作物基因體工程技術的現況、問題與展望

Su-May Yu 余淑美 教授

Institute of Molecular Biology, Academia Sinica 中央研究院分子生物研究所

Plant Genetics Engineering –

Progress, Problems and Prospects

作物基因體工程技術的現況、問題與展望

傳統育種 Conventional breeding

Repeating the process of

Natural mutation

 Selection

 Cross and breeding

 New variety

 Natural mutation

 Selection

 Cross and breeding

 New variety

小麥

番茄

向日葵

For thousands years, human have modified countless numbers of genes in conventional breeding

數千年來，人類已利用育種技術改造無數作物的基因

From Dubcovsky, J. and Dvorak, J. (2007). Genome Plasticity a Key Factor in the Success of Polyploid Wheat Under Domestication. Science. 316: 1862-1866.

抱子甘藍 捲心菜

花椰菜 青花菜

介藍菜 大頭菜

依照市場需要育成各種十字花科蔬菜

Breeding of Brassica to different varieties based on market demands

Burney, J.A., Davis, S.J., and Lobell, D.B. (2010). Greenhouse gas mitigation by agricultural intensification. Proc. Natl. Acad. Sci. 107: 12052-12057.

Population (billions) Crop production (gigaton) Crop area (hectare) 100%

increase

>100%

increase

~20%

increase

人口增加 100% 作物產量增加 100% 栽種面積增加 20%

矮化品種

Dwarf rice/wheat cultivars

+ 氮肥

N fertilizer +

灌溉系統

Irrigation system +

農藥

Pesticides +

機械化

Mechanization

單位面積產量提升，使作物產量足以跟上人口的增加

Crop production has kept pace with population increase due to yield increase

第一次綠色革命

The first ‘Green Revolution”, 1960-2000

Rice Wheat

產量高的半矮性小麥與水稻品種

Lodging-resistant, high-yielding semi-dwarf grain varieties

優良品種，不抗病

Elite variety, disease susceptibale

野生品種，抗病

Wild rice, disease resistant

傳統育種：費時、費力

Conventional breeding: time-consuming and labor-intensive

X

X

X

優良品種，抗病

Elite variety, disease resistant 6-9 years to develop a new rice variety

Mutagens

Physical Chemical Biological

X-ray Transposon

Virus/Phage Ultraviolet light

Nicotine Pesticides

Methane Pollution

Chemical mutagens

Radiation

Breeding by induced mutagenesis 誘導突變育種技術

Commonly used in plants

結果有好有壞！

The result may not always be desirable.

化學、物理學育種：化學藥劑、放射線 使基因突變

Breeding by chemical or physical mutagenesis approaches

突變前 突變後

Chemical mutagens Radiation

Introgression breeding

Transgenesis

Cisgenesis

Precision breeding

Breeding technology 育種技術

基因工程育種技術:

•提高效率與精確度，且節省時間。

•克服種原不足問題。

Genetic engineering:

• Increases efficiency and precision and reduces time of breeding.

• Provides germplasm within or across species for breeding.

基因工程育種 Genetic engineering breeding

Insertion of foreign gene and expression of foreign protein.

基因轉殖，表現外源蛋白質

•Insect/disease resistance

•Drought/salt tolerance

•Increased grain yield

•Increased water/fertilizer use efficiency

基因工程育種：快速、精確

優良品種，不抗病

Elite variety, disease sensitive

野生品種，抗病

Wild rice, disease resistant

優良品種，抗病

Elite variety, disease resistant

Stephanie J. Swenson et al. J Integr Pest Manag 2013;4:D1-D8

Corn earworm Helicoverpa zea (Boddie) is a well- known pest of corn and cotton, but can also be problematic in soybean, particularly in the

southern United States.

玉米穗蟲 危害玉米、棉花、大豆

使用蘇力菌不必安全防護

蘇力菌用在森林及有機蔬果害蟲的生物防治

Bacillus thurigenesis for bio-control of insects in organic farming

ucanr.org

價格高 High cost

Bacillus

thuringiensis expressing Bt toxin

Plant cell expressing Bt toxin

Intestine

Bt蛋白在鱗翅目昆蟲腸胃中與受體結合，造成穿孔，使昆 蟲死亡。其他有益昆蟲及人類腸胃無此受體

The Bt toxin affects only some insects because to be effective it has to be processed and bind to a specific receptor protein.

