BBT210
PLANT TISSUE CULTURE
LECTURE
SUMMARIES
Dr JOHN MOTTLEY
Lecture 1
Introduction to the unit. Review of
unit handbook. Explanation of timetable, staff involved, syllabus, recommended
texts, assessments (essays, prac reports, seminars, SAQ test), importance
of submission deadlines, guidelines for writing practical reports, library
resources, overhead projections, practical schedules.
Lecture 2
Tutorial: Introduction to the in vitro
culture of plants using videos. 1. In vitro techniques for crop improvement:
laboratory requirements and general methods; culture rooms, media composition,
sterilization (autoclaving, surface sterilization, laminar flow cabinets,
explants. meristem culture, clonal propagation, pathogen elimination, embryo
culture, callus and cell culture, staining, plant regeneration, somaclonal
variation, protoplast culture, haploid production. 2. Plants for free:
micropropagation, misting, general techniques, flowers, Kew gardens unit,
use for rare and endangered species.
Lecture 3
Growth of plant cell cultures: general
characteristics of cultured plant cells, artificial nature of their culture
conditions, the need for them to carry out their natural ‘evolved’ functions;
cell division, expansion, differentiation; initiation, characteristics,
use and growth of callus vs suspension cultures. Growth measurement techniques:
fresh and dry weights, cell number, indirect methods: area, volume, medium
conductivity.
Lecture 4
Techniques for helping cultured plant
cells to grow: conditioned medium, feeder systems, microdroplet techniques.
Advantages and disdvantages, setup and uses. Effects on lag, exponential
and linear growth phases. Cell immobilisation. Viability stains for plants
cells: importance, mechanisms, advantages and disadvantages and general
use; non-fluorescent stains (Evan’s blue, Lissamine green, tetrazolium
chloride). Fluorescent stains (fluorescein diacetate, calcofluorowhite).
Lecture 5
The cell cycle of cultured plant cells
and its synchronization: general description of phases in cells in vivo,
modification of phases on culturing plant cells, effect of different treatments,
changes in amount of nuclear DNA. Synchronization of cultured plant cells:
uses, methods available (starvation, use of specific inhibitors), determination
of degree of synchrony (mitotic index, flow cytometry). General principles
of flow cytometry and cell sorting.
Lecture 6
The cryopreservation of plant cells
and tissues: introduction and uses, general principles. Importance of
history and inherent susceptibilities of tissues to low tempertaure stress.
Role of water content of tissues, intracellular and intercellular ice crystal
formation, reduction in water content prior to freezing. Cryoprotectants
- different types and modes of action. Effects of the rate of cooling and
rate of thawing.
Lecture 7
Secondary metabolite production from
plant cell cultures I. Introduction: market values and uses of some secondary
metabolites, flavours, dyes, drugs, perfumes, insecticides. Why plant-derived
chemicals are important. Advantages of producing plant-derived fine chemicals
from plant cell cultures, including constant production under controlled
conditions and the production of novel compounds. Problems encountered
in the large scale production of plant secondary metabolites, including
growth characteristics of plant cells in culture, instability of cell lines,
cell aggregation, need for cell differentiation, product, release, recovery
and purification, susceptibility to shear damage.
Lecture 8
Secondary metabolite production from
plant cell cultures II. Isolation of high producing cell lines: initial
selection of donor plants, optimization of production medium, assay for
secondary metabolite production, determination of stability of cell lines.
Lecture 9
Secondary metabolite production from
plant cell cultures III. Factors affecting production: general principles
relating culture growth to primary and secondary metabolite production.
Environmental factors, including light, temperature, agitation of the culture
vessels. Medium components, including growth regulators, inorganic micro-
and macro-nutrients, carbon sources, metabolite precursors and elicitors.
Lecture 10
Secondary metabolite production from
plant cell cultures IV. Immobilization of cultured plant cells. Uses, gel
and foam entrapment techniques. Effect of immobilization on cultured plant
cells: cell viability, secondary metabolite production through bioconversions,
biosynthesis from distant precursors and de novo synthesis from simple
carbon sources. Disadvantages of immobilization, including loss of cell
activity, product release and recovery and cell detachment.
Lecture 11
Secondary metabolite production from
plant cell cultures V. Bioreactors for plant cell cultures. Need for modification
of existing bioreactors when used with plant cells due to inherent properties
of the cells. Evolution of bioreactor design from shake flasks, through
stirred tanks, air-lift fermentors, coulmn fermentors and fluidized bed
reactors.
Lecture 12
The isolation of plant protoplasts.
Enzymic isolation using cell wall degrading enzymes and a hypertonic medium.
Source material (especially leaves, suspension cultures and microspores).
Procedures involved, including overnight digestion, filtering, pelleting,
washing and sucrose gradients. Determination of protoplast density.
Lecture 13
The culture of plant protoplasts. Importance
of plating density, determination of cell wall synthesis, first cell divisions,
the use of low melting point agarose. The definitions and measurement of
plating density, initial plating efficiency and final plating efficiency.
