morphogenetic studies on haworthia: establishment of tissue culture and control of differentiation

Morphogenetic Studies on Haworthia: Establishment of Tissue Culture and Control ofDifferentiationAuthor(s): K. Kaul and P. S. SabharwalSource: American Journal of Botany, Vol. 59, No. 4 (Apr., 1972), pp. 377-385Published by: Botanical Society of AmericaStable URL: http://www.jstor.org/stable/2441548 .

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Amer. J. Bot. 59(4): 377-385. 1972.

MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION'

K. KAUL AND P. S. SABHARWAL Department of Botany, University of Kentucky, Lexington 40506

AB STRA CT Investigations were carried out on the in vitro morphogenetic responses of inflorescence

segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.

IN VITRO morphogenetic studies on monocotyledons have been less extensive than those on the dicotyledons. In the past few years some investi- gators have reported establishment of monocotyledonous tissue cultures (Morel and Wetmore, 1951; Nickell, 1964, 1967; Carter, Yamada, and Taka- hashi, 1967; Yamada, Tanaka, and Takahashi, 1967; Yatazawa, Furuhashi, and Shimizu, 1967; Wilmar and Hellendoorn, 1968; Sheridan, 1968; Takatori, Murashige, and Stillman, 1968; Majum- dar, 1970a, b; Mullin, 1970; Ziv, Halevy, and Shilo, 1970). However, these reports have hardly ever been followed up by any detailed anatomical study on callus and its subsequent differentiation into organs. Further, a chemical control of or- ganogenesis in these tissues has never been reported. Sometime ago we reported the establishment of a tissue culture of Haworthia (Liliaceae) on modified White's basal medium supplemented with coconut milk (Kaul and Sabharwal, 1970). The present paper is a report on (a) the anatomical studies on callusing and differentiation of Haworthia explants, (b) establishment of callus on a chemically defined medium, and (c) chemical control of root and shoot differentiation.

MATERIALS AND METHODS-Plants of Haworthia variegata L. Bol., H. chloracantha Haw., H. truncata schoenl. x H. setata Haw., H. atrofusca G. G. Smith, H. angustifolia var. albensis (schoenl.) V. Polln., H. maughanii V. Polln., H. sp. aff. baccata, H. sp. nov. (Retusae sect), H. turgida var. pallidifolia G. G. Smith and H. retusa (L) Haw. were used as experimental materials. The explants con- sisted of gynoecia and segments of inflorescence axis without or with flowers. The inflorescence

1 Received for publication 20 September 1971. We thank Professor H. P. Riley for allowing us to use

his personal collection of Haworthia spp.

was cut into approximately one-inch long segments. These were surface sterilized with 4 % sodium hypochlorite solution for 5-10 min and were washed twice with autoclaved double dis- tilled water. Gynoecia were dissected out of surface sterilized flowers or floral buds. The gynoecia and segments of inflorescence were aseptically inoculated either on a modified (Boll and Street, 1951) White's basal medium (WB) or WB supplemented with various concentrations of N-6-benzyladenine (BA), kinetin (K), indole- 3-acetic acid (IAA), a-naphthaleneacetic acid (NAA), casein hydrolysate (CH), and coconut milk (CM).

Experiments for establishment of cultures on chemically defined medium and chemical control of differentiation were carried out on (1) the callus obtained from the explants of Haworthia sp. nov. (Retusae sect) and (2) inflorescence segments and gynoecia of Haworthia sp. nov. (Re- tusae sect) and H. turgida var. pallidifolia G. G. Smith. For these experiments a modified (Kasper- bauer and Reinert, 1965) Murashige and Skoog's medium without or with supplements was used.

The solid media were jelled with 0.8 % agar. These were sterilized by autoclaving at 120 C and 18 psi pressure for 20 min. The pH of both liquid and agar media was adjusted to 5.8 before autoclaving.

The cultures were maintained at 26 1 C under continuous fluorescent white illumination of 225- 250 ft-c.

For anatomical studies, in vitro grown materials were fixed in formalin-acetic-alcohol, dehydrated in an ethyl alcohol-xylene series, and embedded in paraffin. The embedded material was sectioned at 10 and 14 p with a rotary microtome, and the sections were stained with safranin and fast green.

