BIOLOGIA PLANTARUM (PRAHA)

19 (5) : 353~-359, 1977

Regulation of Seed Germination and Polarity in Seedling Development in Orobanche aegyptiaca by Growth Substances

Usm~ KUMAn* and N. S. RANOASWAMu

Dopaxtment of Botany, Universi ty of Delhi, Delhi l l0007, India

Abstract, In Orobanche aegyptiaca PEltS. (Orobanvkaceae) the mature seed is t iny and contains a suBgloboso embryo which is not differentiated into radiole, hypoeoty], plumule, and cotyledons. In aseptic seed cultures on medium TB supplemented with yeast extract or ooeonut milk, bobh roots and shoot originated from the morphological radieular pole of the embryo (monopolax pat tern) . The bipolar w~ode of seedling forma'~ion, t ha t is a shoot originating from the plumular pole and roots from the radieular poke, ensued on the basal medium TI tS a~d on TB supplemented with certain ooneontratio~rs of IAA, kmotin, GA3, or strlgol.

Characteristically in Orobanehaceae and certain other families of parasitic angiosperms the embryo lacks apical meristems and organ differentiation. Such taxa present a problem of morphogenesis -- namely, what is the mode of seed germination in them .~ Al though the l i terature on seed germinat ion is voluminous, all tha t is usually ment ioned of seed germination in parasites is t ha t it is host-dependent . In this review BRow~r (1965) s ta ted t ha t in seeds of parasites " . . . the course of events is similar to t ha t of the other seeds." However, in an exploratory work on Orobanche aegyptiaca one of us (RA~c- oASW~,~Y 1963) demons t ra ted t ha t the shoot apex originated from the radieular pole of the embryo. This paper presents oar detailed investigations on embryo morphogenesis in 0. aegyptiaca in vitro.

Material and Methods

Orobanche aegyptiaca PERS. is a herbaceous, nonchlorophyllous parasite on roots of Petunia, Brassica, Cicer, and several other angiosperms. In the climate of Delhi (India), its fruiting spikes become available during February- March. Therefore, for use round the year, ripe capsules of comparable size were gathered from the middle port ion of the fruiting spikes, and refrigerated in sterilized petriplates at 7--10 ~ Whenever desired, seeds were scooped from different eapsulcs, pooled, and used for culture work.

Before culturing, the refrigerated seeds were surface-sterilized with chlorine water. Following the cus tomary technique of aseptic culture, 50 to

Received Ovtober 2l, 1976; accepted February 3, 1977 * Prezen~ address: 1155 W. Sa~dhurst Drive, St. Paul, Minn. 55123, USA.

~54 U. KUMAR, N. S. RAlqGASWAMY

60 seeds were sown 1-seed-thick on agar nutrient medi~. The following media were tested: (1) White's modified medium (for composition see RA~- GASWAMY 1961), (2) Nitsch's medium (NITSC~ 1951), (3) Murashige and Skoog's medium (Mv~ASHI(~V, and SxooG 1962), and (4) Tepfer's H1 medium containing 4% sucrose (TsrF~.~ et at. 1963}, THS for short. On the basis of results presented in Table 1, TItS was selected as the basal medium. To determine whether all of ~he 15 organic substances contained in THS were necessary, as ma~y as I0 of them were omiteed from it- and the new medium designated TB was then tested before and after supplementing it individttally with IAA, kinetin, GA.% st rigol, yeast extract, and coconut milk. The medium TB comprised the following (rag per 1): (1) the mineral salts:

MgSO4.7 H~O -- 360, Ca(1~03)2.4 H20 -- 260, Na~SO4 -- 200, N a H 2 P O a . 2 H 2 0 - - 165, K N O a - - 8 0 , KC1-- 65, M n S O 4 . H 2 0 - - 3 , Z n S 0 4 . 7 H20 -- 0.50, H.~BOa -- 0.50, CuSO4.5 H20 -- 0.025, and Na~MoO~. 2 1-120 -- 0.025; (2) the iron source FoC6HsO7 . 5 H20 -- 10; (3) amino acid and ,:itamins: glycinc -- 7.50, niacin -- 1.25, th iamine-- HC1 -- 0,26, calcium pantothenate -- 0.25, and pyridoxine - HCI -- 0_25; and (4) the carbon source, sucrose -- 40 000.