Processing

Receptor binding Intestinal

lumen

assembly

After binding, the insecticidal proteins assemble to form a pore in the lining of the insect intestine which kills the insect

Bt 抗蟲蛋白對害蟲有極高專一性

The effect of Bt toxin is highly specific to insect pests

細菌生產

植物細胞生產

鱗翅目昆蟲腸胃受體

穿孔

GE GE

GE

GE

WT

WT

WT WT

抗蟲基改玉米

Insect resistant GE corn

農藥使用量

Insecticide use 抗蟲玉米面積

Areas of Bt corn

基因改良育種：減少殺蟲劑使用

Breeding by genetic engineering: to reduce the use of insecticides

https://www.geneticliteracyproject.org/2015/04/08/gmos-food-and-pesticides-101-no-chemical-flood-but-yields-are-soaring/

• Farmer’s and consumer’s health

• Environmental protection

• Production cost

mRNA干擾，讓不需要的蛋白質不要產生

基因靜默法

Insert double strand (ds) RNA of target gene

No foreign protein is produced

A company in B.C.'s Okanagan region is celebrating today after its genetically modified, non-browning Arctic Apples was approved for deregulation in the U.S.

The Arctic apple doesn't oxidize or turn brown.

加拿大的一家小公司Okanagan利用 基因靜默法抑制蘋果多酚氧化酶的含 量，所生產切開不會褐化的新品種稱 為「北極蘋果」（Arctic apple），最 近也在美國及加拿大獲准商業化。

切開不會褐化的新品種：「北極蘋果」

Brown-toned melanin Polyphenol oxidase

+

Polyphenolics

澱粉經過高溫燒烤或油炸後，所含還原糖會與一種氨基酸─asparagine作用而產生acrylamide，

過量食用可能致癌及傷害神經。因此，如何降低澱粉類食品中asparagine的含量，是作物育種 上很重要的一項工作，尤其在製作麵包的小麥及薯條、薯片的馬鈴薯。

美國愛達荷州Simplot公司「天生馬鈴薯」（Innate potato）降低 asparagine及糖含量

GE potato that does brown when cut or fried, nor does it make acrylamide 馬鈴薯

Acrylamide is formed when Asparagine is heated to high temperatures (like when frying) in the presence of some

sugars. Less Asparagine and sugars means less Acrylamide.

Knockout or alter gene functions

讓不需要的基因突變，產生的蛋白質沒有功能

CRISPR-Cas9 基因編輯系統

Gene mutations are similar to natural mutations 基因突變情況與自然突變類似

CRISPR/Cas9 gene editing system

CRISPR/Cas9 Target gene

Target gene

CRISPR/Cas9

+

Edited target gene

Segregation

CRISPR/Cas9 Target gene

Editing

• Gene knockout is easy.

• Multi-gene editing is possible.

• DNA insertion is still uneasy.

• Weak gene expression could be a problem.

雙孢菇

不易變黑

維持美好外觀 利於機械採收

美國農部通過基改洋菇

基因編輯技術改良

Genome editing allows breeders

to introduce new traits more precisely, and at a faster rate.

Plant Genetic Engineering –

Progress, Problems and Prospects

• Gene mutation

• Gene replacement

• Gene insertion

• Public acceptance of DNA replacement or insertion could still be an issue.

Type of mutation

Phenotype: physical expression of traits

Genotype: sequence of all the genes in a genome

Photo credit LemnaTec; Anderson, L.K., Lai, A., Stack, S.M., Rizzon, C. and Gaut, B.S. (2006). Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes. Genome Research. 16: 115-122.

表現型

Selecting for DNA markers is faster than selecting for phenotype

研究基因型比表現型容易

分子標記輔助育種

Marker assisted selection (MAS) breeding

基因型

Bland Mushy Firm Flavorful and aromatic,

but goes soft quickly

Firm but bland, good for shipping

Wait for plants to produce fruits Test seeds Flavorful and aromatic, but goes soft quickly

Firm but bland, good for shipping

Conventional Backcross Breeding

Marker-Assisted Selection Breeding

Flavorful

Aromatic

Firm

Flavorful Aromatic

* More efficient and precise breeding.

Select the top contenders for continuous breeding

DNA extracted from seeds or young leaves is analyzed to reveal ideal candidates for breeding.

細菌萎凋病在東非造成嚴重香蕉病害

Bacterial wilt destroys banana eastern Africa

www.infonet-biovision.org

www.promusa.org

香蕉是非洲重要糧食之一

Banana is one of major staple foods in Africa

Tripathi, L., Mwaka, H., Tripathi, J.N., and Tushemereirwe, W.K. (2010). Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum. Molecular Plant Pathology 11: 721-731.

基因工程將辣椒的抗病基因導入香蕉

Disease resistant banana by introduction of a pepper gene

Pepper

Disease-resistant gene (Hrap)

• Bananas have a reputation for being difficult to breed.

• No disease resistant gene exist in the banana genome.

