Plantbiotech notes done by Miss Lim


What is plant tissue culture?


 Refers to the sterile, in vitro, cultivation of plant parts such as organs, embryos and seeds, as well as single cells on either solidified or liquid nutrient media.

 Tissue culture: Refers to the aseptic growing of excised plant parts in vitro.

 Unique to plants is the ability of non-dividing cells to revert to an undifferentiated state that has meristematic (capable of cell division) activity. These cells have the ability to divide and differentiate into a whole plant (i.e. exhibits totipotency).

o Clones are genetically identical to the parent and to each other.



Micropropagation

 Plant tissue culture – also known as micropropagation if the technique is used to clonally produce thousands of plantlets.

 Is a vegetative reproduction method (a type of asexual reproduction in plants whereby individuals are obtained without the production of seeds or spores)

 Can be combined with genetic engineering to give rise to new types of plants



Stages in micropropagation

Stage 1 - Initiation of sterile explant culture: the selection of explants, sterilization of tissue surface to prevent contamination, and transfer of explants to nutrient media

(1) Selection of explants

 Plant growth is usually initiated from a piece of tissue or an organ (mature and differentiated) that is removed from a plant (called an explant).

 When grown on a specific nutrient medium in the presence of a specific ratio of growth hormones, the non-dividing cells revert back to an undifferentiated, meristematic state whereby they form callus tissue.

 Callus tissue has the ability to differentiate into a whole plant or plant organ in a process called redifferentiation.

 The ability of a plant cell to give rise to a whole plant through the process of dedifferentiation and redifferentiation is called totipotency  an ability unique to plants.





 Pieces of stem tissue with nodes, flower buds, leaves or tiny sections of the shoot tip meristem and even epidermal tissue can be used.

 Young explants are chosen as they contain a higher proportion of actively-dividing cells that are more responsive to callus initiation efforts.

 Storage organs and cotyledons are also good choices of explants e.g. potato tuber, storage roots of carrots etc., and cotyledons of soy bean.

 The plant that supplies the material, cells or tissues, that is cultured is called the parent plant. This donor plant should be free from diseases and is actively growing.

 The donor plants carries desirable characteristics such as higher yield, better quality fruits or carry genes for resistance to diseases.



(2) Establishing aseptic cultures

 The surfaces of explants (e.g. shoot tip, root tip and very small pieces of leaf and stem) are sterilized using dilute sodium hypochlorite (e.g. Clorox) to kill bacterial and fungal pathogens that may live on the surfaces.



(3) Transfer of explants to nutrient media

 Explants are transferred aseptically to culture vessels containing mineral nutrients, vitamins, carbohydrate sources (e.g. sucrose) and growth regulators (auxins, cytokinins etc.).

 The medium is usually solidified using agar to provide a firm matrix for developing tissues.

 The containers holding the explants are then sealed and incubated for 1 – 9 weeks.

 During this period, a mass of undifferentiated tissue called callus develops.



(4) Rapid multiplication phase

 Calli (singular: callus) can be repeatedly subcultured onto new culture media to obtain required multiplication rates.

 The proportion of cytokinins to auxins must be controlled.

 A higher proportion of auxins is required for proliferation of callus.







Formation of callus:

 Plants form callus in nature in response to plant injury with cells around the injury rapidly dividing.

 The stimuli involved in the initiation of wound callus are endogenous auxin and cytokinin that results in cell division. Invasion by microorganisms and feeding by insects also induce callus formation.

What do we want calli (singular = callus) for?

 They are used because they

o are the fastest method for shoot multiplication and cloning of a plant species.

o have the potential to develop normal roots, shoots and embryoids that can form plants.

o can be a source of protoplasts as they have thinner cell walls that can be digested more easily.

o can be used to screen for cells with special characteristics e.g. herbicide resistance

Establishment of plantlet:

Differentiation: a cellular maturation process; involves changes in patterns of gene expression that affect the structure and functions of cells.

 Rapid multiplication is arrested.

 Process to induce plantlet formation: shoot elongation, root formation, formation of storage organs etc.

 The plant tissue is induced to form plantlets by modifications of the culture medium.

 Different ratios of plant growth hormones induce shoot and root formation.

Stage 2 – Shoot initiation: multiplication of shoot tissue from explants on nutrient media

 To stimulate development of adventitious shoots (shoots from callus), a high cytokinin: auxin ratio is usually used

Stage 3 - Root initiation: multiplication of root tissue from explants on nutrient media

 To stimulate root formation, a high auxin: cytokinin is used.

 Usually the lowering of cytokinins or eliminating it totally and with addition of auxin, will promote root development.

 The callus will differentiate and eventually form plantlets.



Stage 4 – Transfer of plants to sterile soil or other substrate under controlled conditions (acclimatization)

 The plantlets are usually incapable of growing outside of the culture vessel.

