Crops ›› 2017, Vol. 33 ›› Issue (1): 161-167.doi: 10.16035/j.issn.1001-7283.2017.01.029

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Optimization of Virus-Free of Shoot Tip and Virus Detection of Test-Tube Plantlet of Potato

Lu Yanli1,2,Zhou Hongyou2,Zhang Xiaoyu2   

  1. 1Management Center of Emission Right Trading in Inner Mongolia,Hohhot 010011,Inner Mongolia,China
    2Agronomy College,Inner Mongolia Agricultural University,Hohhot 010019,Inner Mongolia,China
  • Received:2016-08-31 Revised:2016-12-28 Online:2017-02-15 Published:2018-08-26

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Abstract:

In order to optimize virus-free technology of potato shoot tip, and to obtain strong virus-free seedlings, the different potato varieties, auxin, L-cysteamine hydrochloride, sterilization time of medium and potato shoots were studied in the experiment. The pH of MS medium was compared for seedling growth and the virus was detected with RT-PCR. Results showed that among 28 potato varieties, some rapidly grew callus and developed into seedlings, others did not. In MS medium containing NAA, the callus formation was faster and bigger, seedling formation was slower and less, but seedlings were stronger. In MS medium containing IAA, no or small calluses were produced, the seedlings formed faster and more, but seedlings were weaker. L-cysteamine hydrochloride could reduce callus brown, increased seedling emergence rate. Sterilization time 20min at 115℃ was the best for medium in tubes. 5-7min and 9-10min with 0.1% mercuric chloride were the best sterilization time for small shoots and big shoots, respectively. The optimum pH of MS medium for seedling growth was 5.8. PVS of 18%, PVY of 12% and PLRV of 5% were detected in different variety seedlings.

Key words: Potato, Virus-free of shoot tip, L-cysteamine hydrochloride, Indoleacetic acid, Culture medium, RT-PCR, Virus detection

Table 1

Medium for inducing shoot tip meristem mg/L"

培养基编号
Medium No.		 IAA NAA 6-BA GA3 L-半胱胺酸盐酸盐
L-cysteamine hydrochloride
1 0.10 - 0.10 0.10 -
2 - 0.10 0.10 0.10 -
3 0.10 - 0.10 0.10 0.10
4 - 0.10 0.10 0.10 0.10

Table 2

Specific primers of coat protein gene of five potato viruses"

马铃薯病毒
Potato virus		 引物
Primer		 序列Sequence Tm值(℃)
Tmvalue
PVX PVX-F 5′-TAGCACAACACAGGCCACAG-3′ 57.4
PVX-R 5′-GGCAGCATTTCAGCTTC-3′ 52.0
PVS PVS-F 5′-TGGCGAACACCGAGCAAATG-3′ 57.4
PVS-R 5′-ATGATCGAGTCCAAGGGCACTG-3′ 60.0
PVY PVY-F 5′-ACGTCCAAAATGAGAATGCC-3′ 53.4
PVY-R 5′-TGGTGTTCGGTGATGTGACCT-3′ 57.8
PSTV PSTV-F 5′-GGATCCCCGGGGAAACCTGGAGCA-3′ 67.2
PSTV-R 5′-GGATCCCTGAAGCGCTCCTCCGAGC-3′ 68.8
PLRV PLRV-F 5′-CGCGCTAACAGAGTTCAGCC-3′ 59.5
PLRV-R 5′-GCAATGGGGGTCCAACTCAT-3′ 57.4

Table 3

Callus growth and seedling emergence in different potato varieties (lines)"

品种（系）Variety (Line) 愈伤组织生长情况Callus growth 植株形成时间Days induced into seedlings(d) 成苗率Seedlings emergence rate(％)
J07-5 出愈快而大,淡绿色 30~35 90
紫花白、克新1号、陇薯
5号、J08-4、底西芮		 出愈快而大,绿色 40~50 30~40
青薯168、水洗红 出愈较快而大,绿色 50~55 20~25
涩皮红、J07-2、J08-2、
J08-3、J07-6、J07-7、
J07-8、J08-1、J07-10		 出愈较快而大,绿色、1/3褐色 50~55 10~20
陇薯7号、陇薯6号、
陇薯3号		 出愈较快而大,绿色、1/2褐色 >60 5~10
J07-3、J07-9、J07-4、费乌瑞它、大西洋、夏坡蒂、LK99、黑美人 出愈较快而大,褐色 不成苗 0

Table 4

Effects of different medium on callus growth and seedling emergence"

处理Treatment 愈伤组织生长情况
Callus growth		 植株形成时间(d)
Days of forming seedling		 成苗率(%)
Seedling emergence rate		 幼苗生长状况
Seedling growth condition
IAA 出愈速度慢,愈伤组织绿色、1/3变褐、小 35~40 61 很细弱,根少且细
NAA 出愈速度较快,愈伤组织绿色、1/2变褐、较大 45~50 34 较壮
IAA+L-半胱胺酸盐酸盐
IAA+ L-cysteamine hydrochloride		 出愈速度较慢,愈伤组织绿色、少部分变褐、较小 30~35 75 较细弱,根少且细
NAA+L-半胱氨酸盐酸盐
NAA+ L-cysteamine hydrochloride		 出愈速度快,愈伤组织绿色、少部分变褐、大 40~45 46 壮

Table 5

Callus growth and seedling emergence at different sterilization time on medium"

温度
Temperature(℃)		 污染率(%)
Contamination rate		 愈伤组织生长情况
Callus growth		 植株形成时间(d)
Days of forming seedling		 成苗率(%)
Seedling emergence rate		 幼苗生长状况
Seedling growth
110 20 大,绿色 45~50 38 较壮
115 0 大,绿色 45~50 45 较壮
121 0 部分较小,变褐色的较多 45~50 35 较弱

Table 6

Callus growth and seedling emergence at different sterilization time on potato shoots"

灭菌时间(min)
Sterilization time		 污染率(%)
Contamination rate		 愈伤组织生长情况
Callus growth		 植株形成时间(d)
Days of forming seedling		 成苗率(%)
Seedling emergence rate
3 50 大,绿色 45~50 12
5 10 大,绿色 45~50 23
7 5 愈伤组织较大,个别的为褐色 45~55 41
9 0 部分愈伤组织小、褐色 45~55 39
10 0 部分愈伤组织小、褐色 45~55 38

Table 7

Growth of potato seedlings in different pH medium"

pH 根鲜重(g/株)
Root fresh weight		 根干重(g/株)
Root dry weight		 地上部鲜重(g/株)
Shoot fresh weight		 地上部干重(g/株)
Shoot dry weight		 茎粗(cm)
Stem diameter		 苗高(cm)
Seedling height		 叶面积(cm2/株)
Leaf area
5.7 0.072b 0.005c 0.132d 0.011c 0.129c 4.73c 2.41c
5.8 0.125a 0.008a 0.222a 0.018a 0.150a 6.77a 4.33a
5.9 0.119ab 0.008a 0.171b 0.014b 0.141b 4.42d 4.25a
6.0 0.089b 0.007ab 0.156c 0.012c 0.153a 4.95c 3.70ab
6.1 0.053c 0.004d 0.159c 0.012c 0.150a 5.73b 3.03b
6.2 0.061b 0.005c 0.148c 0.011c 0.150a 5.24c 1.94c
6.3 0.035d 0.003e 0.014e 0.009d 0.140b 5.19c 2.87b
6.4 0.064b 0.006b 0.134d 0.011c 0.138b 5.40b 1.92c

Fig.1

Detection of RNA quality, and virus by RT-PCR Lanes marked with number are samples; Lane M is DL2000"

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