WO2012109947A1 - 超声波辅助花粉介导植物转基因方法 - Google Patents
超声波辅助花粉介导植物转基因方法 Download PDFInfo
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- WO2012109947A1 WO2012109947A1 PCT/CN2012/000030 CN2012000030W WO2012109947A1 WO 2012109947 A1 WO2012109947 A1 WO 2012109947A1 CN 2012000030 W CN2012000030 W CN 2012000030W WO 2012109947 A1 WO2012109947 A1 WO 2012109947A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Definitions
- the present invention relates to an improved plant pollen-mediated transgenic method.
- Transgenic technology enables excellent gene cross-species communication to achieve targeted and precise improvements in crop quality and yield.
- GM crops have significantly improved yield, stress resistance and nutritional quality compared to traditional crops, and can significantly reduce production costs and reduce environmental pollution in agricultural production.
- crops that are widely used in agricultural production, such as corn, soybean, rapeseed, cotton, and tomato.
- transgenic technology only stays in the laboratory.
- Agrobacterium-mediated method the other is gene gun bombardment. Both of these methods require a lengthy and cumbersome plant tissue culture process, which requires a lot of manpower, material resources, financial resources and time.
- the pollen tube channel method proposed by Zhou Guangyu has been applied in China, and some transgenic lines or varieties have been cultivated by this method.
- the biggest advantage of this method is that it does not depend on tissue culture and plant regeneration.
- the technology is simple, does not require well-equipped laboratories, and is easily mastered by conventional breeders.
- the main disadvantage is that the conversion efficiency is low, and a large number of transformation and treatment progeny need to be screened later. So far, the "bottleneck" problem of plant wrapping and communication engineering is still the plant genetic modification method.
- Sun Yi et al. invented a method for the use of ultrasonic treatment of pollen-mediated plant gene transformation (Chinese Patent No.
- the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide an ultrasonic-assisted pollen-mediated plant transgenic method which can significantly improve the seed setting rate of ultrasonically treated pollen pollinated plants and thereby increase the number of transformants obtained per treatment.
- sucrose solution subjected to aeration and low temperature pretreatment and the whole ice bath operation of the pollen process can maintain a higher proportion of pollen grains to maintain vitality, so that the treated pollen can be imparted to the plant stigma (corn filament) for more High seed setting rate.
- the collection of fresh pollen, storage under low temperature (0-4 ° C) dry conditions and use between 2 and 48 hours are beneficial to maintain pollen vigor.
- the method uses a recombinant plasmid of Agrobacterium carrying an exogenous gene fragment, an Escherichia coli plasmid or other DNA vector as a gene donor, and takes the male gametophyte pollen of the plant as a receptor, and passes through the pollination and fertilization process of the plant.
- Ultrasound-assisted treatment of pollen-mediated gene transfer Using the instantaneous release of high energy and cavitation of the ultrasonic wave, the foreign DNA is first introduced into the plant pollen, and then the pollen tube grows into the female gametophyte sac, and then participates in the formation of the zygote, and finally integrates into the target plant. In the genome.
- Fresh pollen is mixed with the DNA fragment in a ventilated and low-temperature treated 5-50% sucrose solution, and the foreign gene is introduced into the pollen by ultrasonic assist; then the pollen suspension is slightly allowed to stand and the supernatant is discarded.
- the precipitated treated pollen is then applied to the plant pistil stigma with a fine brush or a brush and harvested as the grain matures.
- the seeds harvested after pollination of the transformed pollen are sown in the subsequent growing season, and the transformants are further determined by screening the germinated seeds and seedlings, PCR amplification of the seedling sample DNA, and Southern hybridization.
- the sucrose solution Before adding pollen and exogenous DNA fragments, the sucrose solution needs to be ventilated and pre-treated at low temperature.
- the sucrose solution is continuously ventilated for more than 20 minutes using an air pump to saturate the air (oxygen) content in the sucrose solution while sucrose
- the solution is pretreated in an ice bath or refrigerator at 0-4 ° C; after adding pollen and exogenous DNA fragments, a pollen suspension is formed, and the pollen suspension is always placed at 0-4 ° C during the subsequent operation. In the bath.
- Ultrasonic treatment of the pollen suspension requires ultrasonic treatment of the pollen suspension before and after the addition of exogenous DNA.
- the treatment power intensity is 50-500 W, and the time is 5 seconds to 2 minutes.
- the air pump can be a commercially available small aquarium air pump. '
- Fresh pollen is pollen stored within 5 to 4 ° C for 5 days.
- the treated pollen is applied to the plant stigma and harvested as the grain matures.