 Acclimatization involves slowly weaning the plantlets from a high humidity, low light, warm environment (of the in vitro culture) to what would be considered a normal growth environment of the particular species grown.

 It is grown for 4-8 weeks in a green house as a weaning process.

 The plant is taken out from a culture vessel, the agar washed and the plant soaked in fungicide and then grown in sterile soil.

 Rooting hormones may be used to stimulate root development.

 Plants are eventually transplanted to a uniform medium that adequately supports the plant, and is sufficiently porous to allow adequate drainage and aeration.










Advantages of cloning plants by plant tissue culture (micropropagation)


(1) Rapid multiplication: Plants with desired traits can be multiplied rapidly than can be done using conventional plant breeding techniques, which rely on sexual reproduction.

(2) Genetic uniformity: Since plants produced are genetically identical, they all possess the desirable features of the stock plants. It is difficult to produce plants that breed true (homozygous for the desired traits) when sexual reproduction is used.

(3) Production of disease-free plants: There is a possibility of eliminating viral, bacterial and fungal contamination. Pathogens do not normally penetrate to the tips of meristems usually used for explants.

(4) Takes up little space compared with plants growing in the fields.

(5) Production of rooted plantlets ready for growth (faster), rather than seeds or cuttings.

(6) Production of often, more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods.

(7) Propagation is independent of climatic changes, since tissue culture is carried out in a controlled environment.

(8) Effective means of asexual reproduction in some plant species. Some plants like bananas are sterile. Seeds of some plants such as orchids are difficult to germinate.

(9) Ability to air-freight large quantities of plantlets quickly and efficiently.

(10) Genetic engineering: tissue culture is an important tool for genetic engineering. It is the only viable method for regenerating genetically modified cells or cells after protoplast fusion.



Disadvantages of cloning plants by plant tissue culture (micropropagation)

(1) Contamination: a problem faced by commercial tissue culture laboratories is contamination. Contamination can cause very high losses in a short time. An infected plant sample can produce infected progeny. Stocks are usually checked carefully to prevent this.

(2) High production costs: micropropagation requires sophisticated facilities, sterile laboratory conditions and special nutrient media. It also requires trained personnel (i.e. skilled labour) with specialized skills, and a lot of effort to transfer the plantlets from the laboratory to the soil. Mechanization of the process would eliminate most of the labour cost associated, but this has proven difficult so far despite active attempts to develop this technology.

(3) Losses incurred during transfer of plant material from in vitro conditions to the acclimatization stage.

(4) Higher than acceptable levels of somatic variation / somaclonal variation. Callus cultures sometimes undergo genetic changes.

 Taken from plant biotechnology extra reading materials

Somaclonal variation: the genetic variability produced by plant tissue culture.

 Variability can be exploited to improve characteristics of crop and ornamental plants.

 Examples of somaclonal variants and their improved characteristics obtained in different crop species include:

 Corn – herbicide resistance

 Wheat – grain colour and height

 Sugarcane – sugar content, yield and disease resistance

 A number of different factors influence the presence of somaclonal variation during plant tissue culture. Explant sources, the length of time cells are in culture, the culture conditions such as growth hormone types and concentrations, and selective agents such as herbicides or other toxins that are introduced in low doses influence the degree of success of finding plant variants in culture.



 This variability is caused by changes in the chromosome number and structure of cells of explants being cultured.

 Examples of changes that influence genetic variability include:

1. Chromosome rearrangements

2. Single-gene mutations

3. Gene amplification (increase in gene copy number)

4. The activation of transposable elements.

 The resulting plants differ from the original parent plant (Note: Can be a disadvantage if you want genetically identical plants).

 These mutations occur randomly so that the regenerated mature plant is not genetically identical to the parent tissue.



Plant tissue culture (micropropagation) is a broad term used to define different types of in vitro plant culture. Six different types of in vitro cultures are recognized. Each type can result in a whole plant.

1. Callus culture – culture of differentiated tissue from an explant that dedifferentiates. (Pg. 1-4 of this set of notes)

2. Cell culture – culture of cells or cell aggregates (small clumps of cells) in liquid medium – need not know (need not know)

3. Protoplast culture – culture of plant cells with their cell walls removed (refer to extra readings / plant biotechnology reading)

4. Embryo culture – culture of isolated embryos (refer to extra readings

5. Seed culture – culture of seeds to generate plants (need not know)

6. Organ culture – culture of isolated plant organs such as anthers, roots, buds and shoots (refer to extra reading)

CLASS TEE/SWEATER

Hey 116! 

This post is regarding our class tee. And since nobody has decided on a design (and it's extremely difficult to compromise a design which everyone will love), I have come up with Plan B. That is a class sweater!!!

