Download microinjection as a procedure to deliver small and large molecules

II Congresso Brasileiro de Plantas Oleaginosas, Óleos, Gorduras e Biodiesel
Realização: Universidade Federal de Lavras e Prefeitura Municipal de Varginha
MICROINJECTION AS A PROCEDURE TO DELIVER SMALL AND
LARGE MOLECULES INTO SUNFLOWER PROTOPLASTS
Pedro Canisio Binsfeld 1
Claudio Cerboncini 2
Brigitte Dresen 3
Heide Schnabl 4
ABSTRACT
Microinjection is a reliable method for direct delivering foreign molecules (organelles,
chromosomes, DNA, RNA, proteins, drugs, etc.) into the protoplast via a thin glass capillary
and a micromanipulator. For an efficient microinjection system with Helianthus hypocotyl
protoplast, we combined a simple and efficient protoplast culture system with advanced
procedures of microinjection in order to minimize negative impacts on the cell viability and
their regeneration. For the injection of lucifer yellow, DNA or chromosomes, the protoplasts
were immobilized by embedding them in a thin-layer (120 µm) of 1% agarose in the growth
medium KMAR600. With this method about 40 protoplasts can be injected per hour; in these
microinjected protoplasts, the cells division frequency and microcalli production went up to
70%. For the transformation frequencies, only visual evidence was achieved. The genetic and
molecular verification of transformation and chromosome-mediated gene transfer is in
progress. Another important aspect is that the injection procedure should be as short as
possible in order to avoid cell dehydration and protein denaturation. A well established
microinjection procedure opens new perspectives on the basic sunflower research as well as
genetic transformation procedure.
Keywords: Helianthus; micronuclei; chromosome transfer; plant transformation
_______________________
1
Engenheiro Agrônomo, Doutor e Pós-Doutor em Biotecnologia. Institute for Molecular Physiology and
Biotechnology of Plants, University of Bonn, Karlrobert-Kreitenstr. 13, 53115 Bonn, Germany. E-mail:
[email protected] Corresponding author. Center of Biotechnology, Federal University of Pelotas – UFPel,
Brazil. Campus Universitário, Caixa Postal 354, 96010-900 – Pelotas-RS, Brazil
1
Engenheiro Agrônomo, Doutor em Agronomia. Institute for Molecular Physiology and Biotechnology of
Plants, University of Bonn.
1
Bióloga – Institute for Molecular Physiology and Biotechnology of Plants, University of Bonn.
1
Doutora em Botânica – Institute for Molecular Physiology and Biotechnology of Plants, University of Bonn,
Karlrobert-Kreitenstr. 13, 53115 Bonn, Germany.
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II Congresso Brasileiro de Plantas Oleaginosas, Óleos, Gorduras e Biodiesel
Realização: Universidade Federal de Lavras e Prefeitura Municipal de Varginha
1 INTRODUCTION
The application of biotechnological methods for gene introgression into elite sunflower
genotypes is of high interest for breeding programs that aims to produce superior genotypes to
obtain yield stabilization (Schnabl et al. 2002). Significantly higher yields in sunflower may
be achieved by enlarging the resistance to diseases and other stresses on the existing heterosis
level (Gulya et al. 1997, Seiler & Rieseberg 1997).
Cellular microinjection is a reliable method for direct delivering foreign molecules like
chromosomes, DNA, RNA, proteins, drugs, etc., or for organelle, chromosome and nuclear
transfer into the protoplast via a thin glass capillary and a micromanipulator. It is also an
efficient transformation technique (Griesbach 1987; Vos et al. 1999). To gain insights into
micromanipulation dynamics in Helianthus protoplast, we have undertaken studies to
optimize some microinjection parameters, in order to enable genome manipulations by direct
gene transfer through DNA fragments, metaphase chromosome or micronuclei.
2 MATERIAL AND METHODS
The basic system of the microinjection procedure has been described by Vos et al.
(1999). Hypokotyl protoplasts of sunflower (Helianthus annuus L.), genotype Sunshine which
showed good cytoplasmic streaming, with a dense cytoplasm, at least 40 µm in diameter, a
circular shape and no visible membrane damage were used. Freshly isolated protoplasts were
cultured overnight in liquid KMAR600 medium (Binsfeld et al. 1999). After 24 h the vital cells
were recovered by centrifugation and mixed with an equal volume of agarose (2%) and
embedded in a monolayer (120 µm) as show in Figure 1. For the injection, the coverslip was
removed from the solidified agarose monolayer, and covered with a drop of KMAR medium.
Microinjection was performed under bright field illumination but for the injection of lucifer
yellow, fluorescence illumination was applied. After the injection, the protoplasts were
cultured according to Binsfeld (1999). The osmolarity was adjusted to 600 mOsmol kg H2O-1
with mannitol. For better microcallus development, weekly concomitant to the changes of the
culture medium, the osmolarity was reduced in steps of 100 mOsmol kg H2O-1.