- the seeds obtained after pollination of the transformed pollen are sown, and the germinated seeds and the seedlings thereof are screened according to the screening marker gene carried on the plant transformation vector, and the screening agent may be Not limited to antibiotics or herbicides.
- the DNA of the seedling sample is screened by PCR amplification or Southern hybridization according to the inserted foreign DNA sequence, and the selected transgenic lines are continuously selfed and screened in the subsequent generation until stable homozygous is obtained. Transgenic lines.
- the present invention is an improved ultrasonic-assisted pollen-mediated plant transgenic method, which can significantly increase the seed setting rate of pollinating plants, thereby increasing the number of transformants obtained per treatment.
- the method can directly transfer the foreign gene to the seed genome of the offspring, avoiding the tedious and high-demand plant tissue culture process, and greatly shortening the periodicity of obtaining the transformed seed. It has the characteristics of high gene conversion efficiency, good reproducibility, low probability of chimerism, low cost of ultrasound equipment, no species and genotype dependence, and wide application range. It can improve the seed setting rate of ultrasonic-assisted plant pollen-mediated transgenic method. , thereby increasing the number of transformants obtained per treatment, saving manpower and material resources, and facilitating the application of production practice.
- the germination rate is about 28 °C at room temperature. After pollen soaking in 15% sucrose solution for a corresponding period of time, a small amount of pollen is taken and detected in the medium for 30 minutes.
- the formulation of corn pollen in vitro germination culture medium is: sucrose 15% + boric acid 50 mg / l + calcium chloride 300 mg / l + magnesium chloride 200 mg / l + potassium nitrate 100 mg / l + gibberellin 35 mg / lo in the table is marked with DPS data
- the treatment system Duncan's method multiple comparisons of 5% significant level statistics (the same below).
- Example 2 Sucrose sucrose concentration (osmotic pressure) has a significant effect on pollen germination rate.
- the pollen tube is short and thin.
- the pollen tube is the longest, and the growth is positive.
- the pollen tube is shorter.
- the pollen tube is short and thick, and some are curved and deformed.
- Table 2-2 Pollen germination in different sucrose concentrations of maize (2010.7.15-8.5) Pollen germination rate (%) Pollen germination rate ft) Pollen tube length ( ⁇ m) The pollen swells too much, and the contents are full of
- the tube grows normally. ⁇
- the pollen tube is short and short. - The pollen is almost unbroken, and the middle mountain is soaked
- the sucrose concentration of the suspension is mainly reflected by the osmotic pressure of the solution.
- corn pollen has a higher breakage rate in low concentration ( ⁇ 5%) sucrose solution, regardless of when sampling.
- the pollen integrity rate increases as the sucrose concentration increases.
- high concentration (50%) sucrose solution the pollen germination rate was significantly reduced.
- sucrose concentration of early-seeded corn pollen germination is 20%-30%, and there is still a small amount of germination under 50% sucrose; normal sowing date field corn pollen germination
- the optimum concentration is 15%. More than 20% of the sucrose solution has an inhibitory effect on germination.
- the pollen In the 50% sucrose solution, the pollen has a plasmo separation and no germination.
- the corn pollen sown in the normal season of Datian had a lower rate of swelling at the same sucrose concentration than the earlier sucrose. When the germination rate was similar, the length of pollen tube was longer and the growth of pollen tube was stronger.
- the corn pollen germination medium in the normal sowing date of the region should use a lower concentration of sucrose.
- the phenological conditions in the greenhouse or other low-temperature and humid areas should be higher sucrose concentration, or need to be retested.
- Example 3 Effect of preservation time and conditions on pollen vigor.
- the preservation conditions of corn pollen are as follows: low temperature drying > low temperature humidity > room temperature drying > room temperature humidity, especially in the refrigerator 4 ° C culture dish to preserve pollen is best, has fully met the ultrasonic-mediated transgenic test.
- Example 4 Effect of suspension temperature on pollen germination.
- Pollen "" suspension temperature has a certain effect on pollen germination ability, and lower temperature helps to reduce pollen rupture.
- Example 5 Effect of suspension aeration on pollen germination. It should be pointed out that in pollen-mediated plant transgenic operations, the rapid germination of pollen in the suspension is not conducive to the improvement of seed set rate and conversion rate, because the pollen tube that has already sprouted is easily damaged during the subsequent pollination process, and It is difficult to grow into the stigma after pollination and complete the fertilization process.
- the ideal state of pollen should be that it does not germinate or rupture in the suspension to maintain vitality.
- the germination rate and fertilization rate of such pollen after being applied to the stigma (jade filament) are higher.
