Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CASHEW
axillary buds formed may vary from 1 to 13 with an average of 4–5 buds/culture
in majority (80–100%) of the cultures. Usually 1 or 2 subcultures on expression
medium are required for emergence of axillary buds. Prolonged culture on TDZ
medium beyond 3–4 weeks turns shoots dark and necrotic.
Among all the cytokinins tried TDZ was more potent for shoot bud proliferation
in cashew. However, cotyledonary nodal explants from in vitro source also showed
multiple shoot induction on full-strength MS medium containing 2.2 mg/l BAP, 0.2
mg/l IBA and 1 g/l PVP 360. Initial number of the induced axillary buds varied
between 4 and 8 per culture (Figure 1C) and this increased to many fold (40–60
buds/explant) when the multiple shoot buds were divided and subcultured (3–4
times) on the same medium at 3 week intervals. The elongation of 2 to 3 shoots was
also observed. The long shoots observed in the clumps are harvested and used for
rooting.
2.6. Shoot Bud Elongation
Axillary buds induced on the multiplication medium containing TDZ are to be
cultured on either half-strength semi-solid MS hormone-free medium supplemented
with 0.2% AC and 400 mg/l l-glutamine or on hormone free Raj Bhansali (1990)
semi-solid medium supplemented with 0.2% AC, 500 mg/l glutamine and 500 mg/l
casein hydrolysate for elongation. Multiple shoots with proliferating buds are divided
in 2 to 3 separate clusters and cultured at an interval of 2 to 3 weeks. At each stage
long shoots are harvested and rest of buds were put back on same medium for elongation.
By the end of 8 to 10 weeks the shoot elongation is observed to a frequency of
70–80% (Figure 1D). The shoot buds in cotyledonary nodes elongated faster than
those ones of other explants.
2.7. Root Induction
Long microshoots (>2 cm) obtained are rooted following in vitro method of
culturing on half-strength semi-solid MS medium supplemented with 1 g/l PVP 360,
3% sucrose along with either 5 mg/l of NAA or 2.5 mg/l each of NAA and IBA in
combination. The cultures are incubated in light and with the appearance of root
initials; they are transferred to hormone free liquid half-MS medium containing 1 g/l
AC on a filter paper bridge. Here initiation of rooting is seen as early as 10 days and
up to 40 days. Although the number of roots formed vary (1–6/culture) the majority
has one to two prominent roots growing up to a length of 12–15 cm. The percentage
of rooting varies from 50–80%. Reduction of salt concentration, sucrose and use of
liquid medium are found better for rooting. Shoots harvested from cotyledonary
nodes showed higher percentage of rooting.
Microshoots are also rooted (60%) by pulsing shoots for few seconds either in
10 mM NAA or 10 mM IBA and culturing on hormone free semi-solid half-MS
medium containing 0.2% AC. Ex vitro rooting (12–15%) is also observed when
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