Studies on the Pollen Tube Growth in Six Species of Hibiscus and their Crosses in Vivo

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1 460 Cytologia 23 Studies on the Pollen Tube Growth in Six Species of Hibiscus and their Crosses in Vivo P. Sanyal Received May 13, 1958 Jute Agricultural Research Institute, Barrackpore, India Introduction Almost all the species of Hibiscus are known to produce fibre, but only two species, namely H. cannabinus L. ('Bimlipattam Jute', 'Mesta', 'Kenaf') and H. sabdariffa L. ('Roselle') are commercially exploited for fibre pro duction. Hibiscus cannabinus is characterised by its short vegetative period (3-4 months) with a superior quality of fibre, while H. sabdariffa is a long duration crop (7-8 months) with comparatively inferior quality of fibre, but is a heavy yielder. With the partition of India in 1947, the important jute growing areas went over to Pakistan; consequently there had been a great shortage of raw jute in India for the requirement of the jute mills. As Hibiscus fibres can be easily mixed with jute in different proportions for spinning in jute mills, the shortage of jute was to some extent met by these fibres. Along with the increase in the cultivation of jute, there has also been an increase in the cultivation of Hibiscus fibres especially in areas where jute cannot be grown profitably. As a result the fibres from H. cannabinus and H. sabdariffa became very important in this country as substitutes for jute. Attempts to hybridize cannabinus, 2n=36 with sabdariffa, 2n=72 (Deshpande 1938; Kist and Freiderich 1947 etc.) and also each of these two species with other species of Hibiscus (Toxopeus 1948) for inducing disease resistance and other economic characters were made in the past. All these workers reported the production of only non-viable seeds when cannabinus was crossed with sabdariffa and they considered that the outlook for hybridizing these two species was not promising. Toxopeus (1948) in his preliminary attempts in crossing eight different species of Hibiscus, however, reported the successful hybridization of only between H. cannabinus and H. radiatus Willd, 2n=72 but he did not report any detailed study of this hybrid. After Toxopeus, no further investigation on the interspecific hybridization in Hibiscus have been reported so far. With a view to induce disease resistance in H. cannabinus from other disease resistant species of Hibiscus and also to understand the affinities amongst them, six species of Hibiscus with chromosome numbers (as given by Darlington and Wylie 1955):-cannabinus, 2n=36; sabdariffa, 2n=72,

1958 Pollen Tube Growth in Hibiscus in vivo 461 radiatus, 2n=72; panduraeforrnis Burm, 2n=24; lunariifolius Willd, 2n= 40 and vitifolius L., 2n=34+0-1B were crossed by the present author (1957).

2 1958 Pollen Tube Growth in Hibiscus in vivo 461 radiatus, 2n=72; panduraeforrnis Burm, 2n=24; lunariifolius Willd, 2n= 40 and vitifolius L., 2n=34+0-1B were crossed by the present author (1957). The details of these crossing results and the cytogenetical study of the progenies of the successful hybrids are intended to be published else where. The present paper only describes the results of the pollen tube growth study in these crosses along with the parental species (controls) in vivo and also to investigate the basic cause of incompatibility, if any, due to pollen tube growth in some of these interspecific crosses. Materials and methods Selected flower buds with the stigma at the right stage of receptivity were emasculated and bagged. Controlled pollination were made between the six species of Hibiscus namely, cannabinus, sabdariffa, radiatus, lunarii folius, panduraeforrnis and vitifolius next day. Besides these, controlled self-pollination were also made in all these six species of Hibiscus. After pollination, the flowers were collected at different intervals of time (5, 10, 15, 30, 45 minutes, 1 hr., 1 hr. 15 mins., 1 hr. 30 mins., 2 hrs., 2 hrs. 30 mins. and 3 hrs.); the ovary along with the style and stigma were dis sected out from the rest of the flower and fixed in acetic alcohol (1:3). The dissected pistils were kept in acetic alcohol overnight, rinsed in absolute alcohol and stored in 70 percent alcohol. For clearing and staining of pistils, slight modification were made between freshly preserved (10-15 days storing) and stored materials (beyond 15 days storing) in 70% alcohol. In the freshly stored materials, the styles were dissected from the ovary and transferred to lactophenol (lactic acid one part and phenol one part) after bringing them to water from 70% alcohol through discending alcohol grades. They were then boiled for 1 to 3 minutes till the styles became transparent. After cooling, a style was placed on a clean glass slide. Subsequent staining of the individual style and stigma was done on the slide. The style was stained in 1% acid fuchsin in lactophenol (acid fuchsin-1gm., lactic acid-50cc and phenol-50cc). Stain was placed in sufficient quantity so as to cover the entire length of the style. The style was pressed by a scalpel and slightly warmed over the flame. The excess stain was drained off and the style was again warmed with fresh lactophenol. The excess stain was removed by the same process which continued for 2 to 3 times until most of the superfluous stain was removed from the tissue. The style was then mounted in fresh lactophenol and pressed slightly to expose the pollen tubes. For the stored materials, the styles were brought down to water in the same way as stated above, from 70% alcohol. Before transferring them to lactophenol for boiling, the styles were placed in acidulated water (about 3 4 drops of conc. HCl in 10 cc of distilled water) for five minutes, then thoroughly washed, boiled in lactophenol and stained as above. 30*

