南瓜未授粉子房离体培养及植株再生

doi:10.11983/CBB15004

植物学报 ›› 2016, Vol. 51 ›› Issue (1): 74-80.DOI: 10.11983/CBB15004

南瓜未授粉子房离体培养及植株再生

闵子扬^1,³, 李涵¹, 邹甜¹, 童龙¹, 成娟¹, 孙小武^1,^2,^*()

¹湖南农业大学园艺园林学院, 长沙 410128
²湖南省瓜类研究所, 邵阳 422001
³湖南省西瓜甜瓜研究所, 长沙 410125

收稿日期:2015-01-04 接受日期:2015-04-19 出版日期:2016-01-01 发布日期:2016-02-01
通讯作者: 孙小武
作者简介:
? 共同第一作者
基金资助:
基金项目: 南瓜产业技术研究与示范(No.201303112)

Studies of in Vitro Culture and Plant Regeneration of Unfertilized Ovary of Pumpkin

Ziyang Min^1,3, Han Li¹, Tian Zou¹, Long Tong¹, Juan Cheng¹, Xiaowu Sun^1,2*

¹College of Horticulture, Hunan Agricultural University, Changsha 410128, China
²Hunan Province Melon Research Institute, Shaoyang 422001, China
³Hunan Watermelon and Muskmelon Institute, Changsha 410125, China

Received:2015-01-04 Accepted:2015-04-19 Online:2016-01-01 Published:2016-02-01
Contact: Sun Xiaowu
About author:
? These authors contributed equally to this paper

摘要/Abstract

摘要： 以南瓜(Cucurbita moschata)未授粉子房为外植体, 在离体条件下研究了激素种类、基因型、胚囊发育时期、消毒方式和热激处理时间对胚状体诱导效果的影响, 旨在建立完善的南瓜未授粉子房离体诱导再生体系。结果表明, MS+4.0 mg·L^-12,4-D+0.5 mg·L^-1 NAA+1.0 mg·L^-16-BA和MS+0.04 mg·L^-1TDZ两种培养基胚状体的诱导率都较高, 分别为19.8%和20.1%, 二者相比, 使用TDZ诱导更加简单; 在6个南瓜品种中, 2个生长势较强的品种(雪峰蜜本和鱷鱼南瓜)胚状体诱导率较高, 适合作为离体雌核诱导的实验材料; 开花当天的子房先切片后消毒可以有效降低愈伤组织的形成, 提高胚状体的诱导率; 在黑暗且35°C条件下热激5天有利于子房的转绿和出胚。将子叶胚转移至无激素的MS培养基上得到再生植株并移栽成活。经鉴定有7个再生植株的染色体数目n=x=22, 气孔保卫细胞叶绿体数的平均值为4.28个, 是单倍体植株。

Abstract: Using unfertilized ovaries of pumpkin as explants, we determined the effect of different combinations and concentrations of growth regulators, genotypes, embryo sac development stages, disinfection methods and duration of high-temperature pretreatments on embryoid induction via in vitro culture to establish a regeneration system of unfertilized ovaries of pumpkin. The media MS+4.0 mg·L^-12,4-D+0.5 mg·L^-1 NAA+1.0 mg·L^-16-BA and MS+0.04 mg·L^-1TDZ achieved higher induction rates, 19.8% and 20.1%, respectively, as compared to other media examined, with the TDZ-induced method easier to operate. Among the 6 pumpkin varieties tested, two (Xue feng mi ben and E yu nan gua), with stronger growth, had higher embryoid induction rates and were more suitable to be used as experiment materials. The operation of slicing and then sterilizing the ovaries picked on bloom day decreased the cavernous callus formation effectively and increased the embryoid induction rate. Treatments with darkness and heat shock (35°C) for 5 days could help ovaries turn green and induce embryoids. When moved to hormone-free MS medium, the cotyledon embryos developed to regenerated seedlings and survived after transplantation. Seven regenerated plantlets had chromosome number n=x=22 and an average number of chloroplasts in stomatal guard cells of 4.28, so they were haploid plants.

闵子扬, 李涵, 邹甜, 童龙, 成娟, 孙小武. 南瓜未授粉子房离体培养及植株再生. 植物学报, 2016, 51(1): 74-80.

Ziyang Min, Han Li, Tian Zou, Long Tong, Juan Cheng, Xiaowu Sun. Studies of in Vitro Culture and Plant Regeneration of Unfertilized Ovary of Pumpkin. Chinese Bulletin of Botany, 2016, 51(1): 74-80.

