CULTURE OF OVULES CONTAINING IMMATURE EMBRYOS OF EASTERN COTTONWOOD IN VITRO I

Michael A. Savka, Robert M. Skirvin, Jalmer J. Jokela, and Jeffrey O. Dawson 2

ABSTRACT.--Hybrid ovules containing immature

embryos were excised from capsules on cut branches

of eastern cottonwood (Populus deltoides Bartr.).Ovules in the less than 21-days post-pollination

age-class produced callus only when cultured on amodified Murashige and Skoog medium. Singleshoots developed from 35% and callus developed

from 39% of ovules 21 to 40-days post-pollination.Ovules in the older than 40-days post-pollination

age-class produced only single shoots. Adventi-tious leaves formed on hypocotyls and cotyledons

of some single shoots.

ABBREVIATIONS perlite located in the bottom of the pot. The pot

is then placed in 5 cm of water. Floral buds areBAP = 6-benzylaminopurine; ECM = embryo forced in mid or late winter. After pollination,

culture medium_ MS = Murashi_e and Skoog high 4 to 16 weeks are required for eastern cottonwood

mineral salts; PVP = polyvinylpyrrolidone; SPM = capsules to mature viable seed. Due to this long

shoot proliferation medium, maturation period, capsule mortality can be highand some crosses produce few or no seeds (Farmer

The genus Populj_sscontains some of the most 1976). For instance, at the University of

productive pulpwood, lumber, and fuelwood species Illinois in 1984, 44 poplar crosses were attemptedin the temperate zone. Eastern cottonwood (_. but only II produced seedlings. Mortality coulddeltoides Bartr.) may supply part of the predicted occur at any stage of seed maturation.increase in the need for fiber (Dickmann and

Stuart 1983). In _924, a hybridization program to In order to circumvent the capsule mortalitydevelop _ast-growing poplar hybrids for the United associated with greenhouse crossing, Kouider et

Sta_es was initiated (Schreiner 1937). Subse- al. (1984) developed a tissue culture system toquently other breedin_ programs have been estab- rescue immature embryos of eastern cottonwood. In

fished to produce improved poplar types, the genus Populus, many tissue culture systemshave been investigated (Durzan and Campbell 1974,

Poplar hybridization is commonly performed in Bonga and Durzan 1982). In most studies, the

a greenhouse where cut branches from selected explant tissues have been apical and axillary buds

trees cas be forced to flower and then crossed (Bawa and Stettler 1972, Berbee and Hildebrandtu_der controlled conditions. At the University of 1972, Chalupa 1974, Lester and Berbee 1977,

Illinois. the method "du Plancon en Pots" (Joennoz Whitehead and Giles 1977, Christie 1978, Chalupa

and Vallee 1972) has been used. This method con- 1979, and Kim etal. 1981). Other explants havesists of inserting several female branches down included cambial stem sections (Mathes 1964,

through 15 cm of a rooting medium into 5 cm of Mathes and Einspahr 1965, Wolter 1968, Ahuja1983), root-segments (Winton 1968, 1970a, 1970b),

leaf sections (Bychenkova and David 1978, Wei-lun

et al. 1980, Ahuja 1983), and adventitious rootsIA paper presented at the Fi_tn _L_I ('Briandand Venverloo 1973, Ahuja 1983).

Hardwood Forest Conference held at the University

o:f lllinois, Urbana, IL on April 15-17, 1985. This research was initiated to expand and fur-

ther develop an in vitro system to rescue immature2Spaeth Fellow, Department of Forestry; embryos of eastern cottonwood. Our objectives

Associate Professor of Horticulture, Department of were: (I) to determine the minimum age at which

Horticulture; Associate Professor of Forest ovules containing immature embryos could be

Genetics, and Associate Professor of Forestry, rescued reliably and (2) to document the in vitro

Department of Forestry, University of Illinois, regeneration capacity of ovules from variou---sage-Urbana,IL 61801USA. classes.

