Botanical Gazette 99(3): 630-665 (1938)
GROWTH PATTERNS OF PLANTS DEVELOPED FROM
IMMATURE EMBRYOS IN ARTIFICIAL CULTURE1

H. B. TUKEY
(WITH NINETEEN FIGURES)

Introduction

1Journal Paper no. 254 of the New York State Agricultural Experiment. Station. The writer is indebted to the National Research Council for a grant to carry on this work.

In the course of culturing immature embryos of deciduous fruits excised from the fruit at various stages in development (23), a definite and characteristic type of growth or growth pattern has been observed for the plants. Instead of completing the usual embryonic development from the zygote to the mature seed, as occurs on the parent plant, they have developed in culture into plantlets which exhibit a definite conformation or growth pattern apparently related to the age of each embryo when excised.

Although there are no direct references in the literature to growth patterns, as such, from immature embryos in artificial culture, most workers have called attention to some phase or another of unusual growths. HANNIG (9) experienced difficulty with very young embryos of Cochlearia and Raphanus and reported several anomalous growth forms. Other workers have had similar experience with very small or very young embryos, including STINGL (17) with embryos of Secale, Triticum, Hordeum, and Avena; DIETRICH (3) with Ipomea and Althea; WHITE (27) with Portulaca; TUKEY (18, 23) with Prunus and Pyrus; and LA RUE (11) with Lactuca, Taraxacum, Chrysanthemum, and Zea. In all these instances immature embryos began to grow immediately without first going through the usual embryonic stages.

On the other hand LAIBACH (12), working with partially developed embryos of Linum, found that they did not germinate immediately when excised; but if they were placed in vials on cotton wadding watered by a 10-15 per cent solution of cane sugar, they developed to normal mature embryos which then germinated. Likewise KNUDSON (10) grew orchid embryos unremoved from the seed in culture and eventually these germinated and developed into normal plants. WERKMEISTER (26) found that if poorly developed seeds of Iris were placed on nutrient 10 per cent agar agar with Pfeffer's solution and 0.5 per cent glucose, they gradually imbibed water and developed so that later they could be germinated. All such plants eventually developed normally.

Mature or nearly mature seed has also produced unusual types of growth, but of a different nature from those from very young embryos. FLEMION (4) found that mature but non-after-ripened seeds of Rhodotypos kerrioides developed into plants with a dwarfish appearance, and with short, stocky hypocotyls and internodes, and small, dark green leaves. She later (5) reported a similar type of growth for non-after-ripened embryos of peach, apple, and hawthorn. DAVIDSON (1), in culturing immature peach embryos, described all plants raised in culture as abnormal and dwarfish, having small, wrinkled, and peculiarly curled leaves. VON VEH (25) found that seedlings of apple, pear, quince, plum, and cherry raised from non-after-ripened embryos developed into dwarf plants. LAMMERTS (13) secured similar results with apricot, peach, cherry, rose, and camellia; and GERSHOY (6) with the violet.

Material

Twelve varieties of sweet cherry (Prunus avium L.), five of sour cherry (P. cerasus L.), three of European plum (P. domestica L.), two of American plum (P. americana Marshall), thirty-two of peach (P. persica Batsch.), one of apricot (P. armeniaca L.), five of apple (Malus domestica Borkh.), and four of pear (P. communis L. and P. communis x P. serotina) have been used during five growing seasons, 1932 to 1936 inclusive.

2Supplied through the courtesy of Dr. W. S. REED.
3Supplied through the courtesy of Dr. H. J. HARROLD.

Most of the material was from the varietal orchards of the Experiment Station at Geneva, New York, at which the work was carried on. Fruits for dissection were gathered fresh as needed. For comparative purposes fruits were also secured from Youngstown in Niagara County, New York,2 and from Athens, Georgia.3 Shipments from Youngstown arrived in 18 hours in good condition for dissection. Shipments from Athens, Georgia, consisted of fruiting branches carefully packed in damp moss wrapped in wax paper, the cut ends of the branches being set in a damp sponge of moss. Packed in this way, twenty shipments of five varieties of peaches (covering a range of 49 days in season of fruit ripening), made at weekly intervals from full bloom to fruit ripening, were received in fresh condition within 36 hours of gathering. Material from all three sources was equally viable.

Embryos were dissected at intervals during the growing season, from April to October, covering the stage from zygote to seed maturity. In all, more than 20,000 individual cultures have been made.

Methods

Two procedures of culture have been employed: (A) using a disinfectant; (B) using aseptic conditions. The method involving a disinfectant (18) consisted in gathering the fruit, washing it, and opening it in the laboratory under approximately sterile conditions. The embryos were then submerged for minutes in calcium hypochlorite solution prepared according to WILSON'S formula (31). It contains almost exactly 20,000 p.p.m. of chlorine and has proved most satisfactory. Washing with sterile water following the treatment has proved of no advantage and increases the chances for contamination.

When desirable to avoid the presence of disinfectant in contact with the material or the media, fruits were washed in a 0.1 per cent solution of mercuric chloride, and all operations, including transfer, were done in a transfer room. In some instances, with very young embryos 0.16 mm. in length, dissections were made in broth of the same concentration as the nutrient media upon which the cultures were subsequently grown. That is, in making up the media, several test tubes of solution were set aside and sterilized in which to dissect material later to be cultured in the same media. Such cultures were made on culture micro slides, in hanging drops, and in yeast culture chambers on micro slides, in addition to the larger containers described in following paragraphs. With careful attention to technique, complete freedom from contamination may be secured without recourse to disinfecting agents.

In transferring the embryos from the calcium hypochlorite solution, regular bacteriological technique proved most satisfactory. The small amount of calcium hypochlorite solution introduced with the transferred material had no apparent deleterious effect, while the film of solution aids in preventing contamination during transfer.

For culture chambers, square screw-cap bottles with aluminum metal caps proved superior to other containers tried, such as petri dishes, covered watch glasses, and test tubes. When cultures are maintained for 8 to 24 months, there is less likelihood for the media to dry than with cotton plugged test tubes, and less opportunity for contamination as in the growing of an organism through a cotton plug. Tests with and without caps screwed tight have shown no disadvantage from tight closure, and have the advantage of less contamination over a period of months. For large embryos, as the peach, 1 ounce bottles are good; for smaller embryos, such as the cherry, pear, and apple, the ounce size is adequate.

For a considerable portion of the studies a culture solution has been used which consists of 0.6 per cent agar, 0.5 per cent glucose, and 0.15 per cent of the following salt mixture, designated T:

SALT MIXTURE T

 
10 gm. KCl

Salts ground and mixed, and 1.5 gm. added to 6 gm.
of agar and 5 gm. of glucose in 1 liter of water.

2.5 gm. CaSO4
2.5 gm. MgSO4
2.5 gm. Ca3(PO4)2
2.5 gm. FePO4
2 gm. KNO3

Since the medium has a pH of about 5.5 such a low concentration of agar should be either chilled in a refrigerator or otherwise rapidly cooled following autoclaving.

The salt may be kept ground and mixed in a stoppered bottle and used over a period of months as needed. There has seemed neither advantage nor disadvantage in filtering to remove any cloudiness.

