
Comparis on In Vitro Culture of Mature Embryos of Nelumbo from Eight Provenances Worldwide
Mi Qin, Fengluan Liu, Qingqing Liu, Jianguo Cao, Yuchu Chen, Daike Tian
Comparis on In Vitro Culture of Mature Embryos of Nelumbo from Eight Provenances Worldwide
Establishing a feasible disinfection system for obtaining the germfree mature lotus (Nelumbo) embryos as explants and understanding the differences of in vitro growth of lotus from different provenances is the basis for material selection and evaluation method to construct an efficient in vitro culture and genetic transformation system. Plant preservative mixture (PPM) at different concentrations was used in the pre-culture stage to develop a suitable disinfection system of lotus seeds and a method of acquiring the germ-free lotus embryos. Through this sterilization approach, the in vitro cultured seedlings of lotus from 9 worldwide provenances were successfully obtained. Based on the morphological indexes at different culture periods, the membership function method was used to evaluate the growth differences under the in vitro culture conditions. The results showed that the ideal disinfection system for lotus seeds was 75% alcohol 30 s + 2% PPM 2 h + 0.05% PPM 3-5 d soaking during germination. The in vitro growth differences of the seedlings from the tested lotuses of 8 provenances were mainly observed in the later culture stage, such as the number of leaves, the length and thickness of rhizomes, the number of roots and lateral shoots. The seedlings of lotus from Vietnam and Thailand had the highest growth potential and higher reproduction rate under the same culture conditions. The pre-culture method by soaking lotus seeds with PPM to successfully obtain germ-free lotus embryos has solved the technique problem in disinfecting lotus seeds in the past. The in vitro clones of lotus embryos from the tropical or subtropical regions in both Vietnam and Thailand have stronger growth capacity and therefore are the materials more ideal for establishing regeneration and genetic transformation system of Nelumbo.
Nelumbo / wild type lotus / lotus embryo / sterilization / plant preservative mixture / in vitro culture / membership function {{custom_keyword}} /
