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From Mainberg@aol.com Thu Nov 28 17:13:00 1996
To: kmabie@ibm.net
cc: sro@aloha.net, ahicks@nmt.edu
Subject: Orchid seed disinfection (Part III)
DISINFECTION OF ORCHID SEED
by
Fred J. Bergman
The following study tested the validity of conclusions
drawn by the author from a literature search of orchid
seed disinfection.
METHODS AND PROCEDURE
Analytical Procedure-Available chlorine (AC) was
determined using the iodometric procedure (Snell and
Hilton, 1971), with the sample acidified using acetic acid
(Mellor, 1956). The sodium thiosulfate was standardized
against potassium iodate and the disinfectant solutions
were analyzed prior to every use.
Germination Conditions-The media used throughout the study
was described as solution 5 (Vacin and Went, 1949). Media
was modified by substituting NaFe-EDTA for ferric tartrate
and the addition of 0.1 mg/l of thiamin HCl. The
micronutrients B, Zn, I, Cu, Ni, and Al were added at the
concentrations described as Heller's in Table 2 (Murashige
and Skoog, 1962). The media was prepared using distilled
water with the pH adjusted to 6.0 prior to the addition of
1.2% agar (Sigma Chemical Co. product No. A9915). Culture
was in upright 500 ml Erlenmeyer flasks closed with a
modified Hall stopper (Liddell, 1946), with each flask
containing 160 ml of nutrient solution. Flasks were
sterilized for 30 min in a certified autoclave at 100 KPa.
Following sowing, stoppers were covered with aluminum foil
squares and the flasks were incubated at 30 C, a 16 hr
photoperiod, and 900 lux using cool white fluorescent.
The following conditions were used for disinfection:
(1) a wetting agent, O.T. Clear Aerosol, an ester of
sulfonated bi-carboxylic acid was added at a concentration
of 100 mg/l, (2) disinfectant contact time was 15 min, (3)
5 ml of disinfectant was used with 0.5 mg of seed
(approximately 175), (4) disinfectant was removed by
filtration, and (5) seed were rinsed several times with
sterile distilled water. Seed was transferred to the flask
by punching a hole in the bottom of the filter paper held
in the funnel, and washing the seed directly into the
flask with approximately 1 cc of sterile distilled water.
Seed Source-Seed for most of the program was obtained from
a single capsule of the cross Phalaenopsis (Eunice
Youngman x Bill Wallace) x Virginia Newton. The seed,
collected after the capsule dehisced, was left uncovered
for 24 hr on a clean sheet of paper in a service area
attached to an operating greenhouse. It was expected that
seed so exposed would be contaminated with a natural range
of airborne microorganisms. The extent of contamination
was confirmed by shaking five lots of seed with sterile
water, recovering the seed by filtration, and transferring
the seed to nutrient media. A single control was included
consisting of seed disinfected with a solution of calcium
hypochlorite (CHC). All five water treated flasks showed
multi-point contamination (20 plus colonies per flask)
while the control remained contamination free. The
contaminated seed was stored between sowings in paper
packets over anhydrous silica gel at 4 C.
Some elements of the program provided results requiring
an evaluation of germination and growth. This objective
was met by empirically considering the percent germination
and/or rate of growth. A significant improvement required
that the improvement be obvious. The procedure was
enhanced by controlling the number of seed sown in each
flask and determining their weight. It is acknowledged
that the overall rating procedure is not scientifically
correct, but it is practical and easily performed.
Calcium hypochlorite/concentration effects-It was observed
that 9 of 16 Phalaenopsis crosses that germinated when
disinfected using a 7.1% solution, prepared from old (CHC)
did not germinate when disinfected using a 7.1% solution
prepared using new CHC. The old CHC, which provided good
germination with Phalaenopsis seed, was found to contain
only 18% AC. A 7.1% solution prepared from the old CHC
therefore contained 1.29% AC. This concentration closely
approximates the 1% AC solution used by Wilson (1915). A
series, using CHC which contained 18% AC, was run at
concentrations expressed as % AC of 0.90, 0.72. 0.54,
0.34, 0.18, and 0.09. All flasks were aseptic and
germination improved as the concentration decreased. An
additional 18 flasks were sown using CHC at a
concentration of 0.18% AC. Each flask received individual
units of disinfected seed, while the control received seed
treated with sterile distilled water. All flasks were
aseptic except the control. A solution was prepared using
0.24 g/100 ml of distilled water of new CHC labeled 75 %
AC. Analysis of the filtered solution indicated a
concentration of 1.77g/l AC. Six lots of seed were
disinfected, 3 with the 1.77g/l AC solution and 3 with the
old 1.8g/l AC solution. Germination was uniform indicating
that the 2 solutions were equivalent and that earlier
germination problems resulted from concentration effects,
not the supply of CHC.