- the pollen suspension was subjected to aeration treatment for 20 minutes to allow the pollen portion to be dormant, resulting in less germination and a lower rate of breakage.
- the germination rate is after soaking the pollen for 15 min (25 °C) after 15% sucrose solution after the corresponding ventilation time, and a small amount of pollen is taken in the medium for germination measurement.
- Example 6 Effect of temperature and aeration on pollen germination by ultrasonic treatment.
- the pollen Before pollen treatment, the pollen is suspended in 15% sucrose solution with different pretreatment, and a small amount of pollen is taken in the medium for germination. Immediately after ultrasonic treatment, a small amount of pollen is taken and germinated in the medium. measuring.
- Example 7 Effect of different treatments of pollen on seed setting rate after pollination. After the above differently treated pollen was applied to the corn silk, different seed setting rates were obtained (Table 7).
- Example 8 Conversion rate after pollination of differently treated pollen. Since the transformation vector we used carries the bar gene, this gene confers resistance to the herbicide basta. Therefore, the transformed plants can be initially screened by spraying the herbicide. We planted the corn seeds obtained after the transformation treatment in the test field and sprayed 2% of the basta herbicide when they grew to 5-6 leaves. The results of herbicide screening for transgenic seed seedlings are shown in Table 8.
- the resistant strain rate is: Number of herbicide resistant plants/emergence number.
- PCR positive strain rate PCR positive strain number / emergence number. It can be seen from Table 8 that the TO generation seeds obtained by the above different treatments have no significant difference in the herbicide-tolerant plant acquisition rate. At the 5-leaf stage, leaves were taken from the above herbicide-tolerant plants, total DNA was extracted, and PCR was performed on these DNA samples. The results showed that no matter which method was used to treat pollen, about 20% of PCR-positive strains were obtained. Southern hybridization results confirmed that these PCR-positive plants were all transformants, indicating that the foreign gene can be introduced into the recipient plants by this method.