Lecture 14
Protoplasts as physiological tools.
Including the isolation of cell components (vs tissue maceration), such
as chloroplasts, mitochondria and vacuoles; the study of cell metabolism,
such as cell wall formation, the action of plant growth regulators and
microbial toxins on the plasma membrane, pinocytosis.
Lecture 15
Tutorial: Plant biotechnology on the
internet. Hands-on workshop. Explanation of web browser, URL, bookmarks,
search engines. Examples of some web sites relating to agricultural biotechnology.
Relevant mailing lists, newsgroups, books, journals and periodicals. Bioethics,
international biotechnology associations, regulation and patents, US Federal
government.
Lecture 16
Revision tutorial by all staff involved
with the unit. Hand out of lecture summary to students. Advice on
approach to answering exam questions. Use of past exam questions to illustrate
model answers via outline plans. Question - answer session on queries resulting
from the course material.
PROF.
AV ROBERTS
Lecture 1:
Overview of plant tissue culture
The nature, methods, applications and
history of plant tissue culture are summarized.
Learning outcomes:
-
definitions of in vitro culture
-
concepts of methods, practices and
advantages of plant tissue culture
-
perspectives needed for following
lectures
Lecture 2:
Plant tissue culture media
Plants are maintained, ideally, on a
defined medium. However, if the cultural requirements of a particular species
has not been adequately defined, some ‘undefined’ ingredients such as coconut
milk or casein hydrolysate may improve the growth characteristics. The
inorganic components must include all essential elements as inorganic salts.
Salts usually supply more than one essential element. Attention must, therefore,
be given to the combination and concentration of salts needed to maintain
growth, over the entire culture period, without toxic effects. Oxidized
and reduced forms of nitrogen can induce different growth responses. Iron
must be chelated for availability to the plant at pH 5.2 and above. A carbon
source, usually sucrose, is provided. Plant growth regulators are used
to control development.
Learning outcomes:
-
overview of essential ingredients of culture
media
-
understanding of how modifications of media
may affect growth and development
Lecture 3:
Vegetative growth and propagation in angiosperms
Initial growth of a seedling is from
the primary root and shoot meristems. Plant hormones are influential in
establishing patterns of growth. Secondary meristems which give rise to
adventitious shoots occur naturally in some species. In some other species,
they can be induced in vitro by appropriate methods.
Learning outcomes:
-
an understanding of the development and
characteristics of plant organs and tissues
-
the ability to distinguish, on morphological
criteria, between primary and secondary meristems
Tutorial 1:
Categories of commercially important crops
Horticultural crops are typically propagated
clonally and are suitable subjects for micropropagation. Agricultural,
forestry and plantation crops are typically propagated by seed but, of
these, only agricultural crops are genetically uniform. Clonal propagation
of forestry and plantation crops would have some advantages over seed propagation
and are targets for commercial micropropagation. Seed propagation imposes
certain constraints on plant breeding which may best be solved by plant
tissue culture methods.
Learning outcomes:
-
knowledge of how terms are used to
describe crops
-
understanding of methods of propagation
and how these may constrain breeding objectives
-
understanding the potential role of plant
tissue culture in the various categories of crops
Lecture 4:
Micropropagation: Stages 0-IV
Micropropagation can be divided into
5 stages, each with its own objectives and problems.
Learning outcomes:
-
the ability to distinguish between the 5
stages of micropropagation
-
an understanding of the different challenges
of each of the stages
Lecture 5:
Micropropagation: other considerations
Some species are propagated in unusual
ways in vivo and the best in vitro methods may be similar. For example,
propagation of potatoes by tubers. Some species such as carrots can oil
seed palm can only be clonally propagated by somatic embryogenesis. The
commercial viability of micropropagation is dependent on competitive costing
in relation to in vivo methods. Micropropagation is competitive in only
a relatively small number of species at present.
Learning outcomes:
-
a knowledge of the advantages of microtubers
and the methods used to produce them
-
a knowledge of the methods used to produce
artificial seeds
-
a knowledge of the types of plants that
are commercially micropropagated
-
a knowledge of the actual and potential
scale of the micropropagation industry
Lecture 6:
Micropropagation: forestry
Forests are mainly propagated by seeds.
It is not possible, by this method, to conserve elite clones. Clonal propagation
from cuttings is difficult in some species and presently impossible in
others. However, micropropagation presents opportunities for clonal propagation
of some otherwise recalcitrant species. Many trees can only be propagated
in the juvenile phase but full assessment of their genetic potential can
only be made in the mature phase. One feature of micropropagation is that
it can sometimes induce rejuvenation of explants from mature trees. The
creation of monocultures which are vulnerable to attack by adapted pests
and pathogens is a potential hazard which must be taken into account in
the development of strategies of clonal propagation.