图1 9个不同种源地的野生型莲花特征PLD.普兰店古莲,HLJ.黑龙江莲,BYD.白洋淀红莲,WSH.微山湖红莲,VN.越南莲,TH.泰国莲,IN.印度莲,AU.澳大利亚莲,NLU.美洲莲 |
表1 试验材料的基本信息 |
编号 | 代表种源名 | 种名 | 原产地 | 生态型 | 株型 | 花色 | 瓣型 | 凭证标本/栽培号 | |
---|---|---|---|---|---|---|---|---|---|
中文名 | 拉丁文 | ||||||||
PLD | 普兰店古莲 | 亚洲莲 | Nelumbo nucifera | 中国辽宁大连普兰店区 | 温带型 | 大型 | 红色 | 单瓣 | M385 |
HLJ | 黑龙江莲 | 亚洲莲 | N. nucifera | 中国黑龙江挠力河 | 温带型 | 大型 | 红色 | 单瓣 | M474 |
BYD | 白洋淀红莲 | 亚洲莲 | N. nucifera | 中国河北新安县白洋淀 | 温带型 | 大型 | 红色 | 单瓣 | M282 |
WSH | 微山湖红莲 | 亚洲莲 | N. nucifera | 中国山东滕州市微山湖 | 温带型 | 大型 | 红色 | 单瓣 | TDK1036/M655 |
VN | 越南莲 | 亚洲莲 | N. nucifera | 越南海阳市 | 温带型 | 大型 | 粉色 | 单瓣 | TDK2914 |
TH | 泰国莲 | 亚洲莲 | N. nucifera | 泰国清迈Sarapee区11号公路旁 | 热带型 | 大型 | 白色 | 单瓣 | TDK2782/L140 |
IN | 印度莲 | 亚洲莲 | N. nucifera | 印度Maharashtra邦 | 温带型 | 大型 | 淡粉色 | 单瓣 | TDK1603/M469 |
AU | 澳大利亚莲 | 亚洲莲 | N. nucifera | 澳大利亚北部Mary River附近 | 热带型 | 大型 | 红色 | 单瓣 | L131 |
NLU | 美洲莲 | 美洲莲 | N. lutea | 美洲佛罗尼达迈阿密Okeechobee湖 | 温带型 | 大型 | 黄色 | 单瓣 | TDK642/M519 |
注:M、L开始编号的为荷花资源的栽培编号,TDK开始编号的为标本采集号。 |
表2 不同催芽预培养下白洋淀红莲无菌胚的获得率 |
方法 | 短时消毒步骤 | 长时催芽步骤 | 莲胚无菌率/% | 10 d莲胚正常生长率/% |
---|---|---|---|---|
处理1 | 2% PPM × 2 h | 0.01% PPM × 5 d | 90 | 85 |
处理2 | 2% PPM × 2 h | 0.05% PPM × 5 d | 100 | 95 |
处理3 | 2% PPM × 2 h | 0.1% PPM × 5 d | 100 | 75 |
CK1 | 2% NaClO × 5 min | 无菌水× 5 d | 75 | 25 |
CK2 | 2% NaClO × 10 min | 无菌水× 5 d | 90 | 20 |
表3 培养5、10天时不同种源地莲的成熟胚离体培养幼苗生长比较 |
编号 | 叶数 | 叶短径/mm | 叶柄长/mm | 长小于1 cm根数 | 长大于1 cm根数 | 总根数 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
5 d | 10 d | 5 d | 10 d | 5 d | 10 d | 5 d | 10 d | 5 d | 10 d | 5 d | 10 d | ||||||
PLD | 1.8±0.53a | 2.0±0.00a | 10.2±1.74ab | 7.5±2.60a | 56.6±18.51a | 39.6±12.91ab | 2.8±1.32a | 5.0±1.23a | 0.8±0.58bc | 9.4±1.97a | 3.6±1.57ab | 14.4±2.32a | |||||
HLJ | 2.2±0.17a | 3.0±0.00a | 12.0±0.45a | 9.6±1.04a | 14.5±1.92b | 30.7±4.44ab | 1.8±1.05a | 5.0±0.86a | 0.2±0.17c | 5.7±0.56ab | 2.0±1.83ab | 10.7±1.09ab | |||||
BYD | 1.8±0.25a | 3.0±0.00a | 12.0±0.41a | 13.1±3.37a | 32.8±12.18ab | 44.8±14.96ab | 1.0±0.71a | 5.3±1.44a | 4.0±1.41a | 5.3±1.11ab | 5.0±1.68a | 10.5±0.87ab | |||||
WSH | 1.8±0.20a | 2.4±0.40a | 8.6±1.29ab | 9.1±0.97a | 21.0±9.80b | 31.7±8.10ab | 2.6±0.87a | 5.4±0.51a | 0.2±0.20c | 4.0±1.41ab | 2.8±0.86ab | 9.4±1.12ab | |||||