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Description
超声波辅助花粉介导植物转基因方法 技术领域
本发明涉及一种改良的植物花粉介导转基因方法。
技术背景
转基因技术能够使优良基因跨物种交流, 从而实现对农作物的品质和产量 等性状进行定向、 精确的改良。 转基因作物在产量、 抗逆性和营养品质等方面 较传统作物有显著改进, 还能大大降低生产成本, 减少农业生产中的环境污染。 但目前大面积应用到农业生产中的转基因作物品种只有玉米、 大豆、 油菜、 棉 花、 番茄等少数几种作物, 对绝大多数植物而言, 转基因技术还只停留在实验 室阶段。 目前植物转基因研究主要采用的两种经典方法, 一是农杆菌介导法; . 二是基因枪轰击法。 这两种方法都需要经过冗长繁琐的植物组培过程, 需要花 费大量的人力、 物力、 财力和时间。 一些植物种或品种的组织培养再生困难而 使得这两种方法具有很大基因型依赖性, 因而受到很大的局限。 同时由于植物 组织培养过程中易造成体细胞变异、 再生苗移栽过程中夭亡等, 使本已不高的 转化率又大打折扣, 这些缺陷都大大限制了植物转基因技术的广泛应用。 其他 一些植物转基因方法, 如, 脂质体法、 PEG 法、 电激法、 显微注射法、 超声波 法、 离子束介导法、 激光微束穿孔法、 碳化硅纤维法等, 虽然都有成功的报道, 但均因操作繁琐或效率较低等原因而很少有人使用。 因此简化植物转化方法是 不少相关研究人员的努力方向。
由周光宇提出的花粉管通道法已在我国得到了一定的应用, 并用该法培育 出一些转基因株系或品种。 该法的最大优点是不依赖组织培养和植株再生过程,
技术简单, 不需要装备精良的实验室, 常规育种工作者易于掌握。 其主要缺点 是转化效率低, 后期需要对大量的转化处理后代进行筛选。 迄今, 植物裹.通工 程的 "瓶颈" 问题仍然是植物转基因方法。 孙毅等发明了一种 "利用超声波处 理花粉介导植物基因转化方法" (中国专利号 99121 152.9 ) , 该发明使用超声 波细胞粉碎仪以 200— 300W功率的超声波处理花粉悬浮液, 花粉为新鲜花粉, 溶液为 5— 15 %蔗糖溶液加入不低于 40 μ g/L外源 DNA, 超声波每次处理 5秒, 间隔 10秒, 共处理 5— 8次, 收集超声波处理后的花粉授于植物柱头上, 并从 受体上收获种子, 在后代中筛选转化植株。 该方法不需经过冗长繁琐的组织培 养过程, 具有简便、 有效、 快速和经济等特点, 因此实用性强, 能够与常规育 种方法有机结合被广大作物育种工作者直接应用。 但该方法的主要缺点之一是 授粉后结实率较低, 这显然是由于经过超声波处理的花粉大多数丧失了生活力, 无法正常完成受精过程。 因此, 提高结实率是使该方法具有更广泛应用前景的 关键。
发.明内容
本发明目的是克服上述已有技术的不足, 提供一种可显著提高超声波处理 花粉授粉后植株的结实率、 进而提高每次处理获得的转化体数的超声波辅助花 粉介导植物转基因方法。
实验发现, 在经过通气和低温预处理的蔗糖溶液及整个处理花粉过程的冰 浴操作可以有更高比例的花粉粒保持活力, 因而将处理花粉授到植物柱头 (玉 米花丝) 上, 可以获得更高的结实率。 而且采集新鲜花粉, 低温 (0— 4°C ) 干 燥条件下保存以及在 2〜48小时之间使用均有利于保持花粉活力。
本方法是以携带外源基因片段的农杆菌 Ti质粒、大肠杆菌质粒或其它 DNA 载体为基因供体, 以植物的雄配子体花粉为受体, 在植物传粉受精过程中通过
超声波辅助处理花粉介导基因转移。 利用超声波的瞬间释放高能和空化作用, 先将外源 DNA导入植物花粉中,之后再随着花粉管的生长进入到植物雌配子体 胚囊中, 并进而参与合子的形成, 最终整合到目标植物基因组中。 取新鲜花粉 在经过通气和低温处理的、 5— 50%的蔗糖溶液中与 DNA 片段混合, 通过超声 波辅助作用使外源基因进入花粉中; 然后将花粉悬浮液略微静置后倒掉上清液, 进而用细刷或毛笔将沉淀的处理花粉授到植物雌蕊柱头上, 并在籽粒成熟时予 以收获。 在随后的生长季将转化处理花粉授粉后收获的种子播种, 通过对萌发 种子和幼苗的筛选、 对成苗株样本 DNA的 PCR扩增及 Southern杂交, 进- 步 确定转化株。
在加入花粉和外源 DNA片段前, 需要对蔗糖溶液进行通气和低温预处理, 使用空气泵给蔗糖溶液连续通气 20分钟以上, 使蔗糖溶液中的空气 (氧) 含量 达到饱和状态, 同时将蔗糖溶液置于 0— 4°C冰浴或冰箱中预处理; 加入花粉和 外源 DNA片段后, 形成花粉悬浮液, 并在以后的操作过程中始终将花粉悬浮液 置于 0— 4°C冰浴中。 对花粉悬浮液进行超声波处理, 需要在加入外源 DNA前、 后分别对花粉悬浮液进行超声波处理, 处理功率强度为 50— 500W, 时间为 5秒 一 2分钟。 空气泵可以采用市售的小型水族箱用空气泵。 '
