Micropropagation of Ajuga reptans 'Burgundy Glow'

R. Daniel Lineberger and Audrey Wanstreet


ABSTRACT
Excised shoot tips of Ajuga reptans L. 'Burgundy Glow' proliferate rapidly in vitro on a Murashige-Skoog medium containing either 1.0 mg/liter benzyladenine (BA) or 0.1 mg/liter naphthaleneacetic acid (NAA) and 2.5 mg/liter BA. Shoot proliferation was more rapid on the medium containing 0.1 mg/liter NAA and 2.5 mg/liter BA. Multiple shoots arise from axillary buds, leaf, and root tissues. Cultured shoots vary considerably in size and relative stage of development, with some producing adventitious roots without transferal to a separate rooting medium. Multiplication and subsequent growth of cultured shoots is rapid with as many as 30 salable plants produced per explant in 20 weeks.

INTRODUCTION
Ajuga reptans L. 'Burgundy Glow' is an ornamental ground cover typified by brightly colored foliage which is an irregularly mottled combination of white, green, and pink. The plant is stoloniferous, with the plantlets produced remaining true to type.

Ajuga reptansL. 'Burgundy Glow' was introduced into tissue culture to assess the feasibility of in vitro propagation as an alternative to the maintenance of the large stock plantings which are required for traditional methods of propagation. In addition, this article describes the differentiation of plants from non-meristematic tissue and evaluates the time required to produce salable plants from plantlets established from tissue culture.

MATERIALS AND METHODS
Rapidly growing shoot tips from greenhouse grown plants of Ajuga reptans L. 'Burgundy Glow' were stripped of all leaves larger than 1 cm, washed 10 min in 0.2% Alconox, stirred 15 min in 10% Clorox, and rinsed twice in sterilized distilled water. Sterilized shoot tips were trimmed to 1 cm in length and were placed tip up in a modified Murashige-Skoog medium containing either 1.0 mg/liter benzyladenine (BA) or 0.1 mg/liter naphthaleneacetic acid (NAA) and 2.5 mg/liter BA. The medium also contained 100 mg/liter casein hydrolysate and 7 g/liter agar (pH 5.7) (1).

Cultures were initiated in 25 x 150 mm test tubes and were subsequently transferred to 125, then to 500 ml glass jars as shoot proliferation increased. Cultures were held at 26¡C, and photosynthetically active radiation at 40 micro Einsteins/Msq/sec-was provided by Grolux fluorescent lamps for 16 hr per day.

Following 15 weeks in culture, plantlets were transplanted into Redi-Earth, held for 5 days under intermittent mist with the light intensity at 360µ Einsteins M-2 sec-1, transferred to a greenhouse bench under shade (270 micro Einsteins/Msq/sec) for an additional 5 days, and finally grown in a greenhouse under standard cultural practices (600 micro Einsteins/Msq/sec).

RESULTS AND DISCUSSION
Multiplication of Ajuga reptans L. 'Burgundy Glow' occurred in culture along three separate paths. Initial proliferation resulted from axillary bud growth. Shoots also differentiated directly from leaf tissue where the leaves contacted the medium (Fig. 1).

Figure 1. Shoot differentiation from Ajuga leaf tissue cultured in vitro.

When detached leaves were placed in culture, however, roots first formed at the cut surface of the petiole, and plants subsequently developed directly on this root tissue (Fig. 2).

Figure 2. Close up view of plantlets differentiating from Ajuga root tissue. Note that they appear to arise from cortical tissue.

Upon closer inspection, plants were seen to have originated from the internal tissues within the root and presumably not from the epidermal tissues (Fig. 2).

To quantify the rate of multiplication which occurred in culture, whole cultures were harvested at one time. These cultures contained shoots of a wide range of sized (Fig. 3).

Figure 3. Shoots produced in vitro varied greatly in size, necessitating grading prior to rooting.

Shoots were graded into three groups based on size and degree of adventitious root development. A No. 1 shoot was smallest and did not possess roots at the time of grading, while a No. 3 shoot was actually a small plant with a well developed root system. Larger shoots produced abundant adventitious roots without transferal to a separate root inducing medium, and grew rapidly upon transplanting into a commercial potting mix (Fig. 4)

Figure 4. Large shoots grew more rapidly upon transplanting. Grading prior to transplanting is needed to prevent uneven growth in finished flats.

More shoots were produced at the end of 15 weeks on the medium containing 0.1 mg/liter NAA and 2.5 mg/liter BA than on the medium containing 1.0 mg/liter BA (Table 1).