234

MATERIALS AND METHODS g/h), glutamine (250.0 mg/£), Staba vitamins

(1969), and agar (8.0 g/£). The SPM was further

Controlled pollinations among selected eastern supplemented with BAP at a rate of either 2.0 orcottonwood clones collected from diverse origins 3.0 mg/%. Four weeks later tissue organization

in the Mississippi River Valley were made on cut data was collected.branches 60-120 cm in length maintained in a

greenhouse(Joennozand Vallee1972). Capsules

developingon branches were collectedat various

times from Zto 103 days after pollination.

Throughout the remainder of this paper the term

"embryo" will be used to indicate an ovule and itsembryo.

Capsules were surface sterilized for 10

minutes in a solution of 0.525% NaOCI containing a

drop of Tween 80 per I00 ml used as a surfactant.

Capsules were rinsed twice with sterile distilledwater, 3 minutes per rinse. Capsules in the less

than 20 day post-pollination age-class were smalland individual embryos were not identifiable;

therefore, these capsules were cut into quarters.

Capsule quarters were transferred directly to themedium with the interior of the quarter and their

contents facing upward, away from medium surface.Individual embryos were dissected from capsules in

the 21 to 30 day and older post-pollination age-

classes. Embryos were placed on the surface ofthe medium with their longitudinal axis parallelto the medium surface.

Capsule quarters or embryos were transplanted

to the embryo culture medium (ECM) previously

described by Kouider et al. (1984). The pH was Figure 1.--Tissue development from embryos ofadjusted to 5.6 before autoclaving at I18°C at a eastern cottonwood. (a) callus, (b) globular

pressure of 1 kg/cm 2 for 15 minutes. After auto- callus, (c) single Shoot, (d) single shoot with aclaving, approximately 33 ml of the medium were callused hypocotyl, and (e) single shoot with

dispensed into I00 x 25 mm sterile petri dishes, adventitious leaves derived from the hypocotylThe cultures were grown at about 220C under cool- (all × 15).

white fluorescent light for 16 hours a day. Light

intensity was maintained at a low level ranging To determine the age-class when the embryosbetween 38.0 and 65.6 microEinsteins per square were mature enough to germinate normally, at each

meter per second of photosynthetically active collection date the contents of one capsule wasradiation (400 to 700 nanometers), dissected and plated on moist blotter paper in a

I00 x 15 mm petri dish. The percentage of embryos

After six weeks, the types of tissue that that formed plants was determined on the basis of

developed from the embryos included callus, globu- 32 embryos per capsule (Schreiner 1974). Thelar callus, and single shoots (fig. I, a, b, c). percentage germination obtained in this manner wasWe defined "callus" as a soft translucent mass of compared to that observed in vitro.

cells. "Globular callus" was callus that con-

tained spherical outgrowths. All single shoots To facilitate histological examinations of

had cotyledons (fig. I, c). After ten weeks, the embryos, at each collection date a capsule was

embryos that had developed a single shoot were fixed in FAA (formalin: glacial acetic acid:further evaluated. At that time, three types of ethanol 5:5:90, volume basis). Once we had deter-

shoots were observed: single shoots, single mined the youngest age-class that would yield

shoots with a callused hypocotyl, or single shoots single shoots in vitro, embryos from the fixedwith adventitious leaves derived from the hypo- capsule representing that age-class were passed

cotyl (Fig. I, c, d, e). We defined "adventitious through an ethyl alcohol/xylene series and embed-

leaves" as leaves that failed to grow as a shoot, ded in Paraplast (Monoject Scientific division,The adventitious leaves were normal in appearance Sherwood Medical, St. Louis, MO 63103 USA).

but the associated apical meristem did not elon- Sections, 10-12 micrometers thick, were stained

gate to form a shoot (Fig. I, e.) with Flemming triplestain or safranin and fastgreen (Johansen 1940).

