Chapter L: G. COX, Cornell University

Methods of collecting and storing pollen are of great interest to those engaged in plant breeding. Very little reliable information is available for the various nut species compared with many other horticultural plants. The following preliminary experiments were conducted to obtain data on germination media, forcing methods, and storage conditions for Corylus and Juglans Sieboldiana pollen. The former was mostly from hybrid plants produced by crossing the Rush filbert (Corylus americana) with European varieties.

_The optimum temperature and sugar concentration for germination of Corylus pollen._

The cut ends of Corylus branches with mature catkins collected March 1, 1942 were immersed in water and forced into shedding pollen in a room at a temperature of approximately 20 deg. centigrade. The collected pollen was sifted upon the surface of a thin layer of sugar-agar in petri dishes.

Commercial cane sugar was used in preference to purified sucrose, because other studies have shown it to contain impurities which stimulate pollen germination. A range in sugar concentration from 5% to 55% by weight in 5% intervals was made up in distilled water containing 1.5% agar, heated to boiling and poured into the petri dishes.

The pollen was incubated at 10 deg. C. and at 25 deg. C. on the agar medium for 48 and 24 hours respectively prior to making the germination counts. Pollen was assumed to have germinated if the length of the pollen tube exceeded the diameter of the pollen grain.

At 25 deg. C. germination was prompt and uniform with a maximum of 19.5% at 25% sugar concentration. At 10 deg. C. the rate of germination was very slow and incomplete at the end of 48 hours with a maximum of 9% germination at 35% sugar concentration. For subsequent work a temperature of 25 deg. C. and a sugar concentration of 25% by weight was taken as a standard.

_The effect of temperature and humidity during forcing on the viability of the pollen_

Pollen shed from catkins forced in a warm, dry room (about 75 deg. F.), and in a cool, humid greenhouse (60 deg. F.) gave pollen germinating 36% and 69% respectively, which indicated that the air temperature and humidity surrounding the developing catkins may have considerable effect on the viability of the maturing pollen.

The experiment was repeated by forcing the catkins at 10 deg. C., 18-20 deg. C., and 24-26 deg. C., at two humidity levels. The low humidity level corresponded to the natural room humidity, about 25% and the higher level of nearly 100% was achieved by enclosing the branches with catkins in large sealed cans over a water surface. As soon as a majority of the catkins began to shed their pollen or to absciss their full developed anthers, the catkins were removed and dried on a sheet of smooth paper at room temperature until the pollen was shed. The pollen was then collected and stored at 4 deg. C. until used. The results obtained are given in table 1.

Table 1. Percentage germination after 24 hours of Filbert pollen forced at different temperatures and humidities.

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Temperature

10 deg. C. 18-20 deg. C. 24-26 deg. C.

Low humidity 80 31 7

High humidity 96 60 12
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Later experiments indicate that the pollen viability is greatly lowered if the catkins are removed from the higher humidities prior to the maturity of the anthers as indicated by their tendency to shed their pollen. Apparently the high humidity hinders the dehiscence of anthers and shedding of the pollen grains.

_Effect of catkins extracts on pollen germination_

The failure of pollen to germinate in the catkins at 100% humidity suggested the possibility that the catkin tissue might contain some substance which prevented germination of the mature pollen grains until after it was shed.

Two mature catkins plus remnants of their unshed pollen were ground in a mortar with a small amount of water in clear quartz sand. One cubic centimeter of the resulting turbid suspension was added to 10 cc. of warm fluid agar and mixed by rotating the petri dish.

Pollen which gave a 91% germination on the standard medium showed only 50% germination on this catkin extract. Germination was distinctly abnormal with short stubby pollen tubes, often with numerous nodular swellings. In general the pollen tube grew up into the air away from the surface of the agar, rather than down into it or parallel with the surface as in normal germination.

_Storage of Corylus and Juglans Sieboldiana pollen_

Sulphuric acid solutions to give humidities from 10% to 100% in 10% intervals were made up. The storage chambers consisted of Atlas one-pint, wide-mouth fruit jars. In the bottom of each was placed a small 1-oz. bottle containing 20 cc. of the sulphuric acid solution. The pollen was placed in small glass vials loosely stoppered with cotton.

Two lots of Corylus pollen of 80-1/2 and 96-1/2 initial viability respectively, and one lot of Juglans Sieboldiana pollen of well over 50% viability were used in the experiment. Storage temperatures of 0 deg. 40 deg. and 10 deg. were used.

The Corylus pollen was placed in storage March 20, 1942, and the Juglans April 12, 1942. The pollen was taken out of storage November 28, 1942 and germinated on the standard agar-sugar medium at 25 deg. C. for 24 hours. Results are given in table II.