377



378 AMERICAN JOURNAL OF BOTANY [Vol. 59

TABLE 1. Growth responses of Haworthia explants on WB and WB with supplements

Growth response of gynoecia and inflorescence

Composition of Medium segments

WB No growth WB + NAA (2.0 mg/i) No growth

+ K (0.5 mg/1) WB + BA (0.5 mg/i) No growth WB + IAA (1 mg/1) No growth

+ K (0.5 mg/i) WB + IAA (1 mg/i) No growth

+ K (0.5 mg/i) + CH (1 or2g/1)

WB + IAA (1 mg/I) Callusing and shoot + K (0.5 mg/i) differentiation + CM (20%, v/v)

WB + IAA (1 mg/i) Callusing and shoot + CM (20 %, v/v) differentiation

WB + K (0.5 mg/i) Callusing and shoot + CM (20%, v/v) differentiation

WB + CM (20%, v/v) Callusing and shoot differentiation

WB + CM (10%, v/v) Callusing and shoot differentiation

RESULTS-The growth response of gynoecia and inflorescence segments on different nutrient media containing White's modified nutrients (without or with supplements) has been summarized in Table 1. Presence of coconut milk in these media was necessary for growth of the explants. Ten or twenty percent coconut milk without or with IAA and/or kinetin was equally effective for growth and differentiation.

Responses of gynoecia-On media containing coconut milk, the gynoecia grew 4-6 times their original size within a week after inoculation. In some instances, no further growth took place, and the gynoecia became pale and shrivelled after 2-3 weeks. However, in most of the cultures 4-6 weeks after inoculation, the base of the ovary proliferated into a callus mass (Fig. 1, 7) which differentiated into vegetative buds (Fig. 2). The callus produced from the ovary wall was repeatedly

subcultured on coconut milk medium. It grew as callus for 3-4 weeks, and then differentiation of vegetative buds occurred on the surface of the callus.

The locules were found to be empty in most cases (Fig. 7), but in two pollinated gynoecia of H. variegata which were grown on WB + IAA (1.0 mg/liter), fruit maturation and seed formation occurred. The seeds germinated five weeks after inoculation while still within the capsule (Fig. 3). However, these seedlings did not grow into mature plants and became pale and died when they were 2-3 weeks old.

Responses of inflorescence segments-The inflorescence segments showed two kinds of growth responses:

(1) Production of callus in the axils of non- flower-bearing or flower-bearing bracts (Fig. 5).

(2) Production of callus in extra-axillary position on the inflorescence axis (Fig. 4, 8).

Rarely, on media containing WB + IAA (1 mg/liter) + kinetin (0.5 mg/liter), 4-6 vegetative buds were produced on the inflorescence axis without any callus formation.

The callus in the axils of fertile bracts was produced by the proliferation of the pedicel (Fig. 9). The callus in the axils of sterile bracts was a re- sult of proliferation of the peripheral layers of the inflorescence axis. Numerous meristematic protuberances differentiated on the surface of the callus (Fig. 13). These protuberances later gave rise to vegetative buds (Fig. 12). Callus derived from gynoecia and inflorescence segments con- sisted of parenchymatous cells with a few interspersed tracheidal cells (Fig. 10). There were several pockets of meristematic tissue within the callus mass (Fig. 10). Extra-axillary callus on the inflorescence axis had a well-defined marginal meristem (Fig. 11) in addition to the pockets of meristematic cells. Rarely, root differentiation occurred on extra-axillary callus (Fig. 4).

The vegetative buds which differentiated from the ovary or the inflorescence callus produced roots when subcultured on a coconut milk medium. These plants were successfully transferred to soil (Fig. 6).

KEY TO LABELLING: br, bract; c, callus; fl, flower; fr, fruit; g, gynoecium; i, inflorescence axis; in, inflorescence; Ip, leaf primordium; mh, meristematic hump; mm, marginal meristem; pm, pocket of meristematic cells; r, root; sa, shoot apex; sb, shoot bud; sh, shoot; sl, seedling; tr, tracheidal cell.

