦éÄÇÏ¿© ¾à°£¾¿ ´­·¯¼­ жÞê¿¡ µé¾îÀÖ´Â ÛÆå×äûÀÌ °ÅÀÇ ´Ù ÝÂÝôµÉ ¼ö ÀÖµµ·Ï Çϸ鼭 ìéÓ© Øþж petri-dish(disposable, 6§®éÄ)¿¡ ¿Å°Ü ³ÖÀ½

¢Ñ 5ËÁÀÇ ß²ÊÇÇöó½ºÅ©¿¡ ÛÆå×µÈ Ð¶Þ긦 ´Ù ¸ðÀº ý­, ´Ù½Ã spatula¸¦ ì¦éÄÇÏ¿© ÛÆå×äûÀ» õÌÓÞùÚ ð¶ËÛÇÏ°í ÐÞîûÀ¸·Î ¸·Èù ãËúÐηÀ¸·Î ¿Å°Ü ³ÖÀ½

¢Ñ ÐÞîûÀ» ´Ù½Ã ¸·Àº ý­ ñìÕáÀ» ö´ïÒÇÏ¿© ÛÆå×äûÀÌ Òýá´ ¼¯ÀΠжô÷ÀÇ ñìÕá ÷êäÄ

2.6.2. Enzyme reaction

2.6.2.1. ý£áÈô¥×â

¢Ñ жô÷ÕáÀÌ 10§· Ò®èâ ¹Û¿¡ µÇÁö ¾Ê¾ÒÁö¸¸ 0.1§¢ÀÇ ý£áÈ ô¥×âÇÒ °æ¿ì shaking µî handling¿¡ ¹®Á¦°¡ À־ 1§¢¸¦ жô÷°¡ µé¾îÀÖ´Â ãËúÐη¿¡ ³ÖÀ½

¢Ñ жô÷¸¦ spatula¸¦ ì¦éÄÇÏ¿© Àß Ç®¾î ÁÜ

2.6.2.2. Úãëëë¯Óô

¢Ñ shaking water bath(30¡É)¿¡ ³ÖÀº ý­ 80rpm áÜÓø·Î shakingÇϸç Úãëë ë¯Óô

¢Ñ Øß 20ݸ¶´Ù ²¨³»¾î ãËúÐηÀÇ ¾Æ·¡ Ý»ÝÂÀ» ¼ÕÀ¸·Î ÀâÀº ä 1¡­2õ© µ¿¾È vortex mixingÇÏ¿© ´Ù½Ã shaking water bath¿¡ ³ÖÀ½

¢Ñ 2ãÁÊà µ¿¾È ý£áÈÚãëë ô¥×â

2.6.2.3. Úãëëäû ÕëΦ

¢Ñ ý£áÈÚãëëÀÌ ³¡³­ ý­, 59§­ÀÇ filter mesh¸¦ ì¦éÄÇÏ¿© ÚãëëäûÀ» ÕëΦÇÏ¿© ÐÞîû ãËúÐηÀ¸·Î ¿Å°Ü ³ÖÀ½

¡Ø Ó©, Ðþðí æÚϼ¿¡ µû¸£¸é, áæéÇ Ð¶Þê¿¡¼­ ¾ò¾îÁø protoplastÀÇ òÁÌÓÀº 2¡­10§­ÀÇ ÛôêÌÀ̾úÀ½

¢Ñ Üâ ãùúÐÌ¿Íý´Â filteringÀ» ÇßÀ½¿¡µµ ÝÕϬÇÏ°í ¸¹Àº жÞê°¡ º¸¿©¼­ filtrationÀ» êÓÇÑ mesh size¸¦ ´õ ÁÙÀÏ ù±é©àõÀÌ ÀÖ¾úÀ½(¡Ú 20¡­40§­ Ò®èâ?)

2.6.2.4. îïÚõîÜÀÎ comments

¢Ñ жÞêÀÇ ßæíþ·®ÀÌ ³Ê¹« ÀûÀ½. Çöó½ºÅ© Ò®¿¡¼­ жÞê°¡ ¼­·Î ¾ûÄÑ ÀÖ¾î¾ß¸¸ ô¥×âÇϱ⿡ øµ××ÇÔ

¢Ñ ÓÞÕáÛÆå× ý­, ïÈðúê«À» ¸¹ÀÌ ¸¸µé¾î ÕëΦ¡¤ïÈðúÇÏ°í 7¡­10ìíÊà ÛÆå×µÈ Ð¶Þ긦 ÞÅéÄÇÏ´Â Û°ãÒ óõ÷É ù±é©

2.6.3. Protoplast counting and inoculation

2.6.3.1. úéÚ°Ìð°ú Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ Improved NeubauerÀÇ µÎ grid°¡ Àִ ݻݿ¡ ÇÑ ¹æ¿ï¾¿ protoplast éÁäû ß¾öÇ

¡Ø å³ 2¡­3Ý ÌèΦ ý­ úéÚ°Ìð ¾Æ·¡¿¡¼­ ËþÌð

¢Ñ úéÚ°Ìð 100ÛÃ×Ë¿¡¼­ ã·íÂÇÏ¿© 400¹è â©ñÞ¿¡¼­ ËþÌð

2.6.3.2. Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ 4ËÁÀÇ grid Ò®¿¡ ÀÖ´Â protoplastÀÇ â¦¸¦ countingÇÏ°íÀÚ ÇÔ

¢Ñ #2¿Í #3 ¸ðµÎ ãùð· protoplast´Â 8ËÁ ïïÓøÀÇ grid Ò®¿¡¼­ 1ËÁ ïïÓø¾¿ÀÌ Îºó̵Ǵ ÚãØü, жÞêô÷ ÷òø¸ÀÌ protoplastÀÇ â¦í®¿¡ ÝïÇÏ¿© ¿ÀÈ÷·Á ¸¹ÀÌ Îºó̵ǾúÀ½

¢¡ protoplastÀÇ â¦í®´Â 5.0¡¿105/§¢ ڱػÀÇ â©ñÞ

¢¡ filtration mesh size¿¡ ÓßÇÑ î¢ÍÅ ù±é©

2.6.3.3. dilution of protoplast

¢Ñ osmotic stabilizer êóÙí¿¡ µû¶ó Ï¡ÝÂÇÑ äûô÷ÛÆò¢¸¦ ÊÀÊÀ 0.9§¢¾¿ 5ËÁÀÇ á³û¡ ãËúÐη(mortonîû)¿¡ 1§¢ tipÀ» ì¦éÄÇÏ¿© ¹Ì¸® ÝÂñ»ÇØ ³õÀº ÈÄ, ÛÃ×Ë ¹× osmotic stabilizer êóÙí øúãÆ

¢Ñ ÀÌ ¶§, osmotic stabilizer°¡ ¾ø´Â °Í¿¡ ¸ÕÀú ÝÂñ»Çϸé ÇϳªÀÇ tipÀ¸·Î Í©áÙ ÞÅéÄ Ê¦ÒöÇÏÁö¸¸, ÊÀÊÀ ´Ù¸¥ pipette tipÀ» ÀÌ¿ëÇÏ¿© ñÞÝáÇÔ

¢Ñ 0.1§¢ tipÀ» ì¦éÄÇÏ¿© ý£áÈÚãëëäû¿¡¼­ 0.1§¢À» õÎõóÇÑ ý­ 5.0¡¿104ÀÌ ¾²¿©Áø ãËúÐηÀ¸·Î ¿Å°Ü ³Ö°í ´Ù½Ã Çѹø ±× ýüà·äûÀ¸·Î tipÀ» Çó±¸¾î ³¿. ¿©±â¿¡¼­ ´Ù½Ã 0.1§¢À» õÎõóÇÑ ý­ 5.0¡¿103ÀÌ ¾²¿©Áø ãËúÐηÀ¸·Î ¿Å°Ü ³Ö°í ´Ù½Ã Çѹø ±× ýüà·äûÀ¸·Î tipÀ» Çó±¸¾î ³¿. °°Àº ¹æ½ÄÀ¸·Î 5.0¡¿102, 5.0¡¿101, 5.0¡¿100±îÁö ýüà·.