Table II. The effect of storage temperature and humidity on percentage germination of Corylus and Juglans pollen

------------------------------------------------------------------ Kind of Temperature Degrees Per cent relative humidity Pollen Centigrade 10 20 30 40 50 60 70 80 ------------------------------------------------------------------ Corylus 10 deg. 0 0 0 0 0 0 0 -- Juglans -- 0 -- 0 3 0 0 -- Corylus 4 deg. 0 0 0 0 9.0 0 -- 0 Juglans -- 0 -- 0 -- 0 0 0 Corylus 0 deg. 3.0 1.0 4.5 8.5 0 0 0 0 Juglans -- 0 -- 12.0 -- 12.0 0 0 ------------------------------------------------------------------

This preliminary work indicates that Corylus pollen can best be stored at 0 deg. C. at 30 to 40% relative humidity and Juglans pollen at 0 deg. C. at 40 to 60% relative humidity.

_Summary_

1. The optimum sugar concentration for germination of Corylus pollen is around 25% by weight in 1.5 per cent agar at 25 deg. C.

2. Forcing the catkins at a low temperature (4 deg. C.) and at high relative humidity (80%) favors the development of a high percentage of viable pollen.

3. The catkins contain some substance which when added to the germination media inhibits pollen germination and causes abnormal types of germination.

4. Preliminary results on pollen storage indicate that Corylus americana pollen can be stored for eight months or more in a viable condition at 0 deg. C. with a range of 30 to 40% relative humidity. Juglans Sieboldiana pollen can be stored at 0 deg. C. at 40 to 60% relative humidity. Whether or not pollen stored for this length of time would be effective in plant breeding should be tested by actual trial. The supposition based upon studies with other pollens is that germination tests are a reliable indication of the effectiveness of pollen in fertilization.

Storage and Germination of Nuts of Several Species of Juglans

W. C. MUENSCHER AND BABETTE I. BROWN

_Cornell University, Ithaca, N.Y._

While working on the general problem of the possible toxic effect of the roots of species of Walnut (_Juglans_) upon other plants we have had occasion to germinate the nuts to produce seedlings for experimental use.[1] The storage treatment employed previous to planting the nuts provided a successful method of supplying viable nuts. The simple treatment used, a modification of that suggested by Barton,(2) is briefly described and the results that may be obtained are indicated in a report of some germination data from the plantings of 1943.

The nuts were harvested after they had fallen from the trees and were stored in a cool place as soon as possible thereafter until the time when the husks were removed. Those harvested at Ithaca were put in cold storage at once; those harvested in California or Texas were delayed a few weeks during shipment. The husked nuts were stratified between layers of moist peat 2 cm. thick in two-or five-gallon crocks. The uppermost layer of nuts was covered with peat to a depth of about 10 cm. The nuts were placed in a cold room at 1 to 3 deg. C. in late autumn and left until they were planted, between April 15 and June 2. Nearly all species used germinated well after about five to six months of cold storage.

Table 1 shows the results obtained from treated nuts of ten species of _Juglans_ when they were planted in the open field, in soil in the greenhouse or in moist sphagnum in the greenhouse. While some variation in germination is observed, most of the species gave a good germination under all treatments. The field planted seeds were somewhat slower in appearing above the soil surface than those planted in the greenhouse. This delay may have been caused by the cold rainy weather soon after planting. The firmness of the soil, a clay loam, may also have retarded the emergence of the seedlings.

The germination percentages are based upon lots of 100 nuts except in a few species in which only 50 nuts were used. Differences in the percentage of germination obtained from various plantings of the same species are slight in most species. Even the larger differences in germination obtained in a few species cannot be considered significant but probably indicate variations in the quality of the original lots used.

Summary

Walnuts husked soon after harvest, before they are completely air-dried, and stored in moist peat at 1 to 3 deg. C. for five to six months have their dormancy broken and remain viable for at least three months thereafter. This treatment is effective for all ten species tested. It is probably effective for all species of _Juglans_. This method of handling the nuts has the advantage over outdoor stratifying or autumn planting which often result in much damage or loss of nuts from the activities of rodents.

Table 1. Germination of nuts of _Juglans_ spp. after stratifying in peat over winter, at 1-3 deg.C.

------------------------------------------------------------------------ Per cent germination ------------------------------------- |Date |Planted in| |Planted Kind Source |entered|soil in |Planted |in |in |greenhouse|in field|sphagnum |storage|April 15 |April 24|June 2 -----------------------------------+-------+----------+--------+-------- nigra (Cornell) Ithaca, N.Y. |Oct. 1 | 70 | 80 | 68 nigra (Cayuga) Ithaca, N.Y. |Oct. 1 | 100 | -- | 80 cinerea--Ithaca, N.Y. |Oct. 1 | 60 | 44 | 8 regia (Sorrentina) Chico, Calif. |Nov. 9 | 66 | 48 | 8 regia (Franquette) Chico, Calif. |Nov. 9 | 80 | 36 | -- regia--Chico, Calif. |Nov. 9 | 75 | 46 | -- Sieboldiana--Ithaca, N.Y. |Oct. 1 | 100 | 40 | -- honorei--Chico, Calif. |Dec. 18| 60 | 55 | 46 pyriformis--Riverside, Calif. |Nov. 9 | 10 | 54 | 31 rupestris--Alpine, Texas |Oct. 1 | 40 | 83 | 50 major--Riverside, Calif. |Nov. 9 | 90 | 92 | 66 californica--Pomona, Calif. |Nov. 9 | 62 | 84 | 91 californica quercina--Chico, Calif.|Dec. 18| -- | 18 | 25 hindsii--Riverside, Calif. |Nov. 9 | 50 | 56 | 52 ------------------------------------------------------------------------