All media were jelled with 0.6~/o agar, and adjusted to pH 5.8. The cultures were maintained under 16 h day light (500 to 600 lx), and 25 ~ -4- 2. For each experimen~ 2 to 5 replicates were made; each replicate comprised 24 cultures.

Histologic preparations were made following the customary tectmiquc of paraffin embedding and micr0tomy.

Results

At ma.turi~y the seed is brown-bias-k, approximately 34I ~zm iong, i64 ~zm broad, and light (100 seeds weigh approx. 77 rag, and a capsule contains more than 3000 seeds). I t is endospermous and contains a subglobose embryo. The embryo occupies nearly 0.994 mm s at the micropyle; it lacks radicle, hypocotyl, cotyledons, epicotyl and plumule, and does not show differen- tiation of apical or polar meristems (Fig. 1 A). The pole of the embrvo proximal to micropy]e is the radicular pole, and that distal to micropyle is the plumular pole.

Following the International Seed Testing Association's description (see LA~'o 1965), we have reckoned for Orobanche aegyptiasa the emergence of radicular pole of the embryo as seed g~rmin~tion, and the term seedling t.o mean ~he formation of a shoot, bud (irrespective of its origin) and of one or more roots from a seed.

Table I presents seed germination responses on the four media tested. Obviously, THS was chosen as the basal medium.

On the basal medium (THS) seeds germinated 1 week after culture. During germination the radlcular pole of the embryo grew by cell division and cell enlargement (Fig. I B). This initial growth was accompanied by depletion of starch in the radieular pole; this further distinguished the two poles of the embryo. As growth continued the radieular pole grew out as a 0.2 to 0.3 mm Iong structure herein ~r for convenience, the 'radicnlar cylinder' (Fig. I C). The radieular eytinder was apparent ly root-like and 7 to 9 ceil- thick; it showed a provascalar s trand but bore no root cap or root bai,.s.

SEED GERMINATION AND POLARITY

T ~ 1

Pervenk~ge seed germination on different~ nutrient media

355

,NIedium Days after ~ulture

7 9 12

Whim's 0,0 0.0 2.7 NRseh's 0.0 0.3 2.0 Muraahige sad Skoog's 0.0 0.0 0.0 Tepfer'a Ht (THS) 5.4 24.8 38.6

Eventually the radicular cylinder formed a mass of tissue called the "tu- bercle". When roots differentiated, they invariably originated from the tubercle and resembled those in vivo.

Concomitant with the activity of the radicular pole, the plumular pole also showed significant morphogenesis. I t formed a stem apex overarehed by scale leaves (Figs. 1 D--F). A vascular strand differentiated continuously from the subjacent region of the shoot bud to the base of the tubercIe.

Although not all stages of morphogenesis of the embryo could be observed for an individual seed, the different stages could be simultaneously noticed in any culture. Not all germinated seeds showed synehrony in morphogenesis, neither did all of them reach the final stage of morphogenesis of a bipolar embryo.