¢Ñ ÊÀ ãËúÐη¿¡¼­ 0.1§¢À» õÎõóÇϸé, ÀÌ éÁäû Ò®¿¡ ðíî¤ÇÏ´Â protoplastÀÇ åÖÀº ÊÀ ãËúÐη¿¡ ¾²¿©Áø ¼ýÀÚÀÇ 1/10ÀÌ µÊ

2.6.3.4. ïÈðú

¢Ñ 5.0¡¿100ÀÇ ãËúÐη¿¡¼­ 0.1§¢ÀÇ ÚãëëäûÀ» õÎõóÇÑ ý­, ñÞÝáµÈ ÊÀ ýüà·ÛÃ×Ëܬ·Î 6ËÁÀÇ regeneration plates¿¡ ïÈðúÇÔ. ¡Ø ÊÀ plateÓ× 0.5ËÁÀÇ protoplast ðíî¤

¢Ñ ÊÀ ýüà·ÛÃ×Ëܬ·Î 6ËÁÀÇ plates¿¡ ïÈðú ý­ spreader¸¦ ì¦éÄÇÏ¿© plates À§¿¡ ïÈðúäûÀÌ °ñ°í·ç ÆÛÁú ¼ö ÀÖµµ·Ï ÝÂߤ½ÃÅ´ ¡Ø ÊÀ plateÓ× 5ËÁÀÇ protoplast ðíî¤

¢Ñ °°Àº Û°ÛöÀ¸·Î 5.0¡¿101°ú 5.0¡¿102ÀÇ ýüà·äû¿¡¼­ ÊÀÊÀ 0.1§¢ÀÇ ÚãëëäûÀ» õÎõó, ïÈðúÇÏ°í spreader¸¦ ì¦éÄÇÏ¿© ÝÂߤ½ÃÅ´ ¡Ø ÊÀ plateÓ× 50ËÁÀÇ protoplast ðíî¤

¡Ù 5.0¡¿102¡­5.0¡¿104ÀÇ ãËúÐη¿¡¼­ õÎõóÇÏ¿© ïÈðúÇÏ´Â °ÍÀÌ ¸¶¶¥ÇÏ¿´À¸³ª ã÷â¢

2.6.3.5. Osmotic stabilizer°¡ ¾ø´Â ÛÆò¢¿¡ÀÇ ïÈðúÀ» ¸¶Ä£ ý­, °°Àº Û°ÛöÀ¸·Î osmotic stabilizer°¡ ÀÖ´Â ÛÆò¢·Î ýüà· ¹× ïÈðúÀ» ãùã¿ÇÏ°í plates¿¡ ïÈðúìí, ÛÆò¢Ù£ ÑÀÖâ.

2.6.3.6. ÛÆå×(ÛÆå×è®Óø : 30¡É, 231筃 ÛÆå×ãø)

2.6.3.7. ÛÆå× ý­ ü¬ìã(1999. 3. 1.)¿¡ ëîÇÑ colony ⦠÷÷ܬ Ì¿Íý ËÖãæ×˵µ "0"

3. Tricholoma matsutake Ito et Imai(2ó­ ãËÓñ)

3.1. Hamada¿Í MYG media¸¦ ì¦éÄÇÑ Ð¶ñ»ÀÇ Í©ÓÛ ÛÆå×

3.1.1. Slant ñÞÝá (1999. 2. 19.)

3.1.1.1. ÓßßÚжñ» : 12/25 ïÈðú ý­ Í©ÓÛÛÆå×ÇÏÁö ¾Ê¾Ò´ø жñ»(#1, #4, #5, #6)

- slants : 6 slants/strain, 2 slants/Jap. strain(#51, #70)

- ÊÀ ÛÆò¢Ü¬·Î ÊÀÊÀ 28 slants¸¦ ñÞÝáÇÔ

3.1.1.2. ÛÆò¢ðÚà÷

¡Ø MYG(10§¢/ãËúÐη) ¢Ñ õÅ 280§¢ ù±é©

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1

Distilled water

100§¢

300§¢

2

Malt extract

1g

3g

3

Yeast extract

0.4g

1.2g

4

Glucose

0.4g

1.2g

5

Agar

1.5g

4.5g

¡Ø Hamada(10§¢/ãËúÐη) ¢Ñ õÅ 280§¢ ù±é©

ÛãûÜ

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1

Tap water

100§¢

300§¢

2

Glucose

2g

6g

3

Yeast extract

0.5g

1.5g

4

Agar

1.5g

4.5g

pH = 5.5

¡Ù óÀ½¿¡ Tap water ÓÛãó ñú׶⩸¦ ÞÅéÄÇÏ¿© ´Ù½Ã ð²ðãÇÔ

¡Ø Ðìöâ ñÞÝáÚª

- Pasteur pipette ¹× spoid cap ÊÀ 2ËÁ¾¿ Øþж, ñÞÝá

- ìíÜâжñ» #51, #70 ñé MYG ÛÆò¢ ß¾ÀÇ mycelial cluster û¡à÷ flask ÞÅéÄ

3.1.2. ÛÆò¢ Øþж ¹× slant ñÞÝá

3.1.2.1. ÊÀ ÛÆò¢ ðú׾ܬ·Î 28ËÁ¾¿ ñÞÝá (õÅ 56ËÁ)

3.1.2.2. slant´Â ãùúÐãøÒ® table À§¿¡ À¯¸®Î·À» õîÀ¸·Î ´µ¿©¼­ ñÞÝáÇÔ

3.1.2.3. æ®ÝÂÀÇ 4ËÁ¾¿ÀÇ ÛÆò¢´Â ìíÜâжñ» #51, #70ÀÇ ïÈðú¿¡ ÞÅéÄ

¡Ø ÛÆò¢ ð²ðããÁ´Â MYG¿Í HamadaÀÇ Ï¡ÝÂÀÌ Ê¦ÒöÇϳª Øþж ý­¿¡´Â ¸Å¿ì ¾î·Á¿ò

3.1.3. ïÈðú ¹× ÛÆå× (1999Ò´ 2êÅ 20ìí)

3.1.3.1. ÓßßÚжñ» : #1(petri-dish, Ó¤ìé slant), #4(3ËÁÀÇ petri-dishes),

#5(petri-dish, Ó¤ìé slant), #6(Ó¤ìé slant) ¢Ñ 6 & 6 double slants

#51(MYG liquid), #70(MYG liquid) ¢Ñ 2 & 2 slants and 2 plates

3.1.3.2. From solid media to slants : ËíˬÇÑ Ð¶Þ긦 ¶¼¾î ÇÑ slant¿¡ 2ËÁÀÇ ïÈðúê« ß¾öÇ

¡Ø ÷åÑÀÞÀú£

¢Ñ äûô÷ÛÆò¢ ïÈðú ý­ ¿­Èê ïïÓø±îÁö´Â PDMP¿Í MYGÀÇ ó¬ì¶ïà ̸ۡÀÌ ¾î·Æ°í, ¿ÀÈ÷·Á MYG ÛÆò¢¿¡¼­ÀÇ ßæíþÀÌ ºü¸¥ °Íó·³ ¿©°ÜÁ³À½. ±×·¯³ª, å³ ÇÑ ´ÞÀÌ ÌèΦÇÏÀÚ MYG¿¡ ÝïÇÏ¿© PDMP ÛÆò¢¿¡¼­ÀÇ Ð¶Þê ßæíþÕáÀÌ ´õ ¸¹Àº °ÍÀ» ü¬ìãÇÒ ¼ö ÀÖ¾úÀ½. µû¶ó¼­, ì¤ý­´Â PDMP ÛÆò¢¸¦ äûô÷ÛÆå×ÀÇ ÐñÜâ ÛÆò¢·Î ÞÅéÄ

¢Ñ 3ñÎ ÌèΦ ý­¿¡´Â äûô÷ÛÆò¢ ß¾ÀÇ áæéÇ Ð¶Þê°¡ ѨñéжÞê·Î Û¡Ó¹ÇÏ´Â °ÍÀÌ ³ªÅ¸³²

¢Ñ 100§¢ äûô÷ÛÆò¢·Î´Â 3ñÎ ì¤Ò® ÛÆå×ÀÌ é©Ï´µÇ¸ç, MYG¿¡ ÝïÇÏ¿© PDMP°¡ éÐâ³ÇÔÀ» íÕïÒîÜÀ¸·Î ü¬ìãÇÒ ¼ö ÀÖ¾úÀ½