VN | 1.0±0.32a | 3.0±0.26a | 9.0±1.08ab | 9.1±0.99a | 33.5±10.49ab | 44.1±6.94ab | 0.5±0.50a | 2.8±1.40a | 0.0±0.00c | 2.8±0.79b | 0.5±0.50b | 4.6±2.32b | |||||
TH | 1.3±0.75a | 2.8±0.48a | 11.0±1.58ab | 5.8±3.38a | 44.8±7.04ab | 20.9±12.49b | 1.0±0.58a | 5.5±1.93a | 2.5±1.50ab | 6.5±2.22ab | 3.5±2.06ab | 12.0±4.14ab | |||||
IN | 2.0±0.26a | 2.2±0.49a | 10.5±1.47ab | 9.5±3.50a | 48.2±8.87ab | 70.3±53.25a | 0.3±0.33a | 3.3±1.65a | 0.0±0.00c | 5.3±3.09ab | 0.3±0.33b | 8.5±4.74ab | |||||
NLU | 1.3±0.48a | 2.4±0.40a | 7.0±0.41b | 6.7±2.38a | 26.8±7.95ab | 28.3±13.30ab | 0.5±0.50a | 2.5±1.66a | 0.8±0.75bc | 4.0±2.16ab | 1.3±1.25ab | 6.5±3.66ab | |||||
编号 | 茎节数 | 茎节间长/mm | 茎节间粗/mm | 隶属函数均值 | 综合排名 | ||||||||||||
5 d | 10 d | 5 d | 10 d | 5 d | 10 d | 5 d | 10 d | 5 d | 10 d | ||||||||
PLD | 1.0±0.00a | 2.0±0.00a | 13.2±0.86a | 6.2±0.53ab | 2.2±0.20d | 2.8±0.39a | 0.62 | 0.75 | 1 | 2 | |||||||
HLJ | 1.0±0.01a | 1.0±0.00c | 9.0±1.21a | 5.9±0.64ab | 3.1±0.07ab | 2.9±0.20a | 0.43 | 0.11 | 4 | 8 | |||||||
BYD | 1.0±0.02a | 1.0±0.00c | 12.0±0.00a | 6.9±0.77a | 3.0±0.00abc | 3.0±0.20a | 0.6 | 0.75 | 2 | 1 | |||||||
WSH | 1.0±0.03a | 1.0±0.00c | 9.8±1.50a | 6.4±0.46ab | 2.4±0.40bcd | 3.1±0.11a | 0.27 | 0.6 | 6 | 4 | |||||||
VN | 1.0±0.04a | 1.2±0.17bc | 9.3±1.93a | 4.6±0.38b | 2.5±0.29bcd | 2.8±0.25a | 0.16 | 0.55 | 8 | 5 | |||||||
TH | 1.0±0.05a | 1.0±0.00c | 9.3±1.11a | 4.6±0.41b | 3.4±0.20a | 3.1±0.15a | 0.55 | 0.61 | 3 | 3 | |||||||
IN | 1.0±0.06a | 1.0±0.00c | 10.2±1.01a | 5.2±0.81ab | 2.7±0.21bcd | 2.1±0.78a | 0.33 | 0.54 | 5 | 6 | |||||||
NLU | 1.0±0.07a | 1.4±0.26ab | 9.0±1.58a | 5.6±0.58ab | 2.3±0.25cd | 2.9±0.30a | 0.22 | 0.51 | 7 | 7 |
注:同列中不同小写字母表示差异显著(P<0.05)。PLD代表普兰店古莲,HLJ代表黑龙江莲,BYD代表白洋淀红莲,WSH代表微山湖红莲,VN代表越南莲,TH代表泰国莲,IN代表印度莲,NLU代表美洲莲。下同。 |
表4 培养20天时不同种源地莲的成熟胚离体培养幼苗叶、根和茎的生长指标比较 |
编号 | 叶数 | 叶短径/mm | 叶柄长/mm | 叶柄粗/mm | 长不大于 0.5 cm根数 | 长0.5~1 cm根数 | 长大于1 cm根数 |
---|---|---|---|---|---|---|---|
PLD | 2.3±0.85a | 12.5±0.50ab | 58.2±3.38a | 1.7±0.15a | 5.7±1.76ab | 7.3±2.03a | 4.7±4.00abc |
HLJ | 3.5±0.22a | 13.8±0.94ab | 123.0±9.39a | 1.4±0.08a | 3.5±0.56b | 1.8±0.60b | 6.5±1.43abc |
BYD | 3.8±0.25a | 12.5±1.55ab | 54.1±10.56a | 1.4±0.07a | 5.3±0.63ab | 2.3±1.03b | 5.5±0.50abc |
WSH | 2.4±0.68a | 13.6±1.57ab | 58.3±8.26a | 1.4±0.28a | 9.3±3.38a | 1.3±0.95b | 2.8±0.94bc |