Fig. 1-6. Morphogenetic responses of Haworthia explants.-Fig. 1. Callusing at the base of gynoecium of H. maughanii 40 days after inoculation. X 1.5.-Fig. 2. Differentiation of shoot buds from gynoecium callus of H. maughanii 55 days after inoculation. X 1.5.-Fig. 3. Germinating seeds (still within the capsule) of H. variegata 40 days after inoculation. X 1.5.-Fig. 4. Callus production from the cut end of the inflorescence axis of H. chloracantha 40 days after inoculation. Notice the differentiation of roots on the callus. X 2.-Fig. 5. Callusing and shoot-bud differentiation in the axil of flower-bearing bract of H. maughanii 55 days after inoculation. X 1.5.-Fig. 6. Flowering plant of Haworthia sp. nov. (Retusae sect). This plant had differentiated in vitro from inflorescence axis callus. X 0.4.



April, 1972] KAUL AND SABHARWAL-MORPHOGENESIS IN HAWORTHIA 379

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Fig. 14-17. Establishment of Haworthia tissue culture on a chemically defined medium.-Fig. 14. Shoot differentiation on MS after first subculture from CM medium. X 1.8.-Fig. 15. Shoot differentiation on MS after four subcultures. Notice the small shoot buds. x 1.8. Fig. 16. Callus production from the pedicel on MS. X 1.8.-Fig. 17. Callus production and shoot and root differentiation from gynoecium cultured on MS. X 1.8. All photographs are of cultures of Haworthia sp. nov. (Retusae sect) 60 days after inoculation. See p. 378 for Key to Labelling.

Establishment of Haworthia callus on chemically defined medium-In order to obtain an in vitro chemical control of callusing and differentiation, it was necessary to grow the tissue on a

chemically defined medium. Since some investi- gators (Carter et al., 1967; Yamada et al., 1967; Sheridan, 1968) have been successful in growing monocotyledonous tissues on Murashige and

Fig. 7-13. Morphogenesis of Haworthia tissue culture on coconut milk media.-Fig. 7. L. S. of gynoecium of H. maughanii, showing callusing at the base 40 days after inoculation. Notice the empty locules. X 10.-Fig. 8. L. S. of a callusing inflorescence axis of a hybrid (H. truncata X H. setata) 55 days after inoculation. Observe the extra-axillary position of the callus below the bract. X 6.6.-Fig. 9. L. S. of flower of H. maughanii, showing callus at pedicel 55 days after inoculation. X 10.5.-Fig. 10. Area 'A' in Fig. 7. Most of the callus is parenchymatous with a few meristematic pockets and interspersed tracheidal cells. X 175.-Fig. 11. Area 'B' of Fig. 8 enlarged to show the pockets of meristematic cells and marginal meristem. X 150.-Fig. 12. L. S. shoot buds which differentiated on inflorescence axis callus of H. turgida var. pallidifolia 55 days after inoculation. Note the shoot apex and leaf pri- mordia. X 20.-Fig. 13. Meristematic humps on callus of H. turgida var. pallidifolia 55 days after inoculation. X 40. See p. 378 for Key to Labelling.




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Skoog's medium, we used a modification (Kasper- bauer and Reinert, 1965) of this medium (MS) without or with supplements. Both ovary and inflorescence calli grew and differentiated on MS. For the first subculture the shoot buds differenti- ating on MS were as numerous and as large as those on the coconut milk media (Fig. 14). How- ever, in the subsequent subcultures the size and number of the shoot buds was markedly reduced (Fig. 15). The growth and differentiation of callus occurred on both solid and liquid media during repeated subculturing.

It has also been observed that MS devoid of inositol cannot support the growth of Haworthia tissue. If inositol level in the medium is decreased from the original 100 mg/liter to 75, 50, or 25 mg/liter the growth and differentiation of tissue are adversely affected.

Gynoecia and inflorescence segments from greenhouse-grown plants were inoculated on MS to find out whether callusing and differentiation could be induced. These explants produced callus (Fig. 16, 17). The callus later differentiated into many shoot buds (Fig. 17). Occasionally, root differentiation also occurred on MS (Fig. 17). The mode of callusing and differentiation was similar to that described earlier for coconut milk media.

Chemical control of differentiation-Since both growth and differentiation of Haworthia callus occurred on MS, experiments were conducted to achieve chemical control of root and shoot differentiation by supplying appropriate concentrations of an auxin and a cytokinin in the medium. The in vitro control of differentiation by providing appropriate concentrations of auxins and cytokinins in culture media has been achieved for several dicotyledonous tissues. Haworthia tissue was grown on MS supplemented with 2,4-dichlo- rophenoxyacetic acid (2,4-D) or kinetin-alone or in various combinations.