We can't go to school hot like that (look below). So we constantly try to do something about our uniform look. And I was in Plaza Sing when two girls in Australian International School Singapore uniform walked pass me. Their uniform is a green skirt, white blouse and a sweater with a darker shade of green. And the look is awesome! Very UK-ish. Below I have included many pictures of UK school uniforms to give you guys an example. Models are kind of...no comments. But I'm not asking you to look at them. The point is, LOOK AT THE SWEATER LA.

Girls of St. Trinian. (Obviously, it's not a real school. Just a movie on rebellious girls. That's roughly what every girl in that school wears btw, other than the nerds of course. You can get the movie from me if you want (; )

Heck the blazer k... That's a V-neck sweater on the right.

Okay, I couldn't resist not putting this up. THE GIRLS SO CUTE HORRRRR. Never seen girls so young wear blazer before.. Okay la, I sua-ku okay. BUT SO CUTE HOR!

Okay, many of you probably noticed that ACJC also have a school sweater of the same blue as our skirt. (Got one ACJC imposter in our school, never notice meh? Always wear the ACJC sweater. Aiya, open your eyes bigger, sure can spot her one. I will say no further.) Anyway, naturally the colour to make the sweater would be dark blue. But since ACJC has it already (as with some of our 2008 J2s), I suggest this red!! Imagine, dark red sweater with gold embroidery of the school logo (the crest one. not the cheapo lao pok Y-sigh in the shape of a torch) and behind we can have a smaller embroidery saying Class of 116 (2008) or something like that. SO HOGWARTS GRYFFINDOR!!!! HAHAHAHAHA. Okay, crap. 

Ya la. But you get the point la. We must try to be more daring and try new colours!! Btw, red is also our school colour. So.... Come on guys!! Settle this once and for all! Choose the red sweater! It has many advantages. Like during exams in the hall, can wear to keep warm... Normal days in the lecture theatres also can wear. I don't think it'll get very hot also la. Can wear in the library too. Good right! And when you wear it, it will block our red line in the middle of our shirt which makes us look like ITE students. And look cool at the same time!! Like from a UK-ish school.

The cost of making a sweater should be about $30 I suppose? Don't worry about it... I think the class will be willing to like fork out a little bit for you if you're financially unstable so as to make the cost lesser for you. But those cheapskate people who wanna freeload off the class, don't try to apply. We will han tam you. 

As for the guys who think that wearing a sweater is too gay, YOU'RE WRONG!!! Let me show you some cuties in sweater okay!

See, so cute what. Look at the golden embroidery. Ya. I was thinking of making the school badge in that colour. Instead of what it is now. For those who don't know which school badge I'm talking about, it is this ugly one...

Ya, it's quite ugly. But at least it's a crest!! By changing everything into gold, I'm sure it'll look okay...

Cedric Diggory from Hogwarts Hufflepuff! Everyone in Hogwarts wear a sweater on top of their shirt and on top of that sweater, they have a coat. For girls it's like this = they wear bra then shirt then tie then sweater then coat. But in Singapore, asking us to wear like that is suicide; can call ourselves Walking Sauna already. So, we shall have the sweater only! So sweaters on guys = NOT GAY. But definitely gay in the happy sense.

So guys (and ladies), if you choose this dark red sweater, you will also have to decide on V-neck, round-neck or turtle-neck. Don't gay la. Of course don't try to be funny and choose turtle-neck please. Everyone in 116 will hate you and nobody will wear it in school. 

CHEERS~!

P.S. Basyir's picture is not on the top of the page already. Don't need to prepare barf bag to puke into whenever you visit this blog!

A Belated Post

This is a super belated post but...

HAPPY BIRTHDAY BASYIR!!!!!!!!!

Here are some pictures of Basyir you guys can enjoy (:

Last but not least, my favourite one!!

Teehee.

Okay MISTER B. You're 17 now. A bit late. You're probably slightly younger than most of the girls in your year. But it's okay. They love you!! You can ask neh or pris. I'm sure they love you. Hahaha. But don't give up! Your fluttery eyelashes, your bimbotic handflip thing and everything bimbotic about you will continue to seduce and attract girls from any age group. 

Wheeeeeeeeeeeeeeeeeeeeeeeeeee.

Spread your wings and FLY like a butterfly, B!

Motherlode of Apologies

Hey guys! I'm here to apologise for the major screwup for our CIP outing. To cut the long story short, what happened is that during promos I emailed the coordinator asking if I could send in our class' details after our promos which is the next week. 

Okay. I think you won't be able to see. But he/she (what sex is Kewei?? i have no idea) said "All right. No worries". Then when I did send him/her the details, he/she said that all vacancies have been filled. I think this Kewei is a new volunteer coordinator because the last time I volunteer, I've never heard of this Kewei. He/she has much to learn. OH WELL. Once again, friends, I'm really sorry.

