Proc. Am. Soc. Hort. Science 57: 401-405 (June 1951)
Embryo Culture of Weeping Crabapple
Louis G. Nickell

1 Received for publication August 15 , 1950.

It occasionally happens that work on a plant of horticultural interest is delayed by slowness of seed germination and, less rarely, slow subsequent growth of the seedling. One well-established way of circumventing this difficulty is by direct culture of the excised embryo (9-11, 13, 14). This situation was encountered with a weeping form of crabapple growing in the Brooklyn Botanic Garden. To test the progeny of this weeping crabapple by ordinary means, i.e., waiting for seeds to germinate even under the most favorable conditions, it would have required probably three or four years to determine whether any of the offspring were characterized by the weeping habit. For this reason, embryo culture techniques were employed with the weeping crabapple in an attempt to shorten the period between the ripening of the seeds and the growth of the seedlings to the stage which would show the weeping character, if present.

The material presented in this study represents the results obtained to date with crabapple embryo culture and emphasizes the value of these techniques in the shortening of the time involved.

METHODS AND MATERIALS

Ripened fruits of crabapple were picked from the tree in late October, 1949. In a transfer room, the exterior of the fruits was washed with 70 per cent ethyl alcohol. After evaporation of the alcohol, the fruits were broken in half, exposing the uncontaminated seeds. These were lifted out with sterile forceps, put into a sterile Petri dish, and the seed coats removed. This exposed the embryo which was immediately transferred to a test tube containing a one per cent agar culture medium with the following nutrient materials added: KNO3, 0.002 M; Ca(NO3)2, 0.003 M; KH2PO4, 0.001 M; MgSO4, 0.001 M; CaCl2, 0.003 M; KCl, 0.002 M; MgCl2, 0.001 M; sucrose, 2 percent; thiamin, 100 µgm./liter; pyridoxin, 800 µgm./liter; niacinamide, 800 µgm./liter; and specially added trace elements (in ppm) — B, 0.1; Mn, 0.1; Zn, 0.3; Cu, 0.1; and Fe, 0.5. The pH of this medium is about 4.6 and does not need adjusting. The medium was being used in other experiments with embryo culture and so was convenient to use for these experiments. Since it was found to support satisfactory growth, no attempt was made to study the nutritional aspects of the problem.

By the use of careful bacteriological precautions, it was possible to excise and transfer over 100 embryos in this manner without any contamination. The test tubes containing the embryos were cultured at 25 degrees C and under 500 foot candles of constant fluorescent light of the daylight type.

RESULTS

Regular Development:— Within 24-48 hours after planting the excised embryos on the nutrient medium, germination had begun. Growth of the seedlings was extremely rapid as shown by the diagrams in Fig. 1. After 4 weeks, the seedlings possessed 4-6 leaves and were of sufficient size and vigor to transplant to soil. After transfer to 2 inch pots filled with a sand-loam mixture, the seedlings were placed in an experimental greenhouse with a constant temperature of 25 degrees C and with supplementary fluorescent light during the night. Growth of the seedlings continued, the stems hardened and thickened, and the leaves became dark green. Three months after excision and planting of the embryos on agar, the seedlings had developed a crown of 12-16 leaves (Fig. 2). At this state, growth ceased. However, after a period of 3-4 weeks, new growth took place, and the temporarily dormant shoot again grew rapidly. The size of the seedlings at 5 months is shown in Fig. 3, that at 8 1/2 months in Fig. 4. Control seeds planted in soil started germination after 9 months. At the present time, 5 per cent of these control seeds have germinated, whereas 100 per cent of the excised embryos germinated. (Subsequent embryo culture work, to be reported in detail in a later publication, done in 1950 from June to November, showed that seeds on this tree are after-ripened by mid-October. The control seed and excised embryos used in the present report were, therefore, after-ripened.)

 
FIG. 1. Drawing showing the weeping crabapple fruit, cross-section of the fruit, seed, embryo, and stages in the development of the embryo in sterile culture. When seedlings have reached the stage at the extreme right, they are transplanted to soil in 2 inch pots. (Drawing by M. L. Carroll).

Effect of Thiourea:— When the embryos were first excised, it was not known if they would germinate immediately since the seeds of many rosaceous plants are dormant at maturity (1-8, 15, 16). Therefore, one group of excised embryos was transferred to tubes containing the basal medium plus 0.25 per cent thiourea (12, 16). At this concentration of thiourea, there was some inhibition of germination; of the embryos which germinated in the presence of the compound, the subsequent growth was markedly inhibited as compared with that of seedlings on nutrient agar alone. This type of experiment was not continued since the excised embryos gave 100 per cent germination without any treatment.

 
FIG. 2. Seedlings of weeping crabapple 3 months after excision of embryos. Plants in 2 inch pots. (Photo by Louis Buhle).

Effect of Cutting off the Cotyledons:— Groups of 10-15 embryos which had varying amounts of the cotyledons removed were planted in the basal medium. These groups were: (1) 1/4,  (2) 1/2, (3) 3/4, (4) 7/8, and (5) all of each cotyledon removed. It was found that the removal of part or all of the cotyledons had no effect on the subsequent germination and growth of the seedlings, provided, of course, the stem and root-growing points were not injured. Thus, in the presence of the organic and inorganic compounds in the culture medium, the stored materials in the cotyledons are of no consequence.

Role of Seed Coat in Germination:— Seeds with intact seed coats were transferred to sterile nutrient agar. After one year, none of these has germinated. Another group of seeds with a small incision made through the seed coats was planted in the same manner. After a few days, these had expanded somewhat and that part of the cotyledon which was exposed to the light had turned green. However, the embryo did not break open the very rigid seed coat. Later, when these embryos were removed from their seed coats and examined, it was found that the root had grown in most cases to a length of 30-40 mm. This thin but lengthy root was coiled inside the seed coat. A third group of seeds with a large incision made in the seed coats was planted in the same manner. Those which were deep enougn in the agar for water penetration expanded and pushed out of the seed coats. After the root had entered the nutrient agar, growth was normal.

 
FIG. 3. Weeping crabapple seedling 5 months after start of embryo culture. Plant in 2 inch pot. (Photo by Louis Buhle).
 
FIG. 4. Same seedling shown in Fig. 3, 8 1/2 months after start of embryo culture. Plant in 4 inch pot. (Photo by Louis Buhle).

DISCUSSION AND SUMMARY

The technique of embryo culture was employed in an attempt to shorten the time required for weeping crabapple seeds to germinate and reach suffiecient size to show whether the weeping habit is passed on to the offspring. To date none of the progeny have shown this character. Germination was apparent within 24-48 hours after planting the excised embryos on a synthetic medium in test tubes. Within 3-4 weeks sufficient growth had taken place for the seedlings to be transplanted to soil. By the end of 5 months, the seedlings had gone through two separate peeriods of growth and were 6-7 inches in height. After 8 1/2 months the seedlings were over 3 1/2 feet in height. Seeds planted in soil as controls started germination after 9 months.

Apparently, in the case of this particular crabapple, the seed coat plays a most important role in the delay of germination in after-ripened seeds. If the seed coats are removed and sufficient food material is present and environmental conditions are not adverse, germination takes place immediately and growth is rapid.

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