Protocols

Protocol for producing citrus somaclones via organogenesis

Table 9.10. The mitotic index of different ploidy cells in the same cell population.

Mitotic index (%)

Nucellar seedlings of polyembryonic citrus cultivars grown under aseptic conditions are the most commonly used and most effi cient explant source. Root, epicotyl, hypocotyl and stem internodes are all amenable to adventitious bud induction, but stem intern- odes are most often utilized. Sections approximately 1 cm in length are cultured on DBA3 shoot induction medium (MT basal medium (Murashige and Tucker, 1969) con- taining 13.3 mM 6-benzylaminopurine, 0.045

mM 2, 4-D and 20 ml/l coconut water; Deng et

al., 1992). Two to three 1-month passages on DBA3 may be required to obtain multiple shoots. Shoots are commonly rooted on RMAN rooting medium (half-strength MT basal medium containing 0.11 mM naphtha- lene acetic acid (NAA) and 0.5 g/l activated charcoal; Grosser and Gmitter, 1990). Rooted plantlets are acclimated under low-light humid conditions prior to greenhouse cul- ture. This technique can also be utilized to regenerate plants from enlarged, malformed somatic embryos that fail to germinate.

Protocol for producing citrus somaclones via somatic embryogenesis

Embryogenic callus from many polyembry- onic citrus genotypes (including sweet oranges, mandarins and lemons) can be ini-

Fig. 9.3. The cytogenetic mechanisms and control of chromosome variations in the embryogenic callus of Newhall navel orange. (1) Chromosome lagging at anaphase (1060´). The arrow points to a lagging chromosome. (2) Lagged chromosomes formed a small nuclear body beside the normal nuclei (850´). The arrow points to a mininucleus adjacent to the normal nucleus. (3) Meiosis-like division (920´). (4) Asymmetrical distribution of duplicated chromatins to one protoplast at telophase (520´). (5) Fluorescent observation of a protoplast stained with Hoechst 33258 (520´). The arrow points to the duplicated chromatins distributed to one protoplast. (6) Asymmetrical distribution of duplicated chromatins to one cell at telophase (850´). The arrow points to the duplicated chromatins distributed to one cell. (7) Apoptotic tetraploid cell and normal diploid cell of Newhall navel stained with Hoechst 33258 (320´). (8) Comet electrophoresis of an apoptotic tetraploid cell and a normal diploid cell of Newhall navel (260´). (9) An apoptotic giant protoplast (320´). The arrow points to an apoptotic body budded from the giant protoplast.

(10) An apoptotic giant cell (320´). The arrow points to an apoptotic body budded from the giant cell.

oped ovules (from either developing or mature fruit) on an MT basal medium (Murashige and Tucker, 1969) supple- mented with 5 mg/l kinetin and 0.5 g/l malt extract (V. Sapp, personal communication). Large numbers of ovules should be plated, and 1–3 passages on this medium are gen- erally required to obtain callus induction. Although it is diffi cult and time-consuming to establish friable embryogenic citrus callus cultures, habituated culture lines can be established and maintained indefi nitely on either EME or H+H liquid or solid cul- ture medium (Grosser and Gmitter, 1990). Solid medium cultures require mainte- nance transfers every 4–6 weeks, whereas suspension cultures are generally main- tained on a 2-week cycle.

Citrus somatic embryos are routinely matured on 1500 medium (MT basal medium supplemented with 1.5 g/l malt extract; Grosser and Gmitter, 1990). However, abnormal embryo development occurs frequently. Niedz et al. (2002) rec- ommend the use of cellulose acetate semi- permeable membranes over semi-solid medium to affect the normalizing of citrus embryogenesis positively, thereby improv- ing the effi ciency of plant recovery.

Mature citrus embryos are routinely germinated on MT basal medium supple- mented with 1–3 g/l gibberellic acid (Grosser and Gmitter, 1990). Germinated embryos generally exhibit long, thin and often spindly roots that are inferior for acclimatization. Re-rooting of shoots dis- sected from germinated embryos on RMAN rooting medium (half-strengh MT basal medium containing 0.02 mg/l NAA and 0.5 g/l neutralized activated charcoal) improves acclimatization efficiency (Grosser and Gmitter, 1990).

Somaclones can also be regenerated via somatic embryogenesis from protoplasts (protoclones) isolated from solid or liquid embryogenic citrus cultures. Protoplast- derived populations appear to have the most useful variation. Protocols for produc- ing protoclones and protoplast-derived cybrids can be found in Chapter 10.