In filling the bottles with medium, care has been exercised to place it in the bottom of the bottle by means of a pipette and otherwise to keep the sides and mouth of the bottle free from any medium. Sterilization has been at 15 pounds pressure for 20 minutes.

Unless otherwise indicated, cultures have been grown in daylight in room temperature (ca. 18° C.). Using the technique described, peach embryos have been developed into small plants and maintained in a slowly growing condition in 1 ounce bottles for 17 months.

When plants have developed sufficient root and top growth, they may be shaken from the bottles, together with the agar medium, and planted in sterile quartz sand in inch pots until the plants are sufficiently large to shift to soil in larger pots. Young plants should be carefully shaded and gradually hardened.

AGAR CONCENTRATION.—Tests have been made with agar ranging from 0.5 to 10 per cent. WERKMEISTER (26) has reported good results with Iris seed cultured on 10 per cent agar, but not only has it been difficult to prepare so stiff a gel, but the behavior of similar embryos has been variable. Embryos which develop into plants soon suffer from lack of moisture. Both 1 and 2 per cent concentrations have proved useful, but best results have been secured with 0.6 per cent. At this concentration the agar is sufficiently stiff to support the embryo on the surface, yet availability of water does not prove a limiting factor. Lower concentrations, as 0.5 per cent, have proved difficult to use because of the ease with which they liquefy. LA RUE'S data, using a 0.75 per cent agar, agree closely with these results.

SALT CONCENTRATION.—A wide range of salt mixtures and concentrations have been used with similar results. These include that used by ROBBINS in root cultures and by WHITE in culturing root tips (28, 29); Knop's solution at dilutions used in sand cultures; and formula T, adapted from Crone's nitrogen-free formula.

Although concentrations have been varied from one to ten times a given strength, there has been no appreciable effect so long as the concentrations were not toxic to the plant. These results are in agreement with the previous findings by DIETRICH (3), TUKEY (23), and LA RUE (11), indicating a wide range of tolerance.

PH.—The general purpose formula used in these tests as formula T has a pH of about 5.5. Because the growth of embryos may alter the pH of the media, constant pH cultures were prepared after the formulae of ZINZADZE (32). These ranged from 3.8, 5.2, 6.0, 7.2, 8.2, and 8.6. Embryos grow to small plants on these media, with no deleterious effect other than a slightly chlorotic appearance of leaves of plants grown for 70 days at pH 8.6, and a tendency for the media at 3.8 to liquefy in time owing to acid hydrolysis. The pH of these media remained unchanged throughout the experiment as tested at completion of the growth period.

ORGANIC AND GROWTH-PROMOTING SUBSTANCES.—The addition of various sugars to the media had an appreciable effect upon development, as will be later mentioned; but the chief responses from the addition of organic compounds, from temperatures, and from different photoperiods must be left to another paper.

The following organic compounds and growth-promoting substances have produced no consistent response in the manner and at the concentrations used:

Heteroauxin*
Indoleacetic acid 1 p.p.m.
Indoleacetic acid 1 p.p.m. and glycocoll 100 p.p.m.
Indoleacetic acid 10 p.p.m.
Indoleacetic acid 0.1 p.p.m.
Indolebutyric acid 1 p.p.m.
Indolebutyric acid 1 p.p.m. and glycocoll 700 p.p.m.
Indolepropionic 1 p.p.m.
Indolepropionic 10 p.p.m.
Propionic acid 1 p.p.m.
Propionic acid 10 p.p.m.
Yeast extract 200 PPM.
Yeast extract 10 p.p.m.
Yeast extract 10 p.p.m. and glycocoll 10 p.p.m.
Glycocoll 100 p.p.m.
* Supplied through the courtesy of Dr. F. W. WENT and Dr. K. V. THIMANN.

Growth of normal embryo

The developmental morphology of the embryo, seed, and fruit of the species to be studied has an important bearing upon the culture of excised embryos of deciduous fruits. Using the peach (20) as an example, growth of the carpel is in three stages: stage I, rapid development for 49 to 52 days after full bloom; stage II, retarded development from 5 to 42 days depending upon the variety; and stage III, rapid increase to maturity. During stage I the nucellus and integuments also make rapid increase, reaching maximum size by the time stage II is reached, at which time the stony pericarp begins to form rapidly as sclerenchymatous tissue. The embryo during stage I develops slowly, in common with embryos of most angiosperms, remaining microscopic until the initiation of stage II. At that time it begins a rapid development and reaches maximum size in 12 to 28 days, depending upon the variety, followed by a period of accumulation of storage materials and internal change until maturity.

For other species the growth curves are characteristic. For the sweet cherry, stage I continues to 17 days following full bloom (19), for the sour cherry 21-22 days (21), and for the apricot 42 days (14).

Varieties of peach, cherry, and plum which produce early-ripening fruit fail to develop normally viable seed (19, 20, 21). Embryos of such varieties abort during stage II. The nucellus and integuments which have already reached maximum size collapse upon the partially developed embryo, to give the characteristic shriveled appearance of an abortive seed.

The apple and pear are similar in development but not identical. Growth of the fruit as measured by external measurements is not in the three well defined stages of the fruits of Prunus. The embryo grows similarly, however, in that there is delayed development following full bloom, followed in turn by a period of rapid embryo development. In the apple this rapid development begins 35 to 40 days (15, 16) after full bloom, while for the pear it is about 40 days (15).

Still another characteristic of seed of the deciduous fruits is the high energy content in the form of fat and the after-ripening period necessary for germination. Sweet cherry seed requires 16 weeks under moist conditions at 50° C. to complete after-ripening; peach seed 10 to 12 weeks; apple seed 6 to 8 weeks; and pear seed 4 to 6 weeks. These facts will be discussed more fully in relation to the results secured in culturing embryos at different stages in development.

Results with embryos of peach

The results show a definite and characteristic relation between the stage in development at which an embryo is excised and its subsequent behavior in culture. Embryos of the Elberta peach, received from Athens, Georgia, at weekly intervals in nineteen lots from full bloom to fruit ripening, may be taken as representative, to which other samplings and other classes of fruits will later be referred. For clarity the results with one of several culture media will be considered, namely, that consisting of the nutrient agar given on page 633 with the addition of 10 p.p.m. of dried brewers' yeast. Likewise, unless otherwise noted, procedure A has been used, in which embryos have been treated with calcium hypochlorite solution (2 per cent chlorine) for 5 minutes. The stages in development of pericarp, nucellus and integuments, endosperm, and embryo, the technique used, and the performance of the embryo are given in connection with each sampling.

1. FULL BLOOM: FRUIT IN STAGE I, 3.4 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 0.9 MM.; EMBRYO NOT VISIBLE UNDER DISSECTING BINOCULARS.—The integuments were of waxlike consistency and could be chiseled cleanly from the nucellus by means of dissecting needles sharpened to a knife edge at the points. Embryos were too small to discern, but cultures made of the entire seed dissected aseptically in broth of medium T and cultured in liquid media on micro culture slides increased in length from 0.9 to 1.4 mm. in 9 days, as compared with an increase to 3.0 mm. on the tree.