¢Ñ 3ñÎ ÛÆå× ý­ÀÇ ÛÆå×жÞêÀÇ øÁгòõåÖÀº PDMP´Â 203mg, MYG´Â 126§· À̾úÀ½

¢Ñ 2ñÎ ïïÓøÀÇ ÛÆå× ý­ protoplast preparation ãËÓñ¸¦ êÓÇÑ Ð¶ô÷Õá¿¡ ÓðÓ¹ÇÒ ¼ö ÀÖ´Â Áö ôøÑ¢ ïÈðúÕá¿¡ µû¸¥ ܨûù ÷êäÄ ù±é©

¢Ñ áÜà÷ жñ»(#2)ÀÇ ÌÑéç, Hamada slants¿¡¼­ÀÇ ÛÆå×Àº Øß 1ËÁꟶ´Ù Í©ÓÛÛÆå×À» Çؾ߸¸ ÇÒ °ÍÀ¸·Î »ý°¢µÊ

¢Ñ Ó©, #3, #7ÀÇ ÌÑéç¿¡´Â ßæíþáÜÓø°¡ ÈξÀ ´Ê¾î 2ËÁêÅ ïïÓøÀÇ Ñ¢ÊàÀÌ á¶é©µÊ

3.2. 300§¢ ¡â-flask ¹× shaker¸¦ ì¦éÄÇÑ áæéÇ áÜà÷ ÛÆå×

3.2.1. ÛÆò¢ ñÞÝá (1999Ò´ 2êÅ 22ìí)

3.2.1.1. ÛÆò¢Õá - жñ»Ü¬ 2 flasks

3.2.1.2. ÓßßÚжñ» - #2, #3, #7, #51, #70 ¡Å õÅ 10ËÁ flasks

3.2.1.3. ÛÆò¢ðú×¾ - MYG, 100§¢/300§¢ ¡â-flask

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

1,000§¢

2

Malt extract

1g

10g

3

Yeast extract

0.4g

4.0g

4

Glucose

0.4g

4.0g

3.2.2. Slant mediaÀÇ Ð¶ñ» ì¹ãÕ (1999Ò´ 2êÅ 23ìí)

3.2.2.1. ÓßßÚжñ» - #7, 12/25 ÛÆå× Ð¶ñ»

3.2.2.2. Û°Ûö : 3ËÁ slant¸¦ ÊÀÊÀ ÇÑ ËÁÀÇ flask¿¡ ¿Å°Ü ïÈðú

¢Ñ Crude, medium, fine cutting¿¡ ëîÇÑ ó¬ì¶ ÷÷ܬ ãËÓñ

¢Ñ Crude cuttingÀº åÖÀ» ¸¹ÀÌ ÇÏ°í, fine cuttingÀº åÖÀ» Àû°Ô ÇÔ

¢Ñ Fine cuttingÀº ãÁÊàÀÌ ÈξÀ ¸¹ÀÌ °É¸²

3.2.2.3. ÛÆå× : 1öµ ÛÆå×ãø, äÞÛÆå×, shaking

3.2.3. Liquid mediaÀÇ Ð¶ñ» ì¹ãÕ (1999Ò´ 2êÅ 24ìí)

3.2.3.1. ÓßßÚжñ» - #2, #51, #70 ÛÆå× Ð¶ñ»

3.2.3.2. Û°Ûö : ÊÀÊÀ ÇÑ ËÁÀÇ 100§¢-flask¿¡¼­ жÞ긦 õÎõó, 2¡­3ËÁÀÇ 300§¢-flask¿¡ ïÈðú

¢Ñ #2 : 3ËÁ

¢Ñ #51, #70 : 2ËÁ¾¿

3.2.3.3. ÛÆå× : 1öµ ÛÆå×ãø, äÞÛÆå×, shaking

3.3. 300§¢ ¡â-flask¸¦ ì¦éÄÇÑ áæéÇ ïÚô÷ ÛÆå× 1

3.3.1. ÛÆò¢ ¹× ÐïÎý ñÞÝá (1999Ò´ 3êÅ 1ìí)

3.3.1.1. ÓßßÚжñ» - #2 PDMP ¹× MYG äûô÷ÛÆå× Ð¶ñ»

3.3.1.2. ÛÆò¢ðú×¾ - PDMP, 100§¢/300§¢ ¡â-flask

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

510§¢

2

Potato dextrose broth

2g

10.2g

3

Malt extract

0.3g

1.53g

4

Peptone

0.1g

0.51g

pH = 5.5

3.3.1.3. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

Glass petri-dish

¥õ: 6cm

2

autoclave, 1ËÁ´Â çãÝáéÄ

2

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

6

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

3

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

2

autoclave, 1ËÁ´Â çãÝáéÄ

4

Small spatula

жô÷ïÚ×âéÄ

2

autoclave, 1ËÁ´Â çãÝáéÄ

5

¡â-flask with PDMP

100§¢

1

PDMP 25§¢

6

¡â-flask with PDMP

300§¢

5

PDMP 96§¢

3.3.2. 100§¢ ¡â-flaskÒ® ÛÆå×жñ»ÀÇ Ð¶ô÷Õá ÷êäÄ ¹× жÞêï·Ó¨, ïÈðú (1999Ò´ 3êÅ 2ìí)

3.3.2.1. ÓßßÚжñ» - #2, 2/8 äûô÷ÛÆå× Ð¶ñ»

3.3.2.2. ÊÀ ÛÆå×äû³» жô÷Õá ÷êäÄ

¢Ñ жÞê¿¡ ùßêóµÇ¾î ÀÖ´Â ÛÆå×äûÀ» õöÝÂÈ÷ ð¶ËÛÇÑ ý­ ÐÞîû ãËúÐη¿¡ ¿Å°Ü öàÕá

ðú×¾

PDMP1

PDMP2

PDMP3

MYG1

MYG2

øÁг(§·)

ãËúÐη

13.266

13.415

13.006

13.259

13.391

õÅ Õá

13.508

13.639

13.156

13.396

13.506

жÞêÕá

0.242

0.224

0.150

0.137

0.115

174

¡Ø PDMP´Â å³ ¨ö, MYG´Â å³ ¨÷ÀÇ flask ¹ØØüÀÌ Ð¶Þê¿¡ ëîÇÏ¿© µ¤¿© ÀÖ¾úÀ½

¢Ñ жô÷ÕáÀ» ö´ïÒÇÑ Ð¶Þêô÷´Â Çϳª¾¿ cuttingÀ» ãùã¿ÇÔ

3.3.2.3. жÞêô÷ ï·Ó¨ ¹× ïÈðú

¢Ñ Fine cuttingÀ» êÓÇÏ¿© õÌá³ùÚ 5Ý ì¤ß¾ cuttingÀ» ãùã¿ÇÑ ý­ ÛÆå×äû 25§¢ÀÌ µé¾î ÀÖ´Â 100§¢ ¡â-flask ÇϳªÀÇ ÐÞîû ãËúÐη¿¡ ¿Å°Ü ³ÖÀ½

¢Ñ 100§¢ ¡â-flask Ò®ÀÇ ÛÆå×äûÀ» 4§¢¾¿ õÎõóÇÏ¿© petri dish¿¡ pouringÇÑ ý­ ´Ù½Ã PipetmanÀ» ì¦éÄÇÏ¿© 100§¢ ¡â-flask·Î ¿Å°Ü ³ÖÀ½

¢Ñ 3¡­5Ûã° жÞêô÷¸¦ ¿Å±æ ¶§´Â 300§¢ ¡â-flaskÀÇ ÛÆå×äûÀ» 4§¢¾¿ õÎõóÇÏ¿© petri dish¿¡ pouringÇÑ ý­ ´Ù½Ã PipetmanÀ» ì¦éÄÇÏ¿© 100§¢ ¡â-flask·Î ¿Å°Ü ³ÖÀ½

¢Ñ ïÈðúê«À¸·Î ¸ð¿©Áø жÞêô÷¿¡¼­ 8§¢¾¿ жÞêô÷¸¦ õÎõó, ÊÀ 300§¢ ¡â-flask·Î ïÈðú

¢Ñ ñí, ãùòõîÜÀÎ ïÈðúÕáÀº õÅ ïÈðúê«(=868§·)ÀÇ 8/45(=154§·)°¡ µÇ´Â °ÍÀÓ

¢Ñ ïÈðúÕáÀÇ Ð³ìéûù¸¦ êÓÇÏ¿© ÊÀ ¡â-flask¿¡ 4§¢¾¿ ïÈðúÇÑ ý­, ´Ù½Ã æ½â÷À¸·Î 4§¢¾¿ ïÈðúÇÔ