Table 1.  Comparative Yield of Ajuga Cultures Following 15 Weeks Growth on Murashige 
and Skoog Media Supplemented with 1.0 mg/liter BA or 0.1 mg/liter NAA and 2.5 mg/liter BA.
Percent of Shoots per Grade Medium Shoots per Shoot Tip Explant 1 2 3 1.0 mg/liter BA 10.6 ± 13.9* 28.0 ± 20.3  30 ± 13 40.5 ± 26 0.1 mg/liter NAA 47.6 ± 19.4 70.3 ± 11.4 26.5 ± 9.8 3.1 ± 2.9 +2.5 mg/liter BA *Mean ± standard deviation of shoots per original shoot tip explant. Value
for 1.0 mg/liter BA is the mean of 13 cultures and the mean for 0.1 mg/liter NAA
+ 2.5 mg/liter BA is the average of 8 cultures.
 Percentage of total shoots (± standard deviation) which were assigned to the
stated grade (see text for explanation of the grading system).

The considerable variation noted in the response of individual shoot tips is reflected in the rather large standard deviations about the means of the two treatments. A large percentage of the shoots produced on the medium containing 0.1 mg/liter NAA and 2.5 mg/liter BA were grade 1 (smallest), while relatively few shoots were of grade 3 size (largest). Distribution of shoot sizes from cultures grown on the medium containing 1.0 mg/liter BA was similar for all three grades.

Transplant survival of the cultured shoots was unaffected by the growth regulator content of the tissue culture medium (Table 2).

Table 2.  Transplant Survival, Occurrence of Altered Phenotypes, and Yield
of Salable Plants from Ajuga Cultures Grown for 15 Weeks on Indicated Media. Percentage of Surviving Plants, Salable Transplant Phenotypic 3 Weeks 5 Weeks Medium Survival Variants Post Transplant Post Transplant 1.0 mg/liter BA 83.1 ± 16.7* 26.3 ± 21.3** 55.4 ± 28.5 85.3 ± 17.6 0.1 mg/liter NAA 82.9 ± 11.4 32.1 ± 33.6 19.7 ± 21.5 63.1 ± 13 +2.5 mg/liter BA *Percentage of shoots per culture vessel (± standard deviation) which were
alive 3 weeks after transplanting.
**Percentage of shoots per culture vessel which were not the 'Burgundy Glow'
phenotype (± standard deviation).

However, plants grown from cultures containing 1.0 mg/liter BA were judged to be of salable size and quality earlier than those grown from cultures containing 0.1 mg/liter NAA and 2.5 mg/liter BA (Table 2). The fact that more of the plants grown from cultures containing the lower concentration of cytokinin were salable at both 3 and 5 weeks is a reflection of the observation that a fewer number of larger plants was produced on the lower cytokinin containing medium (Table 1). In terms of the yield of salable plants, more plants were produced by the 0.1 mg/liter NAA-2.5 mg/liter BA treatment (0.63 x 47.6 = 30) than the `.0 mg/liter BA treatment (0.85 x 19.6 = 16.7).

Not all of the plants produced through tissue culture were the 'Burgundy Glow' phenotype. This observation has been reported previously (2), and we have also noted that the Òpink over greenÓ or ÒbronzeÓ sport is the most frequently observed phenotypic variation. Entirely pink plants were also produced in these cultures, but these did not survive transplanting since they lack the capacity for autotrophic growth. Results of this study do not agree with the conclusion of Zilis et al. that lowering the cytokinin concentration in the medium reduced the occurrence of chimeral separation. The percentage of phenotypic variants was very similar in both media tested (Table 2). Since shoots are produced from adventitious meristems from root and leaf tissue as well as lateral buds, only conditions which do not allow adventitious shoot proliferation would give cultures with all true to type plants.

Ajuga reptans 'Burgundy Glow' undergoes rapid proliferation in tissue culture, producing shoots from lateral bud, leaf, and root tissue. Adventitious roots form in vitro on larger shoots without transferal to a separate root inducing medium, and small shoots root successfully following transplanting to a soilless medium under mist. Plants produced through tissue culture grow vigorously in the greenhouse to salable size in as little as 3 weeks. Even though chimeral variation does occur, 68% to 74% of the plants produced are the 'Burgundy Glow' phenotype, and the remainder of the plants could be marketed as a ÒbronzeÓ selection.

LITERATURE CITED
1. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497.

2. Zilis, M., D. Zwagerman, D. Lamberts and L. Kurtz. 1979. Commercial propagation of herbaceous perennials by tissue culture. Proc. Int. Plant Prop. Soc. 29:404-413.

This research was supported in part by a grant from Cuzz-Acres Nursery, Orange, Conn.