2. SIX DAYS AFTER FULL BLOOM: FRUIT IN STAGE I, 7.1 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 2.7 MM.; EMBRYO NOT VISIBLE UNDER DISSECTING BINOCULARS.—Observations were similar to those with sample 1. Since embryos were too minute to be studied in culture no records of growth could be secured, but an entire seed removed aseptically was maintained in liquid medium T for 29 days with daily changes. The nucellus and integuments elongated slightly during this period.

3. THIRTEEN DAYS AFTER FULL BLOOM: FRUIT IN STAGE I, 10.0 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 4.2 MM. EMBRYO NOT VISIBLE UNDER DISSECTING BINOCULARS.—The embryo sac could be removed intact, all but for the micropylar region, which remained firmly affixed to the nucellus and from which it could not be removed without rupturing. Embryos were still too minute to discern in culture. Entire seeds removed aseptically increased in liquid media T from 4.2 to 6.4 mm. in length in 3 days, a rate comparable to the increase on the tree. In one instance two seeds were removed from a single fruit, attached to adjacent edges of the carpel. Cultured on the same slide the one increased from 3.5 to 6.2 mm. in length in 3 days, while the other made no increase.

4. TWENTY DAYS AFTER FULL BLOOM: FRUIT IN STAGE I, 19.0 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 9.0 MM.; EMBRYO NOT VISIBLE UNDER DISSECTING BINOCULARS.—Using a disinfectant and culturing on agar media at this stage resulted in no increase in entire seed; and embryos were too small to be discerned.

5. TWENTY-SEVEN DAYS AFTER FULL BLOOM: FRUITS IN STAGE I, 25.8 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 10.5 MM.; CELLULAR ENDOSPERM 0.16 MM.; EMBRYO NOT VISIBLE UNDER DISSECTING BINOCULARS.—Entire seed treated with a disinfectant and placed on nutrient agar failed to increase in size, but upon dissection it was found that cellular endosperm had increased from 0.16 to 1.3 mm. in 13 days. Perhaps there may have been an increase in the size of the embryo as well, since embryo development follows endosperm development very closely (20), but the embryos were still too small to be observed in culture.

6. THIRTY-FOUR DAYS AFTER FULL BLOOM: FRUITS IN STAGE I, 34.0 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 13.6 MM.; EMBRYO SAC EXTENDING NEARLY TO CHALAZA; CELLULAR ENDOSPERM 1.6 MM. IN LENGTH; EMBRYO 0.16 MM.—The nucellus and integuments are pressed firmly against the ovary wall at this stage, and being tender and turgid are easily broken in opening the fruit. The best procedure was to split the fruit in halves along the ventral suture, the seed then being cut from the side of the carpel to which it was attached. The integuments could be stripped easily from the nucellus with needles and forceps, having passed from the softer, fleshier consistency of previous samples and not yet having reached the condition in which the integuments adhere to the nucellus and tear the nucellus with it. Furthermore, the nucellus was firm throughout and not yet digested by the endosperm.

It appears that the embryo is affixed to the micropylar end of the embryo sac, and the embryo sac in turn is affixed to the nucellus. The embryo sac could be lifted from the nucellus but not without rupturing at the micropylar end and exposing the embryo (figs. 1, 2). It was necessary first carefully to chisel away the nucellus at the micropylar end, after which the embryo sac could be drawn out easily from the surrounding tissue. Isolated embryos failed to develop in culture following treatment with calcium hypochlorite solution (2 per cent chlorine), although when cultured within the seed they differentiated and increased in size to 0.275 mm. in length in 10 days and 1.0 mm. in 77 days, as shown in figures 3-5. Treatment with calcium hypochlorite solution seemed harmful to tissues at this stage.

Figs. 1-11.—Results with peach embryos 34 days after full bloom (all drawings with camera lucida): Fig. 1, embryo sac (es) and embryo (e) dissected from immature seed 10.7 mm. long. Fig. 2, enlarged view of embryo and proximal end of embryo sac showing characteristic failure to remove embryo sac from nucellar tissue without rupturing; 1.0 mm. in length. Fig. 3, enlarged view of embryo, undifferentiated; 0.12 mm. in length. Fig. 4, differentiation of embryo in culture 10 days and increase from 0.12 mm. to 0.275 in length. Fig. '' further differentiation of embryo 77 days in culture but abnormal; length 1.0 mm. Fig. 6, embryo cultured on agar within entire seed for 49 days, increasing from 0.32 mm, to 2.7 in length during that period. Figs. 7-10, anomalous growth of embryos in flowing media (7, 8, increases from 1.2 mm. to 3.8 in length and 4.6 mm. in width in 20 days; 9, same embryo after 166 days; 10 increases from 1.2 mm in length to width of 2.6 mm. in 20 days). Fig. 11, 9-day development of embryo with cotyledons removed, excised from seed 61 days after full bloom; 9 mm. in breadth.

7. FORTY-ONE DAYS AFTER FULL BLOOM: FRUIT IN STAGE I, 40.5 MM. IN LENGTH; NUCELLUS AND INTEGUMENTS 17.1 MM.; CELLULAR ENDOSPERM 3.8 MM.; EMBRYO 0.32 MM.; COTYLEDONS DIFFERENTIATING.—Isolated embryos failed to develop in culture, although when entire seeds were cultured the embryo increased to 1.35 mm. in length in 8 days. In one instance the embryo reached 2.7 mm. in 49 days (fig. )' with spreading and elongating cotyledons and general abnormal growth.

8. FORTY-EIGHT DAYS AFTER FULL BLOOM: FRUIT IN TRANSITION FROM STAGE I TO II, 4.5 MM. IN LENGTH; STONY PERICARP BEGINNING TO HARDEN; NUCELLUS AND INTEGUMENTS NEARLY MAXIMUM SIZE, 19.2 MM.; ENDOSPERM 8.0 MM.; EMBRYO ENTERING PERIOD OF RAPID INCREASE, 1.2 MM.; COTYLEDONS DIFFERENTIATING.—Isolated embryos treated with calcium hypochlorite solution failed to develop further in culture. Embryos dissected in broth of the medium and not treated with a disinfectant showed signs of development by a spreading of the cotyledons within 8 days, and increased from 0.9 to 3.8 mm. in length in 21 days, but failed to develop further. No chlorophyll was formed. In flowing media embryos developed into the anomalous forms shown in figures 7-10.

9. FIFTY-FIVE DAYS AFTER FULL BLOOM: FRUIT IN STAGE II, 46 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM 11.5 MM.; EMBRYOS IN PERIOD OF RAPID INCREASE, 2.0 TO 3.0 MM.—First indications of development following treatment with calcium hypochlorite solution were secured at this stage. In 48 hours after placing on agar the cotyledons had spread to 90 degrees with the central axis, while in 120 hours they had become recurved so that they touched at the tips below the hypocotyl. Chlorophyll formed in the dorsal surface tissue of the cotyledons, and the central axis of the epicotyl lengthened to 1 mm., but remained white throughout (fig. 12).