A review of records of previous sown crosses, all sown
using the 71g/l (10g/140ml) CHC solution, showed a strong
correlation between the age of CHC and the age of the
capsules when they dehisced. Previously, after a new
supply of CHC was opened, for the next 2 years good
germination was obtained only with seed from capsules that
dehisced after 9 or more months. As the CHC aged, seed
from earlier dehiscing capsules germinated. With 7 year
old CHC, seed frequently germinated from capsules
dehiscing after 4 to 5 months after the cross was made. It
appears that as CHC powder ages, the loss in strength
enables younger seed to survive disinfection.
A cross of Phal. Little Pink Doris x Classic Beauty was
made. This cross had been made previously and failed to
germinate disinfected with CHC at 7g/100 ml of water; even
though the seed had tested viable using Lakon's procedure
(1949). The repeat capsule dehisced after 141 days and was
disinfected using both an 1.8g/l AC solution and a 71g/l
AC (10 g/140 ml) solution. The 71g/l treated seed did not
germinate while near 100 % germination was obtained using
the 1.8g/l AC solution.
A cross, Ctna. Keith Roth by C. Priscilla Ward, had
originally produced a capsule that dehisced after 95 days
with a near 100 % viability confirmed (Lakon, 1949). Only
1 seed of 500 had germinated when disinfected using the
traditional 7 % CHC solution. The cross was repeated, and
this time the capsule dehisced after 91 days. Near 100 %
germination was obtained using a 1.8g/l AC solution.
A study of storage conditions on seed longevity,
conducted simultaneously with this study, provided
additional information. Phalaenopsis seed that had been
stored until it had just lost its ability to germinate
using a 7 % CHC solution, germinated when disinfected with
an 1.8g/l AC solution.
Stability of the CHC solutions was checked by using
distilled water to prepare 250 ml of CHC at a
concentration of 3.6g/l AC. Fifteen ml was diluted 1:1
with distilled water and used to sow seed. The remaining
solution was stored in a polyseal capped amber glass
bottle and placed on a workbench. The workbench
temperature during the test period ran between 18 and 21
C. At regular intervals 5 ml samples were withdrawn and
analyzed. Analysis of the CHC solution required 0.51 meq
of thiosulfate at the beginning of the test and 0.505 meq
after 161 days. This indicates a loss of about 1 % in 6
months. The final 15 ml of CHC solution was used to sow
seed, confirming the solution was still an effective
disinfectant. A second stability test conducted during
summer, when temperatures ran between 28 and 35 C, ran for
90 days found no significant change in concentration.
To check the saturation concentration of CHC, 35 g of
CHC powder (70% AC) was ground with 100 ml of distilled,
zero blank water. The slurry was filtered and an aliquot
of the filtrate was analyzed. The saturated solution
contained 21.7 wt%, calculated following the procedure of
Snell and Hilton (1971), almost 3 times the frequently
quoted 7.1%. Such a solution, containing 215g/l AC, would
probably be lethal to seed of any genera.
To determine the optimum concentration for sodium
hypochlorite (SHC), a series of tests were run using the
same experimental conditions employed for CHC. Each test
series included a control using CHC at 1.8g/l AC. The
first run consisted of flasks treated at 1:10, 1:30, 1:50,
1:120, and 1:500. Flasks treated at 1:120, and 1:500 were
contaminated. Next, two flasks were treated with 1:10 SHC
(5g/l AC) and two with 1:50 SHC (1g/l AC). The 5g/l AC was
inferior to the control in both percent germination and
plant vigor, while 1g/l AC appeared equal to the CHC
control.
Seed were treated with CHC and SHC at 1g/l AC at
contact times of 15 and 60 min. The seed treated for 15
and 60 min with CHC and for 15 min with SHC appeared
equal. Germination of seed treated with SHC for 60 min was
inferior to the seed treated with CHC even though the CHC
solution was 1.8 times stronger. Contact times of 10, 20,
and 30 min were studied using CHC and SHC at a pH of 6.0
and a concentration of 175 mg/l AC. There was little
difference between CHC at 10, 20, and 30 min and SHC at 10
min. Seed treated with SHC at 20 and 30 min was definitely
inferior to seed treated with CHC.
