How the Bleaching Process Works
(9 December 2022)
Effect of Bleaching Solution pH
The
point of this whole page is to elucidate just how the process of
bleaching mature Cyp seeds stimulates their germination. That
extended
bleaching of Cyp seeds in hypochlorite bleach greatly improves
germination was discovered independently by Van Waes and Debergh (1986)
in Belgium and Anderson (1989) in Canada, and I have been using such a
treatment ever since I began propagating Cyps in 1989. In this
context, "extended" means bleaching for longer than is necessary simply
for surface sterilization of the seed. For
about as long as I have used the bleaching treatment to improve the
germination of mature Cyp seeds, I have wondered about the mechanisms
involved in its effectiveness. In addition to surface sterilizing
the seed to remove foreign organisms that would infect the cultures,
the bleaching process serves to accelerate germination and to increase
percentage germination, but how?
Early on I wondered whether the high pH of my bleaching solutions had something to
do with improving germination. In 2000-1 I had been working with
seed from a C. pubescens
capsule that had been collected the previous fall, and found that this
particular seed required an unusually long period in the bleach to
germinate
well. With this seed germination was excellent after soaking in a
10% solution of commercial Clorox (6% NaOCl) for 98 minutes in cultures
sown 31 December 2000. The pH of this solution was 12.3.
Did the bleaching solution act through the action of oxidizing
germination inhibitors by free chlorine in the solution, or did the
high pH of the solution somehow remove or inactivate them?
As a first experiment to see
whether the high pH of the bleaching solution played a role in the
process, I made a trial in which I soaked the seed for 82 minutes in a
solution of NaOH with no NaOCl adjusted to pH 12.3. This soaking
was followed by pouring off the NaOH and replacing it with 10%
Clorox (0.6% NaOCl) bleaching solution for 16 minutes to effect surface
sterilization before sowing. Thus the seed was again subjected to
a pH 12.3 solution for a total 98 minutes. Germination of the
seed after this alternate treatment was also excellent; I
estimated it at roughly 70%. Thus bleaching for 98 minutes could
be replaced by soaking in a pH 12.3 NaOH solution for 82 minutes
followed by short bleaching, just long enough to surface sterilize the
seed.
An elementary
chemistry calculation shows that the concentration of NaOH or KOH that
has pH 12.3 is 0.02 N, and subsequently I did trials with soaking
mature seeds of several other Cyp species to see if this pretreatment
could shorten bleaching time. The presoak was effective to
varying degrees; there was appreciable shortening in the
bleaching time for C. candidum, makasin, pubescens, and especially macranthos, but hardly any effect in C. arietinum.
I further found that soaking in 0.02 N NaOH or KOH had no deleterious
effect on the seeds, and so I now routinely soak seeds in 0.02 N KOH
for several hours before bleaching. My choice of KOH over NaOH for routine
soaking is primarily philosophical; I may as well employ a base
with a cation plants can use as opposed to the Na, which they cannot.
Conceptual Model of Bleaching
Since
the early 1990s, I've held a simple conceptual model of what happens
during the bleaching process. In this model, I imagine that the
percentage or the frequency of seeds germinating after a given time in
the bleaching solution follows a normal or Gaussian distribution as
shown at the right. In this figure, the number of seeds
germinating after bleaching time t1
is depicted as the yellow shaded
area under the curve. I have assumed a normal distribution
because so many biological parameters such as the weight or height of
an organism follow this distribution. The mathematical
explanation for the common occurrence of the normal distribution in
biology is that a given biological parameter results from the sum of
many variables so that the normality of the distribution of the
parameter follows from the Law of Large Numbers. Thus I am merely
assuming that the degree of bleaching required to release a seed from
dormancy results from the contributions of many variables including
genetics and mother plant environment. The exact form of the
actual distribution is not that
important to my model, and I have chosen the normal distribution not
only because it may be realized in nature but also because it is easy
to draw.
Bleaching unfortunately also kills seeds, that is,
seeds bleached too long will never germinate. Consequently the time in
the bleaching solution must be chosen so as to be long enough to
promote germination, but not so long as to kill the embryo. In my
model, I also assume that the time required to kill a seed follows a
normal distribution, and the biological argument for that distribution
is the same as above. Thus the frequency of death of seeds during
the bleaching can be depicted as below:

The
results of bleaching can be obtained as a superposition of these two
distributions as shown at the bottom of this section. The
intersection of the curves for these two normal distributions defines
areas that characterize the fate of the seeds being bleached. In
the figure, colors designate these different outcomes. Yellow
represents the seeds that are released from dormancy and will germinate
by bleaching for time t1. Brown indicates seeds that will be
killed by bleaching for time t1. Seeds that would germinate with
longer bleaching than t1 are indicated by orange. Importantly,
there is seen to be a whole class of seeds that cannot be germinated by
bleaching because the bleaching time required to release them from
dormancy is sufficient to kill them. In other words there are seeds that can never be
germinated by bleaching because the bleaching necessary to release the
seeds from dormancy exceeds what the seeds can withstand and remain
viable. This class is represented by
the area in light blue.