10. SIXTY-ONE DAYS AFTER FULL BLOOM: FRUIT IN STAGE II, 46 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM 16.5 MM.; EMBRYO INCREASING RAPIDLY, 5.4 MM.—Chlorophyll formed slowly, appearing first in the surface tissues of the dorsal side of the upper cotyledons. The lower cotyledon, in direct contact with the medium, then frequently elongated 2 to 4 mm. and became spongy. Later the cotyledons spread at right angles to the central axis, followed by chlorophyll development on the ventral surfaces of the cotyledons. The central axis of the epicotyl lengthened to 1-2 mm., but remained white throughout, and the hypocotyl lengthened to 2-3 mm. In one instance, in which the cotyledons were excised by accident, the remaining portion of the embryo developed in 19 days to the anomalous form shown in figure 11.

Fig. 12.—Development of embryos excised 51 days after full bloom, showing greening and spreading of cotyledons and white epicotyl.

11. SEVENTY-THREE DAYS AFTER FULL BLOOM: FRUIT IN STAGE II, 46 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM 17.8 MM.; EMBRYO INCREASING RAPIDLY, 16.4 MM.—Chlorophyll formed rapidly in the cotyledons, which spread at right angles to the central axis, the hypocotyl elongating 2 to 4 mm. with roots developing 10 to 15 mm. in length, and occasionally to 30 mm. The central axis of the epicotyl lengthened 1 to 2 mm. and became green. It terminated in a rosette of six to ten small white appendages 0.5 to 1 mm. in length, much resembling stipules (fig. 13A). The appearance of the epicotyl was that which might be produced by the failure of the internodes of a central axis to elongate and upon which only the stipules had developed and in which no chlorophyll had formed.

FIG. 13.—Typical growth patterns of peach seedlings developing from embryos excised from fruit at following ages after full bloom: A, 73 days; B, 80 days; C, 87 days; D, 94 days.

12. EIGHTY DAYS AFTER FULL BLOOM: FRUIT IN TRANSITION FROM STAGE II TO III, 48.0 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM NEARLY ALL DIGESTED; EMBRYO NEARLY MAXIMUM SIZE, 18.0 MM.—Chlorophyll formed on both dorsal and ventral surfaces of the cotyledons, which separated to 90 degrees with the central axis. The hypocotyl lengthened to 2-4 mm., frequently developing into roots 10 to 20 mm. in length, or even 30 mm. The central axis of the epicotyl lengthened to 7-15 mm. in 21 days and became green, being surmounted by a rosette of eight to twelve anomalous stipule-like appendages 1 mm. in length, but without chlorophyll development (fig. 13B). Along the stem one or two green stipule-like appendages appeared in which chlorophyll developed. The general appearance was as of an elongation for an internode or two of the axis from the sample taken 73 days after full bloom, but with the internodes of the remainder of the stem still unelongated at the apex to give a rosette of white stipules.

13. EIGHTY-SEVEN DAYS AFTER FULL BLOOM: FRUIT ENTERING STAGE III, 50.0 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM NEARLY COMPLETELY DIGESTED; EMBRYO NEARLY MAXIMUM SIZE, 18.8 MM.—Chlorophyll formed on both dorsal and ventral surfaces of the cotyledons, which spread to 90 degrees with the central axis. Roots developed on all specimens and were 10 to 15 mm. in length in 28 days. The central axis of the epicotyl lengthened to 20-22 mm., green throughout, terminating in a rosette of stipule-like appendages mm. in length in which chlorophyll had not developed. True leaves developed in addition to the stipules, although occasionally such leaves were broader than is typical of peach leaves and often had crinkled margins and whitish areas along the edges. The general appearance was as of a still further elongation of the internodes of the short axis from the preceding two samples, the further development of chlorophyll in the stipule-like appendages and the development of leaves (fig. 13C).

14. NINETY-FOUR DAYS AFTER FULL BLOOM: FRUITS IN STAGE III, 51 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM NEARLY ENTIRELY DIGESTED; EMBRYO MAXIMUM SIZE, 19.8 MM.—Chlorophyll formed on both the dorsal and ventral surfaces of the cotyledons, which separated at right angles with the central axis, but the chlorophyll became less abundant than in preceding stages. Roots developed vigorously to 25-30 mm. in length in 20 days. The central axis of the epicotyl elongated to 40 mm., terminated in a rosette of small, green stipule-like appendages 1 mm. in length. Occasionally one or two stipule-like appendages appeared along the stem. They were sometimes entirely green, sometimes white, and sometimes part green and part white. Occasionally also characteristic peach leaves formed 25 to 35 mm. in length, yet these leaves were often broad and crinkled and had whitish areas along the margins. The general appearance was as of a still further elongation of the internodes of the short stem (fig. 13D).

15. ONE HUNDRED AND THREE DAYS AFTER FULL BLOOM: FRUIT IN STAGE III, 52.0 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM ALMOST ENTIRELY DIGESTED; EMBRYO MAXIMUM SIZE, 19.8 MM.—Only a small amount of chlorophyll was found in the cotyledons, which spread at right angles to the central axis. Roots developed vigorously and stem length reached 40 to 45 mm., terminated in a rosette of some whitish and some green leaflike appendages 1 mm. in length. Leaves formed along the stem but were frequently broad and crinkled with whitish areas along the margins. The nodes were still relatively close together, giving the appearance of a dwarfed plant (fig. 14A).

16. ONE HUNDRED AND EIGHT DAYS AFTER FULL BLOOM: FRUIT IN STAGE III, 59.0 MM. IN LENGTH, COLORING; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM ALMOST ENTIRELY DIGESTED; EMBRYO MAXIMUM SIZE, 19.8 MM.—Roots developed vigorously and stems reached 45 to 50 mm. in length with green peachlike leaves. In 17 days the plants were transplanted from the culture bottles to soil. The leaves were often broader than typical peach leaves and the plants were dwarfish. The cotyledons developed no chlorophyll, but served as storage and nutritive organs, gradually becoming shriveled and dried (fig. I4B).

17. ONE HUNDRED AND EIGHTEEN DAYS AFTER FULL BLOOM: FRUIT IN STAGE III, 60.0 MM. IN LENGTH; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM NEARLY ALL DIGESTED AWAY; EMBRYO MAXIMUM SIZE, 19.8 MM.—No chlorophyll found in the cotyledons at this stage. Vigorous root and shoot development occurred so that plants were transplanted to soil in 14 days. Growth was somewhat dwarfish (fig. 14C).

18. ONE HUNDRED AND TWENTY-TWO DAYS AFTER FULL BLOOM: FRUIT IN STAGE III, 63.0 MM. IN LENGTH; "HARD" RIPE; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; ENDOSPERM NEARLY ALL DIGESTED AWAY; EMBRYO MAXIMUM SIZE, 19.8 MM.—Plants developed with vigorous root and shoot growth, although somewhat dwarfish. They were transplanted to soil in 14. days. No chlorophyll formed in the cotyledons.

Fig. 14.—Typical growth patterns of peach seedlings developing from embryos excised from fruit at following ages after foil bloom: A. 103 days; B, 108 days; C, 118 days. D, future development after placing in subdued light at F. for 30 days.

