AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION IN ...

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Dec 12, 2012 (4 years and 6 months ago)

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AGROBACTERIUM
-
MEDIATED GENETIC TRANSFORMATION IN THE MACROSCOPIC MARINE
RED ALGA PORPHYRA YEZOENSIS


Cheney, D., Metz, B., & Stiller, J.

Biology Department, Northeastern University, Boston, MA 02115 USA; Department of Biology, East
Carolina University,
Greenville, NC 27858 USA


Amongst marine algae, stable genetic transformation has only been accomplished in a few unicellular
species (i.e. a few diatom and dinoflagellate species). Here we report on the stable genetic
transformation of a multicellular ma
rine red alga or seaweed,
Porphyra yezoensis
.
Porphyra
, also
known as nori, is one of the most widely eaten and cultivated seaweeds in the world, as well as being
one of the most ancient seaweeds and multicellular eukaryotes that exists today, being at lea
st 500
million years old. Our method of gene transfer is unusual in that it utilizes
Agrobacterium tumefaciens
,
which has not heretofore been used to transform any alga (freshwater or marine) or any marine plant.
Transformation has been confirmed using bot
h GUS (with and without an intron) and GFP reporter
genes coupled to either a heterologous cauliflower mosaic virus (CaMV) 35S promoter gene or one of
two homologous reporter genes we've tested( RPB1 and GAPDH). Transgene expression has been
observed in pr
imary transformants for more than an eight
-
month period, as well as in progeny through
T1 and T2 generations. Evidence of stable genetic transformation will be presented. This gene
transfer system permits for the first time the study of red algal promoter
gene structure and function.
(Supported by NSF)





GENETIC TRANSFORMATION IN THE COMMERCIALLY VALUABLE, MACROSCOPIC, MARINE
RED ALGA
PORPHYRA YEZOENSIS



Authors: Cheney, Donald
(dcheney@lynx.neu.edu)
(1), Jo
hn Stiller(2), and Bradley Metz
(bmetz@lynx.neu.edu)

(1)

Addresses: 1.Marine Science Center, Northeastern University, Nahant, MA 01908, USA;
2.Department of Genetics, University of Washington, Seattle, WA 98195
, USA


In contrast to its broad success and many applications in land plants, stable genetic transformation
has only recently been accomplished in marine algae, and then only in a few unicellular species. Here
we report on the stable genetic transformatio
n of the multicellular, macroscopic marine red alga (or
seaweed)
Porphyra yezoensis. Porphyra
, also known as nori, is one of the most widely eaten and
cultivated seaweeds in the world, as well as being one of the most ancient extant seaweeds or
multicellular eukaryotes (over 500 million years old). Transformation was conducted using a novel
gene delivery system and has been confirmed using both GUS (with and without an intron) and GFP
reporter genes coupled to a heterologous cauliflower mosaic vi
rus (CaMV) 35S promoter gene.
Transgene expression has been observed for over a six
-
month period and in progeny through the T1
and T2 generations. Transgene expression has also been observed using an homologous promoter
isolated from the gene encoding the
largest subunit of DNA
-
dependent RNA polymerase II, RPB1.
The latter promoter (315 bp) differs from the typical land plant promoter in having no TATA or CAAT
elements and in being extremely G + C rich (over 65%) in the 5' flanking sequence. Expression leve
ls
with the RPB1 and CaMV 35S promoters are currently being compared. (Supported by NSF)



Nori blade with "blue" cells expressing the foreign gene GUS after staining with X
-
Gluc

Biomonitoring of the intentional introduction of a nonindigenous, aquacult
ured seaweed (
Porphyra
yezoensis
; Rhodophyta) into Cobscook Bay, Maine


Watson, K. and Cheney, D.

Biology Department, Northeastern University, Boston, MA 02115 USA;


In the early 1990's, PhycoGen Inc. (formerly Coastal Plantations International) began t
he commercial
farming of an introduced species of nori,
Porphyra yezoensis
, in Cobscook Bay, Maine. Permits from
local, state, federal and international agencies were granted based on the presumed inability of this
seaweed to sexually reproduce under Gulf
of Maine temperature regimes. A monitoring program was
begun in 1996 to examine the potential dispersal and establishment of
P. yezoensis

near PhycoGen's
farm sites.
Porphyra

samples were collected from intertidal transects and artificial substrata
constru
cted of Japanese nori netting.
Porphyra

plants were identified to species using morphological
characteristics, as well as isozyme electrophoretic and DNA markers. The Rubisco large subunit
(rbcL) and rRNA ITS1 genes were used to distinguish putative
P. yez
oensis

plants from
morphologically and isozymically similar native species. Our results suggest that
P. yezoensis

only
recruited ephemerally during PhycoGen's summer and autumn growing seasons. We found no
evidence to suggest that
P. yezoensis

was capable
of overwintering or establishing a permanent
population in Cobscook Bay. These results support the permitting process that allowed
P. yezoensis'

introduction and illustrate the value of biomonitoring species introduced for aquaculture.




