2. C HAPTER 17 Tissue Culture: Micropropagation

3. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. C HAPTER O UTCOMES Understand micropropagation history Describe the advantages and disadvantages of micropropagation Recognize the tools and equipment used in micropropagation tony4urban/Shutterstock.com

4. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. C HAPTER O UTCOMES Describe materials used to make growth media for micropropagation Identify the stages of micropropagation culture Discuss the future of tissue culture and micropropagation Explore careers related to micropropagation tony4urban/Shutterstock.com

5. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. I NTRODUCTION Tissue culture Micropropagation Clones Growth medium borzywojo Studio/Shutterstock.com

6. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. H ISTORY 1839: Schleiden and Schwan— Totipotency 1902: Haberlandt—In vitro 1904: Hanning—Embryo cultured 1922: Knudson—Germinated embryos in vitro nakorn/Shutterstock.com

7. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. H ISTORY 1926: Went—First plant hormone discovered 1939: Gautheret, White, and Nobecourt—Constant regeneration and proliferation nakorn/Shutterstock.com

8. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. H ISTORY 1941: Overbeek—Coconut milk added to growth medium 1946: Ball—First entire plant via tissue culture 1962: Murashige and Skoog— First standardized artificial growth medium nakorn/Shutterstock.com

9. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. A DVANTAGES OF M ICROPROPAGATION Uniformity of clones Ability to grow large numbers Produce plants when usually not compatible Praiwun Thungsarn/Shutterstock.com

10. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. A DVANTAGES OF M ICROPROPAGATION Vast quantities of clones from one plant Ability to produce pest-free plants Aid in conservation and replication of rare or endangered plants anyaivanova/Shutterstock.com

11. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. L ABOR AND E QUIPMENT C OSTS Disadvantage—high costs Must have trained technicians Aseptic techniques Clean rooms Specialized tools – Laminar flow hood Requires carefully prepared growing medium Photography by Agri-Starts, Inc.

12. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S UPPLY AND D EMAND Micropropagated materials are costly Plant must be in demand Must be desired in large quantities lobster20/Shutterstock.comDaimond Shutter/Shutterstock.com

13. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. G ENETIC D IVERSITY Cloned plants decrease genetic diversity All clones have same benefits All clones have same disadvantages Chailalla/Shutterstock.com

14. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. E NVIRONMENTAL R EQUIREMENTS Explant – Removed from parent plant – Placed in similar environment Require – Light – Nutrition – Hormones – Moisture – Appropriate temperature – Aseptic environment Photography by Agri-Starts, Inc.

15. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. E NVIRONMENTAL R EQUIREMENTS Maintaining an aseptic environment – Laminar flow hood – Test tubes and petri dishes – Artificial light supply – Growth medium – Isopropyl alcohol solution (70%) – Bleach solution (10%) – Forceps, scalpel, plastic tape

16. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. G ROWTH M EDIA Nutrients Growth regulators Water Sterile Liquid or gel Grow and sustain explant in vitro Recultured luchunyu/Shutterstock.com

17. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGES OF M ICROPROPAGATION Stage 0: Selection and Cultivation of Stock Plants Stage 1: Initiation or Establishment Stage 2: Multiplication Stage 3: Rooting Stage 4: Acclimatization

18. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 0: S ELECTION AND C ULTIVATION OF S TOCK P LANTS Meticulous cultivation of stock plants Ensure disease free No insects Stage 0 limits and prevents contamination

19. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 1: I NITIATION OR E STABLISHMENT Aseptic environment Explant sterilized Explant transferred to in vitro culture anyaivanova/Shutterstock.com

20. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 1: I NITIATION OR E STABLISHMENT Explant material sources – Single cells from plants – Small pieces of plant tissues – Apical meristem – Undifferentiated cell masses Explant must be completely sterilized Jubal Harshaw/Shutterstock.com

21. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 2: M ULTIPLICATION Explants transferred to multiplication medium Must prevent contamination Medium composition – Gel containing vitamins, sugars, and a cytokinin growth regulator – Encourages axillary or adventitious root formation

22. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 2: M ULTIPLICATION Subculturing – Plantlets develop, remain for 5 ‒ 7 weeks – Plantlets can be divided, detached, and cultured again – Somaclonal variation

23. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 2: M ULTIPLICATION Environmental conditions – Plant specific growth requirements – Light quantity – Light quality – Moisture needed – Type and amount of nutrition

24. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 2: M ULTIPLICATION Plantlets ready for transfer to stage 3: – Shoots and leaves present – Rich in green pigment – Do not have any roots Kitto Studio/Shutterstock.com

25. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 3: R OOTING Plantlets transferred to rooting medium Rooting medium – Nutrients – Growth regulators – Sterile aboo2photo/Shutterstock.com

26. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 3: R OOTING Roots develop below medium surface Root presence indicates transplant readiness Time required varies weeks to months

27. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. S TAGE 4: A CCLIMATIZATION Plantlets transferred to a sterile potting medium Acclimatization occurs Roots washed to remove stage 3 medium Photography by Agri-Starts, Inc.

28. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. F UTURE OF T ISSUE C ULTURE AND P ROPAGATION Benefits – Massive replication of plant materials for biopharmaceutical production – Use of explants to determine chemical sensitivity – Create virus free clean stock

29. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. F UTURE OF T ISSUE C ULTURE AND P ROPAGATION Benefits – Chromosome doubling to make infertile seeds fertile – Somatic hybridization – Genetic changes not physically expressed Marcin Gabryelczyk/Shutterstock.com

30. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. C AREER CONNECTION Micropropagation Lab Technician Called culturists Proficient in tissue culture techniques Work in a laboratory Attention to detail and solid record keeping Associate’s degree or equivalent work experience Policies demand ethical behavior

31. Copyright Goodheart-Willcox Co., Inc. May not be posted to a publicly accessible website. C AREER C ONNECTION Cryopreservation Scientist Use of cryogenics Preserve and store germplasm Preserve and store plant tissue culture and seeds National Center for Genetic Resources Preservation (NCGRP) Varied educational training