19. ONE HUNDRED AND TWENTY-NINE DAYS AFTER FULL BLOOM: FRUIT IN STAGE III, 65.0 MM. IN LENGTH, SOFT RIPE; STONY PERICARP HARD; NUCELLUS AND INTEGUMENTS MAXIMUM SIZE, 20.5 MM.; INTEGUMENTS BROWN; ENDOSPERM NEARLY ALL DIGESTED AWAY; EMBRYO MAXIMUM SIZE, 19.8 MM.—Plants developed in 14 days which were only slightly dwarfish, with vigorous root and shoot development. They were transplanted into soil at the end of this time. Chlorophyll failed to develop and the cotyledons became somewhat shriveled, as though material had been utilized from them in the growth of the plant.

FUTURE DEVELOPMENT OF PLANTS

All plants failed to maintain an uninterrupted shoot growth beyond o to 70 mm., as contrasted with plants which develop from after-ripened peach seeds and reach a height of 300 to 400 mm. during the growing season. The cessation of terminal development and failure of the axis to elongate resulted in all cases in plants with at least some degree of dwarfing. Plants from embryos excised at the earliest stages were most dwarfed, and those from later stages least dwarfed. These last appeared similar to those described by FLEMION (5) from non-after-ripened embryos of mature peach seed.

When placed in the greenhouse in soil, the plants remained in this stage of arrested development, the stem became woody and some leaves abscissed as when plants enter a period of dormancy. They were then placed in a nursery cellar which provided subdued light and a temperature of about 45° F. In 30 days the plants were returned to the greenhouse. They immediately resumed rapid growth (fig. 14D), free from any abnormalities, such as characterized the earlier stages of development. Whether a shorter period of time would have accomplished the same results or what other factors might have brought about the same results is not known, since no other treatments were used. Close examination of the new shoot growth showed that it arose not from the terminal bud but from a lateral bud in the axil of one of the leaves near the tip of the shoot, as already described by DAVIDSON (2).

Subsequent behavior of the seedlings was similar to that of seedlings from after-ripened seeds. Planted into the orchard they grew into trees, some of which have borne fruit at 4 years of age. General fruit and tree characters were "normal," the only differences being the variations in individuals to be expected among seedling peaches.

The youngest embryos which were successfully cultured and grown into orchard plants were those excised 73 days after full bloom, and 6 days before the fruit was ripe. Considerable difficulty was experienced with damping-off fungi. Embryos 80 days old from full bloom were raised to orchard plants much more easily but still were difficult to handle because of the fungi. From 94 days to 129 days after full bloom the stand of seedlings was nearly perfect and little difficulty was experienced in growing the plants to orchard trees, some of which have fruited.

Results with other varieties of peaches during different seasons and from different sources

The embryos of the other four varieties of peaches received from Georgia behaved, in general, similarly in culture to the embryos of Elberta. A few points of difference are worth noting. First there was a spread of several days in blooming between the different varieties, in which Greensboro bloomed first and Elberta last. Since the rates of development of the fruit, seed, and embryo are nearly identical for all varieties, it follows that development of Greensboro was several days in advance of Elberta on the same calendar date. For example, 55 days after full bloom, embryos of Greensboro were about one-third maximum size; those of Carman, one-quarter; those of Hiley, one-quarter; those of Belle, one-quarter; and those of Elberta, one-sixth.

The response of the embryo in culture reflected the more advanced development of some varieties over others on the same date. For example, 61 days after full bloom the separation of the cotyledons and the development of chlorophyll was greatest in Greensboro, next greatest in Carman, and least in Elberta. Similarly, 73 days after full bloom, all the embryos of Greensboro and Carman had developed chlorophyll after 10 days in culture, as compared with one-third of the embryos of Hiley, and one-fifth of those of Belle and Elberta. This relationship between varieties continued throughout the season.

Embryos of the early-ripening varieties aborted before they completely filled the integuments. Such embryos, although cultured at intervals for several weeks thereafter, developed only to the degree characteristic of embryos at the time of aborting. That is, embryos of Greensboro which had aborted 61 days after full bloom, and which were cultured 19 days later, developed in culture only to the stage of spreading the cotyledons and forming chlorophyll, as is characteristic of 61-day embryos. Embryos of Elberta, being non-abortive, behaved in culture on this date similar to 80-day embryos, roots frequently forming 5 to 20 mm. in length and the central axis of the epicotyl growing to 7 to 15 mm. in length.

The fact that the samples of peaches received from Georgia were several weeks further developed than those from Geneva, New York, and those from Geneva several days further than those from Youngstown, New York, gave an interesting comparison of embryos of the same variety cultured on the same day but in different stages of development. In all cases the embryos developed characteristic of the stage of development at which they were excised, so that strong shoot and root development appeared from embryos received from Georgia, whereas embryos from peaches from Geneva cultured on the same date developed only short shoot growth, and those from Youngstown developed only to the white rosette stage.

In addition to the varieties mentioned, embryos of the following thirty-one varieties, cultured during the seasons of 1932 to 1936, and secured from both Youngstown and Geneva, New York, representing a wide range of climatic and cultural conditions, seasonal development, and season of fruit ripening, gave substantially the same results: Alexander, Alexander Crosby, Arp, Belle, Blood Leaf, Canada, Carman, Champion, Chili, Crosby, Delicious, Eagle Beak, Early Crawford, Elberta, Foster, Golden Jubilee, Greensboro, Krummel, Lola, Mikado, Morellon, Mountain Rose, Rochester, Rosebud, St. John, Triumph, Troth, Valiant, Veteran, Waddell, and Ward Late.

Results with embryos of cherry, plum, apricot apple, and pear

The results in culturing excised embryos of the sweet cherry, sour cherry, plum, apricot, apple, and pear indicate the same general trend for these classes of fruits as for the peach, the species of Prunus following a little more closely than the apple and pear.

For a given species, the response of the embryos in culture is in accordance with the characteristic curve of growth of the embryo for that species. That is, the embryo of the sweet cherry begins its period of rapid development 17 days after full bloom; the sour cherry 21 days; the peach 49 days; the apricot 42 days; the apple 35-40 days; and the pear 40 days.

Accordingly, embryos of the sweet cherry responded in culture at an earlier calendar date than did embryos of the peach. When excised embryos of the peach had later reached the same stage of development as that of the cherry previously cultured, the growth in culture was similar.

Within a given species, embryos of the various varieties used behaved similarly, with the minor differences to be mentioned in the following paragraphs.

APRICOT

Embryos of the variety of apricot used, Alexander, behaved similarly to those of the peach. They developed vigorously in culture and developed into strong plants.

SWEET AND SOUR CHERRY

Embryos of the cherry responded more slowly in culture than embryos of the peach and the resulting plants were more delicate, less vigorous, and less easily handled. Using the embryo of the Mazzard cherry as the type, embryos excised prior to 27 days after full bloom, which had by that time reached a length of 1.48 mm., failed to develop in culture (fig. 15A). Beginning with embryos 30 days of age from full bloom, when the embryos had reached a length of 2.6 mm. compared with a maximum length of 7.2 mm. at maturity, the first indications of development were secured. The cotyledons of such embryos spread apart but developed no further. Embryos excised 32 days after full bloom, 3.1 mm. in length, developed cotyledons which were thick and green, and two small white recurved leaves (fig. 15B).