References--

1. Brown, Babette I. Injurious Influence of Bark of Black Walnut Roots on Seedlings of Tomato and Alfalfa. Northern Nut Growers Association, 1942: 97-101. 1943.

2. Barton, Lela V. Seedling Production in _Carya ovata_. _Juglans cinerea_ and _Juglans nigra_. Contr. Boyce Thompson Inst. 8: (1) 1-5. 1936

A Key to Some Seedlings of Walnuts

W. C. MUENSCHER AND BABETTE I. BROWN

_Cornell University, Ithaca, N.Y._

While working with the seedlings of several species of walnuts certain diagnostic characters, by which the common species can be separated, became evident. These characters have been used to make a key to seedlings from one to three months of age. This key has been found helpful to us and it is here presented in the hope that it may prove useful to others who need to handle and determine walnuts in the seedling stage.

The key has two main divisions based upon the types of leaves on the main axis. The first division includes three species, _Juglans sieboldiana_, Japanese butternut, _J. cinerea_, American butternut, and _J. regia_, Persian or English walnut, all of which have only compound green leaves. In addition, one or more pairs of minute simple scales or buds occur on the lower part of the stem but above the cotyledons. The second main division includes species in which the seedlings have several simple, alternate, scale-like leaves followed successively by serrate, lobed and finally compound leaves forming a gradual series. This group includes _Juglans rupestris_, Texas black walnut, _J. nigra_, eastern black walnut, _J. honorei_, Ecuador walnut, _J. pyriformis_, Mexican walnut, _J. major_, Arizona black walnut, _J. californica_, California black walnut, and _J. hindsii_, Hind's black walnut.

It is important that the leaves on the primary axis arising from the plumule are examined. If the primary axis is injured secondary shoots may arise from the axils of the cotyledons. These may develop various types of leaves not necessarily like those of the primary axis. The key is based upon seedlings grown in the field and in the greenhouse at Ithaca, New York.

_A Key to seedlings of some species of Juglans_

1. Leaves on the primary axis all compound; 1 to 4 pairs of opposite or subopposite reduced scales or buds sometimes present on the lower axis but above the cotyledons.

2. Scales or buds wanting between the lowest compound leaves and the leaves and the cotyledons _J. sieboldiana_

2. Scales or buds in pairs on 1 to 4 nodes below the compound leaves.

3. Stem with 1 pair of opposite scales or buds near the base; leaflets hairy, serrate _J. cinerea_

3. Stem with 2 to 4 pairs of opposite scales or buds below the compound leaves; leaflets glabrous, entire or denticulate _J. regia_

1. Leaves on the primary axis alternate, forming a gradual series from simple, entire scales to compound leaves; the lower 3 to 8 leaves simple.

4. Lateral veins of leaflets all or mostly all terminating in the notches between marginal teeth _J. rupestris_

4. Lateral veins of leaflets or their main branches all or mostly all terminating in the apex of marginal teeth.

5. Midrib of leaflets glandular hairy.

6. Glandular hairs on midrib of young leaflets interspersed with stellate clusters of gray glandless hairs; lateral leaflets ovate to broadly lanceolate, rugose _J. nigra_

6. Glandular hairs on midrib of young leaflets interspersed with sessile, usually yellow glands; lateral leaflets lanceolate, not rugose _J. honorei_

5. Midrib of leaflets glabrous or nearly so, sometimes with scattered, sessile glands.

7. Leaflets lanceolate, with acuminate apex; rhachis glabrous.

8. Leaflets widest near middle; vein-islets prominently raised; free ends of veins wanting or if present distinct to the apex and mostly unbranched _J. pyriformis_

8. Leaflets mostly widest below the middle; vein-islets not prominently raised; free ends of veins slender, terminating in indistinct branches _J. major_

7. Leaflets ovate or nearly so, with obtuse or acute apex; rhachis somewhat pubescent.

9. Petioles of the 3 lower compound leaves less than 1 cm. long; leaves crowded on a short axis _J. californica_

9. Petioles of the lower compound leaves from 1+ to 3 cm. long; leaves more distant on an elongated axis _J. hindsii_

Further Tests with Black Walnut Varieties