Effect of pH-The unadjusted pH of CHC solutions prepared
using distilled water were 11.7 at 10g/l AC, 10.4 at 3g/l
AC, and 9.5 at 1.5g/l and 175 mg/l AC. Similarly, the pH
of HB solutions were 10.2 at 5g/l AC, 9.75 at 3g/l AC, and
9.2 at 175mg/l AC. To evaluate the lower pH effects, a
series of tests were conducted using solutions of calcium
and sodium hypochlorite. The pH was adjusted to 6.0 (Sweet
and Bolton, 1979) using a buffer. Concentrations from 250
mg/l to 6.5 mg/l AC were examined. Cultures below 50 mg/l
were contaminated while those from 50 to 150 mg/l showed
occasional contamination. Cultures from 175 mg/l and above
were free of contamination. Sowings made using a pH meter
and 0.1 N phosphoric acid required an increase in the
concentration to 250 mg/l to remain aseptic. Probably the
direct results of a low buffering capacity. Continued use
of CHC at a pH of 6.0 and 250 mg/l AC shows a
contamination rate of about 1 %.
Effect of temperature-The temperature effect was evaluated
by sowing seed disinfected at 30 C using hypochlorite
concentrations from 6.7 to 225 mg/l AC. The contamination
pattern was identical to that obtained using room
temperature (18-20 C). In addition, the seed disinfected
at 30 C showed poorer germination and a slower growth rate
than the control disinfected at 20 C.
Contact decomposition of hypochlorites-It is frequently
claimed that rapid decomposition occurs when hypochlorites
contact germination media. Data to support this claim was
not found in the literature. If the decomposition rate is
not rapid, a possible negative effect on germination could
be produced by the resulting increase in contact time.
To estimate the hypochlorite decomposition rate in
contact with media, a lot of seed was added to 3 ml of a
CHC solution containing 1.8 mg/l AC. The solution was
shaken for 15 min, and added to a flask of media. After 1
hr a little over 3 ml of liquid was recovered from the
flask. Analysis showed the solution still contained traces
of AC. On incubation, only six of 150 seed germinated
while the control flask developed normally. The rate of
decomposition of HB was evaluated following the same
procedure using a 1g/l AC solution of HB. The recovered
solution still contained 80 mg/l AC. The flask was
incubated but there was no germination.
The stability of CHC powder is poor. A supply of CHC
used for several years that originally containing 68 % AC
was found to have lost all but 18 % of the AC.
Discussion
Little additional improvement was provided by using
concentrations below 2g/l AC. Most concentrations provided
by the literature clearly exceed what is required for
pathogen free culture. In addition, the use of such high
concentrations is frequently lethal to viable seed.
Contrary to popular belief, hypochlorites apparently do
not decompose rapidly on contact with germination media.
La Garde (1929) reported a rinse as beneficial even though
he sowed seed using a loop. Sweet and Bolton (1979)
obtained optimum germination by removing the disinfecting
solution by filtration followed by three water rinses.
This study found that hypochlorites retain some activity
one hr after contact and that seed remaining in the flask
lost its ability to germinate. These observations
demonstrate that the disinfecting procedure should include
removal of disinfectant and rinsing the seed prior to
sowing.
Orchid literature frequently states that solutions of
calcium hypochlorite are unstable. In reality, it is the
dry calcium hypochlorite that decomposes at a high rate
rather than dilute solutions. Dilute solutions prepared
using distilled water and properly stored have a shelf
life of 6 mo or more, even longer if refrigerated.
It appears that the use of lower concentrations of
calcium hypochlorite may improve germination of seed that
has proven difficult or impossible to germinate in the
past. This suggests that previous sowings, with seed that
appeared viable but failed to germinate using stronger
solutions, should be repeated using calcium hypochlorite
at 2g/l or 175-250 mg/l AC at a pH of 6.
A strong correlation was observed between time required
for a capsule to dehisce and disinfectant concentration.
Seed which dehisced after three to five months, and failed
to germinate when disinfected with 7.1 % CHC, germinated
when disinfected using a 2g/l AC solution. The use of
lower concentrations of CHC with seed from early dehiscing
capsules has the potential to provide significant
improvement in the number of seedlings obtained from ripe
capsules.
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