Protocols for Micropropagation of Woody Trees and Fruits

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4.2. Field Evaluation
V.M. KULKARNI ET AL.
In the first field trial, in principle, each greenhouse hardened plant coming from
mutagenesis experiment is treated as separate genotype. Thus, a replicated field
evaluation experiment is not possible at this stage, and the data are scored on each and
every plant from mutagenized populations. Non-irradiated plants (derived from shoottip
multiples/ECS) of same age are included as standard control.
The shoot-tips from preferably all of the side-suckers from the “selected variant/s”
are appropriately labelled and cultured in vitro for maintenance and multiplication. The
regeneration of plantlets and greenhouse hardening of these populations can be done by
following the above discussed procedures.
In the second and subsequent evaluations, the populations of individual suckers
are maintained separately to obtain sufficient number of plants for subsequent field
evaluation/s. At this stage, a field trial can be laid down either for individual plant
selection (like first field trial) or with appropriate number of replications. The stability
of the selected variants can be confirmed by conducting multilocational trials over
successive generations.
5. NOTES
• Sword suckers and flower buds are collected from a disease-free area and a
reliable source. The materials certified/indexed as disease-free are ideal for
initiating in vitro cultures.
• It is known that the in vitro response of different banana accessions differs
considerably (Kulkarni et al., 2004b) resulting in the genotypic interference. In
general, the genotypes with AAA genome respond better for proliferation as
compared to the ones with B genome.
• As a thumb rule, the subculturing of the shoot-tip multiples shall not exceed the
proliferation beyond 1→1000.
• Delayed subculturing of shoot-tip multiples results in cultures containing single
or unusually elongated shoots (apical dominance) and with lower than normal
proliferation rates. Such cultures are not ideal for mutagenesis because this
would reduce the chances of chimera dissociation.
• The apical dominance can be minimized by timely subculturing the shoot-tip
multiples.
• The embryogenic calli and proembryos can be maintained on BM2G medium by
regular sulbculturing once in 4 weeks. These also can be used for mutagenesis
in vitro, however, owing to their lower regeneration potential (than embryogenic cell
cultures) and also the associated problem of chimera formation, their use for
mutagenesis is not recommended. However, these can be tried if one fails to
obtain embryogenic cell suspensions.
• Subculture of the cell suspensions is done generally by taking 10% of the cell
mass replenished with fresh BM2L medium. Periodic checking for the embryogenic
status (very high proportion of embryogenic cells/clumps that are endowed