Fig. 15.— Growth in culture of sweet cherry embryos excised from fruit at various stages in development, time in days after full bloom: A, 27 days, no development in culture. B, 32 days, cotyledons spreading, thickened, and green, with two small white recurved leaves; C, 34 days, epicotyl terminated in rosette of small, white, stipule-like leaves; D, 36 days, hypocotyl thickened and elongated, roots developing, epicotyledonary axis elongating 2 to 4mm., terminated in rosette of small, white, stipule-like leaves; E, 40 days, roots and small green leaves developing; F, 45 days, epicotyledonary axis elongated, leaves formed; G, 54 days, vigorous plant developments but with broad, crinkled, anomalous leaves and dwarfish habit of growth.

Embryos excised 34 days after lull bloom, which were then 4.42 mm. in length, developed chlorophyll in the cotyledons in culture, the hypocotyl lengthened to 2-4 mm., and the epicotyl developed as a rosette of small, stipule-like leaves in which no chlorophyll formed (fig. 15C). Thirty-six days after full bloom the embryos had reached a length of 5.8 mm. In culture they produced roots 3 to 5 mm. in length, developed chlorophyll in the cotyledons, and produced an epicotyledonary axis 2 to 4 mm. in length, surmounted by, a rosette of stipule-like leaves in which chlorophyll was now developed (fig. 15D). Forty days after full bloom embryos had reached a length of 6.9 mm., which in culture developed roots and small green leaves (fig. 15E). Embryos excised 4 days after full bloom had reached maximum size, and developed in culture into plants with good root development, elongated epicotyledonary axis, and green leaves (fig. 15F).

Embryos excised 54 days after full bloom developed into vigorous plants, but which had broad, crinkled, anomalous leaves quite unlike a typical cherry leaf. Further, the habit of growth was dwarfish. As with the peach, such plants, placed at a temperature of 45° F. for 30 days in subdued light and then returned to the greenhouse, began a new shoot growth which no longer showed any anomalous growth forms or any dwarfish characters  (fig. 15G).

Other varieties of sweet cherry used were Early Purple Guigne, Black Tartarian, Lyons, Seneca, Rockport, Kirtland, Burbank, Oswego, Yellow Spanish, Windsor, and Schmidt.

Sour cherries used were Early Richmond, English Morello, and Brusseler Braune. They behaved similarly to the sweet cherry. The embryo of the sour cherry, however, is less fully developed than that of the sweet cherry on the same date. Accordingly embryos of the sweet cherry in culture were developing into normal plants on the same day that embryos excised from the sour cherry were only beginning to develop chlorophyll in the cotyledons.

As with the peach, the earliest ripening varieties produced abortive embryos. The earliest, Early Purple Guigne, aborted so early that the embryos seldom reached the stage at which they developed in culture. Only occasionally was an embryo of the Early Purple Guigne found sufficiently advanced to develop.

An interesting observation in this connection was made with embryos of varieties ripening in succession and all cultured on the same date, namely, Seneca, Burbank, Knight, and Black Tartarian. The embryos of the earliest ripening variety, Seneca, having aborted at a very early stage, developed only to the degree characteristic of that stage. Embryos of the next ripening variety, Burbank, having aborted at a slightly later stage, developed more fully in culture. Embryos of Knight, which ripens still later, developed still further in culture; and embryos of Black Tartarian, the latest ripening of the group, developed the furthest.

PLUM

Embryos of the varieties of the European plum Oullins, Italian Prune, and Middleburg responded in culture as well as, and similarly to, embryos of the peach. Of these three varieties, Oullins ripens early, Italian Prune in mid-season, and Middleburg late.

Unlike the varieties of peach, which all came into full bloom on the same date, thus giving a similar embryo development for all varieties on a given calendar date, Middleburg blossoms earliest, Oullins next, and Italian Prune last. On the same calendar date, therefore, embryos of Middleburg developed most fully in culture, Oullins next, and Italian Prune least, thus corresponding to the succession in bloom. But, computed on the basis of days following full bloom, embryos of all three developed in culture as characteristic of the interval following full bloom at which they were excised.

No development in culture was observed from embryos excised prior to 61 days from full bloom. Excised at the following intervals after full bloom, the characteristic development of embryos was as follows: 69 days, chlorophyll developed in the cotyledons; 72 days, hypocotyl elongated 2 to 4 mm. and epicotyledonary leaves recurved but remaining white; 78 days, roots developed and a rosette of small whitish stipule-like leaves formed; 83 days, epicotyledonary axis elongated to 2-5 mm. and the rosette of stipule-like leaves becoming green; 86 days, stem elongated to 15 mm.; 90 days, both root and shoot growth vigorous but elongated stem terminating in a rosette of green leaves; 98 days, vigorous plant development.

As in the peach, all plants were dwarfish, and when placed at a temperature of 45° F. in subdued light for 30 days, they began new and normal growth.

Embryos of the American varieties Tecumseh and DeSoto developed in similar stages, but the plants grew more feebly. Tecumseh came into full bloom a few days earlier than DeSoto, so that excised embryos of the former were always a few days in advance of those of the latter and behaved in culture as characteristic of that stage.

APPLE AND PEAR

Embryos of the apple and pear failed to develop as vigorously in culture as did those of the species of Prunus, particularly as those of the peach and apricot. The type of plant into which they developed was characteristic of the age from full bloom at which they were excised, and was similar to those already described for the peach. Shoot development was slender and weak, leaves were small, and roots were slender and lateral roots seldom developed.

The stage of development which embryos of the pear and apple reached on the tree at a given calendar date were sometimes variable, adding to the difficulty of an accurate interpretation of results. Unlike the peach, different varieties of which reach full bloom on nearly the same day, different varieties of apple and pear reach full bloom at successive intervals over a period of a week to 10 days, or sometimes longer. Furthermore, apple flowers are borne in a corymb and those of the pear in a cyme, so that there is a difference in time of bloom of individual flowers in the same cluster. Also, the flowers may be borne terminally, laterally, and on spurs—all on the same tree—and all varying slightly in the time at which they reach full bloom. WHITEHOUSE (30) has shown that fruits which are of the same size in early season develop at the same rate. No doubt tagging of blossoms which bloom on the same day, or hand pollination, would result in more uniform material for culture.

Nevertheless the results were consistent with embryos of the same age. As in the case of the plum, embryos of varieties which reached full bloom earliest were furthest advanced on a given calendar date and responded in culture accordingly. McIntosh, for example, bloomed 6 days earlier than Rome Beauty. Embryos of McIntosh excised on the same day as embryos of Rome Beauty developed into plants more advanced in type. Yet embryos of Rome Beauty excised 6 days later developed into plants of similar type.

APPLE.—Apple varieties used were Red Astrachan, Early Harvest, Maiden Blush, R. I. Greening, McIntosh, and Rome, representing a wide range in season of ripening. Early Harvest embryos were consistently further developed the same number of days after bloom than embryos of the other varieties used. Since the cultivated apple is a combination of several species, this fact may account for the variation; or it may be that embryo development in varieties of apple is not so uniform as in the case of other fruits studied.