新鲜花粉是在 0— 4°C保存 5天以内的花粉。 将处理花粉授到植物柱头上, 并在籽粒成熟时予以收获。 在随后的生长季, 将转化处理花粉授粉后得到的种 子播种, 并根据植物转化载体上携带的筛选标记基因利用筛选剂对萌发种子及 其长出的幼苗进行初步筛选, 所用筛选剂可以是但不局限于抗生素或除草剂。 随后根据插入的外源 DNA序列利用 PCR扩增或 Southern杂交方法对成苗株样 本 DNA进行筛选,并在随后的世代中对筛选到的转基因株系进行连续自交和筛 选, 直至获得稳定纯合的转基因品系。
本发明是经过改良的超声波辅助花粉介导植物转基因方法, 可以显著提高 授粉植株结实率, 进而提高每次处理获得的转化体数。 该方法可将外源基因直 接转移到后代种子基因组中, 避开了繁冗、 操作要求高的植物组织培养过程, 大大缩短获得转化种子的周期性。 具有后代植株中基因转化效率高、 重复性好、 发生嵌合体几率少、 超声波设备便宜和无物种及基因型依赖性、 适用范围广等 特点, 可以提高超声波辅助植物花粉介导转基因方法的结实率, 进而提高每次 处理获得的转化体数, 节省人力和物力, 有利于生产实践的应用。
具体实施方式
下面是本发明为改良原方法应用于玉米基因转化的具体实例:
花粉丧失活力的原因除超声波作用损伤不可避免外, 花粉在悬浮液中也有 一定比例的损伤, 其原因一是花粉破裂, 二是大部分不破裂的花粉也丧失了萌 发能力, 因此从花粉的悬浮条件入手提高花粉的生活力。 悬浮液的三个主要因 素为: 蔗糖浓度 (渗透压) 、 温度和空气 (氧气) 含量。 针对以上因素, 采用 玉米品种郑 58做了下列试验, 结果见表〗〜表 8: 实施例 1: 玉米花粉在悬浮液中浸泡时间对花粉体外萌发有显著影响。
E米花粉在悬浮液中浸泡时间对花粉体外萌发的影响
浸泡
0 min 10 min 20 min 30 min 40 min 50 min 60 min 90 min 120 min 时间
花粉
12.8±2 18.6±2 15.7 ±3 28.2±4 42.3±5 51.1 ±6 56.3±6 63.8±6 78.9 ±'6 破
.29f .88f .10f .81e .57d .37c .85bc .84b .45a 率 (%)
花粉
萌 81.4±5 78.3±5 75.4±5 65.2±6 48.3±5 32.4±5 20.8±4 15.7±3 0.16±0 发率 . .52a .76a .23a .18b .97c .54d .17e .26e .22f
(%)
注: 该萌发率为室温 28°C左右花粉浸泡在 15%蔗糖溶液中静置相应时间后吸取 少量花粉于培养基萌发 30分钟后检测。 玉米花粉体外萌发培养液配方为: 蔗糖 15%+硼酸 50 mg/l+氯化钙 300 mg/l+氯化镁 200 mg/l+硝酸钾 100mg/l+赤霉素 35 mg/lo表中字母标记是采用 DPS数据处理系统 Duncan' s法多重比较 5%显著水 平统计结果 (以下相同) 。
从表 1 可以看出, 随浸泡时间的延长, 玉米花粉的破碎率增高, 萌发率显 著降低。 当花粉在 28°C左右且未做通气处理的悬浮液中浸泡 1小时后, 其萌发 率从 80%迅速降低至 20%, 如浸泡时间达到 120分钟, 仅有极少数的花粉可以 萌发。
实施例 2: 悬浮液蔗糖浓度 (渗透压) 对花粉萌发率有显著影响。
表 2-1 玉米不同蔗糖浓度花粉体外萌发 (2010.5.28— 6.10) 蔗糖浓度 花粉破率 (%) 花粉萌发率 (%)花粉管长度( μ l) 特征描述
花粉胀破多, 内容物散满培
1% 72.5 ±6.85a 7.26±2.37e 200±42. 3f
养基, 花粉管很短, 较细。
5% 60.3 ±6.071〕 11. I ±4.88e 262±48. If 同上. 花粉管细短。
10% 32.9±4.76c 56.5 + 5.69c 671±50. 2d 部分花粉管伸长后乂胀破。
15% 30.8±4. llcd 60.0±5.82bc 1357 ±58 .4c 同上, 花粉管较长。
花粉胀破减少, 花粉 较长,
20% 26.6±3.73cde 71.2±4.71a 1804 ±68 .7 b
生长均匀:平滑且直。
同上, 花粉管最长, 生长正
30% 25.1±3.51def 65.6 ±5.63ab 2058±62 .4a
ITJ。
花粉破后内容物¾一团状,
40% 22.4±3.71ef 42.3 ±5.38d 756 ±5]. 7d
花粉管较短。
花粉胀破少, 内容物团状,
50% 17.9±3.24f 12.2±4.61e 380±45. 9e
花粉管粗短,有的弯曲变形。 表 2-2 玉米不同蔗糖浓度花粉体外萌发 (2010.7.15—8.5) 蔗糖浓度 花粉破率 (%) 花粉萌发率 ft)花粉管长度( μ m) 特征描述