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图/表 7

表1 不同激素配比对胚状体诱导的影响

Table 1 Effects of different hormones proportions on embryoid induction

Hormone components of medium (mg·L^-1)				Number of explants	Number of enlarge- ment ovules	Frequency of ovules turning green (%)	Frequency of embry- oid induction (%)
2,4-D	NAA	6-BA	TDZ	Number of explants	Number of enlarge- ment ovules	Frequency of ovules turning green (%)	Frequency of embry- oid induction (%)
0.0	0.5	1.0	0.0	20	10.0 h	13.7 h	0 i
1.0	0.5	1.0	0.0	20	17.0 g	19.8 g	2.1 h
2.0	0.5	1.0	0.0	20	22.0 f	42.5 d	5.5 g
3.0	0.5	1.0	0.0	20	53.3 d	55.7 bc	12.2 d
4.0	0.5	1.0	0.0	20	100.7 ab	61.5 a	19.8 a
5.0	0.5	1.0	0.0	20	102.0 a	51.7 c	15.2 b
0.0	1.0	1.0	0.0	20	10.3 h	34.3 e	0 i
1.0	1.0	1.0	0.0	20	18.0 g	25.9 f	2.3 h
2.0	1.0	1.0	0.0	20	23.3 f	54.3 c	5.4 g
3.0	1.0	1.0	0.0	20	56.3 c	59.1 ab	11.9 d
4.0	1.0	1.0	0.0	20	101.7 ab	62.0 a	15.5 b
5.0	1.0	1.0	0.0	20	102.7 a	52.0 c	14.2 c
0.0	0.0	0.0	0.02	20	42.0 e	35.7 e	7.4 f
0.0	0.0	0.0	0.04	20	99.0 b	62.3 a	20.1 a
0.0	0.0	0.0	0.06	20	102.0 a	54.3 c	11.2 e

表2 不同基因型对胚状体诱导的影响

Table 2 Effects of different genotypes on embryoid induction

Variety	Growth potential	Number of explants	Number of enlarge- ment ovules	Frequency of ovules turning green (%)	Frequency of embryoid induction (%)
Xue feng mi ben	Strong	20	93.3 b	60.4 c	20.7 a
Ji zao shu mi ben	Weak	20	83.7 c	66.5 ab	10.2 b
Zhong za 1	Weak	20	82.0 c	65.4 bc	8.7 b
Hong li 2	Moderate	20	104.0 a	63.5 bc	10.6 b
E yu nan gua	Strong	20	102.7 a	71.8 a	19.5 a
Huang jin 2	Moderate	20	72.0 d	43.1 d	0 c

表3 胚囊发育时期对胚状体诱导的影响

Table 3 Effects of different development stages of embryo sac on embryoid induction

Sampling time (hours before flower)	Number of explants	Number of enlargement ovules	Frequency of ovules turning green (%)	Frequency of embryoid induction (%)
24	20	83.3 c	55.8 b	11.4 c
12	20	132.0 b	59.5 b	15.3 b
0	20	140.0 a	66.1 a	20.0 a

图1 不同消毒方式和热激时间对胚状体诱导的影响 (A) 先消毒后切片培养3天的子房片; (B) 先切片后消毒培养3天的子房片; (C) 35°C黑暗处理5天的出胚情况; (D) 35°C黑暗处理7天的出胚情况; (E) 25°C黑暗处理5天的出胚情况

Figure 1 Effects of different disinfection methods and high- temperature pretreatment time on the frequency of embryoid induction(A) The ovary disinfected before sliced was cultured for 3 days; (B) The ovary sliced before disinfected was cultured for 3 days; (C) Embryoid induction was treated at 35°C in dark for 5 days; (D) Embryoid induction was treated at 35°C in dark for 7 days; (E) Embryoid induction was treated at 25°C in dark for 5 days

图2 再生植株的诱导形成过程及移栽 (A) 球形胚; (B) 心形胚; (C) 子叶形胚; (D) 具有明显根端和芽端的幼株; (E) 完整再生植株; (F) 移栽16天后成活的再生植株

Figure 2 The induction and transplanting of the regenerat- ed plants(A) Globular embryoid; (B) Heart-shape embryoid; (C) Cotyledon embryoid; (D) Small plants with obvious root end and shoot end; (E) Regenerated plantlet; (F) The survival plants after transplanted for 16 days

表4 不同培养基对胚状体成苗的影响

Table 4 Effects of different media on seedling induction

Medium hormone components (mg·L^-1)			Number of embryoids	Seedling rate (%)
NAA	6-BA	GA₃	Number of embryoids	Seedling rate (%)
0	0	0	12	69.4 a
0	0.5	0	12	19.4 cde
0	1	0	12	13.9 e
0.5	0	0	12	27.8 b
0.5	0.5	0	12	22.2 bcd
0.5	1	0	12	16.7 de
0	0	0.5	12	13.9 e
0	0	1.0	12	25.0 bc
0	0	1.5	12	19.5 cde

图3 再生植株的倍性鉴定 (A) 单倍体再生植株; (B) 正常二倍体组培苗; (C) 单倍体再生植株染色体数目; (D) 二倍体南瓜根尖的染色体数目; (E) 单倍体再生植株气孔保卫细胞的叶绿体数目; (F) 二倍体南瓜气孔保卫细胞的叶绿体数目。Bar=100 µm

Figure 3 The ploid identification of the regenerated plants(A) The haploid regenerated plants; (B) Diploid pumpkin tissue culture seedling; (C) The number of chromosome of the regenerated haploid plantlet; (D) The number of chromosome in the root tip cell of the diploid pumpkin; (E) Number of chloroplasts in stomatal guard cell of the regenerated ha- ploid plantlet; (F) Number of chloroplasts in stomatal guard cell of the diploid pumpkin. Bar=100 µm

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南瓜未授粉子房离体培养及植株再生

Studies of in Vitro Culture and Plant Regeneration of Unfertilized Ovary of Pumpkin

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