VN | 3.1±0.33a | 15.2±2.61ab | 99.8±4.19a | 1.5±0.08a | 3.3±1.20b | 2.2±0.65b | 6.0±2.27abc |
TH | 2.8±1.25a | 17.7±0.33a | 80.4±5.23a | 1.5±0.13a | 4.0±1.00ab | 0.0±0.00b | 11.0±3.00a |
IN | 2.4±0.51a | 12.5±2.93ab | 136.8±8.07a | 1.2±0.16a | 1.3±0.33a | 3.3±1.86b | 9.3±2.73ab |
NLU | 2.7±0.33a | 8.5±1.00b | 25.7±5.65a | 1.2±0.22a | 7.0±2.00ab | 0.0±0.00b | 2.0±0.00c |
编号 | 总根数 | 茎节数 | 茎节间长/mm | 茎节间粗/mm | 隶属函数均值 | 综合排名 | |
PLD | 17.7±1.45a | 2.0±0.00ab | 4.3±0.88ab | 2.9±0.07ab | 0.52 | 4 | |
HLJ | 11.8±1.28a | 2.0±0.00ab | 7.2±0.67a | 3.1±0.17ab | 0.55 | 2 | |
BYD | 13.0±0.71a | 2.0±0.00ab | 6.6±0.59ab | 3.6±0.10a | 0.56 | 1 | |
WSH | 13.3±4.59a | 2.0±0.00ab | 4.1±0.72b | 2.7±0.17b | 0.38 | 6 | |
VN | 11.5±3.06a | 2.5±0.22a | 6.7±0.73ab | 2.9±0.22ab | 0.55 | 3 | |
TH | 11.5±0.50a | 2.0±1.00ab | 5.8±1.17ab | 2.8±0.15b | 0.52 | 5 | |
IN | 14.0±4.04a | 1.0±0.45b | 6.7±1.17ab | 2.9±0.32ab | 0.35 | 7 | |
NLU | 9.0±2.00a | 1.5±0.50ab | 5.8±1.01ab | 3.1±0.22ab | 0.21 | 8 |
表5 培养30天时不同种源地莲的成熟胚离体培养幼苗叶、根和茎生长指标比较 |
编号 | 叶数 | 叶长径/mm | 叶短径/mm | 叶柄长/mm | 叶柄粗/mm | 长不大于0.5 cm根数 | 长0.5~1 cm根数 |
---|---|---|---|---|---|---|---|
PLD | 5.0±1.53ab | 22.6±10.21ab | 18.4±9.05abc | 206.4±16.08ab | 1.2±0.30a | 4.4±1.63a | 2.8±0.17b |
HLJ | 4.3±0.21ab | 29.6±1.36ab | 26.4±1.39ab | 300.4±15.66a | 1.2±0.04a | 2.1±0.58ab | 1.8±0.36bcd |
BYD | 1.5±0.65c | 24.1±9.52ab | 21.6±9.43abc | 128.2±40.33c | 1.4±0.10a | 0.5±0.05b | 7.2±0.41a |
WSH | 2.8±0.86bc | 19.1±4.84ab | 11.3±6.70bc | 101.1±26.81c | 1.1±0.03a | 2.0±1.41ab | 0.79±0.09cd |
VN | 6.0±0.82a | 28.9±1.39ab | 27.5±1.83ab | 196.2±12.75ab | 1.2±0.06a | 3.3±0.67ab | 2.1±0.11bc |
TH | 6.0±0.82a | 33.5±1.06a | 30.3±0.94a | 282.9±19.41a | 1.1±0.04a | 2.8±0.84ab | 2.0±0.02bc |
IN | 4.0±0.41abc | 26.7±1.75ab | 21.9±2.10abc | 201.8±33.84ab | 1.3±0.15a | 1.3±0.75ab | 0.5±0.01cd |
NLU | 3.5±0.50abc | 18.6±2.89ab | 7.8±7.75c | 115.8±48.23c | 1.1±0.28a | 1.5±0.05ab | 0.0±0.00d |
编号 | 长大于1 cm根数 | 总根数 | 茎节数 | 茎节间长/mm | 茎节间粗/mm | 隶属函数均值 | 综合排名 |
PLD | 5.4±2.68ab | 12.6±0.14a | 3.3±0.33b | 5.7±1.20a | 3.2±0.16a | 0.58 | 3 |
HLJ | 6.9±0.82a | 10.5±0.33a | 3.0±0.00bc | 6.1±0.96a | 2.4±0.18ab | 0.56 | 4 |
BYD | 1.0±0.05b | 8.7±0.88ab | 1.5±0.50c | 9.4±3.38a | 2.9±1.14ab | 0.40 | 6 |
WSH | 4.2±0.81ab | 7.0±0.38ab | 2.4±0.25bc | 7.2±1.66a | 1.7±0.18ab | 0.22 | 7 |