3.4. 300§¢ ¡â-flask¸¦ ì¦éÄÇÑ áæéÇ ïÚô÷ ÛÆå× 2

3.4.1. ÛÆò¢ ¹× ÐïÎý ñÞÝá (1999Ò´ 3êÅ 8ìí)

3.4.1.1. ÓßßÚжñ» - #3, #51, #70 PDMP ¹× MYG äûô÷ÛÆå× Ð¶ñ» õÅ 7ËÁ

3.4.1.2. ÛÆò¢ðú×¾ - PDMP, 100§¢/300§¢ ¡â-flask

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

710§¢

2

Potato dextrose broth

2g

14.2g

3

Malt extract

0.3g

2.13g

4

Peptone

0.1g

0.71g

pH = 5.5

3.4.1.3. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

Glass petri-dish

¥õ: 6cm

4

autoclave, 1ËÁ´Â çãÝáéÄ

2

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

8

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

3

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

4

autoclave, 1ËÁ´Â çãÝáéÄ

4

Small spatula

жô÷ïÚ×âéÄ

4

autoclave, 1ËÁ´Â çãÝáéÄ

5

¡â-flask with PDMP

50§¢

2

PDMP 15§¢

6

¡â-flask with PDMP

50§¢

1

PDMP 5§¢

7

¡â-flask with PDMP

300§¢

5

PDMP 95§¢

3.4.2. 100§¢ ¡â-flaskÒ® ÛÆå×жñ»ÀÇ Ð¶ô÷Õá ÷êäÄ ¹× жÞêï·Ó¨, ïÈðú (1999Ò´ 3êÅ 9ìí)

3.4.2.1. ÓßßÚжñ» - #3, #51, #70 2/8 äûô÷ÛÆå× Ð¶ñ»

3.4.2.2. ÊÀ ÛÆå×äû³» жô÷Õá ÷êäÄ

¢Ñ жÞê¿¡ ùßêóµÇ¾î ÀÖ´Â ÛÆå×äûÀ» õöÝÂÈ÷ ð¶ËÛÇÑ ý­ ÐÞîû ãËúÐη¿¡ ¿Å°Ü öàÕá

жñ»

#3

#51

#70

ÛÆò¢ðú×¾

PDMP

MYG1

MYG2

øÁг

MYG

PDMP1

PDMP2

MYG

øÁг

ãËúÐη

13.447

13.661

13.205

13.236

13.898

14.024

13.024

õÅ Õá

13.576

13.741

13.238

13.241

14.053

14.096

13.197

жÞêÕá

0.129

0.080

0.033

0.080

0.005

0.155

0.072

0.173

0.133

¡Ø #51Àº жô÷ÕáÀÌ ÀÛÀº 2ËÁÀÇ cluster·Î¼­ åÖÀÌ Ð¿È÷ Àû¾úÀ½

3.4.2.3. жÞêô÷ ï·Ó¨ ¹× ïÈðú

¢Ñ Fine cuttingÀ» êÓÇÏ¿© õÌá³ùÚ 5Ý ì¤ß¾ cuttingÀ» ãùã¿ÇÑ ý­ 50§¢ ¡â-flask ³»ÀÇ ÛÆå×äûÀ» 5§¢¾¿ õÎõóÇÏ¿© petri dish¿¡ pouringÇÑ ý­ ´Ù½Ã PipetmanÀ» ì¦éÄÇÏ¿© 50§¢ ¡â-flask·Î ¿Å°Ü ³ÖÀ½

¢Ñ 3¹ø° жÞêô÷¸¦ ¿Å±æ ¶§´Â 300§¢ ¡â-flaskÀÇ ÛÆå×äûÀ» 5§¢ õÎõóÇÏ¿© petri dish¿¡ pouringÇÑ ý­ ´Ù½Ã PipetmanÀ» ì¦éÄÇÏ¿© 50§¢ ¡â-flask·Î ¿Å°Ü ³ÖÀ½

¢Ñ ïÈðúê«À¸·Î ¸ð¿©Áø flask¿¡¼­ 10§¢¾¿ жÞêô÷¸¦ õÎõó, ÊÀ 300§¢ ¡â-flask·Î ïÈðú

¢Ñ ïÈðúÕá : ïÈðúê«À» õÌÓÞùÚÀ¸·Î ´Ù ÞÅéÄÇÏ¿´À¸¹Ç·Î, жô÷Õá øÁгö·¿Í °ÅÀÇ °°À½

3.5. 300§¢ ¡â-flask¸¦ ì¦éÄÇÑ áæéÇ ïÚô÷ ÛÆå× 3 (1999Ò´ 3êÅ 12ìí)

3.5.1. ÛÆò¢ ¹× ÐïÎý ñÞÝá

3.5.1.1. ÓßßÚжñ» - slantsÀÇ #7 жñ» 2ËÁ

3.5.1.2. ÛÆò¢ðú×¾ - PDMP, 100§¢/300§¢ ¡â-flask 2°³(ËÁ)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

2¡¿100§¢

2

Potato dextrose broth

2g

2¡¿2g

3

Malt extract

0.3g

2¡¿0.3g

4

Peptone

0.1g

2¡¿0.1g

pH = 5.5

3.5.1.3. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

Glass petri-dish

¥õ: 6cm

2

autoclave

2

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

2

autoclave, 1ËÁ´Â çãÝáéÄ

3

¡â-flask with PDMP

300§¢

2

PDMP 100§¢

3.5.2. 100§¢ ¡â-flaskÒ® ÛÆå×жñ»ÀÇ Ð¶ô÷Õá ÷êäÄ ¹× жÞêï·Ó¨, ïÈðú

3.5.2.1. ÊÀ slants³» жô÷Õá ÷êäÄ

¢Ñ slants õÅ ñìÕá, жÞêô÷ ð¶ËÛý­ ñìÕá ö´ïÒÀ¸·Î üµß© - 2ËÁÀÇ colonies

ãËúÐη ÛãûÜ

#1

#2

øÁг

õÅ ñìÕá(g)

35.870

35.553

жÞêô÷ ð¶ËÛ ý­

35.361

35.191

ãËúÐη + center

35.509

35.252

üÀÕô³ôÀº жÞê

0.361

0.301

0.331

¢Ñ å³ 331mgÀÇ ïÈðúê«ÀÌÁö¸¸ agar¸¦ øÐùßÇÑ ñìÕáÀ̹ǷÎ, ãùòõîÜÀ¸·Î üÀéÄµÈ °ÍÀº 200mg Ò®èâ·Î õÏïÒµÊ

3.5.2.2. жÞêô÷ ï·Ó¨ ¹× ïÈðú

¢Ñ ÊÀ ãËúÐη¿¡ labelÀ» ÀûÀº ý­ õÅ ñìÕá ö´ïÒ

¢Ñ '衁'ÀÚ·Î ÈÖ¾îÁø ÛÜÐÝ켸¦ ì¦éÄÇÏ¿© slants¿¡¼­ áæéÇ Ð¶Þêô÷¸¸À» ±Ü¾î³»¾î Øþж À¯¸® »þ¾Ë·¹¿¡ ¿Å±ä ý­, жÞê°¡ ð¶ËÛµÈ ãËúÐηÀÇ ñìÕá ö´ïÒ

¢Ñ ØüÓﳯÀ» ì¦éÄÇÏ¿© жÞêô÷ÀÇ °¡¿îµ¥¿¡ ÀÖ´Â ±½Àº Ý»ÝÂ(óÀ½¿¡ ïÈðúê«À¸·Î ÞÅéĵǾú´ø Ý»ÝÂ)À» ð¶ËÛÇÔ

¢Ñ °¡¿îµ¥ ݻݿ¡ ÀÖ´ø °ÍÀ» ´Ù½Ã ãËúÐη¿¡ ³ÖÀº ý­ ñìÕá ö´ïÒ

¢Ñ À¯¸® »þ¾Ë·¹ À§¿¡ Àִ жÞêô÷¸¦ Àß°Ô ÀÚ¸§

¢Ñ ÀÚ¸£´Â Ρïï¿¡ ÛÆå×äûÀÇ ìéÝ»(2¡­3§¢)¸¦ õÎõóÇÏ¿© À¯¸® »þ¾Ë·¹¿¡ ôÕÊ¥ÇÑ ý­ Àß°Ô ÀÚ¸§