Supplementation of plant growth regulators was done as described by Wingender et al.
(1996).
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3 RESULTS AND DISCUSSION
Microinjection techniques have been widely used in plant cells research, but in many
cases this technique became unfeasible because of laborious set-up, time consuming
manipulation and expensive and sophisticated manipulation methods (Fig. 2A).
One important factor for easy cell manipulation is the cell size. In our experiments we
found out that selected protoplasts ranging from 40 to 50 µm vastly increased the efficiency
and agility of cell manipulation. In this sense, about 40 protoplasts could be injected per hour
(Fig. 2C). Another important aspect for an efficient plant protoplast microinjection was their
immobilization in one plane (in a monolayer). By embedded cells only one micromanipulator
is necessary, therefore a heated (30-35°C) drop of low-gelling agarose were added to the
isolated protoplasts and carefully dropped in the center of a petri dish and covered with a
coverslip (Fig. 1), to form a homogenous thin layer. The preparation was then placed at 4°C
for 1 min to solidify the agarose and immobilize the protoplasts. The preselected Helianthus
protoplasts were immobilized at densities varying from 10 to 50 protoplasts/mm2, and
covered with KMAR liquid culture medium. After two weeks in culture, the average plaiting
efficiency and microcalli production in different experiments went up to 70% (Fig. 2E).
Petri dish
cover glasses
agarose monolayer
(120 µm)
Cells
Figure 1. Schematic representation showing the agarose monolayer embedding process of
Helianthus protoplasts
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II Congresso Brasileiro de Plantas Oleaginosas, Óleos, Gorduras e Biodiesel
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Figure 2. Microphotographic showing: (A) Structure and equipment for microinjection; (B)
isolated and condensed chromosomes used for microinjection mediated
chromosome transfer; (C) microinjection procedure; (D) plasmolysis and
dehydration of the cell in the agarose monolayer; (E) microcalli developed in
agarose monolayer after 2 weeks in culture.
The suitability of the cells for microinjection was judged visually on the basis of the
presence of cytoplasmic strands and cytoplasm streaming. The best survival rate of the
injected cells was achieved 30 to 60 h after the isolation, when they had partially reformed
their cell wall. The transfer of chromosomes (Fig. 2B) or micronuclei should be done when
the cells undergo the first division. During the microinjection operation, distilled water was
added to replace the evaporated water from the culture medium. This procedure was of
fundamental importance in order to avoid the rapid and irreversible damages caused by
dehydration of the agarose monolayer and respective damage of the embedded cells (Fig. 2D).
In relation to the injection capillaries, they should be sharp enough to penetrate the cell wall
easily and avoid membrane damages. Another important factor observed in our experiments
was that the tip of the capillary should be shorter than 4 mm, since longer tips tend to be too
flexible to penetrate directly the agarose monolayer and the aimed position in the cell. In our
experiments we also noted that a carefully slow injection tend to be more protective for the
cells then a rapid and pulsive injection.
Since internal pressure is directly dependent on the injected volume, this is an important
factor to avoid cell damages. Several reports showed that for cells ranging from 40 to 50 µm
in diameter, the injected volume can range from 0.01 to 4 pl and represent a percentage of the
cell volume from 1 to 20% (Vos et al. 1999). The understanding of the discussed factors
permitted us to develop an efficient injection system for a large range of molecules or
organelles in selected Helianthus protoplasts, as could be demonstrated by cell vitality tests
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Realização: Universidade Federal de Lavras e Prefeitura Municipal de Varginha
by injection of lucifer yellow. Based on the cell vitality and survival rate over 75% after the
injection, we judge this procedure appropriate for efficient manipulation system.
4 CONCLUSION
The emphasis of this article was to analyze some factors that play a fundamental role
in microinjection procedure of plant cells, which has slowly become more and more important
in several plant cell experiments. This procedure permits introduction of any virtually sort of
molecule, at any given moment of the cell cycle. There are many good systems for higher
plant transformation, but microinjection still has the advantage of being a more direct
approach able to transfer large molecules or organelles across cell walls, and opens new
perspectives for useful genetic manipulation, e.g. chromosome, organelle or direct gene
delivering techniques into a receptor cell. In our experiments the Helianthus protoplasts
tolerated microinjection particularly well, with a rapid postinjection recovering of the vital
processes. With these results, it seems evident that microinjection represents an excellent tool
that greatly expands our possibilities to get insight about transformation through direct gene
or genomic manipulation and also genetic and physiological interaction studies.
5 ACKNOWLEDGEMENTS
The authors are grateful to the collaborators and to the Deutsche
Forschungsgemeinschaft (DFG), to the Gemeinschaft zur Förderung der Privaten Deutschen
Pflanzenzüchtung e.V. (GFP), Internationalse Büro (IB/BMBF), Germany and Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo a
Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Brazil, for the financial support.
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Realização: Universidade Federal de Lavras e Prefeitura Municipal de Varginha
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