VN | 7.6±0.56a | 13.1±0.95a | 5.2±0.87a | 11.1±0.84a | 1.6±0.23b | 0.62 | 2 |
TH | 7.2±1.45a | 12.0±0.28a | 5.0±0.41a | 7.8±1.51a | 1.7±0.17ab | 0.68 | 1 |
IN | 8.8±0.84a | 10.6±0.76a | 2.5±0.29bc | 5.6±0.62a | 2.1±0.16ab | 0.54 | 5 |
NLU | 1.5±0.05b | 3.00±0.10b | 3.0±0.00bc | 5.9±0.68a | 3.0±0.35ab | 0.22 | 8 |
表6 不同种源地莲继代培养叶、根、茎指标的差异 |
编号 | 叶数 | 叶展开数 | 叶长径/mm | 叶短径/mm | 叶柄长/mm | 叶柄粗/mm | 总根数 |
---|---|---|---|---|---|---|---|
PLD | 18.7±2.03a | 5.7±1.56b | 31.5±2.22b | 25.8±1.70b | 294.4±23.86cde | 0.8±0.10b | 139.3±15.68a |
HLJ | 10.8±1.11a | 5.5±0.65b | 40.0±3.93ab | 36.1±3.43ab | 326.7±27.78bcd | 1.4±0.08a | 152.3±2.24a |
BYD | 19.5±5.50a | 3.0±1.00b | 44.0±3.21a | 38.7±3.84a | 386.8±43.25ab | 1.1±0.05ab | 92.5±12.5a |
WSH | 21.8±2.29a | 3.0±0.71b | 43.8±3.22a | 37.4±3.59ab | 374.9±39.28abc | 1.4±0.10a | 118.0±4.94a |
VN | 32.1±2.70a | 11.4±0.84a | 38.6±2.24ab | 33.1±2.75ab | 274.4±16.21de | 1.2±0.08a | 218.0±5.24a |
TH | 24.9±9.13a | 12.0±2.11a | 40.8±2.41ab | 38.8±2.43a | 230.0±12.35e | 1.3±0.11a | 176.1±2.84a |
IN | 11.5±0.50a | 4.5±0.50b | 41.5±2.68ab | 36.0±3.15ab | 340.5±30.83bcd | 1.3±0.03a | 99.0±5.00a |
NLU | 11.7±1.67a | 1.3±0.33b | 46.5±2.50a | 34.5±2.50ab | 454.3±25.65a | 1.3±0.05a | 107.0±9.86a |
编号 | 茎节数 | 茎节间长/mm | 茎节间粗/mm | 芽数 | 隶属函数均值 | 综合排名 | |
PLD | 12.0±2.52a | 2.8±0.14c | 2.2±0.19b | 3.7±0.88b | 0.23 | 8 | |
HLJ | 8.8±1.44a | 10.7±2.10b | 3.0±0.19a | 2.0±0.00b | 0.46 | 4 | |
BYD | 7.5±0.50a | 4.1±0.50c | 2.3±0.15b | 3.5±0.50b | 0.40 | 6 | |
WSH | 13.5±1.56a | 11.7±1.77b | 2.4±0.13b | 4.5±1.60ab | 0.53 | 3 | |
VN | 18.4±1.51a | 13.7±1.59b | 1.6±0.13c | 10.6±1.95a | 0.65 | 1 | |
TH | 21.7±9.33a | 9.8±1.16b | 1.4±0.10c | 5.6±1.51ab | 0.58 | 2 | |
IN | 8.0±0.00a | 10.9±1.69b | 2.3±0.35b | 2.0±0.00b | 0.37 | 7 | |
NLU | 7.0±0.57a | 34.6±2.81a | 1.5±0.13c | 1.3±0.33b | 0.42 | 5 |
[1] |
田代科,张大生.莲叶何田田——世界莲研究进展[J].生命世界,2014,6:40-45.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[2] |
王其超,张行言.中国荷花品种图志[M].北京:中国林业出版社,2005:2.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[3] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[4] |
李良俊,何小弟,赵有为,等.莲藕茎尖培养技术的初步研究[J].扬州大学学报:农业与生命科学版,1995,16(3):31-34.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[5] |
