Effect of immersion systems, lighting, and TIS designs on biomass increase in micropropgating banana (Musa spp. cv. Grande naine AAA)
In Vitro Cell.Dev.Biol.-Plant, Mar 13, 2014
Development of in vitro techniques has enabled
rapid clonal propagation, regeneration, and multi... more Development of in vitro techniques has enabled
rapid clonal propagation, regeneration, and multiplication of
genetically manipulated superior clones, production of secondary
metabolites, and ex situ conservation of valuable
germplasm. This has been possible not only due to the refinements
of culture methodologies and applications of cuttingedge
areas of molecular biology but also due to the judicious
inclusion of engineering principles and methods to improve
and refine the system. In the present study, we used engineering
principles and methods to transform basic in vitro techniques
into commercially viable technologies. We investigated
two types of temporary immersion systems (TIS), two
types of lighting, and two different gas exchange systems
during in vitro banana (Musa spp. cv. 'Grande naine') shoot
cultures. After 7 wks, all banana shoots cultured in standard
TIS (5-L glass vessels, type 1) showed superior vegetative
growth for the evaluated parameters. We also found that
illumination provided by light emission diodes (LEDs) was
superior to the use of white fluorescent lamps at the same light
intensity (40 μmol m−2 s−1). Shoots treated with compressed
air for immersion and additional gas exchange during the
culture period in the glass vessel of TIS systems resulted in
higher propagation rates and a larger number of shoots harvested
as well as a larger number of roots formed per shoot
after the 7-wk culture period.
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Papers by Elio Jimenez
rapid clonal propagation, regeneration, and multiplication of
genetically manipulated superior clones, production of secondary
metabolites, and ex situ conservation of valuable
germplasm. This has been possible not only due to the refinements
of culture methodologies and applications of cuttingedge
areas of molecular biology but also due to the judicious
inclusion of engineering principles and methods to improve
and refine the system. In the present study, we used engineering
principles and methods to transform basic in vitro techniques
into commercially viable technologies. We investigated
two types of temporary immersion systems (TIS), two
types of lighting, and two different gas exchange systems
during in vitro banana (Musa spp. cv. 'Grande naine') shoot
cultures. After 7 wks, all banana shoots cultured in standard
TIS (5-L glass vessels, type 1) showed superior vegetative
growth for the evaluated parameters. We also found that
illumination provided by light emission diodes (LEDs) was
superior to the use of white fluorescent lamps at the same light
intensity (40 μmol m−2 s−1). Shoots treated with compressed
air for immersion and additional gas exchange during the
culture period in the glass vessel of TIS systems resulted in
higher propagation rates and a larger number of shoots harvested
as well as a larger number of roots formed per shoot
after the 7-wk culture period.
and chemical mutation induction procedures. Five brown rust resistant mutants with hypersensitive response to Puccinia
melanocephala were selected out of a total population of 11 167 regenerated plants. High selection frequency was obtained
with both mutagenic treatments, although chemical mutagenesis (NaN3) resulted in higher selection frequencies for
brown rust resistance than gamma irradiation (60Co). The brown rust resistant mutants showed variations in molecular,
morphological, and agronomic traits. Traits such as internode shape, bud shape, leaf sheath hairiness, outer auricule shape,
intensity of flowering, stool growth habit, number of stalks per stool, and smut susceptibility were modified in brown
rust resistant mutants. In addition, sugar yield was improved in two mutants with increments in stalk length, stalk number,
and stalk diameter. Mutation induction proved to be suitable for the generation of new sources of brown rust resistance
in sugarcane.
melanocephala is a major disease of sugarcane (Saccharum
spp.). A sugarcane mutant, obtained by chemical mutagenesis
of the susceptible variety B4362, showed a post-haustorial
hypersensitive response (HR)-mediated resistance to
the pathogen and was used to identify genes differentially
expressed in response to P. melanocephala via suppression
subtractive hybridization (SSH). Tester cDNA was derived
from the brown rust-resistant mutant after inoculation with
P. melanocephala, while driver cDNAs were obtained from
the non-inoculated resistant mutant and the inoculated susceptible
donor variety B4362. Database comparisons of the
sequences of the SSH recombinant clones revealed that, of a
subset of 89 non-redundant sequences, 88% had similarity to
known functional genes, while 12% were of unknown
function. Thirteen genes were selected for transcript profiling
in the resistant mutant and the susceptible donor
variety. Genes involved in glycolysis and C4 carbon fixation
were up-regulated in both interactions probably due to
disturbance of sugarcane carbon metabolism by the pathogen.