¢Ñ Àß°Ô ÀÚ¸¥ жÞêô÷¿¡ ÛÆå×äûÀ» ôÕÊ¥(å³ 5§¢)ÇÏ¿© ´Ù½Ã õÎõó, 300§¢ ¡â-flask·Î ïÈðú

3.6. äûô÷ÛÆò¢(PDMP media)¸¦ ì¦éÄÇÑ protoplast ÝÂ×îéÄ Ð¶ñ»ÀÇ ïÈðú 1

3.6.1. ØþжÐïÎý & ÛÆò¢ ñÞÝá (1999. 3. 15)

3.6.1.1. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

3

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

2

Filtering mesh

120§­

3

autoclave on funnel

3

empty ¡â-flask

100§¢

2

4

¡â-flask with 20§¢ PDMP

100§¢

10

5

Glass petri-dish

¥õ: 6cm

3

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

6

Mini spatula

3

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

7

Pipetman tip(ñé, ôìßä)

1.0§¢

2

autoclave, ïÈðúéÄ

3.6.1.2. ÛÆò¢(PDMP medium) - жñ»Ü¬ 5 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

200§¢

2

Potato dextrose broth

2g

4g

3

Malt extract

0.3g

0.6g

4

Peptone

0.1g

0.2g

pH = 5.5

3.6.1.3. ñÞÝá : Øþжô¥×âµÈ ÐïÎý¿Í ÛÆò¢¸¦ ÙíжßÉ¿¡ ³Ö°í í¹èâàÊ lamp¸¦ ÄÑ ³õ¾ÆµÒ

3.6.2. äûô÷ÛÆò¢ ïÈðú - ÙíжßÉ¿¡¼­ íÂåö (1999. 3. 16)

3.6.2.1. shaker¿¡¼­ ÛÆå×ÇÑ #2, #70 300§¢-flasksÀÇ µÎ жñ» üÀéÄ

3.6.2.2. ÛÆå×ßÒ÷¾ ÞÐòØõÉç¯

¢Ñ #7(Tricholoma magnivelare) fine, medium, crude cutting Ì¿Íý ÝïÎò

¢Ñ #2, #70, #51ÀÇ ÛÆå×Ì¿Íý ÝïÎò

¡Ø ÛÆå×ßÒ÷¾, ÞÐòØõÉç¯ìíí® ÔõÀ» ÑÀÖâ ý­ color print filmÀ¸·Î õÉç¯

¢Ñ #7, #51Àº Í©áÙ òÉ÷·Ðï¿¡ ÛÆå×ÇÏ°í, #2, #70Àº Çϳª¾¿ àÔïÒÇÏ¿© trans-culture

3.6.2.3. ïÈðúê« handling

¢Ñ ØþжµÈ ÐÞîû ãËúÐη 2ËÁÀÇ ñìÕá ö´ïÒ ¹× ÑÀÖâ

¢Ñ Mini spatula¸¦ ì¦éÄÇÏ¿© жÞêô÷¸¦ ÛÆå×äûÀ» ð¶ËÛÇϸç À̲ø¾î ³¿

¡Ø Mini spatula´Â 300§¢-flasks¿¡¼­ ÞÅéÄÇϱâ ÍÝÑñÇÏ¿© filtering mesh¸¦ ì¦éÄÇÏ¿© ºó 100§¢-flask¿¡ Á÷Á¢ ºÎÀ¸¸é¼­ filter¿¡ °É¸° жÞê â½ó¢

¢Ñ ØþжµÈ ÐÞîû ãËúÐηÀ¸·Î ¿Å°Ü ³Ö°í ñìÕá ö´ïÒ

ãËúÐη ÛãûÜ

1 - #2

2 - #70

ãËúÐη (g)

13.720

14.162

ãËúÐη + жÞê

15.703

20.451

жÞêô÷ ñìÕá

2.017

6.289

¡Ø filtering(120§­) ý­ ïÈðúÇÑ ãùð· ïÈðúÕáÀº îïô÷ жÞêÕáÀÇ 10¡­20%·Î õÏïÒµÊ

¢Ñ ´Ù½Ã »þ¾Ë·¹·Î жÞêô÷¸¦ ¿Å±ä ÈÄ ØüÓﳯÀ» ì¦éÄÇÏ¿© жÞêô÷¸¦ Àß°Ô ÀÚ¸§

¡Ø ÇѲ¨¹ø¿¡ ¸¹Àº åÖÀ» cuttingÇÏ´Â °ÍÀÌ ÍÝÑñÇÏ¿© ¨÷ ïïÓø¾¿ ³ª´©¾î cutting

¢Ñ õöÝÂÈ÷ жÞêô÷°¡ À߶óÁø ý­, ß²ÊÇ flask ÇÑ ËÁÀÇ îïô÷ éÁäûÀ» filtering mesh (mesh size : 120§­)¸¦ ÷×Φ½ÃÅ°¸é¼­ Àß°Ô À߸° жÞêô÷¸¦ ¸ðÀ¸°í, ´Ù¸¥ 4ËÁÀÇ flasks¿¡¼­µµ 4§¢¾¿ äûô÷ÛÆò¢¸¦ õÎõóÇÏ¿© filter À§¿¡ ºÎ¾î åÖÀ» ñòÊ¥½ÃÅ´

¢Ñ 4°³ÀÇ flasks¿¡ ÊÀÊÀ 8§¢¾¿ ïÈðú (¡Ø ´ÙÀ½¿¡´Â 5§¢éÄ pipette tip ì¦éÄ ù±é©)

¡Ø ïÈðúÕá : 1-1¡­1-4 : ¡Ö 50§·, 2-1¡­2-4 : ¡Ö 300§· *-4´Â Òýá´ ÀûÀº åÖ

3.7. äûô÷ÛÆò¢(PDMP media)¸¦ ì¦éÄÇÑ protoplast ÝÂ×îéÄ Ð¶ñ»ÀÇ ïÈðú 2

3.7.1. ØþжÐïÎý & ÛÆò¢ ñÞÝá (1999. 3. 18)

3.7.1.1. ØþжÐïÎý

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

3

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

2

Filtering mesh

59§­

1

autoclave on funnel

3

Filtering mesh

190§­

2

autoclave on funnel

4

empty ¡â-flask

100§¢

2

5

¡â-flask with 20§¢ PDMP

100§¢

10

6

Glass petri-dish

¥õ: 6cm

3

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

7

Mini spatula

2

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

8

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

2

autoclave, ïÈðúéÄ

3.7.1.2. ÛÆò¢(PDMP medium) - жñ»Ü¬ 5 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

200§¢

2

Potato dextrose broth

2g

4g

3

Malt extract

0.3g

0.6g

4

Peptone

0.1g

0.2g

pH = 5.5

3.7.1.3. ñÞÝá : Øþжô¥×âµÈ ÐïÎý¿Í ÛÆò¢¸¦ ÙíжßÉ¿¡ ³Ö°í í¹èâàÊ lamp¸¦ ÄÑ ³õ¾ÆµÒ

3.7.2. äûô÷ÛÆò¢ ïÈðú - ÙíжßÉ¿¡¼­ íÂåö (1999. 3. 19)

3.7.2.1. shaker¿¡¼­ ÛÆå×ÇÑ 300§¢-flasksÀÇ #2 жñ» 2ËÁ üÀéÄ

3.7.2.2. ïÈðúê« handling

¢Ñ 59§­ filter¸¦ ¾ñÀº 100§¢-flask¿¡ Á÷Á¢ ºÎÀ¸¸é¼­ filter¿¡ °É¸° жÞê â½ó¢

¢Ñ »þ¾Ë·¹·Î ¿Å°Ü ÛÆå×äûÀ» ´­·¯ ð¶ËÛ ý­ Øþж ÐÞîû ãËúÐηÀ¸·Î ¿Å±â°í ñìÕá ö´ïÒ

ãËúÐη ÛãûÜ

1

2

ãËúÐη (g)