Learning outcomes:
-
an understanding of the problems of genetic
improvement of tree populations
-
an understanding of the concept of juvenility
and maturity
-
an appreciation of the advantages and disadvantages
of clonal propagation of trees
Tutorial 2:
Introduction to essay / seminar topics
The tutorial will be used to explain
the scheme described below, allocate titles and discuss expectations of
a model essay.
Each member of the class will take a
title which will be the subject of an essay and seminar presentation. (A
list of suggested titles will be provided but these can be adjusted or
new titles proposed.)
A first draft of the title will be submitted
and the lecturer will make suggestions for improvement. A final draft of
each essay will be copied and circulated to all students. Two weeks later,
the author of each essay will give a seminar presentation (10 min + 5 min
for questions). Each student will give a mark for the essay and presentation
of the other students. These marks will be moderated by the lecturer in
reaching a final assessment.
The intention is that essays will be
prepared which are of high quality in terms of substance and presentation,
that all members of the group will benefit from the work of the others
and that students will cross-examine the presenters to resolve differences
of opinion.
Learning outcomes:
-
clear understanding of the essay/seminar
scheme
-
clear understanding of the required standard
of essay presentation including citation of literature
-
an appreciation of the relevance of
suggested references for each essay title.
Lecture 7:
Adventitious regeneration
Adventitious regeneration may be by
production of adventitious shoots or somatic embryos. The former are unipolar
and the latter are bipolar developments. Both arise form secondary meristems
in which non-vacuolated cells arise from vacuolated cells. In many species,
adventitious regeneration is associated with high frequencies of somaclonal
variants. These are a hazard for micropropagators but are potentially useful
to breeders.
Learning outcomes:
-
an understanding of the defining characters
and origins of adventitious shoots and somatic embryos
-
an understanding of the defining nature
of ‘somaclonal variation’
Lecture 8:
Histogenic layers and chimerism in angiosperms
There are typically 3 histogenic layers
in angiosperms. Meristems in which these layers are genetically different
may give rise to stable production of chimeric plants. The nature of chimerism
may most easily be understood by observing variegations of petal or leaf
colour. The L3 layer gives rise to adventitious roots and the L2 layer
gives rise to germ cells and the L1 layer gives rise to adventitious shoots
in some species. An understanding of chimerism is, therefore, importance
in the propagation and breeding of some species.
Learning outcomes:
-
an understanding of the nature and origins
of periclinal chimeras
-
an appreciation of the implications of chimeras
for the propagation of plants in vitro
-
an ability to predict and solve problems
in the breeding of chimeric plants
Tutorial 3:
Statistical descriptors and analyses
Learning outcomes:
-
revision of the methods of descriptive statistics
-
revision of the t-test method
-
introduction to a method of increasing degrees
of freedom in the t-test
-
introduction to some convenient methods
of calculation
Lecture 9:
In vitro methods applied to plant breeding: general
principles
The income of breeders depends on obtaining
Plant Variety Rights. To obtain these, the conditions of distinctness,
uniformity and stability must be fulfilled, ie. genetic uniformity is required.
The main method of plant breeding is by cross fertilization but when attempts
are made to widen the gene pool of a species by in vitro fertilization,
embryo rescue, somatic hybridization or genetic transformation, in vitro
methods are needed.
Learning outcomes:
-
an overview of objectives of plant breeding
and relevance of plant tissue culture methods to these objectives
Lecture 10:
In vitro methods applied to plant breeding: adventitious regeneration
Adventitious regeneration has a fundamental
role in all plant tissue culture manipulations involving protoplast or
cell culture. Applications include genetic transformation, somatic hybridization
and haploid culture. It is also helpful when non-chimeric plants must be
isolated after mutation breeding or chromosome doubling.
Learning outcomes:
-
an appreciation of the role of adventitious
regeneration in in vitro based techniques with applications in plant breeding
Lecture 11:
In vitro methods applied to plant breeding: F1 hybrid seeds
F1 hybrid seed are obtained by crossing
two inbred lines, one of which must be male-sterile. Male-sterility is
necessary to ensure that the parent that is used as the female does not
fertilize itself. Some plants have natural sources of male sterility but
some important crops do not. A genetic system has been developed that can
be used in several crops (maize, oilseed rape, cotton, cabbage, cauliflower,
brussel sprouts) which is based on transformation with an RNAse gene governed
by a tapetum-specific promotor.
Learning outcomes:
-
familiarity with an example of how in vitro
methods of genetic transformation have been integrated with plant
breeding strategies.
Tutorial 4:
Discussion of practical data (Experiment )
Topics discussed will include the systematic
presentation of data, analysis of data and interpretation of data.
Learning outcomes:
-
how to interpret data and present the final
laboratory report
Seminars (3 h)
Student presentations
Each student will give a presentation
(10 min) on chosen topic (see tutorial 2, above) and answer questions (5
min).
Learning outcomes:
-
experience of the discipline of presenting
a paper and feedback on the clarity of presentation
-
experience of defending opinions when subject
to questioning
-
correction of mistaken views.