3 462 P. Sanyal Cytologia 23 The length of the pistil in all the six species were measured on the basis of 25 pistils for each species at their peak period of flowering. As stated earlier the pollen tube growth was, however, measured only upto the base of the style excluding the portion of the ovary in each case. Observation Pistil length: The average length of the carpels separately for ovary, style, style-arm and stigma and their total mean in each of the six species namely, can nabinus, sabdariffa, radiatus, lunariifolius, panduraeformis and vitifolius are given below in Table 1. Table 1. Comparative length of pistil of six species of Hibiscus in millimetre From table 1 it is observed that H. radiatus has got the maximum length of pistil (27.94mm) and the minium is in H. sabdariffa (12.5mm). It is also noted that due to the variations in the pistil length, the distance travelled by the pollen tubes to reach the base of the style in vivo also varied in different species. Thus in both self-pollination of H. radiatus and its crosses with other species of Hibiscus, the pollen tubes travelled a maximum distance of 23.04mm. Similarly a minimum distance of 7.9mm was travelled by the pollen tubes in the case of H. sabdariffa. Nature of pollen tube growth in the controlled selfings and cross coatings In both selfings and crosses, actual measurement of the length of pollen tubes (average) travelled in each case was recorded with the help of the ocular micrometre. The average length of the pollen tubes were measured on the basis of 15 pollen tubes in each case which could be easily traced. These observations were calculated in/u and further converted in to millimetre readings in Table 2 for convenience. The data on the above pollen tube growth in both selfings as well as the crosses where pollen germination was found to occur (Table 2) have also been graphically represented in Fig. 1. The nature of pollen tubes in both self and cross-matings by no means were of uniform size. A variation in thickness was found to occur in the pollen tubes; some appeared thread-like (4-5/u approximately) under ~90

4 1958 Pollen Tube Growth in Hibiscus in vivo 463 magnification, others as broad as 23.1/u. be full of starch grains and stained deep blue with iodine. In all the selfed styles, in general, it was noted that the pollen grains germinate profuse ly and the number of pollen tubes were more than in the crosses (Figs. 2, 3, 4 and 5). In addition to the above crosses (Table 2) where the pollen tube growth was found to occur, similar records on the The pollen tubes were found to Fig. 1. Graph showing the behaviour of pollen tube growth in different self and cross matings of Hibiscus in vivo. Abbreviations: H. C.=H. cannabinus; H. S.=H. sabdariffa; H. R.=H. radiatus; H. P.=H. panduraeformis; H. L.=H. lunarii folius; H. V.=H. vitifolius; R ~C=radiatus ~cannabinus; R ~S= radiatus ~sabdarifa; S ~C=sabdarifa ~cannabinus; S ~R=sab dariffa ~radiatus; C ~S=cannabinus ~sabdarifa; C ~R=can nabinus ~radiatus. Table 2. Actual length travelled in millimetre by the pollen tubes in both self and cross matings at different intervals of pollination

464 P. Sanyal Cytologia 23 Figs. 2-5. 2, showing pollen tube growth in H. cannabinus (selfed) after 15 minutes of pollination in vivo. ~90. 3, showing pollen tube growth in H.

5 464 P. Sanyal Cytologia 23 Figs , showing pollen tube growth in H. cannabinus (selfed) after 15 minutes of pollination in vivo. ~90. 3, showing pollen tube growth in H. radiatus (selfed) after 45 minutes of pollination in vivo. ~90. 4, showing pollen tube growth (only two tubes shown separately) in cannabinus ~sabdariffa cross after 30 minutes of pollination in vivo. ~90. 5, showing pollen tube growth in radiatus ~cannabinus cross after 30 minutes of pollination in vivo. ~90. pollen germination were kept in the remaining other crosses between different species of Hibiscus employed in the present investigation. These observations were recorded till three hours after pollination at regular intervals but no pollen tube germination was found to occur in the following interspecific cross:- Cannabinus with lunariifolius, panduraeformis and vitifolius; Sabdariffa with lunariifolius, panduraeformis and vitifolius; Radiatus with lunariifolius, panduraeformis and vitifolius; Lunariifolius with cannabinus, sabdariffa and radiatus; Panduraeformis with cannabinus, sabdariffa and radiatus; Vitifolius with cannabinus, sabdariffa and radiatus; Lunariifolius with panduraeformis and vitifolius;

1958 Pollen Tube Growth in Hibiscus in vivo 465 Figs. 6-9. 6, showing pollen tube growth in radiates ~cannabinnus cross after 15 minutes of pollination in vivo. ~90.