抗病 Resistant 感病 Susceptible

Pathogens Pathogens

GE improved Wild type

國立中興大學植病系

葉錫東教授研發之抗病毒木瓜 Virus-resistant papaya developed by Professor Shyi-Dong Yeh at National Chung-Hsing University

• The papaya germplasm has no virus- resistant gene.

• As of 2010, 80% of Hawaiian papaya were genetically modified.

Papaya genome does not have a resistant gene against the ring spot virus 木瓜沒有抵抗輪點病的基因

Papaya ring spot virus disease 木瓜輪點病

木瓜掛蚊帳，防蚜蟲傳病毒 颱風摧毀蚊帳

木瓜輪點病

Plant Genetic Engineering –

Progress, Problems and Prospects

Current 7.6 billion

A population of 9.5 billion and beyond

人類在未來幾十年所面臨的挑戰

The major challenges of human in the next few decades

Energy resource is declining

Population continues to balloon

Food production reaches a plateaus

2050 2050 2050

能源 人口 糧食

Population

Food

2050 2010

How to fill up the huge gap?

如何填補這個大缺口？

Burney, J.A., Davis, S.J., and Lobell, D.B. (2010). Greenhouse gas mitigation by agricultural intensification. Proc. Natl. Acad. Sci. 107: 12052-12057.

Population (billions) Crop production (gigaton) Crop area (hectare) 100%

increase

>100%

increase

~20%

increase

人口增加 100% 作物產量增加 100% 栽種面積增加 20%

矮化品種

Dwarf rice/wheat cultivars

+ 氮肥

N fertilizer +

灌溉系統

Irrigation system +

農藥

Pesticides +

機械化

Mechanization

單位面積產量提升，使作物產量足以跟上人口的增加

Crop production has kept pace with population increase due to yield increase 第一次綠色革命

The first ‘Green Revolution”, 1960-2000

土地惡化

Soil deterioration

鹽化

Alkalization

缺水;沙漠化 Water shortage

Desertification

海岸邊耕地水入侵 Salt water intrusion into coastal farmlands

法國南部靠地中海三角洲 River delta in southern France

溫度增加 1 度對產量的負面影響

Negative impact on crop yields of a 1 °C increase in global temperature

Average of multimethod ensemble.

Several studies have already predicted that climate change could halve the amount of farmland worldwide suitable for growing coffee by 2050, mainly because of increasing temperatures.

• Beer will be in short supply and double in price because of the difficulty of growing barley in a warming world.

• Since beer is the world’s most popular drink by volume, this will have significant social and economic effects, according to the scientists.

• Decreases in beer consumption (for example, − 32% in Argentina) and increases in beer prices (for example, + 193% in Ireland).

June 2, 2015

全球缺水地區

缺水及高溫是全球性問題

Water shortage and rising temperature are global problems

210,000 people affected by lingering drought in SW China.

An boy walks through failed crops and farmland in Afar, Ethiopia. 4/5 of crops in the country have failed. Photograph: Mulugeta Ayene/AP

Drought and rising temperatures ‘leaves 36M people across Africa facing hunger’. March 16, 2016.

Fallow of rice cultivation is a routine policy when water shortage occurs in Taiwan.

基因改良育種：減少肥料造成河川及湖泊優氧化

Genetic engineering: to reduce the use of fertilizers and water eutrophication

細菌、藻類繁衍 Growth of bacteria and algae

非生物性逆境

Abiotic stresses

乾旱 ^Drought 高鹽 ^Salinity 低溫 ^Cold 高溫 ^Heat 水災 ^Flooding

繁殖器官 Reproductive organs

穀粒大小 Grain weight/size

穀粒數目 Grain number/panicle

穗數 Panicle number/plant

形態 Plant architecture

高度 Plant height

分蘗數 Tiller number

分蘗角度 Tiller angle

莖桿厚度 Culm thickness

直立葉片 Erect leaf

根系 Root system

根數目 Root number

根長度 Root length Khush, 1999, Genome 42:646

生物性逆境

Biotic stresses

病害 ^Diseases 蟲害 ^Insects 線蟲 ^Nematodes

決定水稻產量的因子

Factors determine grain yield in rice

環境因子 Environmental

遺傳因子 Genetics

http://irri.org/our-work/research/rice-and-the-

environment/drought-submergence-and-salinity-management

耐旱水稻

Drought-tolerant rice

耐鹽水稻

Salt-tolerant rice

• We do have bred new rice varieties with increased grain yield when grown under normal conditions in field .

• Grain yield is still significantly less under stressed conditions.

• One of the most serious problems is the shortage of graduate students who are interested in plant science research.

糧食安全 Food security

環境永續 Environmental

sustainability

健康糧食 Health food