In drawing this final graph, I positioned the two normal
curves in an arbitrary position along the time axis. The position
of each graph no doubt varies from seeds of one species to another and
probably even from one seed capsule to another for the same species.
Quite possibly there are seeds for which the two curves overlap
to such an extent that bleaching will not be able to germinate any
significant number of seeds.
Another conclusion from this model
is that there exists an optimal bleaching time, that is, the time that
maximizes the yellow area in the graph. Much of the lab work in
germinating Cyp seeds involves trying to find an approximation to this
optimal bleaching time through trial and error. Two or more
samples of seeds from a given capsule may be taken and bleached for
different periods in order to determine a satisfactory bleaching time.
Then the rest of the seeds may be bleached for this "best" time as determined in these trials.

The Actual Effect of Bleaching on Orchid Seed Embryos
In an engrossing study of the effects of bleaching on embryos of the orchid Spathoglottis plicata, Novak
and others (2008) found that cells in the embryos that were killed by
bleaching in NaOCl fluoresced a distinctive green color when
excited by blue light during examination with a digital imaging
fluorescence microscope. This autofluorescence permitted a
determination of the percentage of the cells in the embryo killed by
bleaching for different time intervals in a given concentration of
NaOCl and also for bleaching for a given time interval in different
concentrations of NaOCl. A remarkable result of this work was the
finding that embryos of S. plicata
were able to germinate and grow into seedlings even when somewhat more
than half the embryo was killed by bleaching! The study also found that
bleaching at an appropriate level enhances germination, consistent with
effects seen in studies of other orchid species and in my own work with
Cyps.
The fact that S. plicata
seeds can germinate with a significant number of embryo cells destroyed
no doubt arises as a result of the fact these embryos are relatively
undifferentiated. Orchid embryo cells in general lack such
histodifferentiation, and so it seems probable that embryos of many orchids
including Cyps will germinate and grow into healthy seedlings even
after significant portions of the cells have been killed by bleaching.
Seed Dormancy Mechanisms
Impermeable Seed Coat
Cyps
are temperate climate orchids, and as with many plants in temperate
climates, evolution has bestowed them with a mechanism to avoid
germination at an inappropriate time: seed dormancy. Were the
seeds to germinate in fall, say, the tiny protocorms would need to
survive in frozen ground with no prospect of subsequent growth until
the coming spring.
The exact mechanism or mechanisms of dormancy
in Cyp seeds has long been a subject of speculation. Perhaps the
earliest conjecture about the dormancy mechanism is that the outer seed coat or testa is water repellent and thus
prevents water, essential for germination, from reaching the embryo.
The falsity of this hypothesis can be seen merely by observing
the seeds during bleaching with a good hand lens or low power on a
dissecting microscope. In the seeds of many Cyp species, bubbles
of gas can be seen in the space between the seed coat and the embryo,
the gas being liberated as a result of the chemical reaction between
the bleach and the inner seed coat or the embryo. The bleaching
solution easily enters the opening at the micropylar end of
the outer seed coat (testa) where the seed detached from the placenta. This
opening shows prominently in scanning electron microscope (SEM) photos
of orchid seeds and can also be seen in views with light microscopes at
magnification of 30X or so if the seed is manipulated into the proper
orientation. The photo at the right shows the interior of a
freshly opened C. parviflorum var. pubescens
capsule in which the seeds have already mostly become detached and are
in somewhat randomized orientations. The red circles indicate the
openings in the outer seed coats of some of the seeds. Careful perusal
shows several other seeds with an orientation that allows this opening
to be seen. The seed is indeed sufficiently water repellent that
it can be dispersed in the environment by floating, but if the
seed is trapped underground, soil moisture could very easily enter the
coat through the open end. Thus impermeability of the outer seed coat is not a major hindrance to germination.
While
the outer seed coat itself does not prevent moisture from reaching the
embryo, the embryo sac or inner seed coat, called the carapace by Rasmussen (1995), may very well prevent moisture from entering the embryo itself.
Biochemical Dormancy
Biochemical
means of maintaining seed dormancy in many plants have been studied intensively over
the last several decades, but many details of the mechanisms involved
have only partially been elucidated. Extensive research has
pretty well established that abscisic acid (ABA) and gibberellic acid
(GA) are the primary factors regulating the transition from dormancy
to germination. ABA is responsible for the initiation and maintenance
of seed dormancy and GA is required for the release from dormancy and
the induction of germination. Current studies seek to unravel the
mechanisms by which these substances act at genetic and molecular
levels. At present, ABA is the only endongenous growth regulator
(also known as plant hormone) known to sustain seed dormancy (Liu and
others, 2013).
In numerous temperate plants, the technique of cold,
moist stratification of seeds has been used for many years to break
dormancy in these seeds. Indeed such cold stratification is also
sometimes successful in bringing Cypripedium
seeds to a state at which germination occurs upon bringing cultures of
the seeds up to room temperature. The detailed biochemical pathways
by which cold stratification either reduces the ABA content of seeds or
increases that of GA have yet to be discovered.