于文进,龙明华,徐烔志.莲藕组织培养及快速繁殖试验研究[J].农业研究与应用,1999(2):9-11.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[6] |
柯卫东,彭静,刘玉平,等.试管藕诱导技术研究[J].植物科学学报,2001,19(2):173-175.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[7] |
罗丽萍,郭燕华,蔡奇英,等.经太空诱变的莲藕的组织培养[J].植物生理学报,2004,40(2):201-201.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[8] |
刁英,吴金平,赵玲玲,等.莲胚性愈伤组织诱导研究初报[J].湖北农业科学,2011,50(14):2991-2992.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[9] |
柯善强,洪树荣,倪学明,等.美洲黄莲的外植体培养研究初报[J].园艺学报,1987,14(3):208-212.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[10] |
张建福,王锋.莲藕组织培养与微繁殖技术初探[J].上海农业科技,2002(6):17-18.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[11] |
郑丽. 荷花胚培养与切花保鲜技术研究[D].武汉:华中农业大学,2010.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[12] |
刘建平,王芳,杜彩娴,等.荷花幼胚组织培养技术研究[J].现代农业科技,2016(15):140-140.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[13] |
蔡颖欣,汤宇环,邵晓宇,等.莲子胚培养再生植株及原生质体分离的研究[J].种子,2017,36(3):125-127.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[14] |
邵文莉. 温度对莲藕组培苗生长代谢影响的试验研究[J].农业科技通讯,2013(2):78-80.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[15] |
李佳,赵文龙,周长艳,等.莲藕愈伤组织诱导及组织学观察[J].湖北农业科学,2014,53(3):701-703,717.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[16] |
孔德政. 荷花胚组织培养的初步研究[J].河南科学,2007,25(4):593-595.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[17] |
徐君,李静会,李欣,等荷花顶芽初代组织培养[J].江苏农业科学,2013,41(3):38-39.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[18] |
席嘉宾,陈平,张惠霞,等.中国地毯草野生种质资源耐旱性变异的初步研究[J].草业学报,2006,15(3):93-99.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[19] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[20] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[21] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[22] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[23] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[24] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[25] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[26] |
吕平,张继,檀小辉,等.不同基因型甘蔗离体培养技术研究[J].江苏农业科学,2016,44(10):80-80.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[27] |
张行言,王其超.热带型荷花的发现与荷花品种分类系统[J].中国园林,2006,22(7):82-85.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
{{custom_ref.label}} |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
/
〈 |
|
〉 |