13.660

13.879

ãËúÐη + жÞê

16.562

15.562

жÞêô÷ ñìÕá

2.902

1.883

¡Ø filtering(190§­) ý­ ïÈðúÇÑ ãùð· ïÈðúÕáÀº îïô÷ жÞêÕáÀÇ å³ 20%·Î õÏïÒµÊ

¢Ñ ´Ù½Ã »þ¾Ë·¹·Î жÞêô÷¸¦ ¿Å±ä ÈÄ ØüÓﳯÀ» ì¦éÄÇÏ¿© жÞêô÷¸¦ Àß°Ô ÀÚ¸§

¢Ñ õöÝÂÈ÷ жÞêô÷°¡ À߶óÁø ý­, ß²ÊÇ flask ÇÑ ËÁÀÇ îïô÷ éÁäûÀ» filtering mesh (mesh size : 190§­)¸¦ ÷×Φ½ÃÅ°¸é¼­ Àß°Ô À߸° жÞêô÷¸¦ ¸ðÀ¸°í, ´Ù¸¥ 4ËÁÀÇ flasks¿¡¼­µµ 4§¢¾¿ äûô÷ÛÆò¢¸¦ õÎõóÇÏ¿© filter À§¿¡ ºÎ¾î åÖÀ» ñòÊ¥½ÃÅ´

¢Ñ 4°³ÀÇ flasks¿¡ ÊÀÊÀ 9§¢¾¿ ïÈðú

¡Ø ïÈðúÕá : 1-5¡­1-8 : ¡Ö 150§·, 1-9¡­1-12 : ¡Ö 100§· *-8, 12´Â Òýá´ ÀûÀ½

3.8. Protoplast preparation ¹× trans-culture ñÞÝá (1999. 3. 23)

¡Ø 3/16 ïÈðúÇÑ 100§¢ flaskÀÇ Ð¶ñ»´Â ßæíþÕáÀÌ ³Ê¹« Àû¾î 3/2 ïÈðúÇÑ 300§¢ flask üÀéÄ

3.8.1. ØþжÐïÎý ñÞÝá : 1 жñ»(#2-1¡­#2-5) ô¥×âéÄ - 3 for protoplast, 2 for trans-culture

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

10

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

2

á³û¡ ãËúÐη(morton îû)

íþ : 10cm

36

ËëæðØþж, ýüà· íÂåöéÄ

3

Sterilized petri-dish

¥õ: 6cm

60

(disposable)

4

Sterilized petri-dish

¥õ: 6cm

2

(glass)

5

Pasteur pipette

2

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

6

Spreader

2

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

7

Mini spatula

2

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

8

Long spatula(for 300§¢)

2

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

9

Pipetman tip(á³, üÜßä)

0.1§¢

2

ýüà· ¹× ïÈðúéÄ

10

Pipetman tip(ñé, ôìßä)

1.0§¢

2

ýüà·äû ñÞÝáéÄ

11

Pipetman tip(ÓÞ, ÛÜßä)

5.0§¢

4

plate ñÞÝá, trans-culture

12

Silicon cap(pipetteéÄ)

5.0§¢

2

13

Filtering mesh

40§­

3

autoclave on funnel

14

Membrane filter set

0.45§­

2

autoclave in cap

15

Filtering mesh

190§­

2

autoclave on funnel

16

empty ¡â-flask

100§¢

2

17

¡â-flask with 20§¢ PDMP

100§¢

10

¢Ñ membrane filterÀÇ ¹Ý¦ÀÌ´Â Ý»ÝÂÀÌ À§·Î(¶Ñ²± Ý»ÝÂÀ») ú¾Çϵµ·Ï ³ÖÀ½

3.8.2. Medium

3.8.2.1. Trans-culturing ÛÆò¢(PDMP medium) - 10 flasks (¢Ñ 20§¢/100§¢ ¡â-flask)

ÛãûÜ

ãËå·Ù£

é»Õá/100§¢

ð²íÂ

ÝáÍÅ

1

Distilled water

100§¢

200§¢

2

Potato dextrose broth

2g

4g

3

Malt extract

0.3g

0.6g

4

Peptone

0.1g

0.2g

pH = 5.5

3.8.2.2. Regeneration medium - Hamada medium + sucrose for osmotic buffering

ÛãûÜ

ãËå·Ù£

ÐññÞ

èÐõúÛÆò¢(+)

ÓßðÎÛÆò¢(-)

ÝáÍÅ

ýüà·äû

ÛÆò¢

ýüà·äû

ÛÆò¢

1

Tap water

100§¢

20§¢

150§¢

20§¢

150§¢

1ó­ ñú׶â©

2

Glucose

2g

0.3g

3.0g

0.3g

3.0g

3

Yeast extract

0.5g

0.075g

0.75g

0.075g

0.75g

4

Sucrose

17.1g

2.565g

25.65g

-

-

5

Agar

1g

-

1.5g

-

1.5g

- : Èò»ötape

pH = 5.5

¢Ñ + øöãÆ : sucrose, agar øÐùß 150§¢/300§¢ ¡â-flask ¡æ regeneration medium

sucrose øÐùß 20§¢/100§¢ ¡â-flask ¡æ dilutionéÄ

¢Ñ - øöãÆ : agar øÐùß 150§¢/300§¢ ¡â-flask ¡æ regeneration medium for control

sucrose Ú±øÐùß 20§¢/100§¢ ¡â-flask ¡æ dilutionéÄ

3.8.2.3. Regeneration medium ñÞÝá(pouring)

¢Ñ èÐõúäû øÐùß êóÙí¿¡ µû¶ó disposable petri-dish¸¦ ÞÅéÄ ÊÀÊÀ 30ËÁ¾¿ ñÞÝá

¢Ñ Pipetman°ú ÓÞúþ tip(5§¢éÄ)À» ì¦éÄÇÏ¿© 4§¢¾¿ petri-dish¿¡ ÝÂñ»

¢Ñ ÝÂñ»°¡ ³¡³­ ý­ ½Ä¾î¼­ ͳïÒÀÌ µÇ¸é ¶Ñ²±¿¡ ýüà·ÛÃ×Ë°ú buffer ôÕÊ¥æ¨Üú øöãÆ

¡Ø + øöãÆ : sucrose, agar øÐùß ¡æ regeneration medium

- øöãÆ : agar øÐùß, sucrose Ú±øÐùß ¡æ ÓßðÎÏ¡

3.9. Trans-culture ¹× protoplast preparation (1999. 3. 24)

3.9.1. Trans-culture from one 300§¢-flask to four 100§¢-flasks

3.9.1.1. ÛÆå×ßÒ÷¾ ü¬ìã

¢Ñ ÐÞîûÀ¸·Î ¸·Èù ãËúÐη 5°³ÀÇ ñìÕá ö´ïÒ ¹× ãËúÐη¿¡ ¸ÅÁ÷ÆæÀ¸·Î ÑÀÖâ

¢Ñ 5ËÁÀÇ ß²ÊÇÇöó½ºÅ©¿¡ ÛÆå×µÈ Ð¶Þ긦 ØþжµÈ long spatula¸¦ ì¦éÄÇÏ¿© жÞ길À» ÊÀÊÀ °É·¯³»¾î spatula¸¦ ì¦éÄÇÏ¿© ¾à°£¾¿ ´­·¯¼­ жÞê¿¡ µé¾îÀÖ´Â ÛÆå×äûÀÌ °ÅÀÇ ´Ù ÝÂÝôµÉ ¼ö ÀÖµµ·Ï Çϸ鼭 ìéÓ© Øþж petri-dish(disposable)¿¡ ¿Å±è

¢Ñ ÐÞîûÀ» ´Ù½Ã ¸·Àº ý­ ñìÕáÀ» ö´ïÒÇÏ¿© ÛÆå×äûÀÌ Òýá´ ¼¯ÀΠжô÷ÀÇ ñìÕá ÷êäÄ

ãËúÐη ÛãûÜ

#1

#2

#3

#4

#5

øÁг

õÅ ñìÕá(g)