Unlike in basal medium, in medium TB no seedlings were formed because the plumular pole did not show morphogenesis; neither did the radieu[ar pole. If TB was supplemented individually with certain growth substances the embryo elicited either the monopolar pattern or the bipolar pattern o f producing the seedling,

T A B ~ 2

l~esponses of 5-week-old seed cultures to IAA, kinetin and GAs

Medium Percentage germination

Percentage of germinated seeds which produced

~eedli~ roots only (i. e. roo~s + a shoo~ bud)

TB (control) 14.8 8.3 0.0 TB + IAA 0.1 ppm 34,07 72.3 4,3 TB + IAA 0.5 ppm 44.7 96.7 0.0 TB -4- IAA 1.0 ppm 27.2 91.6 0,0 TB + kinetin 0.5 ppm 1.5 75.0 25.0 TB + kinetin 1.0 ppm 3.5 72.3 18,8 TB -4- kinctin 2.0 pprn 42.20 31.5 15.7 TB + kinetin 5,0 ppm ,51.00 0.0 53.3 TB + GA~ 1.0 p p m 0.0 O.O O.O TB 4- GA~ 2.5 ppm 0.23 0.0 0.0 TB + GAa 5.0 ppm 67.3 15.1 73.4 TB + 0,4. 3 7.5 ppm fi9.5 11.05 75,6 TB + GA~ 10.0 ppm 55.8 18.3 54.9 TB + GA3 15.0 ppm 20.6 60,70 8.3

356 U. KUMAR, N. S. RANGASWAh[Y

Effects of Ytqmt Extract and Coconut Milk

Both yeast extract (5000 mg 1-1) and coconut milk (20% v/w) induced the monopolar pat tern of development of seedling. The radieular pole of the embryo grew further, thus demarcating the plumular pole more distinctly than before germinat~ion (Fig. 2 A, compare with Fig. I A). As growth continued, the radieular pole emerged through the mieropyle as a cylindrical structure simulating a root. Eventually this structure formed the tubercle. Vascular elements differentiated both in the cylindrical structure and in the tubercle, in a slightly advanced stage, primordia of many presumptive roots originated in the tubercle (Fig. 2 B). The plumular pole remained in s i tu and seldom showed any morphogenic activity; instead, a shoot meristem dif- ferentiated in the region of tubercIe near the ruptured testa (Fig. 2 C). Thus, only one pole, namely the radieul:~r pole functioned and gave rise to both roots and shoot bud. Figs. 2 D and E illustrate shoot formation in coconut milk medium.

Effects oi" IAA, Kiuetln, GA~ and Strfool

Certain treatments with IAA, kinetin, GA~, and strigol, released the morphogenic suppression on the plumub~r pole. Consequently, the plumular pole produced a shoot bud and the radicular pole many roots. Thus a con- ventional bipolar root-shoot axis was completed and a seedling formed. Table 2 presents quantitative data on the effects of IAA, kinetin, and GAs.

Of the three concentrations of IAA tested, only 0.1 mg per 1 induced shoot bud differentiation from the plumular pole. Kinetin induced the highest percentage of seed germination and seedIing formation at 5 mg 1-1. GAs (5, 7.5 mg 1-1) induced most effectively both seed germination and seedling formation. However, at 10 and 15 mgl-1 , GA3 promoted rooting and not seedling formation. Like GAa, strigol (0.0i tg) also induced a bipolar pat tern of seedling development..

Ontogenic studies (especially of seeds which germinated in 7.5 and 10 mg per 1 GAs)showed that during the first 7 to 1"2 days of culture the embryo enlarged and its poles became further demarcated. Upon emergence through the micropyLe the radicular pole produced the radicutar cylinder. As in the monopoLar pattern, the radicular cylinder was 7 to 9 cell-thick, and did not bear a root cap or root hairs. Two weeks after culture the radicular cylinder enlarged particularly at its free end to form the tubercle and differentiated a provascular strand. Eventually many roots originated from the tubercle, and both the radieular cylinder and the tubercle lost their identity; vascular elements differentiated also in the plumular pole of the embryo lying in s i tu ; thus the vascular strand became continuous from radicular cylinder to plumular pole; both testa and the remnants of endosperm were cast aside (Fig. 2 F).

