植物生物技術與食品. Transgenic plants why we need them? 吳彰哲 (Chang-Jer Wu) Department of Food Science National Taiwan Ocean University

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1 植物生物技術與食品 吳彰哲 (Chang-Jer Wu) Department of Food Science National Taiwan Ocean University Transgenic plants why we need them? 1

2 Transgenic plants why we need them? 82% of all transgenic food in the world produced by US and Canada 2

3 基因改造生物 基因改造生物 (Genetically modified organisms, GMO) 指該生物的遺傳物質 DNA 是經由除了天然交配或自然發生基因重組以外的方式改造而成的 基因改造食品包含被改造的作物, 改造的微生物以及加工過的食品 生物技術進行作物基因改造優點 減少殺蟲劑的使用, 增加作物本身對病蟲 害的抵抗能力 可以保護土壤表層, 避免因化學融蝕而流 失, 或污染水源 有效利用有限農地 3

4 Herbicides are used for weed control Weeds drastically reduce crop yield and quality Soybean with no herbicides Soybean after herbicides 4

5 Reduction in herbicide usage with resulting from the use of Roundup Ready soybeans (US). From Doane Market Research, Traditional crossbreeding Plant Biotechnology Recombinant DNA techniques 5

6 植物組織培養技術 植物組織培養技術 基因轉殖是必要步驟之一 癒傷組織 組織培養 組織培養 再生植株 紫杉醇 6

7 單倍體培養 植物組織培養技術 體細胞雜種的產生 植物組織培養技術 溶解細胞壁 Potato + Tomato 原生質體 Pomato 原生質体細胞融合 7

8 植物的基因轉殖技術 農桿菌轉殖 農桿菌轉殖法 一種土壤細菌 感染植物傷口 造成植物的腫瘤 8

9 T-DNA of A. tumefaciens is excised and integrates into the plant genome as part of the natural infection process. Any foreign DNA inserted into the T-DNA will also be integrated. 9

10 Ti 質體 (Ti plasmid) DNA between L and R borders is transferred to plant as ssdna; T-DNA encoded genes can be substituted by target genes Ti-plasmid based vectors Binary systems Co-integrated vectors Needs 2 vectors: Disarmed Ti plasmid with gene of interest (no vir genes) Helper vector for infection (with vir genes) Form co-integrated plasmid after homologous recombination on T- DNA Needs 3 vectors Disarmed Ti plasmid capable for infection Intermediate vector with T-region and gene of interest (transferred by conjugation) Helper vector for transfer of intermediate plasmid into A. tum 10

11 Co-integrated vectors (hybrid ti-plasmids) Right now rarely used DISADVANTAGES: 1) Long homologies required between the Ti plasmid and the E. coli plasmids (pbr322 based Intermediate vectors) making them difficult to engineer and use 2) Relatively inefficient gene transfer compared to the binary vector Ti plasmid vector systems are often working as binary vectors DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium. No intermolecular recombination is needed 11

12 GERMINATION The surface-sterilized tomato seeds are placed in a petri dish with nutrient medium for germination CUT EXPLANTS After 7 days, the tomato seedings are at the stage where the cotyledons (explants) can be cut for use in transformation. CUT EXPLANTS The cotyledon explants are cut from the seedlings in liquid MS. The wounded cells made by cutting will be the site of DNA transfer from the Agrobacterium. EXPOSE TO AGROBACTERIUM After overnight preconditioning on the feeder plates, the explants are competent for transformation and are exposed to the Agrobacterium. BLOT EXCESS AGROBACTERIUM Excess Agrobacterium is blotted from the explants on filter paper to avoid excess growth of the bacteria during cocultivation. DNA TRANSFER The explants are co-cultivated with the agrobacterium for 48 hours to allow time for the Agrobacterium to transfer the engineered DNA to the wounded plant cells. 12

13 TOMATO PLANT REGENERATION The explants are placed on a medium containing antibiotics for selection and control of the Agrobacterium. The medium also contains the growth regulator, zeatin riboside. Here the formation of an initial callus can be seen on the explant. The callus occurs at the site of wounding and is a result of stimulated plant cell growth caused by the growth regulator, zeatin riboside. Time point: 3 weeks SHOOTS BEGIN After 6 weeks the cotyledon explants are cut from the calli and discarded. Shoots are beginning to form and are transferred to fresh MSZ media. ROOTING MEDIA The tomato shoots have regenerated from the calli and are ready to be transferred to rooting media. This media lacks zeatin, but contains kanamycin for selection of transformants. Time point: 9 weeks TRANSGENIC PLANTS These are fully differentiated transgenic tomato plants rooting in MS media with kanamycin. Time point: 11 weeks TRANSFER TO SOIL Rooted plantlets are then transferred to boxes containing soil. The plants must be acclimated to the air, or "hardened off". The process takes 4-5 days. Time point: 13 weeks 13