13.209

12.977

13.262

14.025

13.678

жÞê+ãËúÐη

13.432

13.255

13.482

14.283

13.913

жÞêÕá

0.223

0.278

0.220

0.258

0.235

0.2428

ý£áÈ ô¥×â

1.00§¢

1.00§¢

0.74§¢

3.9.1.2. ì¹ï® ïÈðú

¢Ñ ÝïÎòîÜ åÖÀÌ ¸¹Àº °Í 2ËÁ(#2, #4)À» ùêÇÏ¿© 536§·À» ¸¸µç ÈÄ, ÇÔ²² cutting

¢Ñ õÅ 10ËÁÀÇ flasks¿¡ ÊÀÊÀ ïÈðú

¢Ñ cuttingÀº 7Ý ïïÓø ãùã¿ÇÏ¿´À¸¸ç, ïÈðúüù×ËÀº å³ 40%·Î ¿©°ÜÁü

¡Ø ïÈðúÕá : 1-13¡­1-22 : ¡Ö 20§·

3.9.2. Protoplast extraction

3.9.2.1. ý£áÈ ñÞÝá

¢Ñ Water bath¿¡ ¹°À» ä¿ì°í ï³ê¹À» ÄѼ­ è®Óø¸¦ 30¡É·Î settingÇÔ

¢Ñ Clean-bench ¿·ÀÇ table ¼­¶ø¿¡¼­ ñ¼ÞÒÐï ¼¼Æ®¸¦ ²¨³»¾î clean-bench¿¡ ³Ö¾îµÒ

¢Ñ ÕÒÔÐÜÁηÇÏ´ø enzyme(CCZ)À» ²¨³»¾î ãùúÐãø¿¡ ³õ¾ÆµÎ¾î ßÈ访¡¼­ ³ìÀÓ

¡Ø ãùòõîÜÀ¸·Î ý£áÈ°¡ ³ì´Âµ¥ á¶é©µÇ´Â ãÁÊàÀº 10Ý ì¤Ò®À̾úÀ½

¢À 3ËÁÀÇ pre-made¸¦ ÞÅéÄÇÏ¿´À¸³ª 2.74§¢ ¹Û¿¡ ¸¸µé ¼ö ¾ø¾úÀ½

¢Ñ ñ¼ÞÒÐï ¼¼Æ®¸¦ membrane filterÀÇ À­ ݻݿ¡ ֧̿ÇÏ°í ØþжÇÑ ÐÞîû ãËúÐηÀÇ À§¿¡ membrane filter kitÀÌ Ö§Ì¿µÇµµ·Ï ÇÑ ý­, ñ¼ÞÒÐï¿¡ ³ìÀº enzyme solutionÀ» ³Ö°í Á¶½É½º·´°Ô ñ¼ÞÒÐï ÇÚµéÀ» ¹Ð¾î(membrane filter°¡ Âõ¾îÁöÁö ¾Êµµ·Ï) ý£áÈéÁäûÀÌ ØþжµÇµµ·Ï ô¥×â

3.9.2.2. жÞêÕá¿¡ µû¸¥ ý£áÈ á¶é©Õá ͪߩ

¢Ñ #1, #3, #5ÀÇ øÁгÀº 226§··Î ͪߩµÊ

¢Ñ 150¡­200§·¿¡ 1§¢ÀÇ ý£áȸ¦ ô¥×âÇÏ´Â °ÍÀÌ ê«öÎÀ̳ª ͪߩÀÇ øµ××àõÀ» êÓÇÏ¿© ÊÀÊÀ 1§¢¸¦ ô¥×âÇϵµ·Ï ̽ïÒ ¢À ´Ü, #5´Â 0.74§¢¸¸ ô¥×âÇÔ

3.9.3. Enzyme reaction

3.9.3.1. ý£áÈô¥×â

¢Ñ filteringµÈ ý£áȸ¦ 1§¢¾¿ ³Ö¾î ÁÜ

¢Ñ жô÷¸¦ spatula¸¦ ì¦éÄÇÏ¿© Àß Ç®¾î ÁÜ

3.9.3.2. Úãëëë¯Óô

¢Ñ shaking water bath(30¡É)¿¡ ³ÖÀº ý­ 80rpm áÜÓø·Î shakingÇϸç Úãëë ë¯Óô

¢Ñ Øß 20ݸ¶´Ù ²¨³»¾î ãËúÐηÀÇ ¾Æ·¡ Ý»ÝÂÀ» ¼ÕÀ¸·Î ÀâÀº ä 1¡­2õ© µ¿¾È vortex mixingÇÏ¿© ´Ù½Ã shaking water bath¿¡ ³ÖÀ½ ¢¡ 2ãÁÊà µ¿¾È ý£áÈÚãëë ô¥×â

¢Ñ ý£áÈÚãëë ô¥×âÑ¢Êà ñé ýüà·äû ñÞÝá

¢¡ ÊÀÊÀ 18ËÁÀÇ - ¹× + èÐõúéÁäû mortonîû ãËúÐη¿¡ 0.9§¢¾¿ ÝÂñ»

3.9.3.3. Úãëëäû ÕëΦ

¢Ñ ý£áÈÚãëëÀÌ ³¡³­ ý­, 40§­ÀÇ filter mesh¸¦ ì¦éÄÇÏ¿© ÚãëëäûÀ» ÕëΦÇÏ¿© ÐÞîû ãËúÐηÀ¸·Î ¿Å°Ü ³ÖÀ½

3.9.4. Protoplast counting and inoculation

3.9.4.1. úéÚ°Ìð°ú Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ Improved NeubauerÀÇ µÎ grid°¡ Àִ ݻݿ¡ ÇÑ ¹æ¿ï¾¿ protoplast éÁäû ß¾öÇ

¡Ø å³ 2¡­3Ý ÌèΦ ý­ úéÚ°Ìð ¾Æ·¡¿¡¼­ ËþÌð

¢Ñ úéÚ°Ìð 100ÛÃ×Ë¿¡¼­ ã·íÂÇÏ¿© 400Ûà â©ñÞ¿¡¼­ ËþÌð

3.9.4.2. Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ 4ËÁÀÇ grid Ò®¿¡ ÀÖ´Â protoplastÀÇ â¦¸¦ countingÇÏ°íÀÚ ÇÔ

¢Ñ 1ó­ ãËÓñ¿¡¼­¿Í ¸¶Âù°¡Áö·Î protoplastÀÇ åÖÀÌ Ð¿È÷ ÀûÀ½

3.9.4.3. dilution ¹× ïÈðúÀº ãùã¿ÇÏ¿´Áö¸¸ ãùòõîÜÀ¸·Î´Â ÙíëòÚ«

3.10. Protoplast preparation 3ó­ ãËÓñ(without regeneration) ñÞÝá

¡Ø жñ»¿¡ ßÓμ¾øÀÌ ãËÓñ ý£áÈ 2ðú(Abe1, Abe 3)À¸·Î ÊÀÊÀ ãÁÊà 3 ðú×¾

3.10.1. ØþжÐïÎý ñÞÝá : 2 жñ»(#3, #70) ô¥×âéÄ - ÊÀÊÀ 3ÚãÜÖ (1999. 4. 5)

ÛãûÜ

ÐïÎýÙ£

Ó¤êÈ(é»Õá)

â¦Õá

ÝáÍÅ

1

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

2

2ðú×¾ ý£áÈéÄ

2

á³û¡ ãËúÐη(ÐÞîû)

íþ : 10cm

7

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

3

Glass petri-dish

¥õ: 6cm

6

ËëæðØþж

4

Pasteur pipette

2

ËëæðØþж,

5

Mini spatula

6

ËëæðØþж, 1ËÁ´Â çãÝáéÄ

6

Pipetman tip(ñé, ôìßä)

1.0§¢

3

autoclave, ý£áÈ ÝÂñ»éÄ

7

Silicon cap(pipetteéÄ)

5.0§¢

2

autoclave

8

Filtering mesh

190§­

2

autoclave on funnel

9

Filtering mesh

40§­

6

autoclave on funnel

10

Membrane filter set

0.45§­

2

autoclave in cap

¢Ñ membrane filterÀÇ ¹Ý¦ÀÌ´Â Ý»ÝÂÀÌ À§·Î(¶Ñ²± Ý»ÝÂÀ») ú¾Çϵµ·Ï ³ÖÀ½

3.10.2. Enzyme ñÞÝá (1999. 4. 6)

3.10.2.1. Abe series 4ðú×¾ ðàð²


Cell. RS

(§·)

Cell. R10

(§·)

Zymolase

100T(§·)

Glucuronidase624T,341T(§·)

Mannitol

(§·)