花粉胀破多, 内容物散满培
1% 66.4±6.08a 8.69±2.12ef 703 ±54. 2e
养基, 花粉管短, 顶端易破。
尽管花粉破率高, 萌发率低,
5% 5].9±5.88b ]7.8±5.47d 】428±72. 5c
但花粉管生长好, 较 Κ;。
花粉萌发率较高, 花粉管生
10% 40.1±5.24c 67.4±5.72b 2206 ±78. 9b
长势强, 管径均匀平滑。
花粉破率较低, 花粉萌发率
15% 23.8 ±3.83d 80.6 ±4.94a 2625±65. 5a
取 l , 官取 k。
花粉萌发率急剧下降, 花粉
20% 11.4±2. lOe 49.6±5.12c 2285±62. Ob
管生长正常。 ·
同上, 花粉破后内容物呈团
30% 4.07士]. 17f 12.8±5.02de ]188±57. 6d
状。 '
花粉破率低, 内容物 'τ(.丝缕
40% 2.91±1. OOf 2.37土].21f 200 ±46. 3f
状, 花粉管粗短。 - 花粉几乎不破, 中间山现泡
50% 0.50 ±0.47f ' 0 0
状黑圈, 无萌发。 -
悬浮液蔗糖浓度主要体现为溶液的渗透压。 从表 2-1和 2-2中可以看出, 无 论何时取样, 玉米花粉在低浓度 (≤5%) 蔗糖溶液中均破损率较高。 花粉完好 率随蔗糖浓度增高而提高。 但在高浓度(50%)蔗糖溶液中, 花粉萌发率显著降 低。
比较不同时期玉米花粉体外萌发情况, 可见玉米花粉不同品种间对蔗糖浓 度反应基本一致, 但不同物候条件种植的玉米花粉对蔗糖浓度反应有差异。表 1 与表 2分别为早播与大田春播玉米在不同蔗糖浓度培养基上花粉体外萌发结果。 其中早播玉米为 2010年 3月 29日在大棚温室播种, 5月 28日一 6月 10日取粉; 正常播期大田玉米为 4月 29日在有隔离条件的试验地播种, 7月 15日一 8月 5 日取粉。 实验地点为山西太原。 从表 1 和表 2中可见早播玉米花粉萌发最佳蔗 糖浓度是 20%— 30%, 50%蔗糖下仍有少量萌发; 正常播期大田玉米花粉萌发
最佳浓度是 15%, 20%以上蔗糖溶液对萌发有抑制作用, 50%蔗糖溶液中花粉 出现质壁分离, 无萌发。 大田正常季节播种的玉米花粉比早播玉米花粉在相同 蔗糖浓度时胀破率低, 萌发率相近时花粉管长度较长, 花粉管生长势强。 总之, 本地区 (山西太原) 大田正常播期玉米花粉萌发培养基宜采用较低浓度蔗糖, 温室或其他低温潮湿地区物候条件宜采用较高蔗糖浓度, 或者需重新试验确定。 实施例 3: 保存时间和条件对花粉活力的影响。 玉米花粉保存条件优劣依次为低温干燥〉低温潮湿〉室温干燥〉室温潮 湿, 尤其以冰箱 4 °C培养皿内保存花粉最好, 已完全可满足超声波介导转基因 试验用途。 从表 3-1看出早播玉米花粉在体外存活时间短, 只能低温干燥保存 2 h左右, 之后花粉萌发力迅速下降而不宜使用。 大田春播玉米花粉在 4 °C冰箱 培养皿内, 干燥保存可达 5天仍有一定萌发率 (表 3-2) , 并且发现刚采回的花 粉易破萌发率低, 保存 2 h以上萌发率明显提高, 48 h以内花粉具有较高的生活 力。 玉米花粉萌发率及保存时间与当天花粉质量有关。 总之, 大田正常季节 良好物候条件采集到的玉米花粉耐受力强, 花粉保存期长、 胀破少、 ¾粉管生 长势强。
表 3-1 早播玉米不同取粉时间及保存条件的花粉体外萌发率 (%) 取粉时间
保存条件和时间 _:
8: 30 10: 00 11 : 30
即时萌发 18.3±4. 33a 56.9 ±6.72a 69.2±6. 79a
低温千燥保存 2 h 7.30±2. 14b ' 42.2 ±5.88b 68.8.土 6. 45a 低温潮湿保存 2 h 0 15.2±4.13c 35.4 ±5. 62b 室温干燥保存 2 h 0 9.3 ±3.02cd 12.5±3. '67c 室温潮湿保存 2 h 0 0 0
• 低温干燥保存 4 h 0 5.2 ±2.13d 8.26 + 2. 75c
低温干燥保存 6 h 0 0 0
3-2 大田春播玉米 4 °C干燥保存花粉体外萌发
保存时间 0.5 h 2 h 4 h 6 h 24 h 48 h 72 h 96 h 120 h 144 h 花粉破率 48.2±5.2 26.6±3.8 21.5士3.1 lg.4±2.8 16.7±2.5 24.1±3.3 46.3±5.l 52.8±6. 79.8士6. 90.4±7.
(%) 9c 6d 7de I de 5e 4de 5c 28c 91b 87a 花粉萌发率 35.0±4.5 68.3±5.7 75.6±5.4 80.2±5.1 84.3±5.2 72.4±5.9 45.8±45 35.7±4. I5.9±3.
0
(%) 2e 5c 3bc 8ab 7a 4bc 7d 16e 27f 花粉管长度 2000±71 2250±76 2500±73 2750±74 3000±78 1500±72 1000±68 625±52. 250土 41.
