Protocols for Micropropagation of Woody Trees and Fruits

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B. PINTOS ET AL.
Culture density was adjusted to 1–5 × 10 5 microspores/ml by adding medium B
to the pellet obtained after the final centrifugation step. A volume of 1.5 ml was
dispensed into each 35 × 10 mm sterile Petri dishes, leaving about 500 µl to
determine microspores viability and concentration. Seven Petri dishes were used for
each stress treatment.
Table 1. Formulation of culture medium used for the isolation of microspores from anthers of
olive. The culture medium is based on B medium (Kyo & Harada, 1986).
Components
Chemical formula Stock
(g/L)
Macro nutrients, 10 × stock, use 100 ml per L medium
Medium
(mg/L)
Potassium chloride KCl 1.49 149
Calcium chloride-2H2O CaCl2·2H2O 0.146 14.6
Magnesium sulfate-7H2O MgSO4·7H2O 0.25 25
Potassium bi-phosphate KH2PO4· 0.14 140
Other additives
Mannitol 55000
2.1.9. Heat Shock Stress Treatments of the Microspores
Chilling has been used in microspore culture of citrics, apple and cork oak. Low
temperature may trigger the stimulus that changes microspore development from the
gametophytic to the sporophytic pathway, although this mechanism is not well
understood.
High temperatures (32°C or higher) in combination with sucrose starvation
increase the production of embryos. Some species require a combination of heat,
chilling, sucrose starvation and even chemicals to increase the efficacy of embryo
production (Touraev et al., 1997). In this work with olive, seven Petri dishes for
each stress treatment, were placed in heat or cold stress conditions: at 33°C in the
dark for 15 h, at 33°C in the dark for 24 h, at 33°C in the dark for 72 h or at 3°C in
the dark for 96 h. Each treatment was repeated three times. The optimal treatment
for microspore embryogenesis induction was 3°C for 96 h.
The cold shock at 3ºC in darkness for 96 h caused “swelling” of about 16% of
the microspores after 1 day of the culture. Microspore diameter increased by 100–
150% over the original size. “Swollen” microspores continued embryogenic development
(Figure 3 A,B). Heat shock treatments produced “swollen” microspores but at
lower frequency (0.3%). Some reports suggest that low temperatures slow microspore
metabolism and arrest cell cycle in preparation for the resumption of mitosis
once microspore are cultured at normal temperatures (25–28°C) (Zheng, 2003). In
the present study, both low and high temperature pre-treatments induced a switch
from the normal development pathway of microspores to pollen embryogenesis. The
first manifestation of this change is swelling of microspores and cytoplasm structural
reorganization, which were more evident after cold treatment.