6 1958 Pollen Tube Growth in Hibiscus in vivo 465 Figs , showing pollen tube growth in radiates ~cannabinnus cross after 15 minutes of pollination in vivo. ~90. 7, showing the germination of only 1 or 2 pollen grains in can nabinus ~radiatus cross after 10 minutes of pollination in vivo. ~90. 8, showing pollen tube growth in sabdariffa x radiatus cross after 15 minutes of pollination in vivo. ~90. 9, showing pollen tube growth in radiatus ~sabdarifffa cross after 45 minutes of pollination in vivo. ~90. Panduraeformis with lunariifolius and vitifolius; Vitifolius with lunariifolius and panduraefornzis. Position of the pollen tubes in relation to style length in selfings and crosses at different intervals From the above, it is indicated that the length of the pollen tube at different intervals in the self-matings show no germination after first five minutes in all cases excepting in H. radiatus where germination takes place after 10 minutes of pollination. In all cases it was found that within 30 to 45 minutes pollen tubes reach the base of the style. In cross-matings the pollen tubes enter the stigma and stylar tissue after

7 466 P. Sanyal Cytologia 23 five minutes excepting the cannabinus ~radiatus cross where it enters after 10 minutes (Fig. 7) but a slight difference in the behaviour of the growing pollen tubes is noted in the crosses. On the H. cannabinus style, the sab dariffa pollen reaches the base of the style in 30 minutes while the radiatus pollen reaches the full length in one hour. The cannabinus pollen or radiatus pollen on sabdariffa style reaches the base of the style within 15 minutes (Fig. 8) in both the cases showing that sabdariffa style is more favourable for the pollen germination of both cannabinus and radiatus. The pollen of cannabinus also grows favourably and reaches the stylar base within 15 minutes inside the style of H. radiatus thus showing that cannabinus pollen is more receptive to growth in the styles of sabdariffa or radiatus. The pollen of sabdariffa, however, showed delayed growth in the style of radiatus (Fig. 9) where it reaches the base after 45 minutes as compared to in the cannabinus style where it reaches within 30 minutes. It may, therefore, be noted that pollen of sabdariffa is more favourably grown in cannabinus style than in the radiatus style. In case of crosses of lunariifolius, panduraeformis and vitifolius with the above three species namely, cannabinus, sabdariffa and radiatus both as male or female parent there is no germination of pollens on the styles. Discussion and conclusion From the behaviour of pollen tube study in the cross-matings between six species of Hibiscus, it may be said in general that the three species, viz., cannabinus, sabdariffa and radiatus show reciprocal receptivity of pollen germination and growth in the stigma and stylar tissue, thus indicating that no stylar incompatibility is involved. On the other hand, the pollen tubes fail to germinate and do not enter inside the stylar tissue when either the remaining three species namely, lunariifolius, panduraeformis and vitifolius are crossed amongst themselves or in their crosses with cannabinus, sab dariffa and radiatus. The cause of the failure of pollen tube growth may be difinitely attributed to the stylar incompatibility in the latter crosses. In the light of the data of pollen tube growth from the above study between H. cannabinus ~H. sabdariffa and their reciprocal crosses it may be inferred that the cross incompatibility between these two fibre-yielding species of Hibiscus is not due to any hindrance of free pollen germination and growth in the stigma and style. A further investigation on the em bryological study of these crosses was, therefore, undertaken by the present author and its results have been sent for publication elsewhere. Summary Pollen tube growth in vivo of both selfings (controls) and crosses be tween six species of Hibiscus namely, cannabinus, sabdariffa, radiatus, lunariifolius, panduraeformis and vitifolius are reported here.

8 1958 Pollen Tube Growth in Hibiscus in vivo 467 From the behaviour of pollen tube study it is observed that the three species, viz., cannabinus, sabdariffa and radiatus show reciprocal receptivity of pollen germination and growth in the style indicating thereby that no stylar incompatibility is involved in these crosses. In the crosses of other three species, viz., lunariifolius, panduraeformis and vitifolius there is a definite stylar incompatibility between the species themselves as well as their crosses with other three species namely, cannabinus, sabdariffa and radiatus. In the selfed styles no germination of pollen occurs after five minutes of pollination excepting in H. radiatus where germination takes place after 10 minutes of pollination. All the pollen tubes, however, reach the base of the style within 30 to 45 minutes after pollination. In the crosses also the germination of pollen starts after five minutes excepting in the cross cannabinus ~radiatus where it starts after 10 minutes of pollination. The rate of growth of pollen tubes in the self and crossmatings with different species of Hibiscus is also found to be slightly different. Acknowledgement I wish to express my sincere thanks to Dr. B. C. Kundu, Director, Jute Agricultural Research Institute under whose guidance the present investigation was worked out for the D. Phil degree of the Calcutta University. References Darlington, C. D. and Wylie, A. P Chromosome atlas of flowering plants, George Allen and Unwin Ltd. London. Deshpande, R. B Studies in Indian fibre plants. 5. Further studies on the inheritance of certain characters in Hibiscus sabdarijfa L. Indian. Jour. Agr. Sci. 8: *Kist, J. M. and Friederich, J. C De culture van enkele bast en blavezellevende gewassen, Landbouwk Ti jdschr. 9: Sanyal, P Study on the cytological, genetical and cultural aspects of fibre yielding Hibiscus. Dissertation for D. Phil., Calcutta University. Toxopeus, H. J Preliminary account of the results of some species crosses in Hibiscus. Genetica 24: 1: * Publications not seen in original.

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