Root differentiation was induced by 0.2, 0.5, 1.0, and 2.0 mg/liter 2,4-D. Within this range of concentrations, optimum root differentiation occurred on media supplemented with 0.2 or 0.5 mg/liter 2,4-D (Fig. 18, 19). On these media 10-15 roots differentiated in each culture 10 days

after inoculation. Most of the roots differentiated on the callus surface away from the medium but were positively geotropic and grew towards the medium. On 0.2 mg/liter 2,4-D the roots were often green and thick (Fig. 18); on 0.5 mg/liter of 2,4-D the roots were yellowish and thin (Fig. 19). With the increasing 2,4-D concentration in the medium the roots were poorly developed, short, and few in number (Fig. 20). Rooting could not be induced on higher concentrations (4.0 mg/liter and 6.0 mg/liter) of 2,4-D. Shoot differentiation did not occur on media supplemented with 2,4-D only. The callus became pro- gressively friable and pale with increasing amounts of 2,4-D in the media. Rooting was markedly inhibited by kinetin except on a medium containing 0.2 mg/liter kinetin and 0.2 mg/liter of 2,4-D (Fig. 21). On all other combinations of 2,4-D and kinetin (0.2 mg/liter 2,4-D with 0.5, 1.0, or 2.0 mg/liter kinetin and 0.5, 1.0, or 2.0 mg/liter 2,4-D with 0.2, 0.5, 1.0, or 2.0 mg/liter kinetin) root differentiation was completely suppressed and only callus was produced.

Induction of shoots occurred on MS without or with added kinetin (0.2, 0.5, 1.0, and 2.0 mg/ liter). Numerous shoots differentiated, but they were smaller than those which were produced on coconut milk media. A medium with 1.0 mg/liter kinetin proved optimal for shoot differentiation (Fig. 22). Shoot differentiation also occurred on medium with 2.0 mg/liter kinetin, but the shoots were smaller than those produced on the medium containing 1.0 mg/liter (Fig. 23). Higher concentrations (4.0 or 6.0 mg/liter) suppressed shoot formation, and only callus was produced (Fig. 24). All concentrations of 2,4-D (0.2, 0.5, 1.0, and 2.0 mg/liter) completely suppressed shoot differentiation regardless of the kinetin concentration. Unlike 2,4-D media, those containing kinetin induced the formation of nonfriable callus.

Since none of the above media induced both roots and shoots, and root differentiation on MS was only occasional, naphthaleneacetic acid (NAA) was tried instead of 2,4-D. A medium with 0.2 mg/liter NAA and 0.2 mg/liter kinetin induced both roots and shoots on the callus (Fig. 25).

Fig. 18-25. Control of root and shoot differentiation in Haworthia tissue culture.-Fig. 18. Differentiation of well-developed roots on MS + 0.2 mg/liter 2,4-D. X 1.3.-Fig. 19. Root differentiation on MS + 0.5 mg/liter 2,4-D. These roots are thinner than those on 0.2 mg/liter 2,4-D medium. X 1.3.-Fig. 20. Inhibition of root differentiation on MS + 2.0 mg/liter 2,4-D. Notice the few poorly developed roots and the friable callus. X 1.3.-Fig. 21. Differ- entiation of roots on MS + 0.2 mg/liter 2,4-D + 0.2 mg/liter kinetin. This was the only combination of 2,4-D and kinetin which induced organ differentiation. X 1.3.-Fig. 22. The induction of numerous shoot buds without any roots on MS + 1.0 mg/liter kinetin. X 1.8.-Fig. 23. Differentiation of shoot buds on MS + 2.0 mg/liter kinetin. These buds are smaller than those produced on MS + 1.0 mg/liter kinetin. X 1.8.-Fig. 24. Callus formation without any organ differentiation on MS + 6.0 mg/liter kinetin. X 1.8.-Fig. 25. Differentiation of roots and shoots on MS + 0.2 mg/liter kinetin + 0.2 mg/liter NAA. X 1.8. All photographs are of cultures of Haworthia sp. nov. (Retusae sect) 63 days after inoculation. See p. 378 for Key to Labelling.




