Does Frequent Subculturing Cause Tissue-Cultured Plants to Mutate? Understanding Passage Number and Somaclonal Variation
Subculturing is a normal and necessary part of plant tissue culture. Plantlets need fresh medium, additional space for multiplication, and regular transfer to prevent nutrient depletion and the accumulation of metabolic waste inside the culture vessel.
However, subculturing too frequently or maintaining the same culture line through too many passages can create problems. It is important to distinguish between subculturing too soon within each production cycle and continuing the same culture line through a large number of passages, because these two situations involve different mechanisms and risks.
The clearest concern associated with repeated subculture is an increased risk of somaclonal variation, or culture-induced variation.
Research on the banana cultivar ‘Nanicão’, involving nearly 4,000 plants, found off-type rates of approximately 1.3% after five and seven subculture cycles. The rate increased to around 2.9% by the ninth cycle and approximately 3.8% by the eleventh cycle.
This example shows that as a production line is maintained and multiplied for longer periods, the probability of finding off-type plants may increase. Some abnormalities may also remain undetected until the plants are grown in the field, where differences in bunch quality, fruit characteristics, or productivity become visible.
Repeated Subculture Can Affect More Than External Appearance
Changes associated with long-term culture are not limited to visible characteristics.
Genomic studies in cannabis have shown that plants maintained through multiple subculture generations can accumulate DNA differences, and in some systems the frequency of variation has been associated with increasing culture duration or passage number.
However, this relationship is not always linear.
Later studies have suggested that mutation accumulation may be relatively pronounced during the early establishment of a culture line and then slow over time.
It would therefore be incorrect to assume that:
“Every additional subculture cycle causes one additional level of mutation.”
A more accurate interpretation is that passage number should be considered a cumulative risk factor.
Epigenetic Changes Can Also Accumulate
Long-term micropropagation may also produce epigenetic changes, including changes in DNA methylation.
Epigenetic changes affect how genes are switched on and off without necessarily altering the underlying DNA sequence.
This means that a plant may still look genetically similar to the mother plant under some molecular tests while beginning to show differences in:
Growth rate
Environmental response
Flowering
Stress tolerance
Physiological performance
Secondary metabolism
Studies involving several cannabis cultivars have shown that methylation patterns can change with the duration of micropropagation and that different cultivars respond differently.
This is another reason why tissue culture stability cannot be evaluated only by appearance.
Multiplication Capacity May Decline as a Culture Line Ages
Another problem reported in several species is a gradual decline in shoot multiplication capacity after repeated subculture.
Research involving several fruit-tree rootstock genotypes has shown that shoot-forming ability may decline after many consecutive passages even when the same medium and hormone concentrations continue to be used.
For example, a culture line may initially produce five or six shoots per explant but later decline to only two or three shoots per cycle.
This may reflect culture ageing or loss of morphogenic competence.
When this happens, simply increasing BAP or another cytokinin is not always the correct solution. Higher hormone levels may increase abnormal shoots, hyperhydricity, or callus formation without restoring the original multiplication quality.
Subculturing Too Frequently Is a Different Problem
Subculturing very frequently—for example, every one or two weeks—does not have strong evidence showing that it directly causes mutation.
However, it can reduce production efficiency.
Shoots may not have accumulated enough biomass, newly initiated buds may still be underdeveloped, and the culture may be divided repeatedly before it has reached the optimal multiplication stage.
Research involving pineapple has shown that extending the culture period beyond 30 days can, in some passages, help restore shoot formation.
This demonstrates an important point:
The fastest possible transfer schedule is not necessarily the best schedule.
A culture should be transferred when it reaches the appropriate developmental stage, not simply because a fixed number of days has passed.
Callus-Based Systems Carry Higher Risk
The risk of variation becomes greater when multiplication involves callus or disorganized cell proliferation.
In callus cultures, cells undergo developmental reprogramming and repeated cell division before regenerating into complete plants.
Research on Tetrastigma hemsleyanum found increasing molecular polymorphism in callus after repeated subculture. Changes in flavonoid content were also reported after prolonged culture.
This is especially important for medicinal plants or crops whose commercial value depends on specific biochemical compounds.
A regenerated plant may look visually normal while its biochemical profile has changed.
For true-to-type commercial propagation, direct multiplication from shoot tips or axillary buds is generally safer than prolonged callus-based regeneration.
There Is No Universal “Maximum Number” of Subcultures
Passage number cannot be judged using the same limit for every plant.
Some banana culture systems have been maintained for extended periods without detectable polymorphism using the RAPD or ISSR markers applied in those studies.
Likewise, blackberry cultures have been subjected to multiple subculture cycles without detectable differences from the mother plant under the methods used.
This means that a rule such as:
“More than five passages will always cause mutation”
is not scientifically correct.
The appropriate maximum passage number depends on:
Species
Cultivar or clone
Regeneration pathway
Hormone concentration
Callus involvement
Culture duration
Culture conditions
The sensitivity of the quality-control methods being used
A passage limit should therefore be established from actual production data for each crop or cultivar.
Passage Number Should Be Part of Commercial Traceability
For commercial laboratories, passage number should be treated as a critical production record.
A practical system may look like:
Mother Stock → P0 → P1 → P2 → P3 → P4...
Each batch should retain information on its origin and culture history.
The laboratory should monitor:
Multiplication rate
Shoot length
Shoot morphology
Hyperhydricity
Rooting percentage
Root quality
Acclimatization survival
Off-type frequency
Growth after transfer
For example, if P1–P5 consistently produce five healthy shoots per explant with normal morphology, but by P7 multiplication falls to three shoots and abnormal plants begin increasing, the later-generation line should not be used indefinitely.
Instead, production should return to a verified Master Culture and establish a new Working Culture line.
Master Culture and Working Culture Should Be Separated
A robust commercial system should maintain a distinction between long-term reference material and active production material.
The Master Culture should be maintained under controlled conditions and used as the source for new production lines.
The Working Culture is the material repeatedly multiplied for commercial production.
This structure reduces the need to maintain one production line indefinitely through increasing passage numbers.
When multiplication performance declines, abnormalities increase, or culture quality becomes inconsistent, the laboratory can restart from the Master Culture rather than attempting to rescue an ageing line with stronger hormones or more frequent transfers.
Repeated Subculture Is a Risk to Manage, Not an Automatic Cause of Mutation
Frequent subculture does not cause a tissue-cultured plant to suddenly become a completely different cultivar.
A ‘Hom Thong’ banana does not become a Namwa banana simply because it has been transferred many times.
The more realistic concern is that a proportion of plants may gradually develop off-type characteristics, genetic differences, epigenetic changes, reduced multiplication capacity, or poorer performance after prolonged culture.
The most appropriate production principle is therefore not:
“Subculture as quickly and as often as possible.”
It is:
“Subculture at the correct developmental stage, control passage number, monitor culture performance, and restart production from verified Master Culture when the working line begins to decline.”
One banana cultivar may perform best when production is limited to five to seven passages, while another ornamental crop may remain stable for considerably longer.
There is no single passage limit suitable for every plant. The correct limit must be established from cultivar-specific evidence and actual production performance.
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