Genes related with the nascent polypeptide associated
complex, post-translational proteome modulation and
autophagy were transcribed at higher levels in the compatible
interaction. Up-regulation of a putative L-isoaspartyl
O-methyltransferase S-adenosylmethionine gene in the
compatible interaction may point to fungal manipulation of
the cytoplasmatic methionine cycle. Genes coding for a
putative no apical meristem protein, S-adenosylmethionine
decarboxylase, non-specific lipid transfer protein, and
GDP-L-galactose phosphorylase involved in ascorbic acid
biosynthesis were up-regulated in the incompatible interaction
at the onset of haustorium formation, and may
contribute to the HR-mediated defense response in the rustresistant
mutant.
such as digoxin and lanatoside C, which have
been widely applied in the treatment of cardiac insufficiencies.
Elicitation is one of the most effective methods to
enhance the biosynthesis of several secondary metabolites
in medicinal plants. We studied the effect of elicitation
with Chitoplant, Silioplant and methyl jasmonate on
biomass and cardenolides accumulation in shoots of D.
lanata cultivated in temporary immersion systems. Morphological
response of the shoots was influenced by elicitors.
A reduction in length and number of shoots was
evident with all MJ concentrations. Regarding biomass
production, Chitoplant (0.1 g l-1) was found to impact
significantly on fresh and dry weight of the shoots. HPLC
analysis revealed a higher content of lanatoside C compared
to digoxin in all treatments. The highest accumulation
of lanatoside C was achieved with Chitoplant
(0.1 g l-1), which resulted in 316 lg g-DW-1 and with
Silioplant (0.01 g l-1; 310 lg g-DW-1), which accounted
for a 2.2-fold increase in lanatoside C content compared
to non-elicited shoot cultures. Additionally,
elicitation of D. lanata shoots in temporary immersion
systems resulted in an oxidative stress characterized by
hydrogen peroxide and malondialdehyde accumulation.
These observations point to a connection between hydrogen
peroxide generation, lipid peroxidation and cardenolide
accumulation. The optimization of elicitor treatment
and culture conditions for cardenolide production as well
as the advantages of TIS for this purpose are discussed.
causal agent of black leaf streak disease of bananas and
plantains, was transformed with a green fluorescent
protein-carrying construct by using a restriction
enzyme-mediated integration methodology. A quantitative
polymerase chain reaction was adapted to estimate
transgene copy number and pathogenicity assays with
three banana genotypes with dissimilar reactions to M.
fijiensis infection were performed to characterize the
transformants. Transgene insertion varied from one to
five copies per genome among four random selected
transformants. All M. fijiensis strains produced typical
symptoms of the black leaf streak disease on the three
banana genotypes assayed. Interestingly, the GFP-18
transformant showed increased aggressiveness on susceptible
‘Grande naine’ and resistant ‘Yangambi km5’
plants demonstrating that mutation events in M. fijiensis
can increase virulence.
rapid clonal propagation, regeneration, and multiplication of
genetically manipulated superior clones, production of secondary
metabolites, and ex situ conservation of valuable
germplasm. This has been possible not only due to the refinements
of culture methodologies and applications of cuttingedge
areas of molecular biology but also due to the judicious
inclusion of engineering principles and methods to improve
and refine the system. In the present study, we used engineering
principles and methods to transform basic in vitro techniques
into commercially viable technologies. We investigated
two types of temporary immersion systems (TIS), two
types of lighting, and two different gas exchange systems
during in vitro banana (Musa spp. cv. 'Grande naine') shoot
cultures. After 7 wks, all banana shoots cultured in standard
TIS (5-L glass vessels, type 1) showed superior vegetative
growth for the evaluated parameters. We also found that
illumination provided by light emission diodes (LEDs) was
superior to the use of white fluorescent lamps at the same light
intensity (40 μmol m−2 s−1). Shoots treated with compressed
air for immersion and additional gas exchange during the
culture period in the glass vessel of TIS systems resulted in
higher propagation rates and a larger number of shoots harvested
as well as a larger number of roots formed per shoot
after the 7-wk culture period.