Callus or globular callus produced from embryoson ECM were recultured on shoot proliferation

medium (SPM). The SPM consistedof the Murashige RESULTSand Skoog (MS) high mineral salts (1962) supple-mented with myo-inositol (I00.0 mg/_), PVP (150.0 Tissue organization after six weeks varied

mg/_), ascorbic acid (20.0 mg/_), sucrose (30.0 with age-class (fig. 2). Capsule quarters in the

238

2 to lO-day, and [I to 20-day age-classes produced increasing age-class. Adventitious leaves devel-

no shoots on ECM. However, 90 percent of these oped on 3% of the hypocotyl shoots in the 21 to

quarters produced globular callus. A lower per- 40-day age-classeso In the older age-classes, the

centage of the embryos .inthe 21 to 30-day, and 31 development of adventitious leaves increased to anto 40-day age-classes produced globular callus, overall average of 23%. Callus failed to differ-Twenty-eight percent of the embryos in the 21 to entiate shoots. Five percent of the globular

30~day age-class developed a shoot and 83 percent callus of the II to 20-day age-class recultured onof the older than 50-day age-class produced a SPM produced single shoots.

shoot_ On the moistblotterpapera shootdevelopedfrom 5% of the embryos in the 21 to 30-day age-

D _ class and 39% of the greater than 50-day age-class

R Q_c_ embryos(tableI).

_ TABLE 1.--Thepercentage of eastern cottonwood

[] _ ..... embryos of various age-classes which developed ashoot on moist blotter paper and embryo culture

_ '_ media(ECM).

_ Age-class Papera Media Paper Media

! °_ _ (days) (percentage) (number)

2 toI0 - 0 - 155

IIto20 - 0 - 205G

21to 30 5 28 160 240

31to40 7 41 288 256

FIGUkE 2.-_The growth and development of east-

ern cottonwood embryos of various age-classeson 41 to 50 32 68 192 115embryo culture medium (ECM) after six weeks.

> 50 39 83 384 558

Af_:er ten weeks, embryos in the 21 to 40-day aThe percentage Of embryos which produced aage_class had produced either single shoots (52%) shoot on the moist blotter paper was determined by

or a single shoot with callused hypocotyl (45%) assuming 32 embryos per capsule (Schreiner 1974).(flg_ 3). In the 41 to 60-day or older age-class-

es, 75 percent of embryos developed single shoots.

The percent:age of embryos which developed a single Paraffin-embedded, 26-day-old embryos were I/3

shoot w_ th call_ised hypocotyl decreased with to I/2 the length of the ovule (fig. 4).

_ _ FIGURE4.--Longitudinalsectionof a 26-day-_ oldeasterncottonwoodovulecontainingan imma-

! _ tureembryo at an early stageof cotyledonelonga-_ tion(x54).

_o DISCUSSION

° I_ _ _, Embryosof the 2 to I0 day, and ii to 20 day__ age classes developed callus and globular callus_ on ECM. Some of the globularcallus of the II to

20 day age-class developed weak and spindly singleshoots on SPM. Our results are in accord with

those of Wendian et al. (1983) who reported that

FIGURE 3.--The growth and development of sin- embryos of three Populus hybrids (Populus simonii

gle shoots derived from embryos of eastern cotton- Carr. X P. euphratica Oliv., P. simonii Car. X P.

wood of various age-classes on embryo culture lasiocarpa Oliv., and P. simonii Carr. X P. wil--medium(ECM)aftertenweeks.sonii Schned.) taken 15 to 20-days

236

_es post-pollination initially formed callus. Thirty cytokinin and auxin can facilitate simmltaneous

e 21 days later, the callus produced a weak plantlet on shoot and root development from immature embryos.asses, their medium.ased

o di No multiple shoots were observed in studies by LITERATURECITED

bular us or by Wendian et al. (1983) on embryos youngerthan 21-days post-pollination. In contrast, AHUJA, M. R. 1983. Somatic cell differentiationKouider et al. (1984) reported that embryos in the and rapid clonal propagation of aspen. Silva

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_velo pie shoots at a frequency of 80 and i00 percent, BAWA, K. S., and R. F. STETTLER. 1972. Organy age- respectively. Furthermore, we found identified- culture with black cottonwood; Morphogenetic

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