Next,

I have emailed several organisations such as National Parks Board (reforestation), ACRES (the wildlife rescue centre) and ICCS (international coastal clean up singapore). I have considered Hospitals and even IMH but these require a period of commitment and you have to keep going back perhaps once a month to check up on your new friends. So that's not really feasible considering that most of you probably won't want to make the trip so many times plus our other time-consuming commitments like CCAs will not allow us to. If you guys have any requests, just shoot away! Right now I don't really know what you guys wanna do for CIP (other than SPCA! but there's no more vacancies) so I'm just choosing the more interesting ones. Please take into account if there's a period of commitment or if it's a one-time thing yeah.

Or now that promos are OVER, we could start planning our Official Class Outing!

Here are some suggestions:

- cycling/rollerblading 

- zoo (waste $$$$)

- good food marathon

- movie marathon

- beach games @ Sentosa

- bbq

- paintball 

- kayaking

- treetop trail walk (be careful of falling branches)

- ice-skating (suggested by elf which is delph)

Okay guys, start spamming the tagboard!!

AND TO THE PW PPL, 

ALL THE BEST FOR OP!!!!

(guys cut your hair ah)

I'll bring pompom to cheer you guys on!! (just kidding, they'll kick me out of the room la!)

PEOPLE OF 116 LISTEN UP YA'LL.

THE BIG P IS HERE.

U KNOW.that 6 letter word.
that....word.

yes.

P-R-O-M-O-S.
*screams*
*panics*
*faints*

Our first paper is this coming Monday, and yeap, that ain that far awayy.

15th September 2008
H1 General Paper One 0800hr to 0930hr
H1 General Paper Two 1000hr to 1130hr

IF U HAVEN'T ALREADY DOWNLOADED THE PROMO TIMETABLE AND HIGHLIGHTED THE DATES, u've got to be kidding me,seriously.

as of today, september 10th 2008, we have exactly

4 days to GP
26 days to mother tongue and econs(h1 and h2) paper
27 days to maths and lit(h1 and h2) paper
28 days to chem paper one and two, and geog(h2) and history(h2) paper
29 days to biology papers
32 days to chem paper 3
33 days to h1 - geog,history,physics papers

1 week = 7 days
that's about slightly more than 3 weeks, less than a month.

whoa.

*little things we can do as a class*
1. greet each other with a piece of subject information
(eg. heyy tammy mak, DNA stand for deoxyribonucleoacid)
2. start pop quizing each other every spare time we get
(eg. jessica, name me all the elements of the periodic table)
3. start having an open discussion on a topic of a subject,without referring to lecture notes
(eg. Basyir : Market failure is whenn...
Siu Ngee: ..the market fails)
4. start creating songs or raps with the content that we have
(eg. *i really can't think of any at this point*)
5. BOMBARD all your teachers with questions,and i don't care if it sounds silly or not, JUST ASK.
6. Grab every available slots in every teacher's timetable for effective consultations.
(like what tam and i did this afternoon to ms lim...we almost took her meeting slot, but please don't go to that extent).

i hope that enlightened ya'll.
alrite 116,

LET'S DO THIS.

*tam,jess,siu,bas -hope ya'll don't mind that i used your names.nothing intended.but u can try killing me in school if u like)

Welfare Rep
~Your welfare, is in my hands~

Success!

This is what we've all been waiting for!


Teachers' Day Celebration is finally over! Here's a breakdown of what exactly happened.

0700 - I reheated Mr Wong's Kong Ba Bao using my microwave.

0810 -  Mr Loh's GP lecture. Had the Mr. Cool book passed around and making sure that everyone writes a message in it. 

0900 - Mr Loh receives the present!

Mr Loh looking happy.

0905 - The grueling struggle to get the ribbon onto Yang Feng.

Happy Yang Feng (yeah right) ((yang feng! don't hate me!!))

Tada! All ready for Ms Lim.

0915 - While waiting for Ms Lim, we took a few pictures to kill time.

Pris with our Hero of the Day!

(i had to put this picture. Yang Feng's look is priceless and too alluring)

0920 - Ms Lim receives her present!

After that is just some performances by some groups and half-of-the-class outing. 

Yippee.

Note to all by Chemistry Rep Pris:

Chemistry practical tmrw is changed to tutorial. 

Bring Reaction Kinetics.

Holiday Classes

This is a reminder to all that there is Chemistry lesson for Group B and C this saturday. Lessons are pushed forward by an hour, don't forget that! 

So Group B shall report at 8 AM and Group C, 12 NN. 

Also, I've texted Ms Lim and according to her, she's on course so the extra biology lesson will be on Tuesday after school. So guys, NO BIOLOGY CLASSES ON SATURDAY alrights! 

All these information are accurate as of the date of this entry.