Simultaneous with the developmental changes in the radieular pole ot~ the embryo the plumular pole also contributed to the formation of a seedling. At first the plumular pole became characteristically dome-shaped and showed histological organization of a shoot apex (Fig. 2 G). Thc developing shoot apex ruptured the testa and grew beyond it. As growth continued many scale leaf primordia originated from the shoot apex and a vascular strand dif- ferentiated along its longitudinal axis. Eventually the primordia developed into scale leaves (Fig. 2 H).

SEED GERMI~qATION AND POLARITY 357

Discussion

Bl~owN's (1965) statement that in seed germination of parasitic angiosperms " . . . the course of events is similar to that of the other seeds" is not sub- stantiated by the investigations on orobanchaceous parasites. KADRu and Tsw~'IC'S (1956a, b) work on Orobanche crenata impties that only the radicular pole of the embryo becomes active during seed germination. PmVAT (1960) made almost similar observations on O. hsderae. In O. aegyptiaca, RA.W- GASWA•Y (1963) observed that the radicular pole of the embryo produced the shoot bud. RA~C~A~ and RA~GASWAMY (1968) reported a similar phenom- enon for Cistanche tubulosa, another member of Orobanchaceae (see also RA~rGASWAMY 1967). :For Aeginetia indica (also Orobanchaceae) five stages that axe different from the conventional course of seed germination have been enumerated (FsE~c]t and Sn~R~fA~ 1975). However, working on O. ramasa, ABV-S~AKI~A et al. (1970) have reported that " . . . seeds with conspicuous radicles* were considered to have germinated". This is not corroborated by any other investigation including our present work; in fact all other investigations have clearly shown that the embryo of Oro- banche lacks a radicle; therefore the existing concept of seed germination needs to be modified (RA~OASWAMY 1967).

Our study has clearly demonstrated that in vitro the seedling formation in O. aegyptiaca occurs according to either the monopolax pat tern or the bipolar pattern. The monopolar pat tern was evoked on the medium TB supplemented with yeast extract or coconut milk. The radicnlar pole pro- duced the radieular cylinder, the tubercle, and eventually both a shoot bud and many roots. Thus only one pole of the embryo paxticipated in the formation of seedling.

5IaSlt and WXL~]~L~ (1960) found that yeast extract (100 mg per 1) induced seed germination but not seedling formation in O. ramosa. In our cultures of O. aegylatiaca, however, yeast extract (5000 mg per 1) elicited not only seed germination but also the monopolar pat tern of seedling formation. This progressive morphogenesis achieved in our cultures may be due to a 50 fold increase in the concentration of yeast extract in the culture medium.

The bipolar pat tern of seedling formation was characterized by an ex- pression of morphogenesis by both the poles of the embryo, the radicular pole producing roots and the plumu]ar pole a shoot bud. The resultant was a seedling of conventional morphology. What is noteworthy is that in the bipolar pat tern the shoot bud formation was a response definitive to specific growth substances and their concentrations. For example, IAA evoked plumular pole activity at 0.1 mg per l only, whereas kinetin did it at all the four concentrations tested, and GAs induced, at 7.5 mg per 1, the highest percentage (75.6) of bipolar formation of seedlings. At levels greater than 7.5 mg per 1 GAs significantly increased rooting.

Our data on the effects of IAA, kinetin and GAs on seed germination of Orobanche asgyptiaca are somewhat contrary to those of EDWARDS (1972) and GARAS et at. (1974) on that of O. crenata. AIthough GAs is among the most potent seed germination indUcing gibberellins, EDWARDS found that between 1 and 100 mg per 1 GAs did not stimulate seed germination of O. crenata.

3 5 8 U. KUMAR, N. S. RANGASWAMY

G ~ A S st al. have reported that among IAA, kinetin, and GA3 none induced seed germination when supplied without the flax (host) root exuda te ;bu t when supplied previously or simultaneously with the roo~ exudate, gibbereL ]ins increased and kinetin decreased the germination-inducing effect of the root exudate; IAA was ineffective. ABs-SEAKRA et al. (1970) also found that presoaking of 2-year-old seeds of 0. ramosa in GAs solution (100 mg per 1) for I week and then sowing them on flax roo~ diffusate-agar medium resulted in the highest germination (81.7~/o). Stimulation of seed germination and of root growth by GAa have also been reported for citrus, lettuce, ash and douglas fir (RANoASWA~Iu 1961, VAN OVERBEEK 1966, JOHRI and VAR:NER 1967).