MgSO47H2O

(g)

maleate

(g)

pH

AB1

30§¢

-

50

50

50

-

1.47882

0.58035

5.6

AB2

30§¢

50

-

50

50

-

1.47882

0.58035

5.6

ÒØÓø*

0.167%

0.167%

167U

1000U%

-

200mM

17mM

AB3

20§¢

-

50

50

50

50

-

0.58035

5.5

AB4

20§¢

50

-

50

50

50

-

0.58035

5.5

ÒØÓø*

0.25%

0.25%

250U

850U

0.25%

-

25mM

* 1§¢ ÐññÞ ÒØÓø

3.10.2.2. ê«öÎîÜÀ¸·Î 10§¢ ð²ðã ÐññÞ¿¡ µû¶ó ð²ðãÇÏ°íÀÚ ÇÏ¿´À¸³ª pH¸¦ ¸ÂÃߴ Ρïï¿¡¼­ maleic acid°¡ ôÕÊ¥µÊ¿¡ µû¶ó pH´Â 1.5 â©ñÞ¿¡ ¸Ó¹°°í À־ ³Ê¹« ¸¹Àº åÖÀÇ NaOH solutionÀÌ ôÕÊ¥µÇ¾î åÖÀÌ Ü¨ÇÏ°Ô µÇ¾úÀ½

3.10.2.3. µû¶ó¼­, AB1°ú AB2´Â 30§¢ÀÌ ¸¸µé¾îÁ®¼­ ãùð· ðàð²µÈ ý£áÈéÁäûÀº ÙÍøöÇÑ ÒØÓøÀÇ ¨÷, AB3°ú AB4´Â 20§¢ÀÌ ¸¸µé¾îÁ®¼­ ÙÍøöÇÑ ÒØÓøÀÇ ¨ö¿¡ Ó¹ÇÏ´Â ÒØÓø°¡ µÊ

¡Ù ´ÙÀ½¿¡ ð²ðãÇÒ ÌÑéç¿¡´Â, 'maleic acid-NaOH' solutionÀ» 200mMÀÇ ÒØÓø·Î ¹Ì¸® ¸¸µé¾î¼­ pH¸¦ 5.6 Ò®èâ·Î ¸ÂÃá ý­, îïô÷ ðàð²ÄÚÀÚ ÇÏ´Â åÖÀÇ ¨úÀº super quality distilled water, ¨ùÀº (10§¢¸¦ ¸¸µé ÌÑéç 2.5§¢) 200mMÀÇ buffer solutionÀ» ý£áÈ éÁäû ð²ðããÁ ôÕÊ¥ÇÏ´Â Û°ãÒÀ» üÀéÄÇÏ¿© ÇÔ

3.10.2.4. Centrifugation : 1500gÀÇ áÜÓø·Î 10ÝÂÊà 10¡É Ò®èâÀÇ è®Óø¿¡¼­ êÀãýÝÂ×î ý­ ß¾ÔôäûÀ» á³û¡ ãËúÐηÀ¸·Î ¿Å°Ü ºÎ¾î¼­ labeling

¡Ù PipetteÀ» ÞÅéÄÇÏ¿© ß¾ÔôäûÀ» ¶°³»´Â °ÍÀÌ ê«öÎÀÌÁö¸¸, ý£áȸ¦ ÞÅéÄÇÒ ¶§ ùößÈ Øþж membrane filter¸¦ ì¦éÄÇÏ¿© °É·¯¼­ ÞÅéÄÇϹǷΠ±×³É ºÎ¾î¼­ ¿Å±è

3.11. Protoplast preparation (1999. 4. 7)

3.11.1. жÞêÕá ÷êäÄ ¹× жÞêÕá¿¡ µû¸¥ ý£áÈ á¶é©Õá ͪߩ

3.11.1.1. ïÈðúê« handling

¢Ñ 190§­ filter¸¦ ¾ñÀº 100§¢-flask¿¡ Á÷Á¢ ºÎÀ¸¸é¼­ filter¿¡ °É¸° жÞê â½ó¢

¢Ñ »þ¾Ë·¹·Î ¿Å°Ü ÛÆå×äûÀ» ´­·¯ ð¶ËÛ ý­ Øþж ÐÞîû ãËúÐηÀ¸·Î ¿Å±â°í ñìÕá ö´ïÒ

ãËúÐη ÛãûÜ

#3

#70

򋆅 󋞆

1

2

3

1

2

3

ãËúÐη (g)

13.426

13.569

13.675

13.740

14.123

13.594

ãËúÐη + жÞê

13.638

13.806

13.871

13.998

14.450

13.832

жÞêô÷ ñìÕá

0.212

0.237

0.196

0.258

0.327

0.238

3.11.1.2. жÞêÕá¿¡ µû¸¥ ý£áÈ á¶é©Õá ͪߩ

ãËúÐη ÛãûÜ

1

2

3

4

5

6

7

жÞêô÷ ñìÕá

0.212

0.237

0.196

0.238

0.170

0.238

0.177

ý£áÈ á¶é©Õá

1§¢

1§¢

1§¢

1§¢

1§¢

1§¢

1§¢

ý£áÈ ðú×¾

AB1

AB1

AB1

AB3

AB3

AB3

AB3

ô¥×â ãÁÊà

2

3

4

1

2

3

4

¢Ñ 150¡­200§·¿¡ 1§¢ÀÇ ý£áȸ¦ ô¥×âÇÏ´Â °ÍÀÌ ê«öÎÀ̳ª ͪߩ߾ÀÇ øµ××àõÀ» êÓÇÏ¿© ¸ðµÎ 1§¢¾¿ ô¥×âÇÔ

3.11.2. Protoplast extraction

3.11.2.1. ý£áÈ ñÞÝá

¢Ñ Water bath¿¡ ¹°À» ä¿ì°í ï³ê¹À» ÄѼ­ è®Óø¸¦ 30¡É·Î settingÇÔ

¢Ñ ÕÒíúÍ·¿¡ ÜÁηÇÏ´ø enzymeÀ» ²¨³»¾î ãùúÐãø¿¡ ³õ¾Æ äÌïÒûù½ÃÅ´

¢Ñ ñ¼ÞÒÐï ¼¼Æ®¸¦ membrane filterÀÇ À­ ݻݿ¡ ֧̿ÇÏ°í ØþжÇÑ ÐÞîû ãËúÐηÀÇ À§¿¡ membrane filter kitÀÌ Ö§Ì¿µÇµµ·Ï ÇÑ ý­, ñ¼ÞÒÐï¿¡ ³ìÀº enzyme solutionÀ» ³Ö°í Á¶½É½º·´°Ô ñ¼ÞÒÐï ÇÚµéÀ» ¹Ð¾î(membrane filter°¡ Âõ¾îÁöÁö ¾Êµµ·Ï) ý£áÈéÁäûÀÌ ØþжµÇµµ·Ï ô¥×â

¢Ñ filteringµÈ ý£áȸ¦ 1§¢¾¿ ³Ö¾î ÁÜ

¢Ñ жô÷¸¦ spatula¸¦ ì¦éÄÇÏ¿© Àß Ç®¾î ÁÜ

3.11.2.2. Úãëëë¯Óô

¢Ñ shaking water bath(30¡É)¿¡ ³ÖÀº ý­ 80rpm áÜÓø·Î shakingÇϸç Úãëë ë¯Óô

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3.11.2.3. Úãëëäû ÕëΦ

¢Ñ ý£áÈÚãëëÀÌ ³¡³­ ý­, 40§­ÀÇ filter mesh¸¦ ì¦éÄÇÏ¿© ÚãëëäûÀ» ÕëΦÇÏ¿© ÐÞîû ãËúÐηÀ¸·Î ¿Å°Ü ³ÖÀ½

3.11.3. Protoplast counting

3.11.3.1. úéÚ°Ìð°ú Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ Improved NeubauerÀÇ µÎ grid°¡ Àִ ݻݿ¡ ÇÑ ¹æ¿ï¾¿ protoplast éÁäû ß¾öÇ

¡Ø å³ 2¡­3Ý ÌèΦ ý­ úéÚ°Ìð ¾Æ·¡¿¡¼­ ËþÌð

¢Ñ úéÚ°Ìð 100ÛÃ×Ë¿¡¼­ ã·íÂÇÏ¿© 400Ûà â©ñÞ¿¡¼­ ËþÌð

3.11.3.2. Haemacytometer¸¦ ì¦éÄÇÑ protoplast count

¢Ñ 4ËÁÀÇ grid Ò®¿¡ ÀÖ´Â protoplastÀÇ â¦¸¦ countingÇÏ°íÀÚ ÇÔ

¢Ñ 1ó­ ãËÓñ¿¡¼­¿Í ¸¶Âù°¡Áö·Î protoplastÀÇ åÖÀÌ Ð¿È÷ ÀûÀ½

3.11.3.3. dilution ¹× ïÈðúÀº ãùã¿ÇÏ¿´Áö¸¸ ½ÇÁúÀûÀ¸·Î´Â ÙíëòÚ«