0
( u m) .6e .5d .8c .2b .5a .3f ,4e 8h 2i
实施例 4: 悬浮液温度对花粉萌发的影响。
花粉""悬浮液温度对花粉萌发能力有一定影响, 较低温度有助于减少花粉破裂。
表 4 悬浮液温度对玉米花粉体外萌发的影响
温度 35 °C 30 °C 25 。C 20 。C 15 。C 10 °C . 4 °C 花粉破率 ft) 32.2±5.26a 22.4±3.89b '【 8.5±2.87bc 18.4±2.56bc 16.7±2.23cd 15. I±2.75cd 13.3±2. !5d 花粉萌发率 *(%) 64.3±4.66b 76.3±5.19a 78.2±5.31a 74.3±5.07a 79.7土 5.44a 73.4±5.47a 75.8士 5.58a 注:该萌发率是将花粉在相应温度的 1.5%蔗糖溶液浸泡 5分钟后, 吸取少量 花粉于培养基萌发检测。
实施例 5: 悬浮液通气对花粉萌发的影响。 需要指出的是, 在花粉介导植物转基因操作中, 花粉在悬浮液中很快萌发 并不利于提高结实率和转化率, 因为已经萌发的花粉管很容易在随后的授粉过 程中损伤, 而且其在授粉后很难长入柱头中并完成受精过程。 理想的花粉状态 应是, 在悬浮液中既不萌发, 也不破裂而保持活力。这样的花粉被授到柱头(玉 米花丝) 上以后的萌发率和受精率均会较高。 从表 5 中可以看出, 对花粉悬浮 液进行 20分钟的通气处理可使花粉部分处于休眠状态, 使之萌发少且破率低。
表 5 玉米花粉悬浮液通气对花粉体外萌发的影响 *
通气时间 0 min 10 in i n 20 min 30 min 40 m i n 50 m】n 60 min 花粉破率(%) 27.2±4.52 21.7±3.77 16.5±2.45 18.4±2.66 14.7±2.13 16.4±2.73 I3.8±2.24
a b c be c c c
78.4±4.68 56.3±4.26 45.1 ±3.78 51.4±3.85 42.7±3.7I 43.5±2.86 45.3±3.18 花粉萌发率' (%)
a b c b e c c 注:该萌发率是经相应通气时间后的 15%蔗糖溶液浸泡花粉 5 min 后 (25 °C) , 吸取少量花粉于培养基中萌发测量。 实施例 6: 温度和通气对超声波处理花粉萌发的影响。
_超声波处理是使外源基因进入花粉的关键歩骤。 我们的实验 (表 6) 表明, 通气和低温处理可以显著减少花粉破损率和增加花粉在超声波处理后的萌发 率。 经超声波处理后仍有 11.9%的花粉具有萌发能力, 比对照提高了 2.18 倍 (11.9:3.74) 。
不同预处理缓冲液对超声波前后花粉萌发力比较 *
. 注:超声波处理前检测花粉是将花粉悬浮于经不同预处理的 15%蔗糖瘠液中 5 min后, 吸取少量花粉于培养基中萌发测量: 超声波处理后立即吸取少量花粉 于培养基中萌发测量。
实施例 7: 花粉不同处理对授粉后结实率的影响。 将以上不同处理的花粉授到玉米花丝上后, 获得了不同的结实率 (表 7) 。
表 7 不同处理对玉米花粉介导转基因授粉结实率影响 处理 处理穗数 A ' 结籽穗数 B 结实穗率(B/A)% 籽 数 C 毎德结实数(C/A)
means + SI) means + SD 超声波 27 27 27 3 4 4 13.58±1. 14b 17 19 23 0.728±0.113c 超声波 +低温 113 113 113 15 ]7 19 15.04±1.77ab 96 99 87 0.832 ±0.055c 超声波 +通气 315 315 315 51 60 64 18.52±2.15a 392 401 447 1.31 ±0.096b 超声波 +低温
280 280 280 40 41 45 15.0士 2.45ab 439 451 495 ].65±0.10'6'a +通气 从表 7中还可以看出, 通气处理比低温处理的效果明显, 且通气 +低温处理的效 果更加明显, 平均每穗结实数提高了 1.27倍 ( 1.65:0.728) 。 实施例 8: 不同处理花粉授粉后的转化率。 由于我们使用的转化载体上携带有 bar基因,该基因能够赋予转化植株对除 草剂 basta的抗性。 因此, 利用喷洒除草剂的方法可以初步筛选出被转化植株。 我们将转化处理后得到的玉米种子播于试验田中, 并在其长到 5— 6片叶时对其 喷洒 2%的 basta除草剂。 对转基因处理种子幼苗进行除草剂筛选的结果见表 8。
表 8 不同处理花粉授粉后的除草剂抗性株率
注: 抗性株率为: 抗除草剂植株数 /出苗数。 PCR阳性株率为: PCR阳性株 数 /出苗数。
从表 8中可以看出, 以上不同处理所获得的 TO代种子在抗除草剂植株获取 率上没有显著差异。 在 5叶期对以上抗除草剂单株取叶片, 提取总 DNA, 并对 这些 DNA样品做 PCR检测, 检测结果表明, 无论采取哪种方法处理花粉, 均 获得约 20%的 PCR阳性株。 Southern杂交结果证实, 这些 PCR阳性植株均为转 化株, 说明采用本方法确实可以将外源基因导入受体植物。
以上结果说明, 本改良方法在显著提高转基因处理结实率的同时,并没有对 转化率产生明显影响, 从而可以显著提高每次处理获得的转化体数。 .
