Ingrid M. Fordham

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 μM was most effective and it was equivalent to, or better than, 22 μM BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 μM) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO3:NH4 ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 μM TDZ and 1.1 or 5.4 μM NAA, then moving the cultures to 16 h daylight at a photon flux of 60 μmol s-1m-2.

The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of ‘York’ and ‘Vermont Spur Delicious’ apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K+, Mg++ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K+, 1.5 and 3.0 mM Mg++, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K+ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of ‘York’ were obtained on Bacto-agar, while similar but less noticeable effects were obtained with ‘Vermont Spur Delicious’. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.

Use of starch-gelled medium for tissue culture of some fruit crops

Six cultivars of apple and two of red raspberry consistently produced equal or significantly better shoot proliferation on modified Murashige and Skoog medium gelled with a mixture of corn starch and Gelrite than on the same medium gelled with agar. Two pear cultivars grown on starch-Gelrite medium produced hyperhydric shoots and almost no growth, but the addition of a polysaccharide hydric control (‘antivitrifying’) agent to the medium eliminated hyperhydricity. The resulting shoot proliferation equaled or exceeded that on the agar-gelled medium. The starch-Gelrite mixture is easy to prepare and gelling agent costs are only 10–15% of agar, or less if starch is purchased in bulk. Although the opaque gray-white medium makes it more difficult to detect internal contaminants, external contaminants are easily discerned.