Because
of its importance in maintaining seed dormancy, I want to mention a
little bit about the chemistry of ABA. The molecular structure is
shown in the figure at left. Despite the somewhat elaborate
configuration of the molecule, its acidic nature is clear from the
presence of the carboxyl group (C(O)OH) shown in pink in the figure.
Because they are proton donors, carboxylic acids are acidic in
the Brønsted-Lowry
sense. Small carboxylic acid molecules are soluble in water, but
solubility decreases with increasing numbers of carbon atoms.
Organic chemistry tells us that carboxylic acids are generally soluble
in weak solutions of strong bases. Indeed while ABA
is only slightly soluble in water, it is quite soluble in 1 N sodium
hydroxide and also in methanol, ethanol, and the industrial solvent dimethyl
sulfoxide (DMSO), the
solubility in these compounds being roughly 20 to 50 mg/mL.
Several
decades of research on seed dormancy and germination have implicated
ABA as a major player in dormancy. In particular toward the end
of the last century research on the germination of monocot seeds has
associated leaching of
ABA from the seeds with promoting germination. Such a result was
found for barley (Visser and others, 1996) and for wheat (Suzuki
and others 2000). This work on barley germination also seemed to
show that the ABA-perception site resides outside the embryo,
presumably on the embryo surface, and that is the location at which ABA
effects its action to inhibit germination.
In a landmark paper for orchid biology, Lee and others (2015) studied ABA during the development of C. formosanum
(Taiwan lady's slipper) and demonstrated that 1) ABA is the primary
inhibitor of germination of the mature seed; 2) ABA is
synthesized internally in embryo cells during early embryo development,
and 3) as development continues the ABA migrates from within the cells
outward to accumulate in the surface wall of the embryo and the inner
seed coat (carapace) and to some extent in the outer seed coat.
The distribution of ABA in the mature seed was rendered visible both by
immuno-histochemical staining and by immuno-gold labelling. The
photos in this paper showing ABA distribution in the seed by both the
fluorescence of the immuno-labeled ABA and electron microscopy of the
gold-tagged ABA molecules are extremely impressive! I encourage
the serious
Cyp propagator to read this paper, or at the least, peruse the
remarkable photos. Clearly the location of the ABA in the seed
coats and on the embryo surface makes it possible to remove the
inhibitor by bleaching without killing too many embryo cells.
Application to Cyp Seed Germination
Knowledge that ABA is indeed the key inhibitor of germination and the
information about where the ABA is located in the mature seed provide
the basis for understanding the effectiveness of the bleaching
process. Commercial
bleach is not simply a strong solution of sodium hypochlorite, NaOCl.
The Clorox website lists the ingredients as: water, sodium
hypochlorite, sodium chloride, sodium carbonate, sodium chlorate,
sodium
hydroxide, and sodium polyacrylate; the sodium hydroxide is used
to control the pH.
Because
Clorox contains NaOH as well as NaOCl, the effect of the bleach on
orchid seeds is twofold: 1) The NaOCl oxidizes compounds in the carapace
to cause its weakening through corrosion thus allowing the bleach
access to ABA on the embryo surface, and 2) the NaOH in the bleach serves to dissolve
the ABA as the bleach penetrates the carapace. Perhaps the NaOCl also attacks ABA. This picture of
the bleaching process can also explain differences in response of Cyp seeds
to the presoak in NaOH or KOH. Those seeds with relatively thin
or permeable carapace such as C. macranthos
see enhanced germination when the presoak is followed by relatively
short bleaching, while seeds with heavy or impermeable carapace,
especially C. arietinum, show little enhancement of germination with the presoak and still require lengthy bleaching for germination.
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References
Anderson AB. 1989. Asymbiotic germination of seeds of some North American orchids. Pages 75-86 in North American Terrestrial Orchid Propagation and Production Conference Proceedings 1989. Chadds Ford, Pennsylvania.
Lee YI, Chung MC, Yeung EC, Lee N. 2015. Dynamic distribution and the role of abscisic acid during seed development of a lady's slipper orchid, Cypripedium formosanum. Annals of Botany 116: 403-411.
Liu X, Zhang H, Zhao Y, Feng Z, Li Q, Yang HQ, Luan S, Li J, He ZH. 2013. Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis. Proceedings of the National Academy of Sciences 110: 15485-15490.
Novak SD, Pardiwala RS, Gray BL. 2008. A study of NaOCl-induced necrosis indicates that only half of the embryo is required for seedling establishment in Spathoglottis plicata. Lindleyana 21: 32-38.
Rasmussen HN. 1995. Terrestrial orchids--from seed to mycotrophic plant. Cambridge: Cambridge University Press.
Suzuki T, Matsuura T, Kawakami, N, Noda K. 2000. Accumulation and leakage of abscisic acid during embryo development and seed dormancy in wheat. Plant Growth Regulation 30: 253-260
Van Waes JM, Debergh PC. 1986. In vitro germination of some Western European orchids. Physiologia Plantarum 67: 253-261.
Visser K, Vissers APA, Çağirgan MI, Kijne JW, Wang M. 1996. Rapid germination of a barley mutant is correlated with a rapid turnover of abscisic acid outside the embryo. Plant Physiology 111: 1127-1133.