DIscUSSION-Several workers (Morel and Wet- more, 1951; Nickell, 1964; Yatazawa et al., 1967; Takatori et al., 1968; Saka and Maeda, 1969; Kaul and Sabharwal, 1970; Majumdar, 1970a, b; Mullin, 1970; Ziv et al., 1970; Masteller and Holden, 1970; Furuhashi and Yatazawa, 1970) have reported the establishment of tissue cultures of monocotyledons on media supplemented with complex nutrients; e.g., coconut milk, casein hydrolysate, and yeast extract. Recently, there have been some reports on the establishment of monocotyledonous tissue cultures on chemically defined media (Carter et al., 1967; Nishi, Yamada, and Takahashi, 1968; Wilmar and Hellendoorn, 1968; Sheridan, 1968). Our observations show that Haworthia tissue grows and differentiates on Murashige and Skoog's modified medium without any additional supplements. Callus formation on inflorescence segments and gynoecia, and its subsequent differentiation into shoots, was induced on this medium.

Haworthia tissue did not grow on a modified White's basal medium unless the medium was supplemented with coconut milk. Murashige and Skoog's medium, unlike White's medium, contains NH4NO3 and inositol. It seems that inositol and ammonium nitrogen are required for growth and differentiation of Haworthia tissue. Coconut milk has both inositol and ammonium nitrogen (Shantz and Steward, 1964) and, therefore, can support the growth and differentiation of this tissue. On the other hand, casein hydrolysate, which is a rich source of nitrogen but does not contain any inositol, did not support the growth and differentiation of Haworthia callus. These observations seem to explain the failures of monocotyledonous tissue cultures on chemically defined media until the adoption of Murashige and Skoog's nutrients, in- cluding both inositol and ammonium nitrogen.

The in vitro fruit maturation and seed germina- tion in H. variegata reported in this paper, though very infrequent, are of interest. These observations indicate that media composed of White's nutrients without coconut milk do not meet the requirements of growing seedlings of Haworthia. The seeds germinated in two cultures which were devoid of coconut milk. The seedlings grew only for a few days, during which time they most probably got their nutrition from the stored food in the seed. Once this food was exhausted the seedlings became etiolated and died, probably due to nitrogen and inositol deficiency in the medium.

Partial control of organ differentiation in monocotyledonous tissue cultures has been reported by some workers (Carter et al., 1967; Nishi et al., 1968; Wilmar and Hellendoorn, 1968; Sheridan, 1968). They reported induction of shoots and a few roots in various monocotyledonous tissue cultures in absence of any auxin or cytokinin in the culture medium. Observations here, that differentiation of shoots and occasionally of roots occurs

in Haworthia tissue on modified Murashige and Skoog's medium, are in agreement with these reports.

Shimada, Sasakuma, and Tsunewaki (1969) reported establishment of wheat tissue culture and its subsequent differentiation into roots and shoots. These authors could also control differentiation of roots by providing appropriate concentrations of 2,4-D and kinetin in the medium. However, they could not control shoot differentiation. In the present work Haworthia tissue could be grown as callus or root and/or shoot differentiation could be induced by providing appropriate concentrations of auxin (2,4-D or NAA) and cytokinin (kinetin) in the medium. As far as we know, this is the first instance where a chemical control of callusing and root and shoot differentiation in a monocotyledonous tissue has been achieved on a chemically defined medium.

Shoot differentiation in Haworthia was completely inhibited by presence of 2,4-D in the medium. Similarly, root differentiation was inhibited by all concentrations of kinetin, except by 0.2 mg/liter in combination with 0.2 mg/liter 2,4-D. These observations are unlike those made by some other workers. Shimada et al. (1969) reported occasional shoot formation in wheat tissue in presence of up to 0.5 mg/liter 2,4-D. Saka and Maeda (1969) observed root formation in rice tissue cultures in presence of up to 5 x 10-6 M (1.075 mg/liter) kinetin. The differences in responses of these tissues might be due to the different levels of endogenous cytokinins and auxins in these tissues.

The observations made during the present study indicate that root and shoot differentiation in monocotyledonous tissues are controlled by the auxin/cytokinin ratio. These phenomena are well established for dicotyledonous tissues (Skoog and Miller, 1957; Schaeffer and Smith, 1963; Doer- schug and Miller, 1967). Furthermore, these observations show that inositol and ammonium nitrogen are essential nutritional requirements of Haworthia tissue cultures under the experimental conditions of the present study.

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morphogenetic studies on haworthia: establishment of tissue culture and control of differentiation

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