The other varieties of apple developed similarly. No development in culture was observed from embryos excised prior to 39 days from full bloom. Excised at the following intervals after full bloom, the characteristic development was as follows: 45 days, spreading of cotyledons and slight development of chlorophyll; 59 days, cotyledons dark green; 62 days, hypocotyl 2 to 4 mm. long, epicotyl developing into rosette of small, white leaves (fig. 16); 69 days, hypocotyl 6 mm. in length, epicotyl developing as rosette of small white and green leaves (fig. 16); 75 days, roots 10 mm. in length; 86 days, cotyledonary axis elongated to 4-6 mm., surmounted by small rosette of small white and green leaves, roots 20 to 25 mm. in length (fig. 16); 92 days, plants developed sufficiently to be grown in the greenhouse; 99 days, roots 25 mm. in length, epicotyledonary axis 8 mm. in length, surmounted by small rosette of green stipule-like leaves; 10 days, roots 25 to 30 mm. in length, stem 18 mm. with one or two slender leaves 6 mm. long; 120 days, normal but weak root, shoot, and leaf development; 134 days, stem 45 mm. in length, normal but weak shoot and root development.

It should be observed that no plants developed into normal seedling growth unless chlorophyll formed in the epicotyl. Chlorophyll development in the cotyledons and the formation of white leaves were not enough to carry the plants further.

PEAR.—Pear varieties used were Tyson, Seckel, Bartlett, and Kieffer, representing a wide range in season of fruit ripening. The first three are pure Pyrus communis L., the last is a hybrid between P. communis and P. serotina.

No development was observed in culture from pear embryos excised prior to 46 days from full bloom. Excised at the following intervals after full bloom, the characteristic development was as follows: 66 days, cotyledons green; 69 days, cotyledons green, epicotyledonary axis 1 to 2 mm. in length, terminated in small rosette of small white and green leaves (fig. 17A, B, C); 75 days, stem slender and 6 to 8 mm. in length, leaves slender and stipule-like, roots 22 mm. in length, plants developed sufficiently to be grown in the greenhouse (fig. 17D, E, F, G); 81 days, stem slender and 18 mm. in length, leaves slender, roots 40 mm. in length; 97 days, stem weak and slender, 25 mm. in length, leaves normal but small and thin, roots 55 mm. in length (fig. 17H, I).

Fig. 16.—Growth in culture of apple embryos excised from fruit at various stages in development: Top row, 62 days after full bloom; cotyledons green, hypocotyl 2 to 4 mm. in length, epicotyl terminating in rosette of small, white leaves. Middle row, 69 days after full bloom; hypocotyl 6 mm. in length; epicotyl developing as rosette of small white and green leaves. Bottom row, 86 days after full bloom; epicotyledonary axis 4 to 6 mm. in length, surmounted by small rosette of small white and green leaves; roots 20 to 25 mm. in length.

 

Fig. 17.—Growth in culture of pear embryos excised from fruit at various stages in development: A, B, C, 69 days after full bloom; cotyledons green, epicotyledonary axis terminated in small rosette of small white and green leaves; D, E, F, G, 75 days after full bloom; stems slender, leaves slender, roots 40 mm. in length. H,I, 97 days after full bloom; stem weak and slender, leaves normal but small and thin, roots 55 mm. in length.

Results without use of disinfecting agent

While the use of a disinfecting agent lends itself well to large scale methods of embryo culture, the question is immediately raised as to what effect such an agent may have upon the embryos. Of several materials used, namely, zonite, bichloride of mercury, and calcium hypochlorite, the last has proved very close to ideal. It is easily prepared according to the formula of WILSON (31) and as used by KNUDSON (10) for cultures of orchid embryos, giving almost exactly 20,000 p.p.m. of chlorine (2 per cent). It is inexpensive and easily handled, and immersion of embryos for 5 minutes has given almost perfect freedom from contamination.

On the other hand, it has been fairly simple to dissect embryos under aseptic conditions and to place them in culture without the use of a disinfecting agent. With the use of a transfer room, contamination has been reduced to 1 or 2 per cent. DAVIDSON (2) has found a transfer case helpful, and good results may he secured in the open laboratory provided proper precautions are taken. Both aseptic and disinfectant methods have been found practical and useful, depending upon the material used and the objectives desired.

Fig. 18.—Effect of glucose upon apple embryos excised at early stages, showing that glucose is essential to chlorophyll formation and that higher concentrations are best for embryo development at early stages: A, no glucose in medium, no development; B, 0.5 per cent glucose, partial development; C, 2 per cent glucose, most development. Cf. fig. 19.

The behavior of embryos placed in culture without treatment with a disinfecting agent differed in only minor points from the general behavior of embryos treated with calcium hypochlorite, as described in preceding paragraphs. Embryos excised at very early stages of development, 48 days after full bloom or earlier, failed to develop following treatment with calcium hypochlorite solution, although when embryos of this age were dissected in broth of the medium and then placed in culture, the cotyledons had spread apart within 8 days and the embryos increased from a length of 0.9 mm. to 3.8 in 21 days. None developed into greenhouse plants.

With older embryos 51 to 94 days from full bloom, treatment with calcium hypochlorite delayed the formation of chlorophyll in the cotyledons 3 to 6 days, particularly on outer surfaces of the cotyledons, which were most exposed to the action of the material. In some instances, when embryo treatment was extended to 10 or 20 minutes, chlorophyll failed entirely to develop on the dorsal surfaces of the cotyledons, although it later formed on the ventral surfaces after the cotyledons had spread apart.

Fig. 19.—Effect of glucose upon cherry embryos excised at late stages, showing that glucose is inhibiting to the development of late stages: above, 2 per cent glucose in medium, poor growth; below, no glucose in medium, good growth. Cf. fig. 18.

With still older mature embryos, 100 days or more from full bloom, there seemed to be no effect upon development from treatment with calcium hypochlorite solution for 5 minutes.

In general calcium hypochlorite solution has been injurious to very young embryos, retarding to older embryos, and non-injurious to still older embryos.

Relation of medium to age of embryo when excised

The composition of the medium has affected variously the development of embryos excised at different stages. In the results that have been listed as the type in the present paper, the standard medium given in earlier paragraphs has been used. Varying several factors in the medium within the limits described, such as the pH and the composition and concentration of mineral nutrients, has had no appreciable effect upon embryo development. But the concentration of glucose has had a decided effect. In the case of early stages of embryo development, the presence of glucose in the medium is essential to chlorophyll development in the cotyledons. Further, although 0.5 per cent concentration of glucose favors development of both chlorophyll and the embryo, 2 per cent produces a still greater response. By the use of this higher concentration of glucose for embryos excised in early stages of development, still earlier stages can be successfully cultured.

By contrast, in late stages of embryo development, as when embryos have reached maturity, the presence of 2 per cent glucose in the medium is inhibiting. By the use of lower concentrations or by the elimination of glucose, still later stages may be successfully cultured.

By the use of 0.5 per cent glucose for the results presented in this paper, the range has been extended from early to late and has tended to give a more complete story than would otherwise have been the case.