In our work, strigol which is an extremely potent seed germination s t imulant of Striga and is similar in molecular formula to GAs (see COOK st a[. 1966, 1972), induced, like GAs, the bipolar pat tern of seedling formation. I t is said that the seed germination stimulants of Striga and Orobanchs are similar. Based on the extremely high potency of strigol as a seed germination s t imulant and the presence of s~imulants similar to strigol, Cook et al. (1972) have suggested that strigol may be a representative of a new class of plant hormones.

As ia Orobanchaceae, so in Orchigaceae, only one pole of the embryo par- takes in germination (see AaDiwI 1967, RASG.(SWA.~tr I967, T~c~IoJ~mN and NIK~TIC~tEVA 1968). But unlike in Orobanshacsae, in Orchidaceae it is the phimular pole (variously termed as "anterior part", "anterior region") which begins and consummates the activity in producing the seedling. Thus, from a morphogenie standpoint seed germination in acotyledonous taxa such as Orobanshe is neither hypogeal nor epigeal. And in O. aegyptiaca organ differentiation in the embryo is an event of seedling formation and not embryogeny, and the plumular pole of the embryo is morphogenically ar- rested iu viva and therefore seed germination activity becomes the monopoly of the radieular poke. This morphogenic lag could, l~owever, be removed by t reatments with kinetin, GAs, or strigol, and the plumutar pole be made to produce a shoot bud. Thus seed germination in vitro follows two patterns of morphogenesis, monopolar and bipolar, in response to treatments with growth substances.

Acknowledgements

We are obliged to t~he Council of Scientific and Industr ial Research, New Delhi, and to 6he UGC Centre of Advanced St.ady in Botany. Universi ty of Delhi, for financial support.. Our sincere tha~k~ are duo ~o Dr C, E. Cook, R e , a r c h Triangle Inst i tu te , Whitewillo, Nor th Carolina, USA, for the sample of strigol, to Dr K. R. Shivazma for reading through the manuscript , and to Mius bfinakshi Sathi for help with the illustrations.

References

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S E E D G E R M I N A T I O N AND P O L A R I T Y 359

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.Figure8 a~ the end of the isawe.

U. KUMAR, N. S. RANGASWAMY

SEED GERMINATION 1\ND POLARITY

Fig. l. Orobanche aegyptiaca. Seed germination in Tepfer's H 1 medium: A: L.s. seed; the embryo lacks apical meristems and organ differentiation (128. 7 X). B: L.s. seed 7 days after culture; radicular pole of embryo has begun to grow through the micropyle. Note the differences in cell appearance at the two poles. Excepting for the peripheral layer the endosperm has been con­sumed; compare with A (245. 7 x ). C: L.s. seed 2 weeks after culture. A radicular cylinder (rc) has been formed; observe differentiation of provascular strand (pvs), but lack of root cap and root hairs (8.1 X). D: Piumular pole (pp) has already formed a dome comparable to stem apex. Section through tubercle is off-median (88.4 X ). E: Germinated seed 3 weeks after culture show­ing remnant of testa (part of its boundary is accentuated by a broken line), massive tubercle, and shoot (32.5 X). F: Longisection of shoot from 5-week-old culture (up to the level of testa shown in E). Shoot apex, 3 scale leaves, and provascular strand are evident. The arrow-marked strip (bottom right) is remnant of testa and endosperm. Tear in tissue is due to microtomy (31.2 x ).

shoot apex

U. KUMAR, N. S. RANGASW AMY SEED GERMINATION AND POLARITY

J.a !

) ..

A

·ptumular pole

)

G