Biomonitoring
study locations in Cobscook Bay, Maine








PROTOPLAST FUSION AND CARRAGEENAN ALTERATION IN THE RED ALGA
CHONDRUS
CRISPUS


Authors: Bradley R. Metz
(bmetz@lynx.neu.edu)
(1), Donald P. Cheney
(dcheney@lynx.neu.edu)
(1)
and Thierry Chopin (2).

Addresses: 1. Marine Science Center, Northeastern University, East Point, Nahant, MA

2. University of New Brunswick, Centre for Coastal Studies and Aquaculture
, Department of Biology,
Saint John, New Brunswick, E2L 4L5, Canada.


Recently, methods were developed to produce viable protoplasts from spores in red seaweeds. Large
numbers of viable spore
-
protoplasts (>90%) were produced in an agarase
-
based enzyme mixt
ure
without the addition of carrageenase. Spore
-
protoplasts exhibit increased viability and regeneration,
unlike most thallus
-
based protoplasts from morphologically complex
seaweeds. Spore protoplasts were utilized in fusion experiments from both life
hist
ory phases of
Chondrus crispus
.
Successful fusions were observed
microscopically and marked by etching around the fusant. Whole plant
regeneration of etched plantlets was monitored until plants reached 0.5 cm
and were transferred to bubblers. Putative fus
ion products were analyzed
using micro
-
infrared spectrophotometry. One plant, produced in an n+2n
fusion experiment, had a spectra that was similar to
Chondrus

gametophyte controls, except the 805
cm
-
1 peak was absent. Total carrageenan analysis revealed t
hat the galactose: 3,6 anhydrogalactose:
sulfate molar ratio was 1: 0.5: 0.92, unlike a typical

Chondrus

gametophyte of 1: 0.80: 1.27.

Life cycle of the red alga,
Chondrus crispus







Genetic Transformation project
-


Dr. Donald Cheney, Brad Metz, Kathy Watson and John Stiller

(dcheney@lynx.neu.edu)

(bmetz@lynx.neu.edu)

(kwatson@lynx.neu.edu)



Objectives:

To develop genetic transformation techniques in seaweeds and apply them for the
production of specialty compounds

Rationale:

Using genetic transformation techniques, seaweeds such as
Porphyra

can be modified
to produce a variety of valuable products

Status:

Stable expression of GUS reporter gene has been observed through the R2 generation



DEVELOPMENT AND APPLICATION OF GENETIC TRANSFORMATIONS IN SEAWEEDS

Donald P. Cheney, Brad Metz and K
athy Watson. Marine Science Center, Northeastern University,
East Point, Nahant, MA

John Stiller. Department of Genetics, University of Washington, Seattle, WA

Background

Genetic transformation (or engineering) techniques have been around for less than
twenty years, and
yet they have revolutionized agriculture. Today there is hardly a major agricultural crop that has not
been improved using genetic transformation techniques. Because the techniques involve the transfer
of genes from one organism to anothe
r, they permit the introduction of completely new traits into an
organism. The market value of genetically transformed agricultural crops is estimated to be $500
million today and is expected to increase to over $7 billion by 2005. More importantly, this t
echnology
is increasingly being viewed as essential to feeding the world's population in the next twenty years.

By comparison to agriculture, marine aquaculture has been little impacted by genetic transformation to
date. Genetic transformation techniques h
ave only been applied to a small number of marine
organisms, including around a dozen species of finfish, shellfish and unicellular marine algae. Genetic
transformation techniques have not been successfully applied yet to marine macroscopic algae or
seawee
ds, despite their considerable commercial value. In fact, most seaweeds can still only be
improved or modified using classical plant breeding techniques. Because of this, genetic modification
in seaweeds is severely limited in what can be accomplished. The

introduction of completely new traits
into seaweeds and thus, the development of new commercial uses for seaweeds, requires the
application of genetic transformation. The development of such techniques therefore is important,
perhaps even crucial, for the

growth of seaweed aquaculture and biotechnology in this country.