Nevertheless the fact remains that by varying the concentration of glucose and by its absence from the medium, development of embryos has been altered. It is not too much to expect that with a more complete understanding of the chemical make-up of embryos at various stages in their development, and of the nutrients required, and with improved technique and methods of manipulation, still further progress may be made in altering or even eliminating some of the characteristic stages of development of embryos excised at various periods after full bloom and bringing them more nearly to what might be called normal plant development.

Discussion

The culturing in artificial media of immature embryos excised from growing plants has practical application to plant breeding and genetics, as the perpetuation of individuals in a given population which might otherwise fail to survive (12, 18). Further, it provides a method for studying the new sporophyte at earlier stages in development than that provided by the mature seed, and serves to focus attention upon the first expression of the new individual in the embryo rather than upon the plant developed from a mature embryo (22).

Throughout these studies, the stage of development of the embryo when excised from the fruit has overshadowed the other factors considered, but size alone is not a satisfactory criterion of whether or not an embryo can be grown successfully in culture. That chemical composition must be considered as well as size is shown by chemical analyses of developing peach embryos by TUKEY and LEE (24). They have shown that the peach embryo reaches nearly maximum size before any appreciable accumulation of fat (ether extract), nitrogen, and sugars begins. With embryos 17.5 mm. in length the content of fat was 0.40 per cent, of nitrogen 0.68 per cent, and of sucrose 1.06 per cent, whereas at maturity the embryos were only 1.5 mm. greater in length; but the content of fat was 30.67 per cent, of nitrogen 2.60 per cent, and of sucrose 2.32 per cent.

It would seem that a better comparison would be the age of the embryo on the basis of time, the differentiation of the embryo on the basis of morphological characters, or the development of the embryo on the basis of chemical composition.

That some of the youngest embryos did not respond to culture methods is not surprising, but that each age was expressed in a characteristic growth pattern is of particular interest. A similarity is at once recognized between the growth patterns presented in this paper and well known "juvenile" and "adult" forms in other plants. In discussing the differences in the formation of organs at different developmental stages, GOEBEL (7) emphasized the fact that all living things are in a condition of constant change, from earliest to latest stage in development. He cited differences in both function and in conformation of plant parts running through all plant phyla.

These facts emphasize that it is not the genetic make-up of an individual alone which is responsible for the expression of a plant in the formation and function of its various parts. As GOLDSCHMIDT (8) has explained, there are "…..two general notions in regard to the causal understanding of individual development…..One is the notion .... that the action of the genes in controlling development is to be understood as working through the control of reactions of definite velocities, properly in tune with each other and thus guaranteeing the same event always to occur at the same time and at the same place ….. The second notion ….. says that two types of differentiation are closely interwoven in the process of development, namely, independent and dependent differentiation. Independent differentiation means that a once started process of differentiation takes place within an organ or part of the embryo, even if completely isolated from the rest; dependent differentiation, however, requires the presence and influence of other parts of the embryo for orderly differentiation."

Of course it may be that the growth patterns described in this paper are merely the response of the plant to an unnatural and unfavorable environment. In a sense they may be considered maladjusted plants. The younger the embryo, the greater the difficulty in culturing and the greater the abnormal behavior. The older the embryo at the time it is excised from the mother plant, the more fully differentiated it has become and the less easily it is upset or thrown out of balance by an unnatural environment. The facts that a liquid medium is favorable at one stage of development and not at another, and that glucose is favorable at one stage and inhibiting at another, suggest that by providing a more suitable environment, by better technique, and by a better understanding of nutritional requirements, some of these growth patterns may be altered to more nearly the normal for the plant. On the other hand, the fact that even mature seeds must be after-ripened before they develop normally, suggests that there is an internal complex as well as an external environment which must be considered.

The failure of embryos to follow the pattern of embryonic development outside the environment of the mother plant raises the question as to what is the nature of the environment which brings about "normal" embryo development on the plant. The shape of the embryo, it has been shown by HANNIG and others, as well as by these studies, is altered by its surrounding tissue. Does this mean that it is the shape of the campylotropous seed which physically causes a bean embryo to develop its curved shape? Or does it mean that some nutritional factors control the whole?

Finally, from the standpoint of developing mature plants from immature embryos, the facts reported in this paper point toward greatest success by maintaining an embryo in its natural environment or as nearly a natural environment as possible, so that it may follow the normal course of embryonic development before being subjected to a less favorable or less natural one.

Summary

  1. Methods and results are given in culturing embryos of twelve varieties of sweet cherry (Prunus avium), five of sour cherry (P. cerasus), three of European plum (P. domestica), two of American plum (P. americana), thirty-two of peach (P. persica), one of apricot (P. armeniaca), five of apple (Malus domestica), and four of pear (P. communis and P. communis x P. serotina), during five growing seasons, 1932 to 1936 inclusive. More than 20,000 individual cultures have been made. Material has been cultured from Georgia and from three locations in New York State.
  2. Embryos in culture do not pass through the embryonic stages characteristic of embryos on the mother plant. Instead they enter at once into an independent development characteristic of the age of the embryo when excised.
  3. The growth pattern for peach embryos treated with a disinfectant and grown on 0.6 per cent agar media with 0.5 per cent glucose and salt mixture T may be summarized as follows: (A) no development earlier than 51 days of age after full bloom; (B) 51 days, spreading and greening of the cotyledons and small white epicotyledonary leaves; (C) 7 days, cotyledons green, hypocotyl 2 to 4 mm. and roots 10 to 15 mm. in length, central axis of epicotyl 1 to 2 mm. in length terminated by rosette of six to ten small white stipule-like appendages; 80 days, roots 10 to 20 mm. in length, central axis of epicotyl 7 to 15 mm. in length surmounted by rosette of eight to twelve anomalous, white, stipule-like appendages; (D) 87 days, vigorous root development, central axis of epicotyl 20 to 22 mm. in length terminated by rosette of green stipule-like appendages; (E) 94 days, vigorous root development, central axis of epicotyl 25 to 30 mm. in length terminated by rosette of small, green stipule-like appendages, occasional peachlike but malformed leaves; (F) 105 days, vigorous root formation, stem 40 to 45 mm. in length terminated by rosette of small, green stipule-like leaves, with peachlike leaves along the stem; (G) 108 days, vigorous root and shoot development but leaves often broad and crinkled and plants dwarfish; (H) 118 days, vigorous root and shoot growth but dwarfish plants.
  4. After 30 days in subdued light at 45° F., dwarfish plants began normal development and showed no further abnormal behavior.
  5. Embryos of sour cherry, sweet cherry, apricot, plum, apple, and pear behaved similarly with minor differences.
  6. Aseptic methods resulted in earlier response than when a disinfecting agent was used, but growth patterns were similar. Very young embryos were injured by a disinfectant.
  7. Growth patterns were modified by altering the medium, especially glucose, in which at early stages of development glucose was beneficial and at later stages inhibiting.
  8. The data are discussed with reference to physiological changes in the embryo itself, juvenile and adult forms of plants, and general problems of morphogenesis.

NEW YORK STATE AGRICULTURAL EXPERIMENT STATION
GENEVA, NEW YORK

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