14 TO THE GREENHOUSE These transgenic plants are ready for transfer to the greenhouse. Shown are two cultivars of tomato; Moneymaker (left), and Motelle (right). Both contain engineered genes. Time point: 15 weeks A GREENHOUSE OF TRANSGENIC PLANTS. Plants and growth conditions are carefully monitored and controlled. The plants are used for analysis, seed production, or are transplanted to the field. 植物的基因轉殖技術 基因槍 14

15 植物的基因轉殖技術 基因槍 植物病毒感染 植物的基因轉殖技術 15

16 葉綠體基因轉殖 植物的基因轉殖技術 16

17 其他方式 植物的基因轉殖技術 17

18 基因改造食品實例 抗除草劑 抗蟲 抗病 反義 RNA 抗環境 控熟 增加營養成分 產生醫療用途的蛋白質 病毒交互作用 加工品改良 Strategies for Molecular Farming 1. Plant gene expression system Transient transformation Stable transformation Chloroplast transformation 2. Location of trans-gene expression? Whole plant Target specific tissues (e.g. seed, root) 3. Selection of plant species and characteristics Mode of reproduction self/outcrossing Yield, harvest, production, processing 18

19 Why use plants? Advantages Cost reduction Stability Safety Disadvantages Environment contamination Food supply contamination Health safety concerns 19

20 基因改造食品實例 抗蟲基因轉殖煙草乙烯乙烯乙烯乙烯 抑制洋南瓜果實過熟 Wild Type 野生型 Antisense ACC Oxidase ACC X 乙烯 香蕉殖入病原菌抗原作成口服疫苗 抑制果肉軟化的酵素 ( 果膠脢 pectinase) Pectin in cell walls holds Polygalacturonase enzyme Pectin in cell walls softened FlavrSavr gene is antisense to Polygalacturonase enzyme encoding gene 20

21 Vitamin A deficiency affects some 800 million people worldwide Children only: Due to improper immune functioning bob.usuhs.mil/biochem/nutrition/ images/keratomalacia-1.jpg Golden rice Rice normally do not produces vitamine A. On the other hand, rice endosperm anyway contains geranylgeranyl diphosphate (GGDP) (progenitor of vitamine A) GGDP vitamin-a 2 genes from daffodils 1 gene from bacterium Erwinia uredovora Unfortunately, production is too low. All 3 genes are expressed in endosperm (major part of the rice grain) Normal serving of rice (300 g) provides just a few percent of daily diet Anyway, Syngenta supports the humanitarian use of golden rice, for individuals receiving $10,000 or less income from developing country 21

22 Vaccine production in edible plants Examples of edible vaccines ; pig vaccine in corn, HIV-suppressing protein in spinach, human vaccine for hepatitus B in potato. Hepatitis B vaccine in Banana Hepatitis B vaccine now costs $100 to $200 a dose Vaccine banana would cost only a few cents per dose. Just 24 acres of land could produce enough bananas to vaccinate all Mexican children under the age of 5. 22

23 Examples of Current Research Immunogenicity in human of an edible vaccine for hepatitis B (Thanavala et al., PNAS) Expression of single-chain antibodies in transgenic plants. (Galeffi et al., 2005 Vaccine) Plant based HIV-1 vaccine candidate: Tat protein produced in spinach. (Karasev et al Vaccine) Plant-derived vaccines against diarrheal diseases. (Tacket Vaccine) 23

24 基因改造食品的安全性 基因改造食品及作物主要反對原因 不知道由基因改造作物所製造出的食物是否對人體有害? 栽培這些作物是否對生態環境有害? 認為這種改造 基因 " 的技術在本質上是錯誤的, 因此根本排斥這種新技術 轉殖基因在食用後的狀況 基因改造食品的安全性 產生的重組蛋白質是否對人體有害 過敏原常常耐熱 過敏原常常不會被腸道酵素分解 有些過敏原具有特定的胺基酸序列, 可以辨識 改造微生物或再加工過的食品的選用或利用 可以改良發酵的過程 可以增加蕃茄的產量, 並降低損傷率, 同時果膠的含量也增加許多 植物的其他衍生物 24

25 基因改良食品的檢測 偵測蛋白質 檢測 DNA 來源基因的分析主要考量重點 此作物的基因組成有何改變 抗藥基因傳遞 25

26 基因改造作物對生態環境的影響 同種雜交的影響 同類近親植物雜交的影響 對其他昆蟲的影響 害蟲抗毒性的產生 對土壤微生物的影響 解決之道 利用同源重組的技術 利用不稔性的原理 26