Recently, we have initiated an investigation to develop and apply genetic transformation techniques in
the edible red seaweed
Porphyra,

commonly known as nori, which is one of the two most
commercially valuable seaweeds in the world. This study brings together an unique combination of
expertise in the cell culture, development, growth and molecular biology of
Porphyra.

It differs
significantly from previous genetic transformation efforts in
seaweeds, in that we have already
developed two key components for genetic transformation: 1). a novel new method for stable,
heritable gene introduction, and 2). with a collaborator, a homologous promoter. This study should
result in not only the possibil
ity of new commercial uses for
Porphyra
and other seaweeds, but also in
valuable new information about gene structure and function in seaweeds.


Potential applications of genetic transformation in seaweed.

Genetic transformation technology can have signifi
cant commercial and basic science applications.
Possible practical uses of producing transgenic nori we are considering include (but are not limited to):
1). the production of disease
-
resistant strains of nori, 2). the modification of existing biosynthetic

pathways to increase the production of, for example, anti
-
cancer, anti
-
oxidant, and omega
-
3 fatty acid
compounds naturally found in nori, and 3). the production of completely novel compounds, such as
pharmaceutical proteins, antibodies and vaccines.


Safe

Culture of Transgenic Seaweeds

The plants we anticipate producing will be of such high value, that it will be economically feasible to
grow them in a land
-
based aquaculture system, possibly in conjunction with a (transgenic) fish culture
facility where th
ey can remediate and use the nutrients from the fish wastes to support their growth.


Nori blade with "blue" cells expressing the foreign gene GUS after staining with X
-
Gluc





HALOGENATED MONOTERPENE PRODUCTION IN REGENERATED PLANTLET SUSPENSION
CULTURES OF
Ochtodes secundiramea

(Rhodophyta, Cryptonemiales)


Authors: Sanjiv Maliakal, Donald P. Cheney
(dcheney@lynx.neu.edu)
(1) and G
regory L. Rorrer
(rorrerg@che.orst.edu)
(2)


Addresses: 1. Marine Science Center, Northeastern University, Nahant, MA 01908, USA

2. Department of Chemical Engineering, Oregon state University, Corvallis, Oreg
on 97331


Three genera of macrophytic red algae (
Ochtodes, Plocamium, Porteria
) contain novel halogenated
monoterpenes that are potential candidates for pharmaceutical compound development. In order to
develop a biological platform for halogenated monoter
pene production, an
in vitro

culture system was
established for the macrophytic red alga
Ochtodes secundiramea
. Specifically, callus cells were
induced from thallus explants of
O. secundiramea

plants. Shoot primordia regenerated from callus
cells and devel
oped into plantlets. The plantlets were cultivated as a free suspension in aerated, ESS
-
enriched natural seawater, and were vegetatively propagated by cutting the plantlet into smaller
pieces. In bubble
-
aerated culture, each plantlet grew as a symmetrical
array of highly
-
branched shoot
tissues emanting from a common center, ultimately assuming a spherical shape of 20 mm diameter
four weeks after subculture. Specific growth rates of over 20% per day were attained in bubble
-
aerated flask culture at an optimal

temperature of 26 degrees C and photosynthetic saturation light
intensity of 200 micromol photons per m2s. The cultured plantlets contained seven halogenated
monoterpenes, based on GC
-
MS analysis of dichloromethane extracts. Although bromomyrcene was
the
dominant acyclic halogenated monoterpene, several acyclic halogenated monoterpenes, including
chondrocole C and ochtodene, were also produced by the
O. secundiramea

plantlet cultures.
Halogenated monoterpene production was favored at high light near photos
ynthetic saturation and low
nitrate availability, consistent with the carbon
-
nutrient balence hypothesis for secondary metabolite
production.







BIOREMEDIATION OF HEAVY METALS USING THE RED ALGA
PORPHYRA



Kathryn Roberts and Donald Cheney
(dcheney@lynx.neu.edu)

. Marine Science Center, Northeastern
University, East Point, Nahant, MA


Heavy metal contamination, from both natural and anthropogenic sources, is recognized as a major
environmental
concern in marine ecosystems due to the pervasiveness and persistence of the
contaminants. Urban harbors are particularly at risk, with metal concentrations several orders of
magnitude higher than the naturally occuring crustal levels at uncontaminated sit
es. Select metals,
such as Cu and Fe, are naturally present in trace amounts, however, at higher concentrations all
heavy metals are capable of exerting toxic effects on organisms through generalized anesthetic
effects on cell membranes, disruption of esse
ntial enzymes, and displacement of essential ions.
Phytoremediation technology employs vascular plants and microalgae to remediate contaminants from
polluted sites in terrestrial and freshwater environments. Macroalgae are being investigated as
remediators

for marine systems and this study examines the metal concentrating capacity of the red
alga
Porphyra
. Preliminary experiments investigated copper and cadmium uptake by
P. yezoensis

and
future studies will examine interspecific differences in Cu and Cd upt
ake between
P. yezoensis

and
P.
umbilicalis
.