】1
Claims
1、 一种超声波辅助花粉介导植物转基因方法, 其特征是以携带外源基因片 段的农杆菌 Ti质粒、 大肠杆菌质粒或其它 DNA载体为基因供体, 以植物的雄 配子体花粉为受体, 在经过通气和低温处理的浓度为 5— 50%的蔗糖溶液中使花 粉与 DNA片段混合, 通过超声波辅助作用使外源基因转移到受体花粉中; 然后 将花粉悬浮液略微静置后倒掉上清液, 用细刷将沉淀的处理花粉授到植物柱头 上, 在籽粒成熟时予以收获; 在随后的生长季将转化处理花粉授粉后收获的种 子播种, 通过对萌发种子和幼苗的筛选, 对成苗株样本 DNA 的 PCR 扩增及 Southern杂交, 进一步确定转化株。
2、 根据权利要求 1所述的超声波辅助花粉介导植物转基因方法, 其特征是 所述的花粉是在 ()一 4°C保存 5天以内的花粉。
3、 根据权利要求 1所述的超声波辅助花粉介导植物转基因方法, 其特征是 在加入花粉和外源 DNA片段前对蔗糖溶液进行通气和低温处理,使用空气泵给 蔗糖溶液连续通气 20分钟以上, 使蔗糖溶液中的空气含量达到饱和状态, 同时 将蔗糖溶液置于 0— 4°C冰浴或冰箱中预处理;加入花粉和外源 DNA片段后,形 成花粉悬浮液,并在以后的操作过程中始终将该花粉悬浮液置于 0— 4°C冰: i中。
4、 根据权利要求 3所述的超声波辅助花粉介导植物转基因方法, 其特征是 在加入外源 DNA前后, 分别对花粉悬浮液进行超声波处理, 所用超声波功率为 50— 500W, 每次处理时间为 5秒一 2分钟。
5、 根据权利要求 1所述的超声波辅助花粉介导植物转基因方法, 其特征是 根据植物转化载体上携带的筛选标记基因利用筛选剂进行筛选。
6、 根据权利要求 1所述的超声波辅助花粉介导植物转基因方法,.其特征是 根据插入的外源 DNA序列利用 PCR扩增或 Southern.杂交方法进行筛选
7、 根据权利要求 1所述的超声波辅助花粉介导植物转基因方法, 其特征是 对筛选到的转基因株系进行连续自交和筛选, 直至获得稳定纯合的转基因品系。
8、 根据权利要求 5所述的超声波辅助花粉介导植物转基因方法, 其特征是 所用筛选剂为抗生素或除草剂。
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CN109371061A (zh) * | 2018-12-17 | 2019-02-22 | 靖西市秀美边城农业科技有限公司 | 一种提高农杆菌介导的玉米遗传转化效率的方法 |
US20220338432A1 (en) * | 2019-10-01 | 2022-10-27 | Monsanto Technology Llc | Cross pollination through liquid-mediated delivery of pollen to enclosed stigmas of flowers from recipient plants |
CN113774090A (zh) * | 2021-07-20 | 2021-12-10 | 吉林省农业科学院 | 一种借助花粉管通道在玉米中实现基因编辑的方法 |
CN114480477A (zh) * | 2022-02-23 | 2022-05-13 | 吉林农业科技学院 | 一种通过花粉介导转基因提高玉米抗旱性的方法 |
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WO2016032320A1 (en) | 2014-08-29 | 2016-03-03 | N.V. Nutricia | Compressed solid milk tablets and method for making the same |
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CN102127567A (zh) | 2011-07-20 |
CN102127567B (zh) | 2012-06-06 |
US20130232639A1 (en) | 2013-09-05 |
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