and chemical mutation induction procedures. Five brown rust resistant mutants with hypersensitive response to Puccinia
melanocephala were selected out of a total population of 11 167 regenerated plants. High selection frequency was obtained
with both mutagenic treatments, although chemical mutagenesis (NaN3) resulted in higher selection frequencies for
brown rust resistance than gamma irradiation (60Co). The brown rust resistant mutants showed variations in molecular,
morphological, and agronomic traits. Traits such as internode shape, bud shape, leaf sheath hairiness, outer auricule shape,
intensity of flowering, stool growth habit, number of stalks per stool, and smut susceptibility were modified in brown
rust resistant mutants. In addition, sugar yield was improved in two mutants with increments in stalk length, stalk number,
and stalk diameter. Mutation induction proved to be suitable for the generation of new sources of brown rust resistance
in sugarcane.
melanocephala is a major disease of sugarcane (Saccharum
spp.). A sugarcane mutant, obtained by chemical mutagenesis
of the susceptible variety B4362, showed a post-haustorial
hypersensitive response (HR)-mediated resistance to
the pathogen and was used to identify genes differentially
expressed in response to P. melanocephala via suppression
subtractive hybridization (SSH). Tester cDNA was derived
from the brown rust-resistant mutant after inoculation with
P. melanocephala, while driver cDNAs were obtained from
the non-inoculated resistant mutant and the inoculated susceptible
donor variety B4362. Database comparisons of the
sequences of the SSH recombinant clones revealed that, of a
subset of 89 non-redundant sequences, 88% had similarity to
known functional genes, while 12% were of unknown
function. Thirteen genes were selected for transcript profiling
in the resistant mutant and the susceptible donor
variety. Genes involved in glycolysis and C4 carbon fixation
were up-regulated in both interactions probably due to
disturbance of sugarcane carbon metabolism by the pathogen.
Genes related with the nascent polypeptide associated
complex, post-translational proteome modulation and
autophagy were transcribed at higher levels in the compatible
interaction. Up-regulation of a putative L-isoaspartyl
O-methyltransferase S-adenosylmethionine gene in the
compatible interaction may point to fungal manipulation of
the cytoplasmatic methionine cycle. Genes coding for a
putative no apical meristem protein, S-adenosylmethionine
decarboxylase, non-specific lipid transfer protein, and
GDP-L-galactose phosphorylase involved in ascorbic acid
biosynthesis were up-regulated in the incompatible interaction
at the onset of haustorium formation, and may
contribute to the HR-mediated defense response in the rustresistant
mutant.
such as digoxin and lanatoside C, which have
been widely applied in the treatment of cardiac insufficiencies.
Elicitation is one of the most effective methods to
enhance the biosynthesis of several secondary metabolites
in medicinal plants. We studied the effect of elicitation
with Chitoplant, Silioplant and methyl jasmonate on
biomass and cardenolides accumulation in shoots of D.
lanata cultivated in temporary immersion systems. Morphological
response of the shoots was influenced by elicitors.
A reduction in length and number of shoots was
evident with all MJ concentrations. Regarding biomass
production, Chitoplant (0.1 g l-1) was found to impact
significantly on fresh and dry weight of the shoots. HPLC
analysis revealed a higher content of lanatoside C compared
to digoxin in all treatments. The highest accumulation
of lanatoside C was achieved with Chitoplant
(0.1 g l-1), which resulted in 316 lg g-DW-1 and with
Silioplant (0.01 g l-1; 310 lg g-DW-1), which accounted
for a 2.2-fold increase in lanatoside C content compared
to non-elicited shoot cultures. Additionally,
elicitation of D. lanata shoots in temporary immersion
systems resulted in an oxidative stress characterized by
hydrogen peroxide and malondialdehyde accumulation.
These observations point to a connection between hydrogen
peroxide generation, lipid peroxidation and cardenolide
accumulation. The optimization of elicitor treatment
and culture conditions for cardenolide production as well
as the advantages of TIS for this purpose are discussed.
causal agent of black leaf streak disease of bananas and
plantains, was transformed with a green fluorescent
protein-carrying construct by using a restriction
enzyme-mediated integration methodology. A quantitative
polymerase chain reaction was adapted to estimate
transgene copy number and pathogenicity assays with
three banana genotypes with dissimilar reactions to M.
fijiensis infection were performed to characterize the
transformants. Transgene insertion varied from one to
five copies per genome among four random selected
transformants. All M. fijiensis strains produced typical
symptoms of the black leaf streak disease on the three
banana genotypes assayed. Interestingly, the GFP-18
transformant showed increased aggressiveness on susceptible
‘Grande naine’ and resistant ‘Yangambi km5’
plants demonstrating that mutation events in M. fijiensis
can increase virulence.