Herbarium pressing of the red alga
Porphyra umbilicalis




PROTOPLAST ISOLATION AND FUSION OF NORTH ATLANTIC
PORPHYRA

SPECIES AND
P. YEZOENSIS



Katherine Watson
(kwatson@lynx.neu.edu)
, Kathryn Roberts and Donald P. Cheney
(dcheney@lynx.neu.edu)
. Northeastern University, Marine Science Center, East
Point, Nahant, MA 01908.


A protoplast fusion program is underway to p
roduce hybrids between

P. yezoensis

and local species and select for new strains. To acheive this, we use protoplasts
isolated from conchosporangial branch
-
like concholcelis and spores, as well as
blades. To date, protoplasts have been isolated from blades

and conchocelis of
P. yezoensis, P.
purpurea

and
P. amplissima

and from blades and monospores of
P. umbilicalis
. Protoplast
regeneration has been accomplished with protoplasts isolated from all three

Porphyra

species.
Protoplast fusion experiments using t
he chemical fusant polyethylene glycol (PEG) are in progress
and methods for screening somatic hybrids for desirable traits are being developed.

Protoplasts of
Porphyra yezoensis

and
P. umbilicalis

fusing















SEAWEED CULTIVATION AND
BIOTECHNOLOGY IN CHINA



Song Qin, Peng Jiang and
Chengkui Tseng

N0.7 Nanhai Road

Institute of Oceanology, Chinese Academy of Sciences

Qingdao, 266071, P.R.China

sqin@ms.qdio.ac.cn

As early as several hundred years ago, China started cultivation of the seaweed
Porphyra

and
Gloiopeltis
in Fujian province. Now China produces 6 million tons of fresh seaweed via cultivation,
which equals to about 1.0 million tons of dry biomass. In 1950s

scientific methodology of artificial
cultivation of
Laminaria

and
Porphyra
was established, and later,
Undaria
,
Gracilaria

and other
economic seaweeds have been successfully cultivated in China.

Among common seaweeds of China are many species of economic

value. There are 46 genera of
seaweeds including a little over 100 species utilized by Chinese people for food, medicine and
fertilizer and as raw materials in the industrial production of phycocolloids and other valuable
products such as mannitol and iod
ine. Seaweed has been used as traditional Chinese medicine for
long time in China. For example,
Digenea simplex

and
Caloglossa leprieurii
are good antihelminths.

In the term of the traditional seaweed biotechnology, technology applied in utilization of se
aweed
biomass in China has a long history. Advanced seaweed biotechnology initiated in China in late
1950s when Chinese phycologists started work on genetics and genetic breeding of
Laminaria
japonica
, and created a few highly productive strains. In 1970s
tissue culture and cell biotechnology
were employed in the production of new seaweed seedstocks. From 1980s Chinese scholars have
discovered a lot of bioactive substances from seaweed and a few drugs based on seaweed
polysaccharides have been certificated.

Since 1990s genetic engineering of seaweed has been
studied and it is believed that kelp and other seaweeds will become multiple functional marine
bioreactors in fully enclosed facilities which transfoms entrophicated seawater into high value
products.


Copyright © 2001
-

2007


World Aquaculture Society All Rights Reserved.














Source: https://www.was.org/Meetings/AbstractData.asp?AbstractId=14329













Nontraditional nutrients

Farm Chemicals
,

Feb 1999


by
Grady, Tina


Seaw
eed and other sources of nutrients are surfacing as an excellent supplement to traditional
fertilizers.

LTHOUGH there is still much skepticism about their overall benefit to crops, nontraditional nutrient
sources, such as seaweed, are providing options fo
r growers looking to supplement their crop
program with nutrients that traditional fertilizers don't always provide.

For example, seaweeds have been collected and used as soil amendments in agricultural soils for
hundreds of years, and several sources of
seaweed products have been on the market since the
1950s. It has only been within about the last 10 years, however, that a consistent supply of
highquality seaweed products has been commercially available.

Seaweed extracts, along with humic acids, kelp me
als, amino acids, fulvic acid (some scientists
consider this a humic acid since it is usually produced by plant roots), and compost are now being
used as nontraditional nutrient sources.

Other alternative products being used in crop management programs in
clude foliar or applied
fermented nutrient product mixtures, fish hydrolysates and extracts, medicinal plant extracts, sugar,
molasses, and certain biological products rich in selected microbes.

These alternative products are used to enhance traditional f
ertility programs of grower/producer
industries, says Dr. Jeffrey Norrie, agricultural research coordinator for Nova Scotia, Canada
-
based
Acadian Seaplants Ltd., a company which focuses on seaweed biotechnology and other agricultural
research, and botanica
l programs.

Nontraditional products are used for their delivery of various macro
-

and micronutrients,
improvements in cation exchange capacity, the presence of plant growth hormones, and their ability
to improve the plant's own defense mechanisms against
diseases and insects," he says.

Some researchers and experts argue nontraditional products are often regarded as "safer"
alternatives to many chemical products and are held as a more natural remedy for crop ailments.

However, Norrie says, the need for tr
aditional sources of nutrients will not be eliminated.

Similar to the comparison of holistic practices and pharmaceutical medicine, traditional and
nontraditional nutrient sources complement each other
--

making both necessary, he says.

"It makes
fertilizer work more efficiently by stimulating the root growth," says David Williams,
president and chief executive officer of Houston, TX
-
based HumaTech, Inc., which sells humate
-
based nutrients used in nontraditional sources. "They complement; we don't
claim they replace.
When you improve baseline fertility, you improve the ability of the soil and plants so you can achieve
maximum production."

Seaweed As Synergy?

Part of the reason agriculture has been skeptical about the effectiveness of alternative n
utrients is
because the "mode of action" and specific active ingredients in many nontraditional products are
often difficult to pinpoint.

Norrie asserts that "synergistic interactions" between seaweed extract products and conventional
products are routine
ly observed.

"The nutrient content of all these nontraditional products is as varied as the sources from which they
are derived," Norrie says. "And humates are popular because they have many active exchange sites
for holding major actions (for example, ca
lcium, magnesium, potassium)." Whole fish products also
are used because they may be higher in nitrogen and some fatty acid compounds.

"Seaweed," he says, "can make a substantial mineral contribution as well as evoke an effect on plant
growth ... furnishi
ng a wide variety of chemical, physical, or biological improvements to the crop and
its growing environment."

And humic acid and fulvic acid, which are compounding agents, provide natural hormones to plants
that can help to "chelate" or buffer the chemica
ls in the soil to make them more available to the plant,
Williams says.

Modes Of Action

In the 1840s, Justus Von Liebig, noted as the father of chemical fertilizers, found that when water
-
soluble forms of nitrogen, phosphorus, and potassium were added to

soils needing organic matter,
production increased. However, traditional chemical fertilizers speed the natural depletion of the
humus contained in the soil's organic matter. By replacing it, Williams says, there is better nutrient
availability in the soi
l
-

letting the fertilizer work better since humus holds fertilizer available in a
soluble form.

Seaweed extracts and suspensions (produced by extraction of seaweed with either water or an
aqueous alkali such as sodium carbonate or potassium hydroxide) ma
rketed for agricultural and
horticultural use are typically derived from marine brown algae. The brown algae contain several
polysaccharides, some of which are alginates and known to help stabilize clay suspensions once
absorbed, according to information p
rovided by Dr. Henry Lyons, head of development for the
Institute of Technology, Tralee, Ireland.

In Europe and North America, Ascophyllum nodosum is the most commonly used algae and has
been claimed to help prevent compaction of the ground.

While many o
f these nontraditional products are already available, Norrie speculates that the market
sector will continue to develop as growers recognize benefits of using these products in conjunction
with standard nutritional programs
-

and distributors face increas
ing demand.

"Alternative products are becoming more mainstream and are being used increasingly by large
commercial grower enterprises," Norrie says. "Distributors can therefore no longer ignore these
products. If certain products are found to have value i
n specific cases, a judicious and effective
research program should be developed to investigate these effects. At the same time, nontraditional
products will continue to get used in agricultural systems due to their effectiveness on certain crops."


Copyr
ight Meister Publishing Company Feb 1999

Provided by ProQuest Information and Learning Company. All rights Reserved





Source: http://findarticles.com/p